Molecularly Imprinted Polymers 2021

Methods in
Molecular Biology 2359
Antonio Martín-Esteban Editor
Molecularly
Imprinted
Polymers
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Molecularly Imprinted Polymers
Methods and Protocols
Edited by
Antonio Martín-Esteban
Departamento de Medio Ambiente y Agronomía, INIA-CSIC, Madrid, Spain
Editor
Antonio Martı́n-Esteban
Departamento de Medio Ambiente y Agronomı́a
INIA-CSIC
Madrid, Spain
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1628-4 ISBN 978-1-0716-1629-1 (eBook)
https://doi.org/10.1007/978-1-0716-1629-1
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2021
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface
Molecular imprinting is a very powerful approach for the preparation of synthetic polymers
having pre-designed molecular recognition properties. This approach uses a molecular
template to create binding sites in cross-linked polymers by means of free radical polymeri-
zation reactions. Once the molecular template is removed, the polymer matrix contains
cavities complementary in size, shape, and functionalities to the template, thus allowing its
selective rebinding. Due to their extraordinary stability and ease of preparation, molecularly
imprinted polymers (MIPs) have attracted great interest both in fundamental research and
for practical applications.
Accordingly, this book provides laboratory protocols describing the different steps
towards the synthesis of MIPs by different polymerization strategies (Chapters 1–9).
Besides, their use in sample preparation (Chapters 10–15) and their implementation on
the development of sensors (Chapters 16–18) are thoroughly described. Finally, applications
in other areas such as catalysis (Chapters 19 and 20) and the use of bioinformatics and
molecular modeling for MIPs design (Chapters 21 and 22) are also described.
I would like to thank all the authors for their excellent contributions and John Walker
(Editor-in-Chief of the Methods in Molecular Biology series), David C. Casey, and Anna
Rakovsky for their help in the preparation of this book.
Madrid, Spain Antonio Martı́n-Esteban
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Synthesis of Molecularly Imprinted Polymers by Two-Step Swelling

and Polymerization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Jun Haginaka
2 Molecularly Imprinted Polymeric Nanoparticles by Precipitation
Polymerization and Characterization by Quantitative NMR
Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Clovia I. Holdsworth, K. Fremielle Lim,
and Phonlakrit Muang-Non
3 MIP Synthesis and Processing Using Supercritical Fluids. . . . . . . . . . . . . . . . . . . . . 19
Ana I. Furtado, Raquel Viveiros, and Teresa Casimiro
4 Synthesis of Bacteria Imprinted Polymers by Pickering Emulsion
Polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Haiyue Gong, Xiantao Shen, and Lei Ye
5 Restricted Access Molecularly Imprinted Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Mariana Azevedo Rosa, Tássia Venga Mendes,
and Eduardo Costa Figueiredo
6 Synthesis of Double-Layer Imprinted Polymers: BSA Depletion . . . . . . . . . . . . . . 71
Okan Zenger and Gözde Baydemir Peşint
7 Magnetic MIPs: Synthesis and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Rafael da Fonseca Alves, Lucas Neres Chagas da Silva,
Gilberto Matos Neto, Isabela Fernandes Ierick,
Thiago Lima Ferreira, and Maria Del Pilar Taboada Sotomayor
8 Water-Compatible Fluorescent Molecularly Imprinted Polymers . . . . . . . . . . . . . . 97
Huiqi Zhang
9 Generation of High-Affinity Aptamer-MIP Hybrid Nanoparticles . . . . . . . . . . . . . 109
Mark Sullivan, Rachel Hand, and Nicholas Turner
10 Molecularly Imprınted Solıd-Phase Extractıon Sorbents for the Selectıve
Extraction of Drugs from Human Urine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Hasan Basan
11 Molecularly Imprinted Polymers Coupled with Surface-Enhanced
Raman Spectroscopy to Detect Chemical Hazards in Foods . . . . . . . . . . . . . . . . . . 131
Marti Z. Hua, Shaolong Feng, and Xiaonan Lu
12 Molecularly Imprinted Polymer for a Smart Dispersive Micro-Solid
Phase Extraction Technique for Assessing Trace Level Aflatoxins
in Cultured Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
G. D. Thilini Madurangika Jayasinghe and Antonio Moreda-Piñeiro
vii
viii Contents
13 Preparation of Monolithic Fibers in Fused Silica Capillary Molds

for Molecularly Imprinted Solid-Phase Microextraction. . . . . . . . . . . . . . . . . . . . . . 153
Esther Turiel Trujillo and Myriam Dı́az-Álvarez
14 Quick, Easy, Cheap, and Effective Method for the Synthesis
of Rugged Magnetic Molecularly Imprinted Stir-Bars . . . . . . . . . . . . . . . . . . . . . . . 163
Patricia Regal, Monica Dı́az-Bao, and Alberto Cepeda
15 Determination of Neopterin as a Prognostic Indicator
Using Neopterin-Imprinted Cryogel Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Okan Zenger, Burcu Eren, Pırıl Arısoy, Sibel Özdaş,
and Gözde Baydemir Peşint
16 Fluorescence Sensing with Molecularly Imprinted Polymer-Capped
Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Hanieh Montaseri, Heidi Abrahamse, and Patricia B. C. Forbes
17 Dual-Fluorescent Nanoparticle Probes Consisting of a Carbon Nanodot
Core and a Molecularly Imprinted Polymer Shell . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Shan Jiang, Kornelia Gawlitza, and Knut Rurack
18 Molecularly Imprinted Polymer-Based Quartz Crystal Microbalance
Sensor for the Clinical Detection of Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Duygu Çimen, Nilay Bereli, Fatma Kartal, and Adil Denizli
19 Preparing Selective Nanozymes by Molecular Imprinting . . . . . . . . . . . . . . . . . . . . 223
Yuqing Li, Xiaohan Zhang, and Juewen Liu
20 Enzyme-Mimics Molecularly Imprinted Polymers Based on Metal
Complexes: Electropolymerization and Electrocatalytic Application . . . . . . . . . . . 233
Elisabetta Mazzotta and Cosimino Malitesta
21 Using Molecular Dynamics in the Study of Molecularly Imprinted
Polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Gustaf D. Olsson, Jesper G. Wiklander, and Ian A. Nicholls
22 The Search for Peptide Epitopes for Molecular Imprinting Through
Bioinformatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Alessandra Maria Bossi and Laura Pasquardini
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Contributors
HEIDI ABRAHAMSE • Faculty of Health Sciences, Laser Research Centre, University of

Johannesburg, Doornfontein, South Africa
PIRIL ARISOY • Department of Bioengineering, Adana Alparslan Türkeş Science and
Technology University, Adana, Turkey
HASAN BASAN • Faculty of Pharmacy, Department of Analytical Chemistry, Gazi University,
Etiler, Ankara, Turkey
GÖZDE BAYDEMIR PEŞINT • Department of Bioengineering, Adana Alparslan Türkeş Science
and Technology University, Adana, Turkey
NILAY BERELI • Department of Chemistry, Hacettepe University, Ankara, Turkey
ALESSANDRA MARIA BOSSI • Department of Biotechnology, University of Verona, Verona, Italy
TERESA CASIMIRO • LAQV-REQUIMTE, Chemistry Department, NOVA School of Science
and Technology, NOVA University of Lisbon, Caparica, Portugal
ALBERTO CEPEDA • Department of Analytical Chemistry, Nutrition and Bromatology,
Universidade de Santiago de Compostela, Lugo, Spain
DUYGU ÇIMEN • Department of Chemistry, Hacettepe University, Ankara, Turkey
RAFAEL DA FONSECA ALVES • Institute of Chemistry, State University of São Paulo and
National Institute for Detection of Alternative Technologies, Toxicological Evaluation and
Removal of Micropollutants and Radioactives (INCT-DATREM), Araraquara, SP,
Brazil
LUCAS NERES CHAGAS DA SILVA • Institute of Chemistry, State University of São Paulo and
Brazil
ADIL DENIZLI • Department of Chemistry, Hacettepe University, Ankara, Turkey
MYRIAM DÍAZ-ÁLVAREZ • Department of Environment and Agronomy, Instituto Nacional de
Investigacion y Tecnologı́a Agraria y Alimentaria (INIA-CSIC), Madrid, Spain
MÓNICA DÍAZ-BAO • Department of Analytical Chemistry, Nutrition and Bromatology,
BURCU EREN • Department of Bioengineering, Adana Alparslan Türkeş Science and
SHAOLONG FENG • Food, Nutrition and Health Program, Faculty of Land and Food Systems,
The University of British Columbia, Vancouver, BC, Canada
THIAGO LIMA FERREIRA • Institute of Chemistry, State University of São Paulo and National
Institute for Detection of Alternative Technologies, Toxicological Evaluation and Removal
of Micropollutants and Radioactives (INCT-DATREM), Araraquara, SP, Brazil
EDUARDO COSTA FIGUEIREDO • Laboratory of Toxicant and Drug Analysis, Federal
University of Alfenas, Alfenas, MG, Brazil
PATRICIA B. C. FORBES • Faculty of Natural & Agricultural Sciences, Department of
Chemistry, University of Pretoria, Pretoria, South Africa
ANA I. FURTADO • LAQV-REQUIMTE, Chemistry Department, Nova School of Science and
Technology, NOVA University of Lisbon, Caparica, Portugal
KORNELIA GAWLITZA • Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin,
Germany
ix
x Contributors
HAIYUE GONG • Division of Pure and Applied Biochemistry, Department of Chemistry, Lund
University, Lund, Sweden
JUN HAGINAKA • Institute of Biosciences, Mukogawa Women’s University, Nishinomiya,
Japan
RACHEL HAND • School of Pharmacy, De Montfort University, Leicester, UK
CLOVIA I. HOLDSWORTH • Discipline of Chemistry, School of Environmental and Life
Sciences, University of Newcastle, Callaghan, NSW, Australia
MARTI Z. HUA • Department of Food Science and Agricultural Chemistry, McGill
University Macdonald Campus, Sainte-Anne-de-Bellevue, QC, Canada
ISABELA FERNANDES IERICK • Institute of Chemistry, State University of São Paulo and
Brazil
G. D. THILINI MADURANGIKA JAYASINGHE • Trace Element, Spectroscopy and Speciation
Group (GETEE), Strategic Grouping in Materials (AEMAT), Department of Analytical
Chemistry, Nutrition and Bromatology, Faculty of Chemistry, Universidade de Santiago de
Compostela, Santiago de Compostela, Spain
SHAN JIANG • Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin, Germany
FATMA KARTAL • Department of Chemistry, Hacettepe University, Ankara, Turkey
YUQING LI • Department of Chemistry, Waterloo Institute for Nanotechnology, University of
Waterloo, Waterloo, ON, Canada
K. FREMIELLE LIM • Discipline of Chemistry, School of Environmental and Life Sciences,
University of Newcastle, Callaghan, NSW, Australia
JUEWEN LIU • Department of Chemistry, Waterloo Institute for Nanotechnology, University
of Waterloo, Waterloo, ON, Canada
XIAONAN LU • Department of Food Science and Agricultural Chemistry, McGill University
Macdonald Campus, Sainte-Anne-de-Bellevue, QC, Canada
COSIMINO MALITESTA • Laboratorio di Chimica Analitica, Dipartimento di Scienze
e Tecnologie Biologiche e Ambientali (Di.S.Te.B.A.), Universita ` del Salento, Lecce, Italy
GILBERTO MATOS NETO • Institute of Chemistry, State University of São Paulo and National
Institute for Detection of Alternative Technologies, Toxicological Evaluation and Removal
of Micropollutants and Radioactives (INCT-DATREM), Araraquara, SP, Brazil
ELISABETTA MAZZOTTA • Laboratorio di Chimica Analitica, Dipartimento di Scienze
e Tecnologie Biologiche e Ambientali (Di.S.Te.B.A.), Universita ` del Salento, Lecce, Italy
TÁSSIA VENGA MENDES • Laboratory of Toxicant and Drug Analysis, Federal University of
Alfenas, Alfenas, MG, Brazil
HANIEH MONTASERI • Faculty of Health Sciences, Laser Research Centre, University of
Johannesburg, Doornfontein, South Africa; Faculty of Natural & Agricultural Sciences,
Department of Chemistry, University of Pretoria, Pretoria, South Africa
ANTONIO MOREDA-PIÑEIRO • Trace Element, Spectroscopy and Speciation Group (GETEE),
Strategic Grouping in Materials (AEMAT), Department of Analytical Chemistry,
Nutrition and Bromatology, Faculty of Chemistry, Universidade de Santiago de
Compostela, Santiago de Compostela, Spain
PHONLAKRIT MUANG-NON • Discipline of Chemistry, School of Environmental and Life
Sciences, University of Newcastle, Callaghan, NSW, Australia
IAN A. NICHOLLS • Bioorganic & Biophysical Chemistry Laboratory, Department of
Chemistry & Biomedical Sciences, Centre for Biomaterials Chemistry, Linnaeus University,
Kalmar, Sweden
Contributors xi
GUSTAF D. OLSSON • Bioorganic & Biophysical Chemistry Laboratory, Department of

Kalmar, Sweden
SIBEL ÖZDAŞ • Department of Bioengineering, Adana Alparslan Türkeş Science and
LAURA PASQUARDINI • Indivenire Srl, Povo Trento, Italy
PATRICIA REGAL • Department of Analytical Chemistry, Nutrition and Bromatology,
MARIANA AZEVEDO ROSA • Laboratory of Toxicant and Drug Analysis, Federal University of
Alfenas, Alfenas, MG, Brazil
KNUT RURACK • Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin,
Germany
XIANTAO SHEN • Institute of Environmental Medicine, School of Public Health, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan, China
MARIA DEL PILAR TABOADA SOTOMAYOR • Institute of Chemistry, State University of São
Paulo and National Institute for Detection of Alternative Technologies, Toxicological
Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM),
Araraquara, SP, Brazil
MARK SULLIVAN • School of Pharmacy, De Montfort University, Leicester, UK
ESTHER TURIEL TRUJILLO • Department of Environment and Agronomy, Instituto Nacional
de Investigacion y Tecnologı́a Agraria y Alimentaria (INIA-CSIC), Madrid, Spain
NICHOLAS TURNER • School of Pharmacy, De Montfort University, Leicester, UK
RAQUEL VIVEIROS • LAQV-REQUIMTE, Chemistry Department, NOVA School of Science
and Technology, NOVA University of Lisbon, Caparica, Portugal
JESPER G. WIKLANDER • Bioorganic & Biophysical Chemistry Laboratory, Department of
Kalmar, Sweden
LEI YE • Division of Pure and Applied Biochemistry, Department of Chemistry, Lund
University, Lund, Sweden
OKAN ZENGER • Department of Bioengineering, Adana Alparslan Türkeş Science and
HUIQI ZHANG • State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of
Functional Polymer Materials (Ministry of Education), Collaborative Innovation Center
of Chemical Science and Engineering (Tianjin), and College of Chemistry, Nankai
University, Tianjin, P. R. China
XIAOHAN ZHANG • Department of Chemistry, Waterloo Institute for Nanotechnology,
University of Waterloo, Waterloo, ON, Canada
Chapter 1
Synthesis of Molecularly Imprinted Polymers by Two-Step

Swelling and Polymerization
Jun Haginaka
Abstract
Synthesis of a molecularly imprinted polymer (MIP) by two-step swelling and polymerization is described.
Monodisperse, spherical MIP particles, whose diameters are ca. 5–9μm, are prepared using a polystyrene
particle as a shape template and dibutyl phthalate as an activating solvent. The obtained MIPs are suitable
for separation media in liquid chromatography or solid-phase extraction media. Procedures for synthesis of
MIPs and restricted access media (RAM)-MIP, packing of MIPs and RAM-MIPs, and application of MIPs
and RAM-MIPs for selective separation and extraction of a target compound(s) are described.
Key words Molecularly imprinted polymers, Restricted access media, Two-step swelling and poly-
merization, Monodisperse particles, Liquid chromatography, Solid-phase extraction
1 Introduction
There are some methods to prepare microsphere MIPs [1–

3]. Two-step swelling and polymerization method is one of the
methods that give monodisperse, spherical beads.
Two-step swelling was a method for the preparation of mono-
disperse polymer particles with large particles size [4], which are
useful as polymer particle-based separation media in LC [5]. In this
method, first, a slightly water-soluble compound was absorbed
onto seed polymer particles, which have become to absorb water
insoluble, relatively low molecular weight compounds. By this
two-step swelling process, the low molecular weight compounds
formed stable o/w emulsions with a droplet size and narrow size
distribution. This is the reason why we can make monodisperse
polymer particles. The advantages of two-step swelling and poly-
merization method are that it can make monodisperse, spherical
particles for suitable as LC separation media, and easily perform the
surface modification of MIPs in situ [3]. The disadvantage of the
method is that interactions between a template molecule and a
Antonio Martı́n-Esteban (ed.), Molecularly Imprinted Polymers: Methods and Protocols, Methods in Molecular Biology, vol. 2359,
https://doi.org/10.1007/978-1-0716-1629-1_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2021
1
2 Jun Haginaka
functional monomer could be interfered since water is used as a

continuous phase [3]. Though the enantioseparation factor of
racemic naproxen on MIPs for (S)-naproxen prepared by two-step
swelling and polymerization was lower than that prepared by bulk
polymerization in non-aqueous environments, the former gave
higher column efficiency than the latter [6].
Two-step swelling and polymerization was first employed for
the preparation of MIPs for 1,5-diaminonaphthalene and
1,8-diaminonaphthalene using ethylene glycol dimethacrylate
(EDMA) as a cross-linker and acrylic acid as a functional monomer
[7]. A lot of MIPs so far have been prepared by this method
[6, 8]. Those MIPs could be applied to the enantioseparation of
racemic drugs and compounds and selective extraction of a target
compound and its structurally related analogs [3]. Figure 1 illus-
trates the preparation method of MIPs by two-step swelling and
polymerization. Approximately 1μm polystyrene seed particle was
used as a shape template, and dibutyl phthalate was used as an
activating solvent in the first-step swelling step. Next, a dispersion
of porogen and initiator was adsorbed onto the swollen particle in
the second-step swelling. Further, a dispersion of template mole-
cule, functional monomer, and cross-linker was added to the dis-
persion of the swollen particle. After completion of the swelling,
polymerization procedure was started and monodisperse, spherical
MIPs were obtained. As mentioned above, the surface modification
of MIPs prepared by two-step swelling and polymerization could
be easily performed: hydrophilic monomers such as glycerol mono-
methacrylate (GMMA) and glycerol dimethacrylate (GDMA) were
added in situ 4 h after polymerization and then further polymeriza-
tion was carried out for 20 h as shown in Fig. 1. In this method,
restricted access media (RAM)-MIPs [9, 10], which are used for
direct injection of biological and environmental samples without
any pretreatments such as protein precipitation, could be easily
prepared.
(S)-Nilvadipine is one of dihydropyridine calcium antagonists.
MIPs for (S)-nilvadipine were prepared using 4-vinylpyridine
(4-VPY), EDMA, and toluene as a functional monomer, cross-
linker, and porogen, respectively [8]. MIPs prepared using 4-VPY
gave the highest enantioseparation factors (imprinting factors)
among the acidic [methacrylic acid and 2-(trifluoromethyl)acrylic
acid)] and basic (4-VPY and 2-vinylpyridine) functional monomers
tested [8]. 1H-NMR and molecular modeling studies suggested the
intermolecular hydrogen bonding formation between the N-H(S)-
nilvadipine and N4-VPY residues [8].
MIP-based solid-phase extraction (SPE) has leakage problems
that the leaked template molecule prevents the accurate and precise
assay for a template molecule. In order to overcome the problems, a
structurally related analog or a deuterated molecule (a dummy-
template molecule) has been used to prepare MIPs [9, 11–
Synthesis of MIPs by Two-Step Swelling and Polymerisation 3
Fig. 1 Preparation of MIPs and RAM-MIPs by two-step swelling and polymerization
13]. RAM-MIPs for bisphenol A (BPA)-d16 were prepared and

applied to MIP-based SPE of BPA in river water samples [9].
2 Materials
2.1 Reagents Prepare all solutions using ultrapure water (prepared by purifying
deionized water with electrical resistivity of 18 MΩ cm or more at
25 C) and analytical-grade reagents. For LC and LC–MS, respec-
tively, applications, use LC- and MS-grade reagents and solvents.
Prepare all solutions before use and store all reagents and solvents
according to the indication of the manufacturer. MIPs and
RAM-MIPs, respectively, were prepared using a fixed amount of a
cross-linker (25 mmol and 17.5 mol) by changing amounts of a
template molecule and functional monomer. The obtained
amounts of MIPs and RAM-MIPs were ca. 4–5 g.
2.2 Preparation Uniform-sized, polystyrene seed particles utilized as the shape

of Polystyrene Seed template were prepared by emulsifier-free emulsion polymerization
Particles and purified via a modified version of a previously reported
method [14]. The size of the seed particle was ca. 1μm in diameter.
1. Place 85 mL of water in a 200 mL three-neck round-bottom
flask provided with an anchor-shaped stirrer.
2. Add 3.80 mmol/L sodium chloride (see Note 1).
4 Jun Haginaka
3. Add 0.95 mmol/L styrene with stirring at 350 rpm and at

room temperature while nitrogen is bubbled through for
20 min to remove oxygen (see Note 2).
4. Admix a degassed solution of potassium peroxodisulfate (1.03
mno/L) in 65 mL water and then seal the reactor.
5. Start the polymerization for 24 h at 65 C.
6. After the reaction is completed, the particles were purified
through removal of the remaining monomer and salts by
repeated sedimentation in a centrifuge at 3000 g for
30 min and redispersion by sonication in water.
2.3 Preparation 1. Prepare the uniform-sized, polystyrene seed particles as in Sub-

of MIPs heading 2.2.
for (S)-Nilvadipine 2. Prepare a microemulsion from 0.48 mL of dibutyl phthalate as
an activating solvent (see Note 3), 0.02 g of sodium dodecyl
sulfate, and 5 mL of water by sonication (see Note 4).
3. Admix a water dispersion of the uniform-sized, polystyrene
seed particles (0.107 g/mL), 0.83 mL, with a microemulsion
prepared by procedure 2. This first-step swelling was carried
out at room temperature for 15 h with stirring at 125 rpm (see
Note 5) until micro oil droplets completely disappeared.
4. To the dispersion of the swollen particles, add a microemulsion
prepared from 0.375 g of 2,20 -azobis(2,4-dimethylvaleroni-
trile)(ADVN), 5 mL of toluene, 12.5 mL of water and 10 mL
of 4.8% polyvinyl alcohol solution. This second-step swelling
was carried out at room temperature for 2 h with stirring at
125 rpm.
5. To the dispersion of the swollen particles, add a dispersion of
4 mmol of (S)-nilvadipine, 6 mmol of 4-VPY, 25 mmol of
EDMA, 0.02 g of sodium dodecyl sulfate, 12.5 mL of water,
and 10 mL of 4.8% polyvinyl alcohol solution. This third-step
swelling was carried out at room temperature for 2 h with
stirring at 125 rpm.
6. After the third-step swelling was completed, start the polymer-
ization procedure at 50 C under argon atmosphere (see Note
6) with slow stirring for 24 h (see Note 7).
7. Pour the dispersion of polymerized particles into 250 mL of
water to remove the suspension stabilizer (polyvinyl alcohol)
and discard the supernatant after sedimentation of the particles.
Redisperse the polymer particles into methanol and discard the
supernatant again after sedimentation. This procedure was
repeated three times in methanol and twice in tetrahydrofuran
(THF), then the polymer particles were filtered on a membrane
filter and washed with THF and acetone followed by drying at
room temperature.
2.4 Preparation 1. Prepare the uniform-sized, polystyrene seed particles as in Sub-

of RAM-MIPs heading 2.2.
for BPA-d16 2. Admix a water dispersion of 0.085 mL of uniformly sized,
polystyrene seed particles (0.497 g/mL) with a microemulsion
prepared from 0.48 mL of dibutyl phthalate as activating sol-
vent, 0.02 g of sodium dodecyl sulfate, and 10 mL of water by
sonication (see Note 8). This first-step swelling was carried out
at room temperature for 15 h with stirring at 125 rpm until
micro oil droplets completely disappeared.
3. To the dispersion of swollen particles, add a microemulsion
prepared from 0.1875 g of ADVN as an initiator, 1.5 mL of
toluene (see Note 9) as a porogen, 10 mL of 4.8% polyvinyl
alcohol aqueous solution as a dispersion stabilizer, and
12.5 mL of water. This second-step swelling was carried out
at room temperature for 2 h with stirring at 125 rpm.
4. To the dispersion of swollen particles, add a dispersion of
2 mmol of BPA-d16 as a template molecule, 17.5 mmol of
EDMA (see Note 9) as a cross-linker, 4.5 mmol of 4-VPY as a
functional monomer, 10 mL of 4.8% polyvinyl alcohol solu-
tion, and 12.5 mL of water. This third-step swelling was carried
out at room temperature for 2 h with stirring at 125 rpm.
5. After completion of the third-step swelling, start the polymeri-
zation procedure at 50 C under argon atmosphere with stir-
ring at 160 rpm. Four hours after polymerization, add
hydrophilic monomers, 0.25 mL of GMMA, and 0.25 mL of
GDMA, together with 0.01 g of potassium peroxodisulfate as
an initiator to the polymerizing materials. Further polymeriza-
tion was carried out at 70 C for 20 h.
6. The prepared RAM-MIPs were washed and dried as in 2.3.
3 Methods
The prepared MIPs and RAM-MIPs, respectively, were packed into

a stainless steel column (100 mm 4.6 mm i.d. and 10 mm
4.0 mm i.d.) by a slurry packing technique using ca. 1 g and
100 mg of MIPs and RAM-MIPs in a mixture of 6.4 and
0.64 mL of methanol and 3.2 and 0.32 mL of 2-propanol as the
slurry solvent and methanol as the packing solvent at a constant
pressure of 9.8 MPa.
3.1 Separation 1. HPLC conditions: column, MIP for (S)-nilvadipine

of Nilvadipine (100 mm 4.6 mm i.d.); column temperature, 50 C; mobile
Enantiomers phase, H2O – acetonitrile (55:45, v/v); flow rate, 0.5 mL/min;
detection, 236 nm; loaded amount, 0.5μg.
6 Jun Haginaka
Fig. 2 Chromatograms of a racemic mixture of nilvadipine enantiomers (a) and

(S)-nilvadipine (b) on MIP for (S)-nilvadipine. Reproduced by permission of
Copyright © 2003, American Chemical Society [8]
2. Obtain chromatograms of a racemic mixture of nilvadipine

enantiomers and (S)-nilvadipine, respectively (Fig. 2a, b).
3.2 Simultaneous 1. Pretreatment conditions: column, RAM-MIP for BPA-d16

Determination (10 mm 4.0 mm i.d.); column temperature, 40 C; loaded
of Bisphenol A and Its amount, 2 mL of river water sample; washing mobile phase,
Halogenated water–acetonitrile (95:5, v/v) containing 5 mM ammonium
Derivatives acetate for 6 min at a flow rate of 1.0 mL/min.
in River Water 2. Transfer the concentrated BPA and its halogenated derivatives
to an analytical column by a back-flush mode using a mixture of
65 vol% mobile phase A (water) and 35 vol% mobile phase B
(acetonitrile) at a flow rate of 0.2 mL/min.
3. LC conditions: column, Cosmosil 5C18-MS-II
[50 mm 2.0 mm i.d. (Nacalai Tesque, Kyoto, Japan)];
mobile phase, a linear gradient of mobile phase B (35 vol%
for 8 min, increasing from 35 to 80 vol% for 10 min and 80 vol
% for 2 min); column temperature, 40 C; flow rate, 0.2 mL/
min.
4. MS conditions: ionization mode, electrospray (negative); data
collection, selected ion monitoring at m/z 227 for BPA, m/z
233 for BPA-d6, m/z 241 for BPA-d16, m/z 365 for
tetrachloro-BPA (Cl4-BPA) and m/z 542 for tetrabromo-
BPA (Br4-BPA); cone voltage, 40 V at m/z 227, 223, and
241, 50 V at m/z 365 and 60 V at m/z 542; capillary voltage,
3.0 kV; source temperature, 100 C; desolvation temperature,
350 C; flow rate of cone nitrogen, 50 L/h; flow rate of
desolvation nitrogen, 350 L/h.
Fig. 3 Chromatograms of river water sample by a column-switching LC–MS system with RAM-MIP for BPA-d16
as a pretreatment column. BPA-d6 was used as an internal standard. Reproduced by permission of Copyright
© 2006 Elsevier B.V [10]
5. Obtain chromatograms of river water sample by a column-

switching LC–MS system with RAM-MIP as a pretreatment
column (Fig. 3). BPA-d6 was used as an internal standard.
4 Notes
1. Sodium chloride was added to adjust the ionic strength of the

polymerization medium. The ionic strength was the important
factor to determine the size of the seed particle.
2. It is important to remove oxygen completely.
3. 1-Chlorododecane and dibutyl phthalate were used as the
activating solvent. We used the latter.
4. Ultrasonic disruptor provided with a frequency of 20 kHz
[e.g., UD-211 (Tomy Seiko Co., Tokyo, Japan)] was used.
5. A magnetic stirrer was used with a football-type stirring bar.
6. It is essential to polymerize under argon atmospheres.
7. Two hundred-mL three-neck round-bottom flask provided
with an anchor-shaped stirrer was used.
8. In Subheading 2.2, the particle diameter of the obtained MIPs
for (S)-nilvadipine was ca. 5μm. However, the ratio of
8 Jun Haginaka
polystyrene seed particle to dibutyl phthalate was changed to

make MIPs for BPA-d16, whose particle diameter is ca. 9μm.
9. For complete recovery of serum proteins, MIPs for BPA-d16,
which has narrow pore sizes than those of MIPs for
(S)-nilvadipine, were made. The ratio of toluene (progen) to
EDMA (cross-linker) was changed.
References
1. Haginaka J (2009) Molecularly imprinted Comparison of chiral recognition ability with
polymers as affinity-based separation media HPLC chiral stationary phases based on a pro-
for sample preparation. J Sep Sci tein. Anal Chem 75:191–198
32:1548–1565 9. Haginaka J, Sanbe H (2001) Uniform-sized
2. Wang H, Dong X, Yang M (2012) Develop- molecularly imprinted polymers for
ment of separation materials using controlled/ 2-arylpropionic acid derivatives selectively
living radical polymerization. Trends Anal modified with hydrophilic external layer and
Chem 31:96–108 their applications to direct serum injection
3. Orowitz TE, Patria SPPAAA, Rahayu D, Hasa- analysis. Anal Chem 72:5206–5210
nah AN (2020) Microsphere polymers in 10. Sambe H, Hoshina H, Hosoya K, Haginaka J
molecular imprinting: current and future per- (2006) Simultaneous determination of bisphe-
spectives. Molecules. https://doi.org/10. nol A and its halogenated derivatives in river
3390/molecules25143256 water by combination of isotope imprinting
4. Ugelstad J, Kaggerud KH, Hansen FK, Berge and liquid chromatography–mass spectrome-
A (1979) Absorption of low molecular weight try. J Chromatogr A 1134:16–23
compounds in aqueous dispersions of polymer- 11. Andersson LI, Paprica A, Arvidsson T (1997) A
oligomer particles, 2. A two step swelling pro- highly selective solid phase extraction sorbent
cess of polymer particles giving an enormous for pre-concentration of sameridine made by
increase in absorption capacity. Makromol molecular imprinting. Chromatographia
Chem 180:737–744 46:57–62
5. Hosoya K, Fréchet JMJ (1993) Influence of 12. Sanbe H, Haginaka J (2003) Restricted access
the seed polymer on the chromatographic media-molecularly imprinted polymer for pro-
properties of size monodisperse polymeric sep- pranolol and its application to direct injection
aration media prepared by a multi-step swelling analysis of β-blockers in biological fluids. Ana-
and polymerization method. Polym J Sci Part A lyst 128:593–597
Polym Chem 31:2129–2141 13. Nishimura K, Okamura N, Kimachi T, Hagi-
6. Haginaka J, Takehira H, Hosoya K, Tanaka N naka J (2019) Evaluation of molecularly
(1998) Molecularly imprinted uniform-sized imprinted polymers for chlorpromazine and
polymer-based stationary phase for naproxen. bromopromazine prepared by multi-step
Comparison of molecular recognition ability of swelling and polymerization method—the
the molecularly imprinted polymers prepared application for the determination of chlor-
by thermal and redox polymerization techni- promazine and its metabolites in rat plasma by
ques. J Chromatogr A 816:113–121 column-switching LC. J Pharm Biomed Anal
7. Hosoya K, Yoshizako K, Tanaka N, Kimata K, 174:248–255
Araki T, Haginaka J (1994) Uniform-size 14. Smigol V, Svec F, Hosoya K, Wang Q, Fréchet
macroporous polymer-based stationary phase JMJ (1992) Monodisperse polymer beads as
for HPLC prepared through molecular packing material for high-performance liquid
imprinting technique. Chem Lett chromatography. Synthesis and properties of
23:1437–1438 monodisperse polystyrene and poly(methacry-
8. Fu Q, Sanbe H, Kagawa C, Kunimoto KK, late) latex seeds. Angew Makromol Chem
Haginaka J (2003) Uniformly sized molecu- 195:151–164
larly imprinted polymer for (S)-nilvadipine.
Chapter 2
Molecularly Imprinted Polymeric Nanoparticles

by Precipitation Polymerization and Characterization
by Quantitative NMR Spectroscopy
Clovia I. Holdsworth, K. Fremielle Lim, and Phonlakrit Muang-Non
Abstract
An optimized synthetic methodology for the preparation of highly homogeneous MIP nanoparticles by the
precipitation method is presented. A quantitative 1H NMR method that was developed to estimate
template incorporation, polymer composition and conversion, and binding capacities and selectivities is
also described. While the experiment presented here is exemplified by an MIP formulation using ()-
propranolol as the template, methacrylic acid as the functional monomer and ethylene glycol dimethacry-
late as the crosslinker, the methods and techniques are applicable to other precipitation MIP systems.
Key words Precipitation MIP, MIP microspheres, MIP nanospheres, q-NMR, Propranolol MIP,
q-NMR analysis of propranolol
1 Introduction
Molecular imprinting is a simple and effective method of imparting

highly specific and selective recognition sites in synthetic polymers
by immobilizing a template molecule (usually also the target mole-
cule) within a rigid polymer matrix. At its simplest, the template
(T) molecules and functional monomers (FM) are allowed to self-
assemble and interact in solution in the presence of an excess
amount of crosslinker prior to polymerization, usually by the radi-
cal process. The template-moulded cavities within the polymer
matrix are capable of selectively rebinding the template/target
molecules. Conventionally, molecularly imprinted polymers
(MIPs) are prepared with minimal amounts of porogenic solvent
(i.e., bulk imprinting) resulting in monoliths that require grinding
and sieving. However, MIPs can be prepared more conveniently
using higher volumes of solvent [1–4], allowing insoluble nano- to
micro-particulate MIPs to form and settle out of solution, by a
number of methods such as emulsion polymerization [5],
https://doi.org/10.1007/978-1-0716-1629-1_2,
9
10 Clovia I. Holdsworth et al.
suspension polymerization [6], multi-step swelling [7], and precip-

itation polymerization [8–13]. In these processes, formation of the
polymers is due to continuous addition of monomer to the grow-
ing polymeric chain forming short chain of oligomers (i.e., nucle-
ation). These chains will continue to add monomer units
(polymerization) until they become insoluble in the porogen [3].
Among the methods used to prepare MIP microspheres, pre-
cipitation polymerization is the most widely used because of its
convenience in preparation since this involves a one-pot synthesis
of polymers [1, 8–10, 14]. However, literature has indicated that
this method is susceptible to changes in polymerization conditions
which affect the properties and the binding efficiency of the
imprinted polymers [15–21].
Here, we present an optimized synthetic methodology for the
preparation of MIP nano- to microspheres by the precipitation
method. Our methodology has been based on a number of studies
employing various templates and considering a number of factors
affecting the synthesis including the formulation (i.e., feed compo-
sition), initiator concentration, the nature and volume of the poro-
genic solvent, the reaction temperature, and other synthetic
conditions. It is noteworthy that while the imprinting and binding
efficiencies, template selectivity, and physical properties of the
resulting MIPs could vary from template to template, the precipi-
tation imprinting synthetic method we have adopted resulted in the
best performing MIPs from all systems investigated and highly
homogeneous particle size distribution [22, 23]. Additionally, we
also present a quantitative 1H NMR method that was developed to
estimate imprinting efficiency (i.e., template incorporation), poly-
mer composition (including template-functional monomer ratio)
and conversion, and binding capacities and selectivities. While the
experiment presented here is exemplified by an MIP formulation
using ()-propranolol (PNL) as the template, methacrylic acid
(MAA) as the functional monomer and ethylene glycol dimetha-
crylate (EGDMA) as the crosslinker, these methods and techniques
are applicable to other precipitation MIP systems and are discussed
in Subheading 4.
2 Materials
2.1 Polymerization 1. Monomers: Methacrylic acid as functional monomer (FM) and

ethylene glycol dimethacrylate (EGDMA) as crosslinker (XL).
Monomers are purified by passing through a basic aluminum
oxide column to remove inhibitors (see Note 1).
2. Initiator (I): 2,20 -azobisisobutyronitrile, AIBN (see Note 2).
AIBN is recrystallized from methanol prior to use.
Precipitation MIPs and q-NMR Characterization 11
3. Template (T): ()-Propranolol (PNL). PNL is commercially

available in its acid form and has to be converted to the neutral
base form prior to use (see Note 3).
4. Porogenic solvent: Acetonitrile, analytical grade used as
received.
5. Oven or thermal bath with temperature control (see Note 4).
6. 10-mL test tubes fitted with rubber septa.
7. Ultrapure nitrogen gas for purging polymerization mixture (see
Note 5).
8. Long needles that can penetrate rubber septa for nitrogen
purging.
2.2 Template 1. Methanol:acetic acid mixture, 9:1 by volume. Analytical grade

Extraction methanol is used as received.
2. Stirrer and stirrer bars.
3. 50-mL centrifuge tubes with lids.
4. Centrifuge, at least 1500 G.
5. UV–Visible spectrophotometer.
2.3 Quantitative 1. 400 or 600 MHz NMR machine.

1
H NMR 2. 5-mm NMR tubes.
3. Co-axial insert for 5-mm NMR tubes.
4. 0.25 μm syringe filters.
5. External standard or calibrant: 2,4-dinitrophenol (DNP). One
(1.00) mL of 10.00 mM DNP in DMSO-d6 inside a co-axial
insert (see Note 6).
6. 1.5-mL microcentrifuge tubes.
7. 2.00 mM PNL stock solution (see Note 7).
3 Methods
3.1 Polymerization 1. Mix 14.2 μL (0.17 mmol) MAA, 157.2 μL (0.83 mmol)
EGDMA, 32.8 mg AIBN, and 10 mL acetonitrile in a small
beaker or flask (see Note 8).
2. Fill two 10-mL test tubes, fitted with rubber septa, each with
5 mL of the reaction mixture. Add 5.3 mg of PNL to one test
tube—this is the MIP formulation. The other test tube is its
non-imprinted equivalent, i.e., the NIP (see Note 9).
3. Purge the capped reaction mixtures with nitrogen gas for
10–15 min (see Fig. 1). Adjust the flow of the nitrogen gas
between slow and moderate and avoid vigorous gas bubbling.
Fig. 1 Setup for polymerization and nitrogen gas purging
4. Polymerize the reaction mixtures for 24 h in a water bath or an

oven at 60 C without agitation (see Note 10). Quench the
polymerization by placing in an ice bath.
3.2 Template 1. Remove the solvent from the MIP particles by drawing it out
Extraction slowly using a Pasteur pipette (see Note 11). Keep the solvent
in a tightly closed vial for q-NMR analysis (Subheading 3.3.1)
if required.
2. Transfer the precipitated MIP into a 50-mL centrifuge tube.
Add a stirrer bar of appropriate size and 3–5 mL of the metha-
nol:acetic acid mixture. Tightly close the lid and stir between
200 and 300 rpm for 6–12 h (see Note 12) and centrifuge at
1500 G for 10 min. Remove the supernatant.
3. Wash the MIP particles twice with 3 mL pure methanol for 3 h
with stirring between 200 and 300 rpm, centrifuge at 1500 G
for 10 min, and remove the methanol supernatant.
4. Repeat the above procedure using 1 mL of methanol, centri-
fuge and transfer the methanol supernatant to a cuvette.
5. Check for PNL by running the spectrum of the methanol
supernatant between 180 and 600 nm using a UV–Visible
spectrophotometer (see Note 13). Use pure methanol as a
blank.
6. If a peak is observed at 340 nm, then PNL is likely to still be

leaching out from the MIP. Repeat procedure steps 2, 3, 4, and
5 until no peak is visible at 340 nm.
7. Wash the extracted MIP with diethyl ether and place it in a
vacuum oven at 40 C for exhaustive drying.
8. Carry out the same extraction and washing procedure (steps 1,
2, 3, and 7) for the non-imprinted polymers.
3.3 Quantitative 1H 1. Transfer 0.50 mL of the MIP or NIP pre-polymerization feed

NMR Analysis solution (from Subheading 3.1, steps 1 and 2) to a 5-mm
NMR tube (see Note 15).
3.3.1 Determination of
Template Incorporation and 2. Filter the solvent collected from the post-polymerization mix-
Polymer Composition (See ture (from Subheading 3.2, step 1) using a 0.25 μm syringe
Note 14) filter (see Note 16). Transfer 0.50 mL of the MIP or NIP
pre-polymerization feed solution to a 5-mm NMR tube.
3. Run the 1H NMR spectra of the original pre-polymerization
feed, MIP or NIP, and the corresponding post-polymerization
solutions (see Note 17). In each case, make sure that the coaxial
insert containing the DNP calibrant is fitted inside the
NMR tube.
4. Integrate the peaks of interest (see Fig. 2) for quantitative
analysis as follows: hydrogens HE1 and HE2 for EGDMA (see
Note 18), hydrogen HM for MAA, HP for PNL (see Note 19),
and HS for the DNP external standard.
5. Calculate the response factor for each component using their
initial concentrations and the integration of the selected hydro-
gens from the 1H NMR spectrum of the pre-polymerized feed
according to the following equation:
I x CS N S
F ¼ ∙ ∙ ð1Þ
I S Cx N X
where F ¼ response factor, IX ¼ integration of the compo-
nent’s peak of interest, IS ¼ integration of the calibrant’s peak
of interest, CS ¼ concentration of the calibrant, CX ¼ concen-
tration of the component, NS ¼ number of protons responsible
for the calibrant’s chosen signal, and NX ¼ number of protons
responsible for the component’s chosen signal.
6. Calculate the concentration of the residual components post-
polymerization from the 1H NMR spectrum of the post-
polymerization solution according to the following equation
and parameters described in Eq (1).
I x CS N S
CX ¼ ∙ ∙ ð2Þ
IS F NX
The difference between the concentrations of the mono-
mers and template in the initial feed and the residual
Fig. 2 An example of a 1H NMR spectrum used for q-NMR
components post-polymerization is a measure of the polymer

conversion and composition and template incorporation,
respectively.
3.3.2 Binding Tests (See 1. Weigh 2.0 mg of MIP and NIP into 1.5-mL microcentrifuge
Note 20) tubes. Add 1.0 mL of 2.0 mM PNL and incubate, with shak-
ing, for 2 h (see Note 21).
2. Centrifuge (at 1500 G) the mixtures for 10 min and transfer
0.5 mL of the supernatant into 5-mm NMR tubes. Also trans-
fer 0.5 mL of the 2.0 mM PNL stock solution into another
NMR tube.
3. Run their 1H NMR spectra with the DNP calibrant in a coaxial
insert.
4. Integrate the peaks of interest (see Fig. 2): HP for PNL and HS
for the DNP calibrant. Calculate the response factor F for PNL
from the 1H NMR spectrum of the PNL stock solution using
Eq. (1) and the concentration of free unbound PNL in the
MIP and NIP samples from the 1H NMR spectra of their
supernatants using Eq. (2).
4 Notes
1. The column can be prepared using a disposable syringe (50-mL

or smaller depending on the amount of monomer required)
plugged with a small ball of cotton. This method works for all
liquid monomers even with the viscous crosslinkers, ethylene
glycol dimethacrylate (EGDMA) and trimethylolpropane tri-
methacrylate (TRIM).
2. Reversible addition-fragmentation transfer (RAFT) initiators
can also be employed. If a RAFT initiator is employed, add
10 mol % AIBN with respect to the RAFT agent. While not
fully investigated, we have found that commercially available
RAFT initiator 4-cyano-4-(phenylcarbonothioylthio)-
pentanoic acid does not form polymers in the presence of
propranolol as template.
3. ()-Propanolol hydrochloride is converted to ()-propanolol-
free base by addition of excess NaHCO3, typically four times
that of PNL-Cl. Briefly, 1 mmol (250 mg) of PNL-Cl dissolved
in 10 mL of water is mixed with 4 mmol (336 mg) of NaHCO3
dissolved in 10 mL of water. The solution is stirred overnight,
i.e., 12 h, to ensure complete conversion to PNL base. Precipi-
tation or cloudiness is observed due to the formation of the
water insoluble PNL-free base. PNL is extracted (in a separa-
tory funnel) three times with 10 mL dichloromethane or until
the water layer is no longer cloudy. After drying the dichlor-
omethane extract in MgSO4 and filtering it off, the solvent is
removed by rotary evaporation to afford the PNL-free base.
4. Polymerization can also be conducted at room temperature by
UV irradiation (200–400 nm) using a water-jacketed 450 W
medium pressure mercury vapor quartz UV lamp. To maintain
the temperature lower than 30 C (between 22 and 27 C in
our case) during the reaction, the UV lamp and reaction test
tubes are immersed in a water bath. The temperature of the
water bath can easily be kept low by using a 5-L (or larger)
water bath.
5. In-house nitrogen gas can be used for conventional radical
polymerization, but we found that ultrapure nitrogen gas is
necessary for RAFT polymerization. The freeze-thaw method
is not used because all polymerization reactions are conducted
in test tubes with no side-arms for attachment to vacuum.
6. The external standard and the analytes of interest must have
peaks that are not overlapping and can be integrated with
precision for quantitative analysis. Choose the appropriate
external standard concentration by obtaining test NMR spectra
of the external standard at various concentrations in the pres-
ence of the least and most concentrated analyte solutions to be
used for the q-NMR.
7. The concentration of the rebinding stock solution may vary. It

is recommended to run binding tests to determine the appro-
priate concentration to use. Choose the concentration that will
bind 20–80% of the original amount.
8. For multiple synthesis and if Subheading 3.3.1 is planned, it is
recommended that a stock solution be prepared. In accordance
with the optimized method, all formulations contain 1 mmol
total monomer (tM ¼ MAA + EGDMA) per 10 mL of aceto-
nitrile porogen while the FM:XL (i.e., MAA:EGDMA) mole
ratio is kept at 1:5. A 1:5 I:tM mole ratio is used as our previous
studies showed this ratio to be optimal for MIP
preparation [23].
9. The T:FM:XL (i.e., PNL:MAA:EGDMA) is kept at the most
common mol ratio of 1:4:20. The expected polymer conver-
sion for this formulation is at least 80% (~70 mg). It is advisable
to prepare the MIP and NIP at the same time so they can both
be subjected to similar conditions.
10. Previous studies by our group have shown that agitation by
stirring or rolling does not result in the formation of particles
and discrete nanoparticles are formed simply by allowing the
polymers to settle out of solution freely with no agitation [22].
11. If the solvent looks cloudy and not all polymer particles have
precipitated, transfer the entire content of the test tube into a
50-mL centrifuge tube and centrifuge for 10 min at 1500 G, or
until the polymer particles are separated, before removing the
solvent.
12. We found this method to be more efficient and more conve-
nient than Soxhlet extraction.
13. Alternatively, 1H NMR can also be used. A few drops [5–10] of
the methanol supernatant are added to 0.5 mL of CDCl3
(or DMSO-d6) in a 5-mm probe NMR tube. The presence of
PNL is indicated by peaks at 1.5 ppm, between 3 and 4.5 ppm
and between 7 and 8.5 ppm. Note that peaks attributable to
methanol is observed between 3 and 4 ppm. It is recom-
mended to run the 1H NMR spectra of pure methanol and
PNL first.
14. This analysis requires the pre-polymerization and post-
polymerization solutions. Take this analysis into account, if
required, when preparing the monomer feed stock solution
for NIP and MIP. The polymer composition and template
incorporation can be deduced from the difference between
the amounts of components in the initial pre-polymerized
feed and the residual components left in solution after
polymerization.
15. The pre-polymerization solution can be stored in the refrigera-

tor for a few days before NMR analysis. Note that, if the stock
solution contains an initiator, polymerization can occur, so it is
recommended that the NMR analysis be run as soon as
possible.
16. Filtration is recommended to separate oligomeric fractions that
dissolve in acetonitrile from the residual monomers.
17. It is not necessary to add deuterated NMR solvent to the
solution to be analyzed. The external standard/calibrant is
already in d-DMSO. Acetonitrile is visible as a broad peak at
~2.5 ppm but does not pose a problem so long as the signals
used for q-NMR are far from this section.
18. In some cases, the vinyl peaks of both EGDMA and MAA
overlap. To estimate the integration of the vinyl peak attribut-
able only to MAA, the integration of the O-CH2 signal HE2 of
EGDMA is subtracted from the total integration of the vinyl
peaks.
19. The hydrogen meta from HP can also be used to measure PNL
concentration.
20. This method is also applicable to cross-binding and selectivity
tests provided the analogues have signals unique from the
calibrant.
21. Separate binding tests showed the optimum binding time for
PNL to be 2 h.
Acknowledgments
The authors acknowledge the technical assistance of Monica Ros-

signoli (q-NMR), Kit-yi Tang (particle size measurements by DLS),
David Phelan, and Yun Lin (SEM). KFL is grateful for the Univer-
sity of Newcastle International (UNIPRS) and Central (UNRSC)
Postgraduate Research Scholarships.
References
1. Chen L, Xu S, Li J (2011) Recent advances in polymerization. Macromolecules 37
molecular imprinting technology: current sta- (26):9746–9752
tus, challenges and highlighted applications. 4. Vasapollo G, Del Sole R, Mergola L, Lazzoi M,
Chem Soc Rev 40(5):2922–2942 Scardino A, Scorrano S, Mele G (2011) Molec-
2. Sellergren B (ed) (2000) Molecularly ularly imprinted polymers: present and future
imprinted polymers: man-made mimics of anti- perspective. Int J Mol Sci 12:5908–5945
bodies and their application in analytical chem- 5. Tovar G, Brunner H, Krauter I, Landfester K,
istry. Elsevier Vaihinger D (2002) Molecularly imprinted
3. Bai F, Yang X, Huang W (2004) Synthesis of polymer nanospheres as synthetic affinity
narrow or monodisperse poly(divinylbenzene) receptors obtained by miniemulsion polymeri-
microspheres by distillation–precipitation sation. Macromol Chem Phys 203:1965–1973
6. Mosbach K, Mayes AG (1996) Molecularly 15. Golker K, Karlsson B, Olsson CG, Gustaf D,
imprinted polymer beads: suspension polymer- Rosengren AM, Nicholls IA (2013) Influence
ization using a liquid perfluorocarbon as the of composition and morphology on template
dispersing phase. Anal Chem 68 recognition in molecularly imprinted polymers.
(21):3769–3774 Macromolecules 46(4):1408–1414
7. Haginaka J, Hoshina K, Sambo H (2007) 16. Komiyama M, Takeuchi T, Mukawa T, Asa-
Molecularly imprinted polymers for triazine numa H (2003) Molecular imprinting: from
herbicides prepared by multi-step swelling and fundamentals to applications. Wiley-VCH
polymerization method. their application to 17. Danielsson B (2008) Artificial receptors. Adv
the determination of methylthiotriazine herbi- Biochem Eng Biotechnol 109:97–122
cides in river water. J Chromatogr A 18. Martin-Esteban A (2013) Molecularly-
1152:130–137 imprinted polymers as a versatile, highly selec-
8. Ye L, Cormack PA, Mosbach K (1999) Molec- tive tool in sample preparation. Trends Anal
ularly imprinted monodisperse microspheres Chem 45:169–181
for competitive radioassay. Anal Commun 36 19. Perez-Moral N, Mayes A (2018) MIP formats
(2):35–38 for analytical applications. In: Piletsky S,
9. Ye L, Cormack PA, Mosbach K (2001) Molec- Turner AP (eds) Molecular imprinting of poly-
ular imprinting on microgel spheres. Anal mers. CRC Press Taylor & Francis Group,
Chim Acta 435(1):187–196 Boca Raton, pp 64–74
10. Ye L, Mosbach K (2001) Molecularly 20. Puoci F, Cirillo G, Curcio M, Parisi O,
imprinted microspheres as antibody binding Iemma F, Picci N (2011) Molecularly
mimics. React Funct Polym 48(1–3):149–157 imprinted polymers in drug delivery: state of
11. Zhang H, Ye L, Mosbach K (2006) art and future perspectives. Expert Opinion
Non-covalent molecular imprinting with Drug Delivery 8(10):1379–1393
emphasis on its application in separation and 21. Long Y, Philip JYN, Schillén K, Liu F, Ye L
drug development. J Mol Recognit (2011) Insight into molecular imprinting in
19:248–259 precipitation polymerization systems using
12. Bompart M, Haupt K, Ayela C (2012) Molec- solution NMR and dynamic light scattering. J
ular imprinting. In: Haupt K (ed) Topics in Mol Recognit 24(4):619–630
current chemistry, vol 325. Springer-Verlag, 22. Lim KF, Hall AJ, Lettieri S, Holdsworth CI
Berlin Heidelberg, pp 83–110 (2017) Assessment of the imprinting efficiency
13. Yan H, Row K (2006) Characteristic and syn- of an imide with a “stoichiometric” pyridine-
thetic approach of molecularly imprinted poly- based functional monomer in precipitation
mer. Int J Mol Sci 7(5):155–178 polymerisation. J Mol Recognit. https://doi.
14. Yoshimatsu K, Reimhult K, Krozer A, org/10.1002/jmr.2655
Mosbach K, Sode K, Lei Y (2007) Uniform 23. Lim KF, Holdsworth CI (2018) Effect of for-
molecularly imprinted microspheres and nano- mulation on the binding efficiency and selectiv-
particles prepared by precipitation polymeriza- ity of precipitation molecularly imprinted
tion: the control of particle size suitable for polymers. Molecules 23:2996. https://doi.
different analytical applications. Anal Chim org/10.3390/molecules23112996
Acta 584(1):112–121
Chapter 3
MIP Synthesis and Processing Using Supercritical Fluids

Ana I. Furtado, Raquel Viveiros, and Teresa Casimiro
Abstract
Supercritical fluid technology provides a clean and straightforward way for the preparation of high affinity
polymeric materials. Molecularly Imprinted Polymers (MIPs) as dry, free-flowing powders are obtained in a
one-step synthetic route yielding molecular recognition materials for several applications. Herein, we
describe the experimental procedures involved in the scCO2-assisted MIP development: synthesis, template
desorption, impregnation, and membrane preparation. MIP applications are described putting in evidence
the advantages of MIP development using supercritical fluid technology.
Key words Molecularly imprinted polymers, Supercritical CO2, High-Pressure processes, Green
Technology, Drug delivery, Purification devices, Membranes, Sensing
1 Introduction
In the past few decades, the interest in Molecular Imprinting Poly-

mers (MIPs) has increased sharply because of their promising
applications such as separation processes, drug delivery, sensing
devices, biomimetic detection, and catalysis [1, 2]. MIPs are syn-
thetic polymers, obtained by Molecular Imprinting Technique
(MIT), that have affinity cavities for a specific template molecule.
MIPs are commonly referred as plastic antibodies, since mimic
molecular recognition similarly to a natural antibody, with their
high-affinity sites complementary to the template, in terms of
chemical functionality, size, and shape [2, 3]. The MIT involves
the polymerization of a functional monomer, a crosslinking agent,
and an initiator, using a porogenic solvent, in the presence of a
template. Functional monomers are chosen to interact with the
template molecule since the formation of a stable template-
monomer complex is fundamental for the success of the molecular
recognition process. The template is surrounded by monomers and
the complex is “frozen” by copolymerization with the crosslinking
agent. The polymer obtained is a three-dimensional macroporous
matrix possessing complementary cavities template shaped. The
https://doi.org/10.1007/978-1-0716-1629-1_3,
19
20 Ana I. Furtado et al.
removal of the template molecule from the polymer is performed

by washing the polymer with a solvent, leaving these cavities readily
available to rebind with template and analogue molecules in future
applications [1, 2].
Nowadays, sustainable technologies have been gaining atten-
tion especially greener alternatives which are aligned with Sustain-
able Development Agenda 2030. Moreover, in the near future it
can be potentially implemented at industrial scale [4]. One of the
alternatives is the replacement of hazardous organic solvents by
green technologies, such as supercritical fluids (SCFs). SCF is a
highly compressible gas, above the critical temperature and pres-
sure of the fluid but below the pressure required to condense it
from the fluid to the solid state [5]. Due to their interesting proper-
ties, scCO2 is attracting interest in the materials production, in
particular MIP synthesis and processing. These properties include
its accessible critical point (critical temperature and pressure 31 C
and 73 bar, respectively, Fig. 1 [6]), it is available in high purity at
low cost, chemically inert, can be easily removed without any
additional energy input, is non-toxic and non-flammable, thus it
is considered a green solvent [6]. Moreover, scCO2 has liquid-like
densities, gas-like viscosities and diffusivities [6], thus being a very
attractive medium to perform chemical processes, such as
dry-cleaning processes [7], chemical reactions [8–12], impregna-
tions [13–15], and extractions [16]. Finally, scCO2 density is easily
tuned by simply adjusting pressure and temperature, as a conse-
quence, it is possible to control the properties that are density
dependent such as solubility [6, 11, 17, 18] (Fig. 2).
To the best of our knowledge, the first MIP produced using
scCO2 technology was reported by Duarte et al. in 2006 in the field
of drug delivery [14]. Since then, scCO2 has been used as solvent
for several applications [19] (Fig. 3 [20]), such as drug delivery
[14, 15], sensors [21, 22], and for separation and purification
processes, including desulfurization post-reaction purification
[23], HPLC processes as stationary phase [24], and active pharma-
ceutical ingredients (APIs) [3, 25, 26]. The scCO2 allied with MIT
has proven to have advantages in the production of MIPs compared
to the conventional strategies. The main advantages are: its poro-
genic solvent [6]; scCO2 contributes to an apolar and aprotic
medium, which stabilizes hydrogen bonds between template and
functional monomers [27]; by simple depressurization of the sys-
tem, no solvent residues are found in the final product [28]; MIPs
are usually obtained as dry, free-flowing powders (Fig. 4 [29]), i.e.,
ready to use, revealing to be a fast way to obtain synthetic materials
with molecular recognition ability [19]. Moreover, scCO2 has near-
zero surface tension, high diffusivity, and low viscosity [30] which
explain the high capacity of scCO2 to remove the template from
imprinted matrices, with higher efficiency than traditional organic
MIP Synthesis and Processing Using Supercritical Fluids 21
Fig. 1 Phase diagram of carbon dioxide (adapted from [6])
Fig. 2 Density vs pressure diagram of carbon dioxide and its dependency on the
temperature (adapted from [6])
solvent processes [31]. The extraction is ten-fold more efficient

than traditional methods [31].
MIP development involves the optimization of several experi-
mental variables. The key parameters are the nature of the mono-
mer and template, the monomer/template ratios, the crosslinking
degree, and the porogen. The most common functional monomers
for MIP synthesis in scCO2 are acrylates, vinylpyridines, and acry-
lamides, commercially available at low cost [19]. There are several
strategies to produced MIPs based on the type of interaction
between functional monomer and the template molecule, such as
Fig. 3 MIPs synthesized in scCO2 for different applications [20]
Fig. 4 Physical appearance of the synthesized MIP powder under scCO2
covalent, non-covalent, semi-covalent (by covalent and

non-covalent interactions) and metal ion-mediated imprinting
[1]. Among these, the non-covalent strategy is still by far the
most frequently adopted approach for the preparation of MIPs in
scCO2 [19]. However, a semi-covalent MIPs with molecular rec-
ognition towards bisphenol A was already reported in scCO2
[32]. The most used crosslinkers are ethylene glycol dimethacrylate
(EGDMA) [19], divinylbenzene (DVB), among others [33]. The
polymerization of monomers used in molecular imprinting in
scCO2 are radical thermal initiated, most commonly by 2,2-
0
-Azobis(2-methylpropionitrile) (AIBN) [1] which has its optimal
initiation temperature at 65 C [34]. Regarding the morphology of
the produced polymeric particles, the pore size and total surface
area can be tuned by the composition of the reactants in the
porogenic solvent, but can also be tuned by the CO2 pressure

changes, thus the combination of these parameters has a strong
impact in the particle’s morphology [30]. Moreover, pre-designed
MIPs using computational approaches that use virtual libraries
containing functional monomers and combinatorial approaches
has revealed to be a faster strategy for the selection of the best
monomers and ratios for a specific template molecule
[35]. Recently, computational tools were applied to MIP synthesis
in scCO2 revealing to be a very interesting approach to produce
high-affinity and selectivity materials, avoiding the screening exper-
imental tests [36].
The main challenge in the use of scCO2 technology is the fact
that high molecular weight macromolecules—except silicate [37]
and fluorinated [38] compounds—as well as polar molecules such
as biomolecules [39, 40], typically have negligible solubility in
scCO2. To circumvent this, typically a small amount of an organic
solvent is added as co-solvent to enhance the dissolution since most
organic solvents are soluble in scCO2. In some cases, the addition
of a co-solvent in the polymerization step has a significant effect on
MIPs performance [3]. On the other hand, if the solvent interferes
too much in the interaction between functional monomer tem-
plate, the specific site will be less effective in binding [1].
The scCO2-assisted MIP synthesis is carried out inside a
stainless-steel reactor typically with sapphire windows that allow
to visually follow the reaction. The reactants are introduced, and
then scCO2 is added to the reactor up to the desired pressure. The
initial phase of the polymerization is homogenous, i.e., all the
reagents are dissolved in scCO2, but as soon as the resulting poly-
mer reaches a certain molecular weight threshold, it precipitates
(Fig. 5), which is typical of a precipitation heterogeneous reaction,
obtaining a MIP as dry powder composed by nano- to micropoly-
meric particles [19].
Batch polymerization reactions, as mentioned above, also could
be used for other processes, such as surface imprinting, production
of MIPs as support particles, and imprinted polymeric membranes.
An example of surface imprinting using scCO2, is the synthesis of
an MIP layer on a pre-functionalized surface. For example, core
silica beads with molecularly imprinted shells were successfully
developed for acetamide [25]. Another strategy to prepare affinity
materials using supercritical fluid technology is the production of
molecularly imprinted membranes by scCO2-assisted phase inver-
sion, by blending the MIP particles with a commercially available
polymer to produce polymethyl methacrylate (PMMA) composite
membrane with recognition ability to certain analyte (e.g., bisphe-
nol A) [41].
Herein, we will focus on the procedures for free-radical hetero-
geneous polymerization in scCO2, being the most used strategy in
scCO2-based polymerization by the scientific community
Fig. 5 High-pressure reactor at: (a) initial homogeneous condition and (b)
polymer inside the cell
[19, 20]. A deep knowledge on the methodology is well described

for a better understanding on the green MIT process.
ScCO2-assisted MIP development involves: (a) synthesis,
(b) template desorption, (c) impregnation, (d) and production of
ready-to-use device (membrane/particles/columns). A landscape
of the applicability of these smart materials are described.
2 Materials
ScCO2 operates at high pressures (above 200 bar), thus it is man-

datory to use high-pressure equipment and follow the appropriate
safety rules for these type of equipment. Check the maximum
operating temperature and pressure of the high-pressure equip-
ment and never use conditions above those indicated by the manu-
facturer. The protective glasses and gloves should be part of the
operator’s daily routine. In addition, the installation must be under
a fume hood, in order to ensure that inhalation of toxic substances
is prevented.
All reagents are analytical grade. Store all reagents according to
the indication of the manufacturer and follow all waste disposal
regulations when disposing of waste materials.
2.1 MIP Synthesis Material required for the synthesis of approximately 1 g of MIP in
in scCO2 scCO2.
1. High-pressure material: 33 mL high-pressure cell with sapphire
windows or steel windows (the reactor, see Note 1); O-rings;
valves; fittings.
2. Pressure transducer.
Fig. 6 Scheme of the experimental apparatus for the MIP synthesis in scCO2 (a, b) and for the scCO2-assisted
template desorption (a–c). (1) CO2 supply; (2) cryostat; (3) CO2 liquid pump; (4) column packed with molecular
sieve; (5) water bath; (6) high-pressure cell; (7) heating bath circulator; (8) magnetic stirrer; (9) Expansion
vessel; (10) Freezing mixture; (11) MIP-Packed Column; V01–V05: high-pressure valves; W1 – compressed air
supply; P1 – outlet chain; PT – pressure transducer
3. Magnetic stirrer and Teflon-coated magnetic stirring bar.

4. Water bath with temperature control (thermostat) with a heat-
ing bath circulator to keep the bath temperature
homogeneous.
5. CO2 liquid pump and compressed air.
6. CO2 with purity higher than 99.998%.
7. Cryostat.
8. Column packed with molecular sieve (to remove residual water
molecules from CO2 source).
9. Schlenk tube.
10. Freezing mixture (ice). An assembly is depicted in Fig. 6.
11. Template, e.g., Subheading 3.5.1: metronidazole (MZ); Sub-
heading 3.5.2: acetamide (ACET); Subheading 3.5.3: bisphe-
nol A (BPA).
12. Functional monomer(s), e.g., Subheading 3.5.1 itaconic acid

(ITA); Subheadings 3.5.2/3.5.3: acid methacrylate (MAA);
Subheading 3.5.2: methacrylamide (MAM).
13. Crosslinker, e.g., ethylene glycol dimethacrylate (EGDMA).
14. Radical initiator, e.g., 2,20 -Azobis(2-methylpropionitrile)
(AIBN).
15. Co-solvent (if necessary), e.g., Subheading 3.5.2: acetonitrile.
The synthesis of approximately 1 g of non-imprinted polymer
(NIP) in scCO2 requires the previously material, except no addition
of the template.
2.2 ScCO2-Assisted Materials required for the template desorption from the polymeric
Template Desorption matrix to enable empty binding sites for further rebinding
processes.
1. High-pressure material: 33 mL high-pressure cell with sapphire
windows or steel windows (see Note 1); O-rings; valves;
fittings.
2. High-pressure steel tubular packed column (ID 7 mm, 15 cm
length).
3. Pre-synthesized MIP.
homogeneous.
7. CO2 with purity higher than 99.998%.
8. Cryostat.
9. Column packed with molecular sieve (to remove residual water
molecules from CO2 source). An assembly is depicted in Fig. 6.
10. Co-solvent, e.g., Subheading 3.5.2: ethanol; Subheading
3.5.2: acetonitrile; Subheading 3.5.3: methanol.
2.3 ScCO2-Assisted Materials required for the impregnation of compounds in the

MIP Impregnation polymeric matrix (MIP or NIP).
1. All apparatus described in Subheading 2.1.
2. Template, e.g., Subheading 3.5.2: metronidazole.
3. Macroporous support (e.g., stainless-steel mesh 0.2–0.5)
(Fig. 7).
4. Cellulose membrane dialysis (cut off 3.5 kDa).
5. Desorbed MIP using procedure adopted in Subheading 3.2 or
NIP using procedure adopted in Subheading 3.1.
a Cellulose membrane
loaded with polymer
b
Cellulose membrane
loaded with polymer
Macroporous support
Drug
Fig. 7 (a) General representation for the scCO2-assisted impregnation. The cellulose membrane loaded with
polymer placed on the cell’s top compartment of the high-pressure cell and the drug in the bottom
compartment, the drug solubilizes in scCO2, and the impregnation starts. After the end of the process, by
the depressurization, the non-impregnated drug precipitates and CO2 is released. (b) High-pressure cell
experimental apparatus
2.4 ScCO2-Assisted Materials required for the molecularly imprinted membranes using
Membranes an scCO2-assisted phase inversion method.
Preparation
1. High-pressure material (Fig. 8) [42]: 33 mL cylindrical cell
with an internal mechanism to homogenously disperse: a
porous structure that supports a bed of Raschig rings, in
order to disperse CO2 in the top of the casting solution; and
in the bottom of the cell a sapphire window (diameter of
2.4 cm) sealed with a Teflon O-ring (68 mm diameter and
1 mm height) and a support to the membrane formation;
valves; fittings.
homogeneous.
5. CO2 with purity better than 99.998%.
6. Cryostat.
7. Back-pressure regulator which separates the CO2 from the
solvent. An assembly is depicted in Fig. 9 [43].
Fig. 8 High-pressure cell used for membrane preparation [42]
8. Solvent, e.g., Subheading 3.5.3: dimethylformamide (DMF).

9. MIP or NIP.
The PMMA membrane was prepared using the same procedure
except that no pre-synthesized polymer, nor MIP neither NIP, was
added to the casting solution.
(4)
CO2
Vent
(1) V02
Refrigeration
fluid
(3)
V01 V03
(5)
(2)
Thermostated water bath

T
Controller
Fig. 9 Scheme of the experimental apparatus for the scCO2-assisted membrane formation [43]: (1) Gilson
305 piston pump; (2) temperature controller; (3) high-pressure cell; (4) pressure transducer; (5) back pressure
regulator; V01–V03: high-pressure valves
2.5 Applications Materials required for the development of MIPs for drug delivery
in scCO2 and their experimental test (in vitro drug release experi-
2.5.1 Drug Delivery MIPs
ment). All apparatus for the MIP development, and correspondent
NIP, is described in Subheadings 2.1, 2.2, and 2.3.
In Vitro Drug Release The drug release assessment is based on the comparison between
Experiments the results of the experiment by the produced MIP and their
counterpart non-imprinted polymer (NIP).
1. Cellulose membrane dialysis (cut-off 3.5 kDa).
2. Impregnated MIP or impregnated NIP.
3. Glycine–HCl acid buffer solution pH 2.2: deionized water,
glycine (99% purity), HCl (36.5–38.0%), and pH meter.
4. Phosphate-buffered saline (PBS) pH 7.4: deionized water and
PBS tablets.
5. 1 mL aliquots.
2.5.2 MIPs All apparatus for the acrylate and acrylamide-based MIP develop-
for Purification Processes ment, and correspondent NIP, in scCO2 are described in Subhead-
ings 2.1 and 2.2. The apparatus required for the synthesis of a MIP-
layer at silica beads, and the correspondent NIP layer, is also men-
tioned in Subheadings 2.1 and 2.2. Herein, pre-functionalized
core-shell silica beads were used.
Materials required to assess the affinity and selectivity of the

produced acrylate and acrylamide-based MIPs designed for API
purification processes are described below. The MIP-layered silica
beads are designed to build a gravity-driven API purification pro-
cesses; therefore, the solid-phase extraction (SPE) experiments
were performed in a column operating in a gravitational mode
without any vacuum system. Both affinity and selectivity assessment
are based on the comparison between the results of the experiment
by the produced MIP and their correspondent non-imprinted
polymer (NIP).
Static Binding Tests 1. Desorbed MIP or NIP.

2. Cellulose membrane dialysis (cut off 3.5 kDa).
3. Control shaker with an orbital movement.
4. Phosphate buffered saline (PBS) pH 7.4: deionized water and
PBS tablets.
5. Template, e.g., acetamide.
6. Syringe and 0.20μm filter syringe.
Solid Phase Extraction 1. Desorbed MIP or NIP.

(SPE) Experiments 2. Vacuum system.
3. SPE tubes of 3 mL with filter of 0.2 mm (diameter: 1 cm, bed
height: 1.5 cm).
4. Schlenk tube.
5. Freezing mixture (ice). An assembly is depicted in Fig. 10.
6. Template, e.g., acetamide.
7. Solvent, e.g., acetonitrile.
2.5.3 ScCO2-Assisted All apparatus for the production of molecularly imprinted mem-
Preparation of a PMMA brane and their counterpart non-imprinted membrane are
Composite Membrane described in Subheadings 2.1, 2.2, and 2.4.
3 Methods
3.1 MIP Synthesis 1. Apparatus assembly (Fig. 6), keep all valves closed.
in scCO2 2. Open the CO2 bottle and the compress air stream, turn on the
cryostat and the thermostat of the water bath at the desired
temperature (optimal initiation temperature of the initiator
used, e.g., AIBN it is 65 C).
3. Close one of the reactor windows and introduce all amounts of
reactants (template, functional monomer(s), crosslinker, initia-
tor) into the reactor, with the addition of a volume using
volumetric pipette in the case of liquid reagents and with the
Fig. 10 Scheme of the SPE experiments and the experimental apparatus. (1) SPE
tubes pack with polymer; (2) Visiprep SPE Vacuum Manifold; (3) Schlenk tube;
(4) freezing mix (ice); (5) Vacuum pump
help of sample holders for solid reagents, weighing the last ones
on an analytical balance (see Note 2).
4. Introduce magnetic stirring bar into the reactor and close the
other window.
5. Make sure that the reactor windows and valves are tightly
closed (V03; V04).
6. Couple the reactor to the high-pressure installation.
7. Pressurize around 10 bar of CO2 by opening the inlet valve
system (V01) and then closing the inlet reactor valve (V03);
when the pressure transducer display achieves 10 bar, close the
inlet reactor valve; depressurize the system by opening the
reactor outlet valve (V04), in order to inert the cell and then,
close the reactor outlet valve (V04).
8. Pressurize around 50 bar of CO2 (CO2 bottle pressure) by
opening the inlet reactor valve (V03); when the pressure trans-
ducer display achieves 50 bar, close the inlet reactor valve;
Certify that there is no leakage in the connections with the
help of soapy water (see Note 3).
9. Close the previous valve (V03) and compress CO2 to the
desired pressure through a CO2 liquid pump, typically
200–250 bar.
10. High-pressure cell is immersed in the thermostatic water bath.
11. Open gradually the inlet reactor valve (V03) and check the
pressure on the pressure transducer display; when the desired
pressure is reached, close the previous valve (V03); check the
initial pressure, stirring and that if there is no leakages in the
reactor or valves; thus, the polymerization reaction starts in the
batch system (see Note 4); close the previous valves (V01; V03)
that are opened (V01) and turn off the pump; the batch
reaction will occur under stirring during 24 h, and the polymer
grows.
12. After 24 h of reaction, a homogeneous crosslinked polymer is
obtained (Fig. 5).
13. Put a Schlenk tube on a freezing mixture and couple the
Schlenk tube to the reactor outlet valve (V04). The polymer
is slowly washed with fresh high-pressure CO2 for 1 h in order
to remove unreacted reactants. The Schlenk tube will trap the
unreacted residues and the CO2 will be vented. A continuous
CO2 flow is passed at 200–250 bar, typically up to 10 mL/min
through a CO2 liquid pump by opening the inlet valves of the
system V01, V03, and the outlet valve of the system V04.
Constantly, check the pressure, in this step a continuous flux
is required in order to remove all unreacted residues.
14. After 1 h, close the inlet valves of the system (V01; V03) and
turn off the pump, so the system is slowly depressurized.
15. Depressurize the system, i.e., when it is reaching zero pressure,
close the outlet valve (V04) and decouple the reactor from the
high-pressure apparatus.
16. Collect the polymer from the high-pressure reactor (Fig. 4).
The non-imprinted polymer (NIP) is synthesized following the
previous procedure (Subheading 3.1) with the exception that no
template is added to the reaction.
3.2 ScCO2-assisted 1. Apparatus assembly (Fig. 6), keep all valves closed.
Template Desorption 2. Open the CO2 bottle and the compress air stream, turn on the
cryostat and the thermostat of the water bath at the desired
temperature.
3. Introduce a small amount of co-solvent (usually 3 mL of
co-solvent) into 33 mL stainless steel high-pressure cell
through the use of a volumetric pipette.
4. Load the tubular column with the synthesized MIP.
5. Couple the tubular column in the previous high-pressure cell
and couple both to the high-pressure installation.
6. Pressurize around 50 bar of CO2 (CO2 vapor pressure) by
opening the inlet valves of the system (V01; V03) and the
outlet reactor valve (V04), keeping the V05 closed; when the
pressure transducer display reaches 50 bar, close the inlet reac-
tor valve (V03).
7. Certify that there is no leakage in the entire system (high-
pressure cell and packed column) with the help of soapy water
(see Note 3).
8. Compress CO2 up to the desired pressure through a CO2

liquid pump (usually 200–250 bar).
9. High-pressure cell and packed column are immersed in the
thermostatic water bath at temperature of 40 C. The high
diffusivity of CO2 promotes the efficient template removal
(see Note 5).
10. Open the inlet reactor valve (V03) and then open gradually the
outlet column valve (V05); thus, the continuous process of
template desorption from the polymeric matrix begins, check
the pressure on the pressure transducer display; check if there is
no leakage in the reactor or valves. In this process, the CO2 is
bubbled through the cell containing the co-solvent (bottom to
top) and the mixture scCO2-co-solvent was passed through the
tubular reactor in continuous mode for 3 h. Constantly check
the pressure; in this step, a continuous flux is required in order
to remove all the templates from the matrix.
11. After 3 h, the inlet valves of the system (V01; V03) are closed
and the compressor is turned off, so the system is slowly
depressurized.
12. When the system is totally depressurized, i.e., reaching zero
pressure, close the outlet valves (V04; V05) and decouple the
reactor and the packed column from the high-pressure
installation.
13. Collect the MIP desorbed polymer from the packed column.
3.3 ScCO2-Assisted The procedure adopted is very similar to the polymerization under
MIP Impregnation scCO2 (Subheading 3.1), with exception of the following steps:
3. Close one of the reactor windows and introduce the
macroporous support to divide it into two compartments and
prevent physical contact between the drug and the polymer (MIP
or NIP) (Fig. 7). Introduce the drug in the bottom compartment,
under the porous support and in enough quantity to obtain
medium saturation at pressure and temperature impregnation
conditions.
4. Load the MIP (typically 100 mg of MIP) into the cellulose
membrane, place on the cell’s top compartment, and close the
other window.
10. High-pressure cell is submerged in the thermostatic water
bath at 40 C.
12. After 24 h of impregnation, the system was quickly depres-
surized, and no more steps are required. Collect the impregnated
polymer from the high-pressure reactor.
Fig. 11 Physical appearance of the prepared molecularly imprinted membrane

using an scCO2-assisted phase inversion method
3.4 ScCO2-Assisted 1. Installation assembly (Fig. 9), keep all valves closed.
Membranes 2. Open the CO2 bottle and the compress air stream, turn on the
Preparation cryostat and the thermostat of the water bath at the desired
temperature.
3. Prepare the casting solution.
4. Load the casting solution (approximately 400 mg) using a
pipette by spreading over the sapphire window into a Teflon
cap in order to produce membranes with thickness ranging
from 450 to 550μm [43] and placed inside the high-pressure
vessel.
5. Immerse the high-pressure vessel into the thermostatted
water bath.
6. CO2 is added by opening the inlet system valve (V01) and the
inlet high-pressure vessel (V02) until the desired pressure, with
an exact flow (typically CO2 flow of 9.8 g/min during 2/3 h)
using a piston pump.
7. After reaching the normal operational pressure (typically
200 bar), the supercritical solution passes through a back-
pressure regulator which separates the CO2 from the solvent
used in the casting solution.
8. At the end, the system is slowly depressurized (typically during
20/30 min) by opening the outlet system valve (V03) and
closing the inlet valves (V01; V02).
9. Collect the thin homogeneous membrane from the high-
pressure vessel (Fig. 11).
3.5 Applications A pH-responsive MIP-based itaconic acid (ITA):Ethylene Glycol

Dimethacrylate (EGDMA) was developed as a potential body-
3.5.1 Drug Delivery MIP
friendly oral drug delivery system for metronidazole (MZ) in
(See Note 6)
scCO2. The MIP synthesis and the desorption of the template
follow the Subheadings 3.1 and 3.2, respectively. In this example,
MIPs are produced using two different molar ratios, 1:5:25 and
1:5:50, as template:functional monomer: crosslinker (MZ:ITA:
EGDMA), and 1 wt% of the radical initiator AIBN. Thus, in the
MIP synthesis procedure, 1 mmol of the template molecule MZ,
5 mmol of the functional monomer ITA, 25 mmol and 50 mmol of
the crosslinker agent EGDMA and the initiator are introduced in a
33 mL stainless steel high-pressure cell (see Note 7). The CO2 is
added up to 210 bar and reactor is immersed in a thermostatted
water bath at 65 C (optimal initiator temperature of the
AIBN) [15].
MZ was desorbed from the MIP matrices using 3.3 mL of
ethanol as co-solvent by the 3 h continuous process at 40 C and
the system pressurizes with CO2 up to 210 bar [15]. The poly
(ITA-co-EGDMA) matrices have been prepared in scCO2 in high
yields (90%, determined gravimetrically) as dry, free-flowing pow-
ders. As particle size distribution was heterogeneous for each con-
dition, obtain MIP microparticles with an average particle size
diameter of 6–8μm. Finally, MIPs were impregnated with MZ
using scCO2 at 200 bar of CO2 and 40 C during 24 h (Subheading
3.3).
Further, the MZ delivery profile was followed by in vitro drug
release experiments.
In Vitro Drug Release Drug release profile is evaluated at sink conditions. Commonly, the
Experiments impregnated MIPs and their respective controls are used to release
the drug, in a simulated oral administration situation.
1. Put MIP (or the control: NIP) into 100 mL of Glycine–HCl
acid buffer solution pH 2.2 for 3 h.
2. Immersion in 100 mL of PBS (pH 7.4) for 5 h.
3. Set the temperature at 37 C (mimicking our internal body
temperature) and withdraw 1 mL aliquots at time intervals and
add the same volume of fresh medium to the solution.
4. In this case, to quantify the amount of MZ, the 1 mL aliquots
collected over the time samples were analyzed in UV–Vis by a
spectrophotometer at 275 nm for pH 2.2 and 318 nm for PBS
(pH 7.4).
5. For example, drug transport profile, through the synthesized
polymeric networks, at different pHs, can be modeled using
the semi-empirical Korsmeyer–Peppas eq. [44]:
Mt
¼ k∙t n
M1
where Mt is the absolute cumulative amount of drug
released at time t, M1 is the total amount of drug released
from polymer samples, k is the diffusion coefficient that reflects
the structural and geometric characteristics of the device, and
n is the release exponent, which gives a better understanding
on the specific transport mechanism.
3.5.2 MIP for Purification Acrylate and acrylamide-based monomers are used to develop
Processes MIPs in scCO2 for a model pharmaceutical impurity, acetamide
(ACET), for the application in Active Pharmaceutical Ingredient
Acrylate (API) manufacturing processes. The MIP synthesis and the desorp-
and Acrylamide-Based tion of the template follow the Subheadings 3.1 and 3.2, respec-
MIPs for a Model tively. MIPs are produced using a molar ratio 1:4:20 as template:
Pharmaceutical Impurity functional monomer: crosslinker (ACET: MAA or MAM:
(See Note 8) EGDMA), 1 wt% of the radical initiator AIBN, with or without
acetonitrile (0.5 mL) as co-solvent (see Note 9). Thus, 1 mmol of
the template molecule—ACET, 4 mmol of the functional mono-
mer MAA or MAM, 20 mmol of the crosslinker agent EGDMA and
the initiator are introduced in a 33 mL stainless steel high-pressure
cell. The CO2 is added up to 210 bar and reactor is immersed in a
thermostatted water bath at 65 C [3]. All polymers were obtained
in high yields (>90%, determined gravimetrically) as dry, free-
flowing white powders with similar morphology as polymeric
microparticles, which is consistent with scCO2-assisted polymeriza-
tions. ACET was desorb under scCO2 conditions by using 3 mL of
acetonitrile as co-solvent at 40 C with CO2 up to 210 bar [3]. The
affinity and the selectivity of these molecularly imprinted materials
were assessed through comparison with their respective
non-imprinted polymer. A significant effect on MIP performance
was observed by the addition of a co-solvent to the polymerization
step [3].
Static Binding Tests 1. Weight 20 mg of MIP (or NIP) and place into cellulose
membrane.
2. Introduce the membrane containing MIP in 50 mL of template
solution (e.g., solutions with concentration range from 10 to
250 ppm of ACET in acetonitrile).
3. Introduce the previous solution with membrane containing
MIP on an orbital shaker with 100 rpm stirring during 24 h.
4. In this case, samples are filtered and analyzed by HPLC.
5. The binding capacity, Q, is calculated by using the expression
below:
ðC 0 C Þ∙V
Q ¼
W
where C0 and C are the template concentrations in the

solutions which were measured initially and after sorption,
respectively; V is the volume of the solution, and W is the
sample weight.
Solid Phase Extraction 1. Weight 20 mg of MIP and pack into empty columns.
(SPE) Experiments 2. Condition the column with 3 mL of solvent (acetonitrile).
3. Pass 10 mL of template solution or analogue molecules solu-
tion through the MIP-SPE cartridge (e.g., solution of contain-
ing 250 ppm of acetamide, benzamide, and pivalamide in
acetonitrile), by using an SPE vacuum system (Fig. 10).
4. Collect the solution samples. The samples were analyzed
by HPLC.
5. Between each solution in this specific sequence, wash the col-
umns three times with 10 mL of solvent (acetonitrile).
6. Binding capacity, Q, is calculated by the previous equation,
where C0 and C are the template concentrations in the solu-
tions which were measured initially and after the extraction,
respectively; V is the extract volume of the solution, and W is
the sample weight packed in the SPE column.
MIP-Layered Silica Beads MIP-layered silica beads with affinity for a model pharmaceutical
for a Model Pharmaceutical impurity, ACET, are developed in scCO2. This process is very
Impurity (See Note 10) similar to the polymerization under scCO2 (Subheading 3.1),
except in the step of introducing the reagents into the reactor,
that is, in addition to the necessary reactants for the MIP surface:
Thereby, in the MIP synthesis procedure, 0.5 g of
pre-functionalized silica, 1 mmol of the template molecule ACET,
4 mmol of the functional monomer MAA, 20 mmol of the cross-
linker agent EGDMA, and 1 wt% of the radical initiator AIBN are
introduced in a 33 mL stainless steel high-pressure cell. The CO2 is
added up to 210 bar and reactor is immersed in a thermostatted
water bath at 65 C [25].
ACET-desorption in scCO2 is performed as described in Sub-
heading 3.2. For that, core–shell MIP beads were loaded and
compacted in a tubular column and was used 3 mL of acetonitrile
as co-solvent at 40 C and CO2 added up to 210 bar in continuous
flux [25].
These MIP-layered silica beads are designed for gravity-driven
API purification processes; therefore, the affinity and selectivity of
these molecularly imprinted materials were assembled by gravity-
driven column experiments. It is very similar to the SPE experi-
ments although no need to apply pressure or vacuum since the
experiment is operated in a gravitational mode.
Gravity-Driven Column 1. Weight 396.5 mg of core–shell MIP (or NIP) beads and pack
Experiments into blank columns.
2. Condition the column with 10 mL of solvent (acetonitrile).
3. Pass 10 mL of template solution or analogue molecules solu-
tion through the MIP gravity-driven cartridge (e.g., solution
containing 250 ppm ACET and 3500 ppm API in acetonitrile.
4. Collect the solution samples. Samples were analyzed by HPLC.
5. Between each step solution in this specific sequence, wash the
columns three times 10 mL of solvent (acetonitrile).
6. The binding capacity, Q, is calculated by the previous equation,
where C0 and C are the template concentrations in the solu-
tions which were measured initially and after the extraction,
respectively; V is the extract volume of the solution, and W is
the sample weight packed in the gravity column.
3.5.3 ScCO2-Assisted Briefly, to produce the PMMA membranes, the casting solution
Preparation of a PMMA with 30 wt% of polymer blend consisting in 70:30 of PMMA and
Composite Membrane MIPs or NIP (previously synthesized for Bisphenol A – BPA), in
5 mL of DMF, was loaded into a Teflon cap and placed inside the
high-pressure cell, following the procedure described in the Sub-
heading 3.4. The MIP and NIP were obtained by the procedure
described in the Subheading 3.1. The MIP synthesis under scCO2
was used as a molar ratio of 1:5:25 of BPA:MAA:EGDMA (i.e.,
0.49 mmol of the template molecule BPA, 2.54 mmol of the
functional monomer MAA, 12.88 mmol of the crosslinker agent
EGDMA, and 1 wt% of the radical initiator AIBN, at 65 C, and the
CO2 was added up to 210 bar). The NIP synthesis were produced
under the same conditions without addition of the template.
Imprinted and non-imprinted polymers were obtained as dry,
free-flowing powders in high yields (~99%, determined gravimetri-
cally). The membrane production was performed at 45 C and CO2
was added until an operational pressure of 200 bar was reached.
Pressure was set at 200 bar by means of a back-pressure regulator,
which separates the CO2 from the DMF present in the casting
solution. All the experiments were performed with a CO2 flow of
9.8 g/min for 3 h. At the end of the production membrane process,
the system was slowly depressurized during 20 min. A thin homo-
geneous membrane composed of PMMA (pure PMMA) or MIP
(PMMA MIP) or NIP (PMMA NIP) was obtained. The pure
PMMA membrane was prepared using the same procedure (Sub-
heading 3.4), except that no pre-synthesized polymer (MIP or
NIP) was added to the casting solution. The incorporation of
highly crosslinked polymeric particles with molecular recognition
ability in the porous structure PMMA composite not only
enhanced considerably the affinity of membrane to the template
but also increased the permeability of the PMMA membrane [41].
4 Notes
1. Processes with pressures above 300 bar is advisable to use steel

windows.
2. In the introduction of the reactants into the reactor, the most
volatile reagents are the last ones to be introduced, to avoid
mass losses.
3. If there are soap bubbles, it is a sign of leakage; depressurize
and check if it is tightly closed; otherwise, replacement of the
material is required.
4. Typically, the pressure increases in the initial phase of the MIP
synthesis since the pressure increases with temperature (tem-
perature of the water bath (65 C) is higher than room tem-
perature). Check that the pressure does not increase above
400 bar. In the event of this, depressurize the system for safety
reasons by opening gently the outlet reactor valve (V04);
5. ScCO2 properties can be easily tuned by adjusting the pressure
and/or temperature. For example, the density of the supercrit-
ical fluid can vary from the density of gas to the density of liquid
depending on the pressure or temperature applied. This varia-
tion can affect the solvation capacity of CO2 which can be
exploited in desorption processes to desorb selectively the tem-
plate molecule from the MIP [6].
6. Application of the MIP in drug delivery using for the release of
metronidazole is described as an example. These procedures are
generic and can be adapted to other analytes and systems.
7. To obtain MIPs in high yields, an optimization study is
required to choose the best parameters that are critical for the
system. In the MIP-drug delivery example, it is evaluated the
MIP performance using different crosslinker agent degrees.
When the crosslinking degree is doubled, the particle size
diameter increases. In the impregnation process, both polymers
obtain higher drug uptake ability with metronidazole, reveal-
ing loadings of 18–61 wt% in MIPs, compared to 7–20 wt% in
the respective control. The MIP using the molar ratio 1:5:25
load three-folds more drug impregnated than the MIP that
uses double crosslinking degree, as a consequence, after 4 h at
pH 2.2, the first MIP released almost two-folds more drug than
the second MIP in drug release tests [15].
8. MIP-based purification devices are used for the selective
removal of pharmaceutical impurity, acetamide, is described as
an example of MIP development under scCO2. The assessment
experimental tests to the application are generic, and it can be
adapted to other analytes, concentration ranges and mediums.
9. To obtain MIPs in high yields, an optimization study is

required to choose the best parameters that are critical for the
system. The acetamide-MIP example evaluates the perfor-
mance using monomers of different nature (acid monomers
and basic monomer) and the effect of using co-solvent.
10. MIP-layered silica beads for the selective removal of pharma-
ceutical impurity, acetamide, are described as an example of
surface molecular imprinting technique in scCO2. In this
example, silica beads were pre-functionalized using the method
of grafting to (3-(Trimethoxysilyl) propyl methacrylate/etha-
nol (10% v/v) in scCO2 and grafting from using plasma tech-
nology. Then, the MIP-layered silica beads for acetamide were
synthesized using scCO2 technology, as described [25]. Appli-
cation of purification devices using MIPs silica beads as station-
ary phase for the detection and retention of pharmaceutical
impurity, acetamide, is described also as an example. These
procedures are generic and can be adapted to other analytes,
systems, concentration ranges, and mediums.
Acknowledgments
The authors would like to thank financial support from Fundação

para a Ciência e a Tecnologia, Ministério da Ciência, Tecnologia
e Ensino Superior (FCT/MCTES), Portugal, through projects
PTDC/QEQ-PRS/2757/2012, PTDC/EQU-EQU/32473/
2017 (funded by national funds through FCT/MCTES -PID-
DAC), and principal investigator contract IF/00915/2014
(T.C.). The Associate Laboratory for Green Chemistry - Clean
Technologies and Processes - LAQV is financed by national funds
from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/
2020) and co-financed by the ERDF under the PT2020 Partner-
ship Agreement (POCI-01-0145-FEDER – 007265).
References
1. Vasapollo G, Del Sole R, Mergola L, Lazzoi for Sustainable Development’, 2015. [Online].
MR, Scardino A, Scorrano S, Mele G (2011) Available: https://sustainabledevelopment.un.
Molecularly imprinted polymers: present and org/post2015/transformingourworld
future prospective. Int J Mol Sci (Accessed 30 Sep, 2020)
12:5908–5945 5. Rehman I, Darr J, Moshaverinia A (2008)
2. Belbruno JJ (2019) Molecularly imprinted Supercritical fluid processing, encyclopedia of
polymers. Chem Rev 119:94–119 biomaterials and biomedical engineering, 2nd
3. Viveiros R, Lopes MI, Heggie W, Casimiro T ed. - four volume set, G. L. B. Gary Wnek,
(2017) Green approach on the development of pp. 2522–2530
lock-and-key polymers for API purification. 6. Boyère C, Jérôme C, Debuigne A (2014) Input
Chem Eng J 308:229–239 of supercritical carbon dioxide to polymer syn-
4. Division for Sustainable Development Goals, thesis: an overview. Eur Polym J 61:45–63
‘Transforming our world: the 2030 Agenda
7. Sousa M, Melo M-J, Casimiro T, Aguiar- 20. CleanMIPTech, https://sites.fct.unl.pt/clean-

Ricardo A (2007) The art of CO2 for art con- mip-tech/home. [Online]. Available: https://
servation: a green approach to antique textile. sites.fct.unl.pt/clean-mip-tech/home
Green Chem 9(9):943–947 (Accessed 21 Jul, 2020)
8. Gang Z, Huan-feng J, Ming-Cai C (2003) 21. Rebocho S, Cordas CM, Viveiros R, Casimiro
Chemical reactions in supercritical carbon T (2018) Development of a ferrocenyl-based
dioxide. ARKIVOC 2:191–198 MIP in supercritical carbon dioxide: towards an
9. Medina-Gonzalez Y, Camy S, Condoret JS electrochemical sensor for bisphenol A. J
(2014) ScCO2/green solvents: biphasic Supercrit Fluids 135:98–104
promising systems for cleaner chemicals 22. Lourenço A, Viveiros R, Mouro A, Lima JC,
manufacturing. ACS Sustain Chem Eng Bonifácio VDB, Casimiro T (2014) Supercriti-
2:2623–2636 cal CO2-assisted synthesis of an ultrasensitive
10. Licence P, Ke J, Sokolova M, Ross SK, Poliak- amphibious quantum dot-molecularly
off M (2003) Chemical reactions in supercriti- imprinted sensor. RSC Adv 4:63338–63341
cal carbon dioxide: from laboratory to 23. Ferreira JP, Viveiros R, Lourenço A, Soares da
commercial plant. Royal Soc Chem 5:99–104 Silva M, Rosatella A, Casimiro T, Afonso CAM
11. Casimiro T, Banet-Osuna AM, Ramos AM, (2014) Integrated desulfurization of diesel by
Nunes Da Ponte M, Aguiar-Ricardo A (2005) combination of metal-free oxidation and prod-
Synthesis of highly cross-linked poly(diethy- uct removal by molecularly imprinted poly-
lene glycol dimethacrylate) microparticles in mers. RSC Adv 4:54948–54952
supercritical carbon dioxide. Eur Polym J 24. Soares da Silva M, Vão E, Temtem M, Mafra L,
41:1947–1953 Caldeira J, Aguiar-Ricardo A, Casimiro T
12. Skouta R (2009) Selective chemical reactions in (2010) Clean synthesis of molecular recogni-
supercritical carbon dioxide, water, and ionic tion polymeric materials with chiral sensing
liquids. Green Chem Lett Rev 2:121–156 capability using supercritical fluid technology.
13. Kikic I, Vecchione F (2003) Supercritical Application as HPLC stationary phases. Bio-
impregnation of polymers. Curr Opin Solid sens Bioelectron 25:1742–1747
Stat Mater Sci 7:399–405 25. Viveiros R, Dias FM, Maia LB, Heggie W,
14. Duarte ARC, Casimiro T, Aguiar-Ricardo A, Casimiro T (2017) Green strategy to produce
Simplı́cio AL, Duarte CMM (2006) Supercrit- large core–shell affinity beads for gravity-driven
ical fluid polymerisation and impregnation of API purification processes. J Ind Eng Chem
molecularly imprinted polymers for drug deliv- 54:341–349
ery. J Supercrit Fluid 39:102–106 26. Esteves T, Viveiros R, Bandarra J, Heggie W,
15. Marcelo G, Ferreira IC, Viveiros R, Casimiro T Casimiro T, Castelo F (2016) Molecularly
(2018) Development of itaconic acid-based imprinted polymer strategies for removal of a
molecular imprinted polymers using supercriti- genotoxic impurity, 4-dimethylaminopyridine,
cal fluid technology for pH-triggered drug from an active pharmaceutical ingredient post-
delivery. Inter J Pharm 542:125–131 reaction stream. Sep Purif Tech 163:206–214
16. Gandhi K, Arora S, Kumar A (2017) Industrial 27. Soares da Silva M, Nobrega FL, Aguiar-
applications of supercritical fluid extraction: a Ricardo A, Cabrita EJ, Casimiro T (2011)
review Industrial applications of supercritical Development of molecularly imprinted
fluid extraction: a review. Inter J Chem Stud co-polymeric devices for controlled delivery of
5:336–340 flufenamic acid using supercritical fluid tech-
nology. J Supercrit Fluids 58:150–157
17. Hebb AK, Senoo K, Bhat R, Cooper AI (2003)
Structural control in porous cross-linked poly 28. Soares da Silva M (2011) PhD Thesis. Devel-
(methacrylate) monoliths using supercritical opment of molecularly imprinted polymers
carbon dioxide as a “pressure-adjustable” using supercritical fluid technology. Universi-
porogenic solvent. Chem Mater dade Nova de Lisboa, Faculdade de Ciências
15:2061–2069 e Tecnologia
18. Clifford AA, Pople K, Gaskill WJ, Bartle KD, 29. Furtado AI (2019) MSc Thesis. Development
Rayner CM (1997) Reaction control and of smart polymers using clean technologies for
potential tuning in a supercritical fluid. Chem application in bio-purification processes, Facul-
Comm 6:595–596 dade Ciências e Tecnologia, Universidade
Nova de Lisboa
19. Viveiros R, Rebocho S, Casimiro T (2018)
Green strategies for molecularly imprinted 30. Beckman EJ (2004) Supercritical and near-
polymer development. Polymers 10:1–27 critical CO2 in green chemical synthesis and
processing. J Supercrit Fluids 28:121–191
31. Ellwanger A, Berggren C, Bayoudh S, 38. Li H, Xu A, Zhang Y (2007) Synthesis of fluor-

Crecenzi C, Karlsson L, Owens P, Ensing K, opolymers in supercritical carbon dioxide.
Cormack P, Sherrington D, Sellergren B Progr Chem 19:1562–1567
(2001) Evaluation of methods aimed at com- 39. Ribeiro dos Santos I, Thies C, Richard J,
plete removal of template from molecularly Meurlay D, Gajan V, Vandevelde V, Benoit
imprinted polymers. Analyst 126:784–792 J-P (2010) A supercritical fluid-based coating
32. Soares da Silva M, Viveiros R, Aguiar-Ricardo- technology. 2: Solubility considerations. J
A, Bonifácio VDB, Casimiro T (2012) Super- Microencaps 20:97–109
critical fluid technology as a new strategy for 40. Vedaraman NG, Brunner G, Srinivasa
the development of semi-covalent molecularly Kannan C, Ramabrahmam BV, Rao PG
imprinted materials. RSC Adv 2:5075–5079 (2004) Solubility of N-CBZ derivatised amino
33. Ye L, Yoshimatsu K, Kolodziej D, Da Cruz FJ, acids in supercritical carbon dioxide. J Supercrit
Dey ES (2006) Preparation of molecularly Fluids 30:119–125
imprinted polymers in supercritical carbon 41. Soares da Silva M, Viveiros R, Coelho MB,
dioxide. J Appl Polym Sci 102:2863–2867 Aguiar-Ricardo A, Casimiro T (2012) Super-
34. ‘Oil soluble Azo initiators – AIBN’. Pure Wako critical CO2-assisted preparation of a PMMA
Chemical Corporation composite membrane for bisphenol A recogni-
35. Nicholls IA, Andersson HS, Charlton C, tion in aqueous environment. Chem Eng Sci
Henschel H, Karlsson BCG, Karlsson JG, 68:94–100
O’Mahony J, Rosengren AM, Rosengren KJ, 42. Temtem M, Casimiro T, Mano JF, Aguiar-
Wikman S (2009) Theoretical and computa- Ricardo A (2008) Preparation of membranes
tional strategies for rational molecularly with polysulfone/polycaprolactone blends
imprinted polymer design. Biosens Bioelectr using a high pressure cell specially designed
25:543–552 for a CO2-assisted phase inversion. J Supercrit
36. Viveiros R, Karim K, Piletsky SA, Heggie W, Fluids 43:542–548
Casimiro T (2017) Development of a molecu- 43. Temtem M, Casimiro T, Aguiar-Ricardo A
larly imprinted polymer for a pharmaceutical (2006) Solvent power and depressurization
impurity in supercritical CO2: rational design rate effects in the formation of polysulfone
using computational approach. J Clean Prod membranes with CO2-assisted phase inversion
168:1025–1031 method. J Memb Sci 283:244–252
37. Hoefling TA, Newman DA, Enick RM, Beck- 44. Siepmann J, Peppas NA (2012) Modeling of
man EJ (1993) Effect of structure on the drug release from delivery systems based on
cloud-point curves of silicone-based amphi- hydroxypropyl methylcellulose (HPMC). Adv
philes in supercritical carbon dioxide. J Super- Drug Deliv Rev 48:139–157
crit Fluids 6:165–171
Chapter 4
Synthesis of Bacteria Imprinted Polymers by Pickering

Emulsion Polymerization
Haiyue Gong, Xiantao Shen, and Lei Ye
Abstract
Molecularly imprinted polymers have been studied for a long time and have found useful applications in
many fields. In most cases, small organic molecules are used as templates to synthesize imprinted polymers.
In contrast to low molecular weight targets, large biological molecules and cells are more challenging to use
as templates to synthesize cell-recognizing materials. This chapter presents an interfacial imprinting method
to synthesize bacteria-recognizing polymer beads using Pickering emulsion polymerization. The tendency
of bacteria to reside between two immiscible liquids is utilized to create surface-imprinted binding sites on
cross-linked polymer microspheres.
Key words Bacteria, Molecular imprinting, Pickering emulsion, Chitosan, Surface imprinting
1 Introduction
Pickering emulsion is a special type of discrete liquid mixture

stabilized by small solid particles that accumulate at the interface
between the two immiscible liquid phases [1]. Compared with
other types of emulsions, Pickering emulsion is significantly more
stable and can be kept for very long period without obvious phase
changes [2, 3]. A number of solid particles are known to stabilize
Pickering emulsions. For example, silica nanoparticles can be easily
synthesized, modified, and are environmentally friendly, therefore
are one of the most widely studied particle emulsifiers for preparing
Pickering emulsions [4, 5]. Magnetic nanoparticles, because of
their magnetic responsibility and low toxicity, are widely used to
prepare Pickering emulsions for biomedical applications [6]. Car-
bon nanotubes have also attracted lots of attention as new particle
emulsifiers in the last few years because their larger surface area and
abundant active sites provide many interesting possibilities of the
Pickering emulsions [7]. Nature-derived particle stabilizers are
becoming increasingly popular reagents because of their prominent
https://doi.org/10.1007/978-1-0716-1629-1_4,
43
44 Haiyue Gong et al.
biocompatibility and biodegradability [8]. The diversity of the

emerging particle emulsifiers has offered many exciting opportu-
nities to exploit Pickering emulsions in applied sciences.
Microbial cells, as one special type of the natural particles, can
serve as emulsifiers to stabilize Pickering emulsions [9]. Since
microorganisms such as bacteria and yeast are now widely utilized
in food, cosmetics, and pharmaceutical industries, new Pickering
emulsions stabilized by these microorganisms have a great potential
to contribute to improving quality and productivity of many indus-
trial processes. Microorganism cells can be modified via physical
and chemical treatments to alter their surface properties and topo-
graphical structures [10, 11], and Pickering emulsions derived
from different modified microorganism cells have been introduced
into several industrial applications [12–14].
Molecularly imprinted polymers can be synthesized by Picker-
ing emulsions in different manners. In a typical oil-in-water emul-
sion, the oil phase containing the monomers are polymerized to
form cross-linked polymer beads, where the molecular templates
are either embedded inside the polymer beads or located on the
bead surface. By removing the templates under an appropriate
condition, specific binding sites are formed with their size, shape.
and 3D structures mirroring the corresponding templates
[15]. When microorganisms are used to stabilize a Pickering emul-
sion composed of hydrophobic monomer droplets dispersed in
water, the microbial cells can act as template to generate surface-
imprinted sites after the monomer droplets are cross-linked to form
solid polymer beads. For many practical applications, making
imprinted binding sites accessible is critical to achieving fast analyt-
ical results and simplified regeneration and recycling of precious
materials [16–21].
Using bacteria as surface template in an oil-in-water Pickering
emulsion, surface-imprinted polymer beads have been obtained by
Pickering emulsion polymerization. The synthetic process of self-
assembling bacterial-chitosan complex, the establishment of the
complex-stabilized Pickering emulsion, and the subsequent
imprinting reaction is schematically shown in Fig. 1 [22]. To
anchor cationic chitosan onto the polymer beads, chitosan is first
modified with acryloyl chloride to introduce C¼C bonds before it
is added to the bacteria template (Fig. 2). The self-assembly of
bacteria-chitosan complex is driven by electrostatic interaction
between the negatively charged bacteria and the positively charged,
acryloyl-functionalized chitosan [11]. In this way, the bacteria-
chitosan complex acts as particle emulsifier to stabilize an oil-in-
water Pickering emulsion, and the bacteria play the role of cellular
template to generate surface imprinted recognition sites on the final
cross-linked polymer beads.
Bacteria-Imprinted Polymers 45
Fig. 1 Interfacial bacteria imprinting by Pickering emulsion polymerization.

Reproduced from ref. [22] with permission from Wiley-VCH Verlag GmbH &
Co. KGaA
Fig. 2 Synthesis of N-acrylchitosan pre-polymer. The molar ratio between the amino groups in chitosan and
acryloyl chloride was 5:1. Reproduced from ref. [22] with permission from Wiley-VCH Verlag GmbH & Co. KGaA
2 Materials
2.1 Stock Solutions 1. 1 M NaOH solution: weigh 4.0 g NaOH and dissolve it in
100 mL H2O.
2. 2 M NaOH solution: weigh 8.0 g NaOH and dissolve it in
100 mL H2O.
3. Lysogeny broth (LB) medium: weigh 1.0 g tryptone, 0.5 g

yeast extract, 1.0 g NaCl, and 0.1 g glucose and dissolve the
reagents in 100 mL H2O. Adjust the pH to 7.0 with
1 M NaOH.
4. 100 mg/mL ampicillin stock solution: weigh 0.2 g ampicillin
sodium salt and dissolve in 2 mL H2O. Store in 20 C freezer.
5. Phosphate-buffered saline (PBS, pH 7.2, 0.1 M): weigh 4.0 g
NaCl, 0.1 g KCl, 0.72 g Na2HPO4, and 0.12 g KH2PO4 and
dissolve in 400 mL H2O. Adjust the pH to 7.2 with hydro-
chloric acid (HCl, 37%) and add H2O to a total volume of
500 mL.
6. 2 mM NDA solution: weigh 1.9 mg naphthalene-2,3-dicar-
boxaldehyde and dissolve in 5 mL methanol.
7. 1 mM KCN solution: weigh 1.3 mg KCN and dissolve in
20 mL H2O.
8. 8% aqueous acetic acid solution: pipet 153 μL acetic acid into
2 mL H2O.
9. 0.03% acetic acid solution: pipet 9 μL acetic acid into 30 mL
H2O.
10. Washing solution: add 96 mL acetic acid and 10 g sodium
dodecyl sulfate (SDS) into 1 L H2O.
11. Escherichia coli (E. coli, TG1) and Micrococcus luteus (M. luteus)
cells are cultured in LB medium at 37 C with shaking.
2.2 Chemical 1. Initiator: Benzoyl peroxide.

Synthesis 2. Cross-linker: Trimethylolpropane trimethacrylate (TRIM),
divinylbenzene (DVB).
3. Prepare an aluminum oxide (Al2O3) column by adding Al2O3
into a ϕ 1 10 cm glass column.
3 Methods
3.1 Preparation 1. Pipet 10 μL ampicillin stock solution into 10 mL LB medium.

of Microorganism 2. Add E. coli or M. luteus cells into 10 mL of LB medium.
Samples
3. Cultivate the LB medium for 17 h at 37 C in a shaking
incubator.
4. Pipet 0.5–1 mL of the culture medium in step 3 and 100 μL of
ampicillin stock solution into 100 mL of LB medium in a
500 mL flask.
5. Cultivate at 37 C in a shaking incubator to give a final OD600
of 7.
6. Centrifuge the cell culture at 5000 g for 5 min.
7. Wash the cells twice with PBS buffer and collect by

centrifugation.
8. Resuspend the cells in PBS buffer and adjust the optical density
to OD600 ¼ 2.
3.2 Synthesis 1. Solution A: weigh 1.61 g chitosan (MW ¼ 50,000–190,000)

of N-acrylchitosan and dissolve in 40 mL N,N-dimethylacetamide (DMAC) con-
(NAC) Pre-polymer taining 2 mL triethylamine.
2. Solution B: pipet 161 μL acryloyl chloride into 5 mL DMAC.
3. Deoxygenate solution A by bubbling with argon for 10 min at
0 C (see Note 1).
4. Add solution B dropwise into solution A (see Note 2).
5. Stir the mixture at 0 C for 4 h and 25 C for another 20 h.
6. Wash the NAC in DMAC, dichloromethane, and methanol,
respectively.
7. Recover the NAC by filtration and vacuum drying to obtain a
powder.
8. Confirm the pre-polymer NAC by Fourier transform infra-red
(FT-IR) spectroscopy (Fig. 3).
9. Confirm the acrylation degree by fluorescence emission mea-
surement (Fig. 4). Weigh 8 mg of chitosan or NAC powder and
disperse it in a mixture of 1 mL NDA solution and 1 mL KCN
solution. Stir the mixture at room temperature for 1 h. Wash
the derivatized chitosan and NAC with water and methanol.
Filter and dry NDA-KCN derivatized product in a vacuum
chamber. Weigh 4 mg NDA-KCN derivatized chitosan or
NAC, dissolve it in 1 mL of aqueous acetic acid solution (8%)
and dilute the solution 500 times before the fluorescence emis-
sion is measured. Compared to NDA-treated chitosan (8 mg/
L), the fluorescence intensity of NAC treated with NDA
(8 mg/L) is 16% lower, indicating that 16% of the primary
amine on chitosan is acrylated. In order to ensure a strong
electrostatic interaction between bacteria and NAC, the degree
of acrylation is controlled at a low level. However, the acryloyl
groups in NAC are enough to co-polymerize with the mono-
mers in the oil phase.
3.3 Synthesis 1. Add 10 mL TRIM into a separatory funnel. Add 5 mL of 2 M

of Bacteria-Imprinted NaOH solution to extract the inhibitor. Collect the organic
Polymer (BIP) Beads layer, dry it over MgSO4, and filter.
2. Pass 10 mL of DVB through the Al2O3 column to remove the
inhibitor.
3. Weigh 9 mg NAC and dissolve in 30 mL acetic acid solution
(0.03%) (see Note 3).
Fig. 3 FTIR spectra of chitosan and NAC. The signal at 1550 cm1 is the C¼C
stretching peak. Reproduced from ref. [22] with permission from Wiley-VCH
Verlag GmbH & Co. KGaA
Fig. 4 Fluorescence spectra of NDA-KCN derivatized chitosan (1) and NAC (2).
The concentrations of NDA-KCN derivatized chitosan and NAC were both
8 mg L1. The excitation wavelength was 422 nm. Reproduced from ref. [22]
with permission from Wiley-VCH Verlag GmbH & Co. KGaA
4. Pipet 0.3 mL of the NAC solution and 0.9 mL of E. coli or

M. luteus suspension, mix and incubate for 30 min in a 2 mL
Eppendorf tube to form the water phase (see Note 4).
5. Mix 0.5 mL TRIM, 0.5 mL DVB, and 31 μL N,N-dimethyla-
niline (DMA). Add 6.2 mg of BPO into the mixture. Mix and
incubate for 30 min in a 5 mL tube to form the oil phase.
6. Mix the oil phase and the water phase. Shake vigorously by
hand for 2 min to give a stable Pickering emulsion.
Fig. 5 SEM images of polymer beads imprinted with bacteria before (a, b) and
after (c, d) removal of the E. coli template. Reproduced from ref. [22] with
permission from Wiley-VCH Verlag GmbH & Co. KGaA
Fig. 6 Uptake of E. coli (a) and M. luteus (b) cells by E-BIP and M-BIP beads. Conditions: Polymer beads
(50 mg) in PBS buffer (pH 7.2; 2 mL), incubated with bacteria at 4 C for 3 h. Reprinted with permission from
(see ref. [22]) © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
7. Keep the Pickering emulsion at room temperature for 24 h

without agitation (see Note 5).
8. Wash the polymer beads with the washing solution and water
for six times, respectively. This step is used to remove the
bacteria template.
9. Wash the polymer beads with methanol twice and dry the BIP
beads in a vacuum chamber. For rod-shaped E. coli cells, the
beads are named as E-BIP; for spherical M. luteus cells, the
beads are named as M-BIP. Analyze the BIP by scanning elec-
tron microscopy (SEM) imaging (Fig. 5).
10. Confirm bacteria recognition by equilibrium binding analysis

(Fig. 6).
4 Notes
1. Solution A is kept at 0 C during the deoxygenation to avoid

evaporation.
2. Solution B is added slowly to avoid excessive heat generation
due to the exothermic reaction.
3. Acetic acid is added to increase the solubility of NAC.
4. The bacteria-NAC complex is formed via electrostatic interac-
tions between the negatively charged bacteria and the positively
charged NAC.
5. NAC located at the oil–water interface and the cross-linker in
the oil phase (TRIM and DVB) copolymerize to form the solid
polymer beads.
Acknowledgments
This work was supported by the Swedish Research Council

(VR) and the Swedish Research Council for Environment, Agricul-
tural Sciences and Spatial Planning (FORMAS).
References
1. Yang Y, Fang Z, Chen X, Zhang W, Xie Y, 5. Chevalier Y, Bolzinger MA (2013) Emulsions

Chen Y, Liu Z, Yuan W (2017) An overview stabilized with solid nanoparticles: Pickering
of Pickering emulsions: solid-particle materials, emulsions. Colloid Surface A 439:23–34
classification, morphology, and applications. 6. Lin Z, Zhang Z, Li Y, Deng Y (2016) Magnetic
Front Pharmacol 8:287 nano-Fe3O4 stabilized Pickering emulsion liq-
2. Marku D, Wahlgren M, Rayner M, Sjöö M, uid membrane for selective extraction and sep-
Timgren A (2012) Characterization of starch aration. Chem Eng J 288:305–311
Pickering emulsions for potential applications 7. Leclercq L, Nardello-Rataj V (2016) Pickering
in topical formulations. Int J Pharm 428:1–7 emulsions based on cyclodextrins: a smart solu-
3. Matos M, Laca A, Rea F, Iglesias O, Rayner M, tion for antifungal azole derivatives topical
Gutiérrez G (2018) O/W emulsions stabilized delivery. Eur J Pharm Sci 82:126–137
by OSA-modified starch granules versus 8. Cheng H, Li Z, Li Y, Shi Z, Bao M, Han C,
non-ionic surfactant: Stability, rheological Wang Z (2020) Multi-functional magnetic
behaviour and resveratrol encapsulation. J bacteria as efficient and economical Pickering
Food Eng 222:207–217 emulsifiers for encapsulation and removal of oil
4. Zhao X, Yu G, Li J, Feng Y, Zhang L, Peng Y, from water. J Colloid Interf Sci 560:349–358
Tang Y, Wang L (2018) Eco-friendly Pickering 9. Srivastava SP, Singh HD, Baruah JN, Krishna
emulsion stabilized by silica nanoparticles dis- PV, Iyengar MS (1970) Physicochemical stud-
persed with high-molecular-weight amphi- ies on the water-yeast cells-gas-oil system. J
philic alginate derivatives. ACS Sustain Chem Appl Chem 20:105–108
Eng 6:4105–4114 10. Firoozmand H, Rousseau D (2016) Microbial
cells as colloidal particles: Pickering oil-in-
water emulsions stabilized by bacteria and of the new psychoactive substance “Mephe-
yeast. Food Res Int 81:66–73 drone”. Biosens Bioelectron 119:163–169
11. Wongkongkatep P, Manopwisedjaroen K, 17. Bie Z, Chen Y, Ye J, Wang S, Liu Z (2015)
Tiposoth P, Archakunakorn S, Boronate-affinity glycan-oriented surface
Pongtharangkul T, Suphantharika M, imprinting: a new strategy to mimic lectins for
Honda K, Hamachi I, Wongkongkatep J the recognition of an intact glycoprotein and its
(2012) Bacteria interface Pickering emulsions characteristic fragments. Angew Chem Int Edit
stabilized by self-assembled bacteria–chitosan 54:10211–10215
network. Langmuir 28:5729–5736 18. Cumbo A, Lorber B, Corvini PFX, Meier W,
12. Destribats M, Rouvet M, Gehin-Delval C, Shahgaldian P (2013) A synthetic nanomaterial
Schmitt C, Binks BP (2014) Emulsions stabi- for virus recognition produced by surface
lised by whey protein microgel particles: imprinting. Nat Commun 4:1503
towards food-grade Pickering emulsions. Soft 19. van Grinsven B, Eersels K, Akkermans O,
Matter 10:6941–6954 Ellermann S, Kordek A, Peeters M,
13. Frelichowska J, Bolzinger MA, Valour JP, Deschaume O, Bartic C, Diliën H, Steen
Mouaziz H, Pelletier J, Chevalier Y (2009) Redeker E, Wagner P (2016) Label-free detec-
Pickering w/o emulsions: drug release and tion of Escherichia coli based on thermal trans-
topical delivery. Int J Pharm 368:7–15 port through surface imprinted polymers. ACS
14. Sharma T, Kumar GS, Chon BH, Sangwai JS Sensors 1:1140–1147
(2015) Thermal stability of oil-in-water Pick- 20. Kunath S, Panagiotopoulou M, Maximilien J,
ering emulsion in the presence of nanoparticle, Marchyk N, S€anger J, Haupt K (2015) Cell and
surfactant, and polymer. J Ind Eng Chem tissue imaging with molecularly imprinted
22:324–334 polymers as plastic antibody mimics. Adv
15. Ou H, Chen Q, Pan J, Zhang Y, Huang Y, Qi X Healthc Mater 4:1322–1326
(2015) Selective removal of erythromycin by 21. Kupai J, Razali M, Buyuktiryaki S, Kecili R,
magnetic imprinted polymers synthesized Szekely G (2017) Long-term stability and reus-
from chitosan-stabilized Pickering emulsion. J ability of molecularly imprinted polymers.
Hazard Mater 289:28–37 Polym Chem 8:666–673
16. Razavipanah I, Alipour E, Deiminiat B, Rou- 22. Shen X, Svensson Bonde J, Kamra T, Bülow L,
naghi GH (2018) A novel electrochemical Leo JC, Linke D, Ye L (2014) Bacterial
imprinted sensor for ultrasensitive detection imprinting at Pickering emulsion interfaces.
Angew Chem Int Edit 53:10687–10690
Chapter 5
Restricted Access Molecularly Imprinted Polymers

Mariana Azevedo Rosa, Tássia Venga Mendes,
and Eduardo Costa Figueiredo
Abstract
The use of conventional molecularly imprinted polymers (MIPs) for biological sample preparation is a
difficult procedure due to the presence of high concentrations of proteins which can obstruct the selective
binding sites, decrease the adsorption capacity, and compromise the analytical validation. In this way,
modifications of conventional MIPs have been carried out in order to give them the ability to exclude
macromolecules. Superficial coverings with hydrophilic groups and/or proteins have been the main
procedures to obtain these restricted access molecularly imprinted polymers (RAMIPs). These materials
have been efficiently used for the selective extraction of small molecules from untreated complex matrices
(e.g., blood, plasma, serum, and milk), without the need of a pre-deproteinization step. In this chapter, we
describe a generic synthesis protocol to obtain RAMIPs as well as the assays to evaluate the protein
exclusion efficiency and possible applications in offline and online procedures.
Key words Restricted access molecularly imprinted polymer, Hydrophilic groups, Bovine serum albu-
min, Protein exclusion, Macromolecules exclusion
1 Introduction
The use of molecularly imprinted polymers (MIP) in biological

sample preparation (e.g., blood, plasma, serum, milk, among
others) is not a simple task due to the presence of macromolecules
(such as proteins, polypeptides, and lipids) which can be sorbed
onto the polymer surfaces and minimize selectivity for the target
molecule [1]. To solve this problem, additional sample preparation
steps, such as the protein precipitation technique, have been carried
out before the molecularly imprinted solid phase extraction
(MISPE). However, this procedure can result in some problems
such as sample dilution, pH changes, loss of analytes through
Mariana Azevedo Rosa and Tássia Venga Mendes contributed equally to this work.
https://doi.org/10.1007/978-1-0716-1629-1_5,
53
54 Mariana Azevedo Rosa et al.
co-precipitation, and ionic suppression in mass spectrometry due to

the presence of residual precipitant agents [2].
Haginaka et al. [3] proposed, in 1999, MIPs that are able to
exclude macromolecules. Today, these are called restricted access
molecularly imprinted polymers (RAMIPs). The exclusion mechan-
isms of RAMIPs were inspired by the concept of restricted access
materials (RAMs) by the presence of physical (pores) and/or chem-
ical (hydrophilic network) diffusion barriers [4–6]. Low molecular
weight target molecules can penetrate through these barriers and
be captured in the selective binding sites, whereas macromolecules
are excluded. Additionally, the protective barriers can also prevent
the influence of water in the selective interactions between target
molecules and RAMIP [3, 7, 8]. Conventional MIPs can be con-
verted to RAMIPs through the modification of their external sur-
faces with hydrophilic monomers, proteins, or a combination
between both [4, 5], as shown in Fig. 1.
The hydrophilic monomers are incorporated in the MIP during
its synthesis and create a dense region of hydroxyl groups on the
material surface. Monomers like glycidyl methacrylate (GMA) and
3-glycidyloxypropyltrimethoxysilane (GPTMS) become hydro-
philic after a chemical reaction (opening of their epoxide ring).
Other monomers, such as glycerol monomethacrylate (GMMA),
glycerol dimethacrylate (GDMA), and hydroxymethylmethacrylate
(HEMA) are primarily hydrophilic due to their free hydroxyl
groups. GMA is one of the most often used monomers because of
its low interference in the polymerization complex formation
[9]. In its turn, the BSA layer is incorporated after polymerization
and with a dry and clean polymer [4, 10, 11]. Unlike what occurs
with the hydrophilic groups, the BSA layer is not going to build any
chemical bond with the polymer surface. In place of this, a cross-
linked BSA capsule is formed outside of the MIP. Glutaraldehyde
has been used as a crosslinker reagent to bond the BSA molecules
by reaction with their amino terminal groups [12, 13]. The high
instability of the imines obtained requires their conversion to amine
groups by using sodium borohydride as a reducing agent, as shown
in Fig. 2 [8, 10, 11]. The RAMIPs generally are able to exclude
80–100% of the amount of proteins present in the samples [4, 10,
11, 14].
The RAMIPs can be employed as sorbents in traditional
MISPE [1, 15–18] or in its variants, such as microextraction
using fibers [11, 19], microextraction using packed sorbents [20],
in-tube microextraction [21], and dispersive extraction [10, 22,
23], as well as in column switching liquid chromatography [3, 5,
7, 12, 13, 24–27]. The online method makes possible the direct
injection of complex matrices into liquid chromatographic (LC)
systems. Therefore, sample preparation and analyte determination
are performed in sequential steps with minimum sample manipula-
tion by the analyst.
Restricted Access Molecularly Imprinted Polymers 55
Fig. 1 General synthesis scheme to obtain RAMIPs. The polymer surface can be modified with hydrophilic
monomers (HM) and/ or with bovine serum albumin (BSA). Reproduced from Ref. [4] with permission from The
Royal Society of Chemistry
Fig. 2 Bovine serum albumin (BSA) coating reactions. (1) interaction between glutaraldehyde (crosslinking
agent) and a BSA molecule; (2) bonding effect from glutaraldehyde on BSA molecules forming the crisscrossed
BSA layer; (3) addition of sodium borohydride to reduce imines to amines
In this chapter, a generic procedure to obtain RAMIPs by using

hydrophilic monomers and BSA is demonstrated. Additionally,
some strategies to verify the RAMIP’s protein exclusion ability are
presented, as well as some possible analytical applications.
2 Materials
All of the solutions must be prepared on the same day that they are
to be used. The aqueous solutions should be prepared using ultra-
pure water (conductivity of 18 MΩ-cm at 25 C) and the buffers
should be stored in a refrigerator (4 C), if necessary. After promot-
ing the reactions, the reagents residues must be disposed of appro-
priately. Due to the addition of the modifiers, the MIP surface
modifications can result in different amounts of final material.
2.1 RAMIP 1. Template: any target molecule for which the RAMIP will be
Synthesis: Hydrophilic selective.
Monomer Grafting 2. Functional monomer: methacrylic acid (MAA) or 4-vinyl pyri-
dine (4-VP), for basic or acidic molecules, respectively.
3. Radical initiator: 4,40 -Azobis (4-cyanovaleric acid) (ABCVA)
or 2,20-Azobis (2-methylpropionitrile) (AIBN).
4. Porogenic solvent: acetonitrile, methanol, chloroform, among
others.
5. Three neck round bottom flask.
6. Nitrogen gas.
7. Glycerin bath with temperature control.
8. Condenser.
9. Thermometer.
10. Stoppers.
11. Magnetic stirrer and a stir bar.
12. Glycidyl methacrylate (GMA) monomer (see Note 1).
13. Drying oven.
2.2 Template 1. Cleaning solutions: methanol:acid acetic (9:1 v/v) and

Removal from RAMIP methanol.
2. Glass tubes.
3. Ultrasonic bath.
4. Centrifuge or a magnet (for magnetic RAMIPs).
5. Drying oven.
2.3 Epoxide Ring 1. Perchloric acid water solution (10% v/v) (see Note 2).
Opening 2. 100 mL glass flask.
3. Shaker or stirrer with a stir bar.
4. Vacuum filter apparatus (Büchner filter, paper filter, Kitasato,
and vacuum pump) or a centrifuge or a magnet.
5. Water to wash the material.
2.4 Coating 1. 1% (w/v) BSA aqueous solution prepared in a 0.05 M phos-

with Bovine Serum phate buffer, pH 6.0.
Albumin 2. 5% (w/v) aqueous glutaraldehyde solution.
3. 1% (w/v) sodium borohydride aqueous solution.
4. Water to wash the material.
5. Drying oven.
2.4.1 Covering the MIP 1. Empty conventional solid phase extraction (SPE) cartridge.
Using a Solid Phase 2. Frits.
Extraction Cartridge
3. Manifold® system.
4. Vacuum pump.
2.4.2 Covering the MIP 1. Glass tube.

Using a Glass Tube 2. Ultrasonic bath.
3. Shaker.
4. Magnet (in magnetic MIP cases).
2.5 Protein The protein exclusion test is performed to appraise the RAMIP
Exclusion Test capacity to exclude macromolecules. The analyst can choose among
different strategies such as using columns [4, 10, 12, 26, 27] or
cartridges [14, 16, 18, 28] filled with RAMIP, as well as in a solid
phase microextraction procedure in case of RAMIP fibers [11]. The
material is placed in contact with an aqueous protein solution
(normally BSA), and the protein recovery is correlated to the
RAMIP exclusion efficiency (see Note 3).
2.5.1 In Column Packed 1. Empty column (4–50 mm 4.6 mm i.d.).

with RAMIP 2. Frits or guard filters.
3. 44 g L1 BSA aqueous solution (prepared in 0.05 M phosphate
buffer, pH 7.0).
4. Mobile phase: 0.01 M phosphate buffer, pH 7.0.
5. LC system with an ultraviolet (UV) detector.
2.5.2 In Solid Phase 1. Empty conventional SPE cartridge.

Extraction Cartridges 2. Frits.
3. Manifold® system.
buffer, pH 7.0).
5. Quartz cuvettes.
6. UV spectrophotometer.
2.5.3 For Solid Phase 1. Glass inserts.

Microextraction RAMIP 2. Shaker.
Fibers
buffer, pH 7.0).
4. Bradford reagent.
5. Quartz cuvettes.
3 Methods
3.1 RAMIP The methods will be described in chronological order from the
Synthesis: Hydrophilic RAMIP synthesis until the material evaluation.
Monomer Grafting Usually, the molar ratio between the GMA (see Note 4) and the
functional monomer is 1:1. However, 2:1 proportions can also be
employed [8, 24, 28].
GMA can be added in a single-step synthesis when all the
reagents are placed together and the polymerization occurs for
24 h, or after a pre-polymerization stage when an MIP is formed.
Both procedures are described below. Figure 3 shows a schematic
setup for the synthesis of RAMIP particles.
3.1.1 RAMIP Obtained 1. In a 250 mL three neck round bottom flask, dissolve the
with Hydrophilic Monomer template (0.2 mmol) and the functional monomer
in a Single-Step Synthesis (1.2 mmol) in the porogenic solvent (40 mL) (see Note 5).
2. Add the radical initiator (0.15 mmol), crosslinking agent
(7.0 mmol), and GMA (1.2 mmol) into the synthesis flask.
3. Purge the mixture with nitrogen for 10–20 min and seal the
flask.
4. Connect the flask to a condenser.
5. Immerse the flask in a glycerin bath at 60–80 C and maintain it
under agitation for 24 h. The selected temperature depends on
the radical initiator.
6. Dry the material at 60–70 C for 24 h.
3.1.2 RAMIP Obtained The pre-polymerization methodology is used to avoid possible

with Hydrophilic Monomer interference from some hydrophilic monomers in the formation
Added After of imprinting binding sites (see Note 6).
a Pre-polymerization Step
1. Pre-polymerization: Add the template (0.2 mmol), functional
monomer (1.2 mmol), and porogenic solvent (40 mL) to a
three neck round bottom flask (see Note 5). React for about 4 h
under stirring, at room temperature.
Fig. 3 Schematic setup for the synthesis of RAMIP particles
2. After that period, add the radical initiator (0.15 mmol), cross-
linking agent (7.0 mmol), and GMA (1.2 mmol) into the
medium.
3. Purge the mixture with nitrogen for 10–20 min and seal the
flask.
4. Connect the flask to a condenser.
5. Immerse the flask in a glycerin bath at 60–80 C and maintain it
under agitation.
6. The polymerization proceeds for 24 h (see Note 7).
7. Dry the material at 60–70 C for 24 h.
3.2 Template 1. Transfer the synthesized material to glass tubes (about 500 mg
Removal from RAMIP of the material in each tube) (see Note 8).
2. Add 10 mL of a 9:1(v:v) methanol:acetic acid solution.
3. Place the tubes into an ultrasonic bath for about 1 h (see Note
9).
4. Separate the material using a centrifuge and dispose of the
supernatant.
5. Repeat steps 2, 3, and 4 until the template molecule is totally
removed from the RAMIPs.
6. Wash the material with pure methanol to remove the acid

residue from the RAMIP.
7. Let the polymer dry at 70 C for 24 h.
8. This cleaning step can be evaluated by an analytical instrument
if necessary (see Note 10).
3.3 Epoxide Ring 1. Place 400 mg of dried MIP in a 100 mL glass flask with 25 mL
Opening of perchloric acid solution (see Note 2).
2. Let the reaction occur for 24 h under agitation and at room
temperature.
3. Filter the particles or separate them with a centrifuge.
4. Wash the RAMIPs with water until the acid solution is
completely removed.
A scheme of the RAMIP external layer after the epoxide
ring opening process is shown in Fig. 4.
3.4 Coating Coating with BSA can be applied on dried MIPs or RAMIPs with
with Bovine Serum hydrophilic groups (obtained with hydrophilic monomers or
Albumin monomers that become hydrophilic after opening the epoxide
ring). The BSA covering process can be performed using a solid
phase extraction cartridge [4, 8, 13, 19, 28] or a glass tube [10, 11,
20, 23].
3.4.1 Covering the MIP 1. Place 500 mg of the material in a 5 mL conventional and empty
Using a Solid Phase SPE cartridge (use a bottom frit).
Extraction Cartridge 2. Couple the cartridge to a Manifold® system.
3. Percolate about 20 mL of 1% BSA solution (see Note 11).
4. Add 25 mL of 5% (w/v) glutaraldehyde aqueous solution to
the material and stop the flow. Let this solution stand in contact
with the polymer for 5 h (see Note 12).
5. Afterwards, percolate the 5% (w/v) aqueous glutaraldehyde
solution.
6. Percolate 10 mL of 1% (w/v) aqueous sodium borohydride
solution through the polymer at 1 mL min1.
7. Wash the polymer with water to remove any reagent residues.
8. Dry the material at 60 C for 24 h (see Note 13).
3.4.2 Covering the MIP This method has been used for magnetic MIP particles and MIP
Using a Glass Tube fibers. To develop a magnetic MIP, Fe3O4 nanoparticles should be
previously synthetized and incorporated to the polymer. These
particles will bring a magnetic characteristic to the MIP [10]. On
the other hand, the fibers can be synthesized on surface-modified
stainless-steel fibers or inside glass capillary tubes and applied in
solid phase microextraction analyses [11, 19].
Fig. 4 Arrangement of the external layer with hydroxyl groups after the epoxide ring opening process
3.4.3 Procedure 1. Add 500 mg of a magnetic MIP (M-MIP) into a 50 mL

for Covering glass tube.
Magnetic MIPs 2. Place 20 mL of 1% (m/v) BSA aqueous solution in the same
glass tube.
3. Sonicate the tube for 10 min.
4. Leave the tube on standby for 10 min after the sonication.
5. Place a magnet outside of the wall tube and the M-MIP will be
attracted to it.
6. Discard the supernatant.
7. Disperse the polymer in 5 mL of 25% (m/v) glutaraldehyde
aqueous solution and promote a gentle and continued agita-
tion (shaker) or just let it stand in contact with the material for
5 h (see Note 12).
8. After 5 h, the supernatant should be discarded following the
steps 5 and 6.
9. Disperse the material in 10 mL of 1% (m/v) sodium borohy-
dride aqueous solution under agitation (gentle and continued)
for 15 min (see Note 14).
10. Separate the RAMIP from the supernatant as in steps 5 and
6 and wash it with water several times to remove any reagent
residues.
11. Dry the material at 60 C for 24 h (see Note 13).
3.4.4 Procedure 1. Place the MIP fiber in a glass tube containing 10 mL of 1% BSA
for Covering MIP Fiber solution.
2. Slightly shake the tube for 20 min.
3. Discard the BSA solution.
4. Add 10 mL of a 25% glutaraldehyde aqueous solution to the

same tube containing the fibers and leave the tube on standby
for 5 h (see Note 12).
5. After 5 h, discard this solution and add 10 mL of 1% sodium
borohydride aqueous solution to the glass tube. The fibers
need to be immersed in the solution for 20 min.
6. Discard this solution after the 20 min and wash the RAMIPs
with water several times.
7. Dry the fibers at 60 C for 24 h (see Note 13).
3.5 Protein The strategies to perform the protein exclusion assays are described
Exclusion Tests in the following topics and demonstrated in Fig. 5.
3.5.1 Column Packed 1. Fill a column (4–50 mm 4.6 mm i.d.) with the synthesized
with RAMIP RAMIPs. Use frits or guard filters to confine the material.
2. Place the RAMIP column at the chromatographic column spot
into an LC system.
3. Use the mobile phase with a 0.3–1.0 mL min1 flow rate
during all the assay.
4. Inject 10 μL of the BSA aqueous solution directly into the
system. Use 255 nm as the selected wavelength.
5. The obtained peak area refers to the BSA signal from the
RAMIP exclusion.
6. Change the filled RAMIP column to a connector
(non-column) in the LC system.
7. Repeat the steps 3 and 4.
8. The obtained peak area refers to the 100% BSA signal.
9. Compare both peak areas (obtained with and without the
column) to calculate how much protein percentage the
RAMIP was able to exclude.
3.5.2 Solid Phase 1. Fill empty SPE cartridges with RAMIPs (100–500 mg). Use
Extraction Cartridges frits to delimit the material space (bottom and top).
2. Couple the cartridges to a Manifold® system.
3. Percolate the BSA solution (about 2.0 mL).
4. Collect the eluates.
5. Quantify total proteins in the eluates by UV or Bradford
method [29] (see Note 15).
6. For the 100% BSA signal, use the prepared BSA solution.
Repeat step 5.
7. Compare both signals to calculate the percentage protein
exclusion.
Fig. 5 Procedures to evaluate the RAMIP’s macromolecules exclusion capacity: (a) using a column packed
with RAMIPs, (b) using an empty solid phase extraction cartridge packed with the RAMIPs, and (c) using a solid
phase microextraction RAMIP fiber
8. The BSA concentration can also be determined by an analytical

curve (see Note 16).
3.5.3 Solid Phase 1. Add around 200 μL of the BSA solution at a known concentra-
Microextraction RAMIP tion into a glass insert.
Fibers 2. Place one fiber into the insert.
3. Sonicate for about 10 min.
4. Separate the fiber from the supernatant.
5. Analyze the proteins in supernatants by UV or Bradford
reagent.
6. For the 100% BSA signal, use the prepared BSA solution (with-
out extraction).
7. Compare both signals to calculate the percentage protein
exclusion.
8. The BSA concentration can also be determined by an analytical
curve (see Note 16).
Fig. 6 Possible applications of RAMIPs in sample preparation procedures. (a) solid phase extraction in
cartridge, (b) magnetic dispersive solid phase extraction, (c) microextraction by packed sorbent, (d) solid
phase microextraction, (e) offline in-tube solid phase microextraction, (f) column switching liquid chromatog-
raphy, (g) online in-tube solid phase microextraction. (AA: autosampler, D: detector V: valve, W: waste)
3.6 Possible The RAMIPs are versatile materials that can be employed in differ-
Applications ent strategies for sample preparation, as demonstrated in Fig. 6 and
described below.
3.6.1 Solid Phase 1. Pack 100–300 mg of RAMIPs in an empty SPE cartridge. Use
Extraction in Cartridge frits to confine the material (see Note 17).
2. Couple the cartridge to a Manifold® system.
3. Follow the SPE steps. Use constant flow rate.
(a) Activation: pure methanol and then ultrapure water are
generally used to activate the RAMIPs.
(b) Loading: percolate the sample through the cartridge.
(c) Washing: water or methanol:water (4:6 v:v) can be

employed in this step.
(d) Eluting: normally methanol:acid acetic (9:1 v:v) solution
is used for analytes elution.
4. Collect the eluent.
5. Dry the eluent and dissolve the residue in a small volume of
mobile phase or organic solvent.
6. Perform the developed chromatography method.
3.6.2 Magnetic 1. Place 10–20 mg of the magnetic RAMIP (M-RAMIP) particles

Dispersive Solid Phase in a glass tube and add the biological sample (0.5–1.0 mL) (see
Extraction Note 18).
2. Shake the tubes: the agitation time should be sufficient to reach
the equilibrium.
3. Separate the RAMIP particles with an external magnet.
4. Wash the particles with ultrapure water and dispose of the
water.
5. Disperse the M-RAMIP in the eluting solution: methanol or
methanol: acetic acid (9:1 v:v) solution can be used in this step.
6. Separate the eluate using a magnet.
organic solvent or mobile phase, if necessary.
8. Perform the developed chromatography method.
3.6.3 Microextraction by 1. Fill a micro-column with 1–4 mg of RAMIP. Use frits to

Packed Sorbents confine the material (see Note 17).
2. Connected the column to the needle of a micro-syringe.
3. Perform the microextraction by packed sorbent cycle:
(a) Conditioning: ultrapure water is used in this step.
(b) Sample loading: cycles of aspersion/dispersion to
pre-concentrate the analytes.
(c) Washing: ultrapure water is a related solvent for this step.
(d) Eluting: methanol:acetic acid (9:1 v/v) solution is used
for analytes elution.
4. Collect the eluent.
organic solvent, if necessary.
6. Perform the developed chromatographic method.
3.6.4 Solid Phase 1. Place the RAMIP fiber in contact with the sample: dip the fiber
Microextraction in a 1.5 mL tube containing the sample and shake the tube (see
Note 19).
2. Desorb the analytes from the fiber: acetonitrile and methanol:

acetic acid (8:2 v:v) solution were the reagents reported in the
literature to perform this step. The desorption can also be
directly in the gas chromatography.
organic solvent or mobile phase, if necessary.
3.6.5 In-Tube Solid 1. Connect the RAMIP capillary to a micro-syringe (see Note 20).
Phase Microextraction 2. Perform the solid phase microextraction procedures:
(a) Conditioning: pure methanol and then ultrapure water
are employed in the column conditioning.
(b) Sample loading: drawn/eject cycles to pre-concentrate
the analytes.
(c) Washing: ultrapure water is generally used in this step.
(d) Eluting: methanol:ethanol (1:1 v:v) solution is used for
analytes elution.
3.6.6 Column Switching 1. Pack a lab-made or commercial pre-column holder with about
Liquid Chromatography 30–70 mg of the RAMIP (see Note 17).
2. Be aware of the necessity to place frits and/or guard filters in
both sides of the column, confining the material within a
certain space (see Note 21).
3. Place the RAMIP column into the LC system with the analyti-
cal column and perform the developed method. The sample is
injected, and the analytes are retained in the RAMIP column
while the interferents flow to the waste. Concomitantly, the
analytical column is conditioned with the mobile phase. The
switching valve commutes, and the analytes are eluted from the
RAMIP column through the analytical column. After a specific
moment, the valve returns to the initial position and the
RAMIP column is cleaned (see Note 22), while the analytes
are chromatographic separated.
4 Notes
1. Other hydrophilic monomers can also be used, such as GPTMS

[14, 22], GDMA [3, 4, 7, 12, 20, 21, 23, 24, 27, 30, 31],
GMMA [3, 7, 25, 26, 30, 31], and HEMA [4, 12, 20, 23, 24,
27].
2. Sulfuric acid water solution (10% v/v) can also be used for the
epoxide ring opening. In this case, the procedure is performed
at a temperature of 60 C [18, 28, 32].
3. The exclusion test can also be made using a blank matrix and an
LC-UV system by comparing the chromatograms obtained
with extractions using a conventional MIP and its RAMIP.
4. This general RAMIP synthesis protocol was stipulated based on
different papers in the literature [8, 9, 15, 16, 18, 28, 32, 33]
and the precipitation polymerization methodology was related.
However, it is worth pointing out that MIPs synthesized by any
method can be converted to RAMIPs.
5. An ultrasonic bath can be used to improve the process.
6. When using primarily hydrophilic monomers (e.g., GMMA,
GDMA, HEMA) the pre-polymerization step is usually
demanded.
7. In some protocols, the polymerization step occurs for 10 h
instead of 24 h [1, 17].
8. Soxhlet extraction can also be used for template removal [1, 9,
14, 16, 17, 33].
9. A shaker can be used instead of the ultrasonic bath.
10. An ultraviolet-visible spectrophotometer, a liquid
chromatography-ultraviolet detector, or a liquid
chromatography-mass spectrometry can be employed to mon-
itor the template content in the used cleaning solutions.
11. Do not let the material dry during the process.
12. This is the time necessary to promote the binding between the
BSA molecules.
13. Do not use a higher temperature than 60 C. This can promote
BSA degradation and diminish the performance of the RAMIP
for excluding proteins.
14. Do not shake it hard because the solution can form bubbles
and the covering can be harmed. Also, it is harder to handle the
material when this happens.
15. Other methodologies using SPE or matrix dispersive solid
phase extraction can also be performed:
(a) The amount of BSA in the eluate can be determined by
combustion using an elemental analyzer. The total con-
tent of nitrogen is given, and this value may be converted
into protein content by using conversion factors [17].
(b) Comparison of analyte recoveries using MIPs and
RAMIPs in samples fortified with different concentrations
of macromolecules [22].
16. To build the analytical curve, the absorbance measurements of
different BSA standard solutions need to be made.
17. It is important to uniformly pack the cartridge/column with
the RAMIPs to avoid the formation of preferred pathways that
can give unreliable results. The material needs to be compacted

between the filters to avert mobility when the mobile phase
flows.
18. The M-RAMIPs were also employed in the treatment of envi-
ronmental matrix. In this case, 135 mg of the material were
shaken with 90 mg of soil samples [22].
19. There is a described methodology in which the authors
exposed the RAMIP fibers into livers of rats in vivo [19].
20. In the online in-tube solid phase microextraction, the RAMIP
capillary can be connected to a switching six-port valve in the
LC system (Fig. 6g). Firstly, the sample is percolated through
the capillary and the drawn/eject cycles are performed. At the
same time, the analytical column is conditioned with the
mobile phase. Afterwards, the RAMIP is cleaned with a weak
solvent to remove the reminiscent interferents. The retained
analytes are eluted from the RAMIP capillary to the analytical
column when the valve switches. Then, the valve returns to the
initial position and the analytes are analyzed, while the RAMIP
is conditioned for the next extraction cycle.
21. Frits usually have larger pore diameters than guard filters, so
they provide lower pressures, and it is important to evaluate if
there is any material leakage from the RAMIP column.
22. Cleaning the RAMIP column is performed during the online
solid phase extraction cycle. This step is important due to the
possibility of remaining interferents in the RAMIP column
increase the system pressure along the analyses.
Acknowledgments
The authors are thankful to the Fundação de Amparo à Pesquisa do

Estado de Minas Gerais (FAPEMIG, Belo Horizonte, Brazil); the
Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico
(CNPq, Brasilia, Brazil); and the Coordination for the Improve-
ment of Higher Education Personnel (CAPES, Brasilia, Brazil) for
financial support, as well as The Royal Society of Chemistry for the
reproduction of Fig. 1 (from ref. [4]). Mariana Azevedo Rosa and
Tássia Venga Mendes contributed equally to this work.
References
1. He J, Song L, Chen S et al (2015) Novel 10. Mendes TV, Franqui LS, Santos MG et al
restricted access materials combined to molec- (2020) Synthesis and characterization of a
ularly imprinted polymers for selective solid- new magnetic restricted access molecularly
phase extraction of organophosphorus pesti- imprinted polymer for biological sample prep-
cides from honey. Food Chem 187:331–337. aration. Mater Today Commun 24:101002.
https://doi.org/10.1016/j.foodchem.2015. https://doi.org/10.1016/j.mtcomm.2020.
04.069 101002
2. Majors RE (1991) An overview of sample prep- 11. Abrão LCDC, Figueiredo EC (2019) A new
aration. LC-GC 9:16–20 restricted access molecularly imprinted fiber for
3. Haginaka J, Takehira H, Hosoya K, Tanaka N direct solid phase microextraction of benzodia-
(1999) Uniform-sized molecularly imprinted zepines from plasma samples. Analyst
polymer for (S)-naproxen selectively modified 144:4320–4330. https://doi.org/10.1039/
with hydrophilic external layer. J Chromatogr C9AN00444K
A 849:331–339. https://doi.org/10.1016/ 12. de Lima MM, Vieira AC, Martins I et al (2016)
S0021-9673(99)00570-1 On-line restricted access molecularly imprinted
4. Santos MG, Moraes GDOI, Nakamura MG solid phase extraction of ivermectin in meat
et al (2015) Restricted access molecularly samples followed by HPLC-UV analysis. Food
imprinted polymers obtained by bovine serum Chem 197:7–13. https://doi.org/10.1016/j.
albumin and/or hydrophilic monomers’ exter- foodchem.2015.10.082
nal layers: a comparison related to physical and 13. de Oliveira Isac Moraes G, da Silva LMR, dos
chemical properties. Analyst 140:7768–7775. Santos-Neto ÁJ et al (2013) A new restricted
https://doi.org/10.1039/C5AN01482D access molecularly imprinted polymer capped
5. de Faria HD, de Abrão LCC, Santos MG et al with albumin for direct extraction of drugs
(2017) New advances in restricted access mate- from biological matrices: the case of chlor-
rials for sample preparation: a review. Anal promazine in human plasma. Anal Bioanal
Chim Acta 959:43–65. https://doi.org/10. Chem 405:7687–7696. https://doi.org/10.
1016/j.aca.2016.12.047 1007/s00216-013-7275-5
6. Faraji M, Yamini Y, Gholami M (2019) Recent 14. Zuo H, Lin Y, Ma X et al (2019) Preparation of
advances and trends in applications of solid- a novel restricted access material combined to
phase extraction techniques in food and envi- core-shell magnetic molecularly imprinted
ronmental analysis. Chromatographia polymers for determination of dimethyl phthal-
82:1207–1249. https://doi.org/10.1007/ ate in soils. Soil Sediment Contam An Int J
s10337-019-03726-9 28:529–546. https://doi.org/10.1080/
7. Haginaka J, Sanbe H (2000) Uniform-sized 15320383.2019.1633272
molecularly imprinted polymers for 15. Du B, Qu T, Chen Z et al (2014) A novel
2-arylpropionic acid derivatives selectively restricted access material combined to molecu-
modified with hydrophilic external layer and larly imprinted polymers for selective solid-
their applications to direct serum injection phase extraction and high performance liquid
analysis. Anal Chem 72:5206–5210. https:// chromatography determination of
doi.org/10.1021/ac0005215 2-methoxyestradiol in plasma samples. Talanta
8. Santos MG, Tavares IMC, Barbosa AF et al 129:465–472. https://doi.org/10.1016/j.
(2017) Analysis of tricyclic antidepressants in talanta.2014.05.005
human plasma using online-restricted access 16. Zhao S, Wei C, Sun Z et al (2019) Preparation
molecularly imprinted solid phase extraction of restricted access media-molecularly
followed by direct mass spectrometry identifi- imprinted polymers for the detection of chlor-
cation/quantification. Talanta 163:8–16. amphenicol in bovine serum. J Anal Methods
https://doi.org/10.1016/j.talanta.2016.10. Chem 2019:1–12. https://doi.org/10.1155/
047 2019/7930102
9. Puoci F, Iemma F, Cirillo G et al (2009) New 17. Zuo HG, Yang H, Zhu JX, Ding Y (2017)
restricted access materials combined to molec- Preparation of a novel RAM-MIP for selective
ularly imprinted polymers for selective recogni- solid-phase extraction and gas chromatography
tion/release in water media. Eur Polym J determination of heptachlor, endosulfan and
45:1634–1640. https://doi.org/10.1016/j. their metabolite residues in pork. Anal Meth-
eurpolymj.2009.01.021 ods 9:6009–6018. https://doi.org/10.1039/
C7AY01941F
18. Wang X, Zhao L, Sun Z, Gong B (2019) Prep- 26. Li X, Zhou M, Turson M et al (2013) Prepara-
aration of ofloxacin-restricted access media–- tion of clenbuterol imprinted monolithic poly-
molecularly imprinted polymers for its mer with hydrophilic outer layers by reversible
selective recognition of ofloxacin in milk sam- addition–fragmentation chain transfer radical
ples. Chromatographia 82:1041–1050. polymerization and its application in the clen-
https://doi.org/10.1007/s10337-019- buterol determination from human serum by
03738-5 on-line solid-phase e. Analyst 138:3066.
19. Wang D-D, Gao D, Huang Y-K et al (2019) https://doi.org/10.1039/c3an36801g
Preparation of restricted access molecularly 27. da Silva KKMS, Boralli VB, Wisniewski C, Fig-
imprinted polymers based fiber for selective ueiredo EC (2016) On-line restricted access
solid-phase microextraction of hesperetin and molecularly imprinted solid-phase extraction
its metabolites in vivo. Talanta 202:392–401. of selective serotonin reuptake inhibitors
https://doi.org/10.1016/j.talanta.2019.05. directly from untreated human plasma samples
016 followed by HPLC-UV analysis. J Anal Toxicol
20. de Oliveira HL, Teixeira LS, Dinali LAF et al 40:108–116. https://doi.org/10.1093/jat/
(2019) Microextraction by packed sorbent bkv121
using a new restricted molecularly imprinted 28. Sun Z, Liu H, Zhou Y et al (2019) A restricted
polymer for the determination of estrogens access molecularly imprinted polymer coating
from human urine samples. Microchem J on metal–organic frameworks for solid-phase
150:104162. https://doi.org/10.1016/j. extraction of ofloxacin and enrofloxacin from
microc.2019.104162 bovine serum. RSC Adv 9:27953–27960.
21. Souza ID, Melo LP, Jardim ICSF et al (2016) https://doi.org/10.1039/C9RA04143E
Selective molecularly imprinted polymer com- 29. Bradford MM (1976) A rapid and sensitive
bined with restricted access material for in-tube method for the quantitation of microgram
SPME/UHPLC-MS/MS of parabens in breast quantities of protein utilizing the principle of
milk samples. Anal Chim Acta 932:49–59. protein-dye binding. Anal Biochem
https://doi.org/10.1016/j.aca.2016.05.027 72:248–254. https://doi.org/10.1016/
22. Liang T, Chen L, Ma Y (1609) Mesoporous 0003-2697(76)90527-3
structured molecularly imprinted polymer with 30. Sambe H, Hoshina K, Haginaka J (2007)
restricted access function for highly selective Molecularly imprinted polymers for triazine
extraction of chlorpyrifos from soil. J Chroma- herbicides prepared by multi-step swelling and
togr A 2020:460453. https://doi.org/10. polymerization method. J Chromatogr A
1016/j.chroma.2019.460453 1152:130–137. https://doi.org/10.1016/j.
23. de Oliveira HL, Pires BC, Teixeira LS et al chroma.2006.09.003
(2019) Novel restricted access material com- 31. Sanbe H, Haginaka J (2003) Restricted access
bined to molecularly imprinted polymer for media-molecularly imprinted polymer for pro-
selective magnetic solid-phase extraction of pranolol and its application to direct injection
estrogens from human urine. Microchem J analysis of β-blockers in biological fluids. Ana-
149:104043. https://doi.org/10.1016/j. lyst 128:593–597. https://doi.org/10.1039/
microc.2019.104043 b301257n
24. Santos MG, Tavares IMC, Boralli VB, Figueir- 32. Zhou Y, Liu H, Li J et al (1613) Restricted
edo EC (2015) Direct doping analysis of beta- access magnetic imprinted microspheres for
blocker drugs from urinary samples by on-line directly selective extraction of tetracycline vet-
molecularly imprinted solid-phase extraction erinary drugs from complex samples. J Chro-
coupled to liquid chromatography/mass spec- matogr A 2020:460684. https://doi.org/10.
trometry. Analyst 140:2696–2703. https:// 1016/j.chroma.2019.460684
doi.org/10.1039/c4an02066a 33. Parisi OI, Cirillo G, Curcio M et al (2010)
25. Yang M, Zhang Y, Lin S et al (2013) Prepara- Surface modifications of molecularly imprinted
tion of a bifunctional pyrazosulfuron-ethyl polymers for improved template recognition in
imprinted polymer with hydrophilic external water media. J Polym Res 17:355–362.
layers by reversible addition-fragmentation https://doi.org/10.1007/s10965-009-9322-
chain transfer polymerization and its applica- 7
tion in the sulfonylurea residue analysis.
Talanta 114:143–151. https://doi.org/10.
1016/j.talanta.2013.03.078
Chapter 6
Synthesis of Double-Layer Imprinted Polymers: BSA

Depletion
Okan Zenger and Gözde Baydemir Peşint
Abstract
A sensitive, rapid, and cost-effective method for quantitative analysis of proteins (e.g., detection, purifica-
tion, depletion) for a wide variety of purposes is required in a number of areas, such as immunodiagnostics
and biotechnology. Double-layer imprinting technique, which is carried out via polymerization of polymer
solution with higher monomer concentration, covering and filling the supermacroporous structure of a
pre-synthesized cryogel column with a lower monomer concentration, thus improving the surface area and
adsorption capacity of final product, is a brand new approach for the application of cryogels in molecular
imprinting technology. Within the scope of this chapter, BSA is selected as a model protein for the
application of double-layer imprinting protocol. In this chapter, synthesis of double-layer BSA-imprinted
and non-imprinted cryogel columns (BSA-DLIP and DLNIP, respectively) are described. In addition,
characterization of synthesized columns and BSA depletion studies from aqueous solutions are described in
detail, as well as selectivity of BSA-DLIPs for BSA, against competitors.
Key words Depletion, Molecular imprinting technique, Cryogel, Double-layer imprinted polymer,
Bovine serum albumin, High-abundant proteins, Advanced adsorption capacity
1 Introduction
Detection, purification, quantification, and depletion of proteins is

of great importance in a number of areas, such as proteomics,
biochemical analysis, immunodiagnostics, and biotechnology
[1]. A sensitive, rapid, and inexpensive method for quantitative
analysis of proteins for a wide variety of purposes, is highly desirable
[1, 2]. As in all proteomic studies of blood plasma, high-abundant
proteins, e.g., albumin, hemoglobin, interfere with the analysis of
low-abundant and disease-related proteins. Depletion of high
abundant proteins is often the initial step in serum- and plasma-
based proteomics studies where discovery of potential biomarkers is
the main goal, which are often in low abundance and critical for
diagnosis and prognosis of certain diseases (e.g., cancer types,
cardiovascular diseases) [3, 4].
https://doi.org/10.1007/978-1-0716-1629-1_6,
71
72 Okan Zenger and Gözde Baydemir Peşint
Cryogels are affinity matrices synthesized at temperatures

below 0 C via cryotropic gelation process. Owing to their perfect
spongy morphology and supermacroporous structure, it is possible
to study with viscous or heavy fluids with particles, such as milk and
blood without blocking the column [5, 6]. Cryogels have drawn
attention by their relatively reduced flow resistance, shorter diffu-
sion distance, etc. features for both adsorption and desorption
processes. However, cryogels still need to be improved in terms
of surface area and adsorption capacity [6–8].
In molecular imprinting technique, target molecule is used as
the template during the synthesis of the polymeric system, in order
to create specific cavities for the attachment of the desired molecule
[2]. Recognition and specific adsorption properties of the molecu-
larly imprinted polymers (MIPs) depend on the physical and geo-
metrical characteristics including size, shape, and overall charge, as
well as the chemical properties of the template [9–11]. Molecularly
imprinted cryogels have come into prominence by combining the
advantages of molecular imprinting technique and cryogels [11].
Approximate pore size of a given cryogel is primarily affected by
preparation temperature, monomer concentration, rate of cooling
(types of cooling, i.e., liquid cooling or air cooling, concentration
of initiator system), presence and concentration of additives, e.g.,
NaCl, CaCl2, polymerization inhibitor and the solvent [12]. Even
though morphology of the cryogel network does not essentially
depend on monomer concentration, it can be clearly seen that
monomer concentration is closely related to pore size; increasing
monomer concentration results in a remarkable reduction in poros-
ity, when other parameters (temperature, rate of cooling, etc.) are
kept constant [12–14]. Under certain conditions, following the
synthesis of first molecularly imprinted cryogel with lower mono-
mer concentration, thus with larger pore sizes; performing a second
imprinting process upon the macroporous structure of the first
layer of cryogel may be an effective and innovative approach,
which we called double-layer imprinting technique. Therefore,
the synthesis of affinity systems with larger surface area, thus higher
adsorption capacity (much more adsorption cavities specific to
template molecule) for the molecule of interest can be achieved
[2, 15–19].
Serum albumin is one of the most studied proteins in mamma-
lian plasma [20, 21]. Bovine Serum Albumin (BSA) is a type of
monomeric multi-domain albumin which is synthesized by liver
and is one of the most abundant proteins in bovine serum, it
constitutes about half of serum proteins [22, 23]. BSA has a molec-
ular weight of about 67 kDa and exists as a single polypeptide chain
[24]. This molecule exhibits an extraordinary ligand binding capac-
ity and serves as carrier protein for many endogenous and exoge-
nous compounds such as neutral and weak acidic metabolites, fatty
acids, and steroids [22]. Therefore, BSA is selected as a model
Double-Layer Imprinted Polymers 73
protein for the application of double-layer imprinting technique

and is employed as the template molecule.
In this chapter, synthesis of double-layer BSA-imprinted and
double-layer non-imprinted cryogel columns (BSA-DLIP and
DLNIP) are described: monomer concentrations for these two
layers are 5% and 12% (w:w), respectively. In addition, characteriza-
tion of synthesized columns and their use in adsorption studies of
BSA from aqueous solutions are described in detail, as well as
selectivity of BSA-DLIP columns for BSA against competitors.
2 Materials
Prepare all solutions using ultrapure water with a conductivity of

18 MΩ-cm at room temperature and analytical grade reagents.
Prepare all solutions before use, store all reagents according to
provided information, and dispose waste materials abiding by
waste disposal regulations. Given amounts of reagents are for
synthesizing approximately 0.5 g (dry weight) of each BSA-DLIP
(see Note 1). Phosphate-buffered saline (PBS) solution is used in
adsorption studies to provide physiological conditions.
2.1 Synthesis 1. Template molecule: Bovine Serum Albumin (BSA).

of BSA-Specific 2. Functional monomer: 1-Vinylimidazole (VIM).
Double Imprinted
3. Plain monomer: 2-Hydroxyethyl methacrylate (HEMA).
Polymers (BSA-DLIPs)
4. Cross-linker: Methylene Bisacrylamide (mBAA).
5. The initiator/catalyst system for the initiation of free radical
polymerization: Ammonium persulfate (APS) (for stock solu-
tion (1%, w:v) dissolve 100 mg of APS in 1 mL UW, see Note 2)
and N,N,N0 ,N0 -tetramethylene diamine (TEMED).
6. Ultrapure Type 1 water (UW).
7. Cryostat.
8. Magnetic stirrer and magnetic fish.
9. Ice bath.
10. 10 mL syringes (id. 14.5 mm).
11. Parafilm.
2.2 Cleaning 1. Methanol/UW solution (30:70, v:v).

the Columns 2. Elution agent: 0.5 M NaCl in UW.
and Template Removal
3. Peristaltic pump.
Studies
4. Spectrophotometer able to handle nanovolumes.
2.3 Characterization 1. Scanning Electron Microscopy (SEM).

Studies 2. Mercury porosimetry Brunauer-Emmet-Teller (BET).
3. Lyophilizer.
4. Electronic balance.
5. Filter paper.
2.4 Adsorption 1. PBS, pH 7.4: Dissolve 7.19 g NaCl, 1.56 g KH2PO4, 6.74 g
Studies anhydrous Na2HPO4, 8.45 g Na2HPO4·2H2O, and 17.01 g
Na2HPO4·12H2O in 1 L of UW. The final concentrations are
123 mM sodium chloride, 11.5 mM potassium phosphate, and
47.5 mM sodium phosphate.
2. BSA (2 mg/mL in PBS).
2.5 Selectivity 1. Template molecule: BSA.

Studies 2. Competitors: Bovine Hemoglobin (Hb) and Bovine Immuno-
globulin G (IgG) solutions (2 mg/mL, 5 mL each) in PBS and
also a solution of BSA and competitors together (4 mg of each
molecule in 6 mL of in PBS).
5. SDS-PAGE reagents and equipment for further assessment of
selectivity.
2.6 Desorption 1. Desorption agent: 0.5 M NaCl in UW.

Studies 2. Peristaltic pump.
3 Methods
3.1 Synthesis For the first layer of BSA-DLIP with 5% monomer concentration
of BSA-DLIPs (w:w),
1. Solution A: Add 0.65 mL of HEMA into 5 mL of UW
(Solution A).
2. Solution B: Dissolve 137.5 mg of MBAA in 10 mL UW (see
Note 3).
3. Add Solution A into Solution B in a beaker. Insert the beaker
into the ice, making sure that the magnetic fish is under the
influence of the magnetic stirrer.
4. Prepare BSA-VIM pre-complex (see Fig. 1) by dissolving
27.5 mg BSA molecule and 30 μL VIM functional monomer
in 1 mL UW in an Eppendorf tube.
Fig. 1 Representative illustration of the complexation between Bovine Serum Albumin (BSA) [25] and
1-Vinylimidazole (VIM) molecules
5. Add the pre-complex into the HEMA-MBAA solution (Solu-

tion A + Solution B).
6. Add 35 μL APS and 20 μL TEMED into the final solution and
mix the total polymer solution for 5 min on ice.
7. Divide the final polymer solution equally into 10 mL syringes
(id. 14.5 mm) with closed outlets at the bottom (3 mL polymer
solution to each syringe).
8. Fix the syringes in an upright position and leave them in the
cryostat at 16 C for at least 8 h to complete the cryogelation
process.
9. After the polymerization process, take cryogel columns from
the freezer and allow them to reach room temperature.
10. Thaw the water content frozen in the columns at room tem-
perature in order to obtain interconnected macroporous
structure.
11. Keep one of the columns with 5% monomer concentration (w:
w) for SEM and BET analysis.
For the second layer of BSA-DLIP with 12% monomer con-
centration (w:w),
1. Apply the same procedure for the synthesis of the second layer
of the cryogel with 12% monomer concentration, except that
the amount of ingredients were doubled, excluding UW and
BSA-VIM pre-complex
(a) Solution A: 1.3 mL of HEMA dissolved in 3.5 mL of UW.
(b) Solution B: 275 mg of MBA dissolved in 10 mL of UW
(see Note 3).
(c) BSA-VIM pre-complex: 27.5 mg of BSA and 30 μL of

VIM in 1 mL of UW.
(d) Initiator/catalyst system: 70 μL APS and 40 μL TEMED.
2. Divide the polymer solution equally onto the first layer of
cryogel columns in 10 mL syringes (id. 14.5 mm) with closed
outlets at the bottom (3 mL polymer solution to each, 12 mL
in total) and allow them to swell by the action of polymer
solution (supermacropores of the first layer of BSA-DLIP will
allow the second polymer solution to fill out and cover the
structure of the first layer.
3. Pour the rest of the polymer solution (3 mL) into a separate
syringe to be used in SEM and BET analysis.
4. Fix the syringes in an upright position and leave the polymeric
system in the cryostat at 16 C for at least 8 h to complete the
cryogelation process in order to obtain BSA-DLIPs via double-
imprinting technique (see Fig. 2).
For control,
5. Prepare double-layer non-imprinted columns (DLNIPs) via
double-layer imprinting procedure, but in the presence of
VIM only, instead of BSA-VIM pre-complex.
3.2 Cleaning 1. Wash the columns with methanol/UW solution (30:70, v:v) in
the Columns order to remove unreacted monomers and other undesired
and Template Removal components in a continuous system at a flow rate of 1.0 mL/
Studies min at 25 C for 2 h using a peristaltic pump.
2. Take 1 mL of sample from the elution agent as the initial
solution.
3. Pass 5 mL of the elution agent (0.5 M NaCl) through
BSA-DLIP columns in a continuous system at a flow rate of
1.0 mL/min at 25 C for 2 h using a peristaltic pump.
4. Take 500 μL of sample from the elution final solution.
5. Analyze BSA elution amount by measuring absorbance value of
initial and final samples at 280 nm using a spectrophotometer,
after each elution cycle. Repeat the elution step until no BSA
molecule is measured in the final solution (see Fig. 3).
6. Wash template-free BSA-DLIP columns with UW in a contin-
uous system at a flow rate of 1.0 mL/min at 25 C for 2 h using
a peristaltic pump.
7. Store all the columns in UW at 4 C until use.
3.3 Characterization 1. Carry out a SEM analysis (see Note 4) for the determination of
Studies surface properties of the synthesized materials (see Note 5).
Fig. 2 Schematic setup for the synthesis of BSA-DLIPs via double-imprinting technique
2. Analyze the materials via mercury porosimetry Brunauer-

Emmet-Teller (BET) analysis for the calculation of surface
area of synthesized materials (see Notes 5 and 6).
3. Evaluation of swelling properties.
(a) Dry a BSA-DLIP column using a lyophilizer (see Note 7)
and weigh dried column (Wo).
Fig. 3 Schematic representation of template removal studies
(b) Immerse BSA-DLIP in UW and leave the column at room

temperature for 24 h.
(c) Remove excess water around BSA-DLIP using a piece of
filter paper and weigh the column (Ws).
(d) Weigh dried and wet cryogel at equilibrium and use the
following equation in order to calculate the swelling ratio
(SR) of BSA-DLIP.
SR ð%Þ ¼ ½ðW s W 0 Þ=W 0 100 ð1Þ

(e) Swell BSA-DLIP to equilibrium and weigh the swollen
column (mswollen). Then, squeeze the column (see Note 8)
and weigh (msqueezed). Calculate macroporosity using the
following equation:

Macroporosity% ¼ mswollen msqueezed =mswollen 100 ð2Þ
(f) Calculate the polymerization efficiency of BSA-DLIPs by
the total amounts of reagents used in polymerization (mt)
and the weight of dried Neo-MIP (mdry) in grams.

Polymerization yield ð%Þ ¼ mdry =mt 100 ð3Þ
(g) Use the same procedure and calculations for the determi-
nation of swelling properties and polymerization yield of
DLNIP.
3.4 Adsorption 1. Prepare 6 mL of BSA (2 mg/mL in PBS, pH 7.4) and take

Studies 1 mL of initial sample.
2. Pass 5 mL of BSA solution (2 mg/mL in PBS, pH 7.4) through
BSA-DLIP and DLNIP in a continuous system at a flow rate of
1.0 mL/min at 25 C for 2 h using a peristaltic pump.
3. Analyze BSA adsorption of materials by measuring absorbance
value of initial and final adsorption samples using a spectropho-
tometer at 280 nm.
4. Calculate the adsorption capacity (Q, mg/g) using the follow-
ing equation:
Q ¼ ½ðC i C f Þ V =m dry ð4Þ

In Eq. (4), Ci and Cf refer to the equilibrium BSA concentra-
tion (mg/mL) in adsorption solution before and after passing
through BSA-DLIPs, respectively. V is the volume of the adsorp-
tion solution (mL) and mdry is the dry weight of BSA-DLIPs (g).
3.5 Selectivity Selective BSA adsorption properties of BSA-DLIPs are analyzed,

Studies performing a selectivity experiment against competitor molecules:
Hb and IgG.
1. Prepare separate solutions of BSA, Hb, and IgG with the
concentration of 2 mg/mL in 6 mL of UW and also a solution
of BSA and competitors together (BSA-Hb-IgG solution: 4 mg
of each molecule in 6 mL of UW).
2. Pass 5 mL of each solution through BSA-DLIP columns in a
continuous system at a flow rate of 1.0 mL/min at 25 C for
2 h using a peristaltic pump.
3. Take 1 mL of sample before and after adsorption for each
solution.
4. Use a spectrophotometer for the determination of the concen-
tration of absorbed molecules at 280 nm.
5. Calculate the distribution coefficient (Qd, mL/g) according to
the following equation:
Q d ¼ ½ðC i C f Þ=C f V =m dry ð5Þ

6. Calculate imprinting factor (IF) for BSA and competitors using
the following equation, in order to evaluate selective depletion
properties of BSA-DLIPs
IF ¼ Q d BSADLIPs =Q d DLNIPs ð6Þ
Fig. 4 Experimental setup of selectivity studies for sample preparation in order to use in SDS-PAGE
In this equation, Qd BSA-DLIP and Qd DLNIPs refer to the

distribution coefficients of BSA-DLIPs and DLNIPs,
respectively.
7. Selectivity of BSA-DLIP columns for the template molecule is
evaluated by interpreting the selectivity coefficient (k) of com-
petitors. Calculate k values of each competitor according to the
following equation
k ¼ Q d BSADLIPs ðtemplateÞ=Q d BSADLIPs ðcompetitorÞ ð7Þ
QBSA-DLIPs(template) and QBSA-DLIPs(competitor) refers to
the distribution coefficients of the template and each competi-
tor molecule in the BSA-DLIPs.
8. Perform a suitable SDS-PAGE protocol, employing the
adsorption initial and final samples of BSA-Hb-IgG solution
(Ai and Af), the desorption final solution (Df), and a proper
protein ladder (see Note 9), for further assessment of the
selective adsorption properties of synthesized materials (see
Fig. 4).
3.6 Desorption For the desorption of adsorbed BSA and competitor molecules,
Studies
1. Take 1 mL of sample from the desorption agent as the initial
solution.
2. Pass 5 mL of the desorption agent through BSA-DLIP col-
umns in a continuous system at a flow rate of 1.0 mL/min at
25 C for 2 h using a peristaltic pump, in order to desorb
adsorbed molecules.
3. Take 1 mL of sample after the desorption process.
4. Measure absorbance value of initial and final solutions using a
spectrophotometer at 280 nm, for the determination of the
concentration of desorbed molecules in the desorption
medium.
5. Calculate desorption rate using the adsorbed and desorbed
molecule concentrations using the following equation:
Amount of molecules in desorption medium

DR ð%Þ ¼ 100 ð8Þ
Amount of the adsorbed molecules
6. After each desorption step, wash BSA-DLIPs and DLNIPs with
excess UW in a continuous system at a flow rate of 1.0 mL/min
at 25 C for 2 h using a peristaltic pump (see Note 10).
4 Notes
1. Double-imprinted polymers of which the synthesis protocol is

described in this chapter, can be synthesized for a wide variety
of purposes, employing different types of molecules as the
template. Nevertheless, the experimental conditions (i.e., tem-
perature, flow rate, buffers, and solutions) may vary from one
molecule to another and should be optimized according to the
molecule of interest. This protocol has been carried out under
different experimental conditions and the optimized protocol
has been given within the scope of this chapter.
2. It is recommended to prepare the APS solution just a few hours
before use and store it at 4 C. Rest of the APS solution should
be used within a month at most. APS solutions prepared more
than 1 month ago may result in insufficient polymerization.
3. Ultrasonic water bath is recommended to employ for complete
dissolution of MBAA in UW.
4. When collecting samples for SEM from dried columns, it is
important to take a piece from inside, instead of taking samples
from external parts. Samples from the outer surface which is
artificially smooth, may be misleading. Internal macro- and
microporous structure is reflected best, only when the outer
part of the material is cut and the inner sample is taken.
5. Samples for SEM and BET analysis are given below:

Sample 1. Cryogel column with 5% monomer concentration
(w:w).
Sample 2. Cryogel column with 12% monomer concentration
(w:w).
Sample 3. BSA-DLIP.
Sample 4. DLNIP.
6. Increase in surface area of the double-layer imprinted columns
can be evaluated by comparing to that of a single-layer (with 5%
or 12% monomer concentration, w:w). This information may
serve as proof for the success of novel double-layer imprinting
technique.
7. It is highly recommended to dry columns using lyophilizer
rather than oven. Lyophilizer will draw off all the water content
inside the columns without disrupting the porous structure, so
that open macro- and micropores can be clearly observed in the
SEM image.
8. Squeeze columns gently and avoid disrupting the column.
9. Further assessment of the selective adsorption properties of
BSA-DLIPs for BSA can be achieved by performing a suitable
SDS-PAGE protocol, with a monomer concentration of
10–12% (w:v) and employing a protein ladder within the
range of 10–200 kDa, since molecules of interest have molecu-
lar weights within this range (BSA: 67 kDa, Hb: 16 kDa 4
subunits, IgG: two identical light chains about 25 kDa and two
identical heavy chains about 50 kDa). Samples to run in
SDS-PAGE should be as follows:
Sample 1. Protein ladder within the range of 10–200 kDa.
Sample 2. BSA-Hb-IgG solution (Ai).
Sample 3. Adsorption final solution of BSA-Hb-IgG solution
(Af).
Sample 4. Desorption final solution of adsorbed input 4 (Df).
Sample 5. BSA solution, 2 mg/mL (optional).
Sample 6. Hb solution, 2 mg/mL (optional).
Sample 7. IgG solution, 2 mg/mL (optional).
10. Wash columns with methanol/UW solution (30:70, v:v)
before storage and store all the columns in UW at 4 C.
References
1. Zhang Y, Guo YM, Xian YL, Chen WW, Zhao 2. Mosbach K, Ramström O (1996) The
YY, Jiang XY (2013) Nanomaterials for ultra- emerging technique of molecular imprinting
sensitive protein detection. Adv Mater and its future impact on biotechnology. Nat
25:3802–3819 Biotechnol 14:163–170
3. Jesus J, Fernandes R, Pessoa G, Raimundo I, 15. Ye L, Cormack P, Mosbach K (1999) Molecu-

Arruda M (2017) Depleting high-abundant larly imprinted monodisperse microspheres for
and enriching low-abundant proteins in competitive radioassay. Anal Commun
human serum: An evaluation of sample prepa- 36:35–38
ration methods using magnetic nanoparticle, 16. Say R, Birlik E, Ersöz A, Yılmaz F, Gedikbey T,
chemical depletion and immunoaffinity techni- Denizli A (2003) Preconcentration of copper
ques. Talanta 170:199–209 on ion-selective imprinted polymer microbe-
4. Schalich KM, Herren AW, Selvaraj V (2020) ads. Anal Chim Acta 480:251–258
Analysis of differential strategies to enhance 17. Andac M, Say R, Denizli A (2004) Molecular
detection of low-abundance proteins in the recognition based cadmium removal from
bovine serum proteome. Anim Sci J 91(1): human plasma. J Chromatogr B 811:119–126
e13388. https://doi.org/10.1111/asj.13388 18. Ersöz A, Say R, Denizli A (2004) Nickel
5. Lozinsky V, Galaev I, Plieva F, Savina I, (II) ion-imprinted solid phase extraction and
Jungvid H, Mattiasson B (2003) Polymeric preconcentration in aqueous solutions in
cryogels as promising materials of biotechno- packed-bed columns. Anal Chim Acta
logical interest. Trends Biotechnol 502:91–97
21:445–451 19. Daniel S, Babu PEJ, Rao TP (2005) Precon-
6. Perçin I, Baydemir G, Ergün B, Denizli A centrative separation of palladium(II) using
(2013) Macroporous PHEMA-based cryogel palladium(II) ion-imprinted polymer particles
discs for bilirubin removal. Artif Cells formed with different quinoline derivatives and
Nanomed Biotechnol 41(3):172–177 evaluation of binding parameters based on
7. Dainiak M, Plieva F, Galaev I, Hatti-Kaul R, adsorption isotherm models. Talanta 65
Mattiasson B (2005) Cell chromatography: (2):441–452
separation of different microbial cells using 20. Murozuka T, Moriwaka M, Ito H, Sekiguchi S,
IMAC supermacroporous monolithic col- Naiki M, Tanaka K, Aoyama T, Komuro K
umns. Biotechnol Prog 21(2):644–649 (1990) Bovine albumin-like protein in com-
8. Kumar A, Srivastava A (2010) Cell separation mercial human albumin for clinical use. Vox
using cryogel-based affinity chromatography. Sang 59(1):1–5
Nat Protoc 5(11):1737–1747 21. Balkani S, Shamekhi S, Raoufinia R, Parvan R,
9. Sellergren B (2001) Molecularly imprinted Abdolalizadeh J (2016) Purification and char-
polymers: Man-made mimics of antibodies acterization of Bovine Serum Albumin using
and their applications in analytical chemistry, chromatographic method. Adv Pharm Bullet
1st edn. Elsevier, New York 6(4):651–654
10. Martin-Esteban A (2013) Molecularly- 22. Lindsey BJ (1996) Amino acids and proteins.
imprinted polymers as a versatile, highly selec- In: Bishop ML, Duben-Engelkirk JL, Fody EP
tive tool in sample preparation. TrAC Trends (eds) Clinical chemistry: principles, techniques,
Anal Chem 45:169–181 and correlations. J. B. Lippincott Co, Philadel-
11. Baydemir G, Denizli A (2015) Heparin phia, pp 169–214
removal from human plasma using molecular 23. Fanali G, di Masi A, Trezza V, Marino M,
imprinted cryogels. Artif Cells Nanomed Bio- Fasano M, Ascenzi P (2011) Human serum
technol 43:403–412 albumin: from bench to bedside. Mol Asp
12. Okay O, Lozinsky V (2014) Synthesis and Med 33(3):209–290
structure-property relationships of cryogels. 24. Morrisett JD, Pownall HJ, Gotto AM Jr
In: Okay O (ed) Polymeric cryogels, advances (1975) Bovine serum albumin. Study of the
in polymer science, vol 263. Springer Interna- fatty acid and steroid binding sites using spin-
tional Publishing, Switzerland, pp 103–157 labeled lipids. J Biol Chem 250(7):2487–2494
13. Ozmen MM, Okay O (2005) Superfast respon- 25. Image of RCSB PDB (rcsb.org) of PDB ID:
sive ionic hydrogels with controllable pore size. 4F5S (Bujacz A, Bujacz G (2012) Crystal
Polymer 46(19):8119–8127 structure of bovine serum albumin DOI:
14. Bat E (2016) Hydroxyethyl methacrylate- https://doi.org/10.2210/pdb4f5s/pdb) cre-
based nanocomposite hydrogels with tunable ated with The PyMOL Molecular Graphics
pore architecture. JOTCSA 3(3):607–622 System, Version 2.0 Schrödinger, LLC
Chapter 7
Magnetic MIPs: Synthesis and Applications

Rafael da Fonseca Alves, Lucas Neres Chagas da Silva,
Gilberto Matos Neto, Isabela Fernandes Ierick, Thiago Lima Ferreira,
and Maria Del Pilar Taboada Sotomayor
Abstract
Magnetic molecularly imprinted polymers (MMIPs) are constructed based on the blending of inorganic
nanoparticles with molecularly imprinted polymers (MIPs). MMIPs are synthesized in a core-shell format in
which inorganic nanoparticles are applied as the core part of the material while selective polymeric layers are
used as the shell covering the surface of the core area. In essence, MMIPs thus reflect a combination of the
best characteristics of both inorganic nanoparticles and MIPs, where the specificity of cavities imprinted on
the MIP is merged with superparamagnetic properties of the nano-magnetite. The synergic combination of
the two distinct materials facilitates the process of extracting analytes from complex samples. Owing to their
suitable characteristics, MMIPs have become widely used in different areas of analysis.
Key words Molecularly imprinted polymers, Magnetic nanoparticles, Magnetic molecular imprinted
polymer, Solid-Phase Extraction, Electrochemistry
1 Introduction
A wide range of magnetic nanoparticles have been applied as core

materials for the synthesis of MMIPs; among these magnetic nano-
particles, iron oxides have been most widely used because of their
suitable properties, including super-paramagnetism and high sta-
bility. Maghemite (γ-Fe2O3) and magnetite (Fe3O4) oxides are
found to be primarily endowed with the aforementioned proper-
ties; in addition to that, these oxides exhibit low toxicity and proven
biocompatibility.
The synthesis of magnetite nanoparticles can be performed in
several ways. Of the wide range of techniques applied toward the
synthesis of magnetite nanoparticles, the polyol and
co-precipitation methods have become the most commonly used.
Under the co-precipitation technique, iron (II) and iron (III)
https://doi.org/10.1007/978-1-0716-1629-1_7,
85
86 Rafael da Fonseca Alves et al.
chloride salts are used for the precipitation of magnetite in alkaline

medium [1].
The main advantages of magnetite nanoparticles are that they
have suitable surface features that allow easy modification and
encapsulation of the particles, and this protects them from degra-
dation, thus maintaining their magnetic response. These modifica-
tions may range from simple functionalization of the functional
groups on the magnetite surface to adhesion of organic molecules,
polymers, and biological compounds [1, 2]. A combination of
magnetite nanoparticles with molecularly imprinted polymers
leads to the development of magnetic MIPs, which are synthesized
by the core-shell format, in which the magnetic core is coated with
a selective polymer layer (shell) [1, 3].
In analytical separation methods, the magnetic polymer is used
mainly in solid-phase extraction (SPE) as the adsorbent material.
Another advantage of magnetic MIPs is that, unlike the cartridge
(which is less simple), magnetic MIPs are applied directly in the
sample without prior treatment. Furthermore, the polymer is easily
separated from the analysis solution by applying an external mag-
netic field, such as a magnet. Generally, magnetic MIPs can be
synthesized by the following methods: (i) bulk polymerization;
(ii) polymerization by suspension; and (iii) polymerization by pre-
cipitation. Bulk polymerization is performed in a homogeneous
system under nitrogen (N2) or argon (Ar) gas flow in the absence
of oxygen; the process is induced by heating or ultraviolet
(UV) light. Although this method of polymerization is simple and
straightforward, it has some unpleasant disadvantages; the applica-
tion of the method may give rise to non-uniform particles, with
irregular sizes and cavities.
Under polymerization by suspension, the reaction occurs in
two solvents with opposite polarities. The first step of the synthesis
involves the solubilization of all the reagents required for the
reaction to occur in a nonpolar organic solvent. Thereafter, the
reaction mixture is added to water containing a surfactant agent;
this water is used in order to prevent coagulation of the monomer
droplets during the process.
Polymerization by precipitation is quite analogous to bulk
polymerization. Under polymerization by precipitation, the prepa-
ration of the MIP involves the use of higher volumes of solvents;
the application of high amounts of solvents helps prevent the
formation of a polymeric block. The quantity of solvents applied
in the precipitation method is approximately five times greater than
the quantity applied in the bulk method. Among the advantages of
the precipitation method are that one does not need to use stabi-
lizers; apart from that, the method allows one to obtain more
homogeneous particles [1].
Thus, a combination of the best properties of the magnetic
material with those of the MIP in the conduct of the synthesis leads
Magnetic MIPs 87
to the formation of magnetic MIPs with excellent properties.

Compared to conventional MIPs, magnetic MIPs are found to be
more advantageous in the sense that they are characterized by a
relatively higher ratio between the surface area and the volume; this
has to do with the fact that the core area is composed of a nanopar-
ticle and the MIP acts like a shell. The synergic effects resulting
from the combination of the two distinct materials help increase the
accessibility of the analyte to the selective cavity, thus enhancing the
binding kinetics.
2 Materials
All solutions must be prepared immediately before application,

using ultrapure water (resistivity 18 MΩ cm at 25 C) and analyt-
ical grade reagents. Reagents should be stored according to the
manufacturer’s instructions.
2.1 Co-precipitation 1. Pre-reaction mixture: Dissolve 1.72 g of FeCl2.4H2O and

Method (Fe3O4) (See 4.72 g of FeCl3∙6H2O in 80 mL of H2O.
Note 1) 2. Oxidizing agent: Concentrated NH4OH.
3. Mechanical propeller shaker.
4. 250 mL glass reactor connected to nitrogen line.
5. Buchner funnel.
6. Neodymium magnet.
7. Drying oven.
8. Fe3O4 is finally obtained.
2.2 Silanization 1. Pre-reaction mixture: Dissolve 300 mg of Fe3O4 in 1:1 v/v of

of Fe3O4 absolute ethanol/H2O.
2. Oxidizing agent: Concentrated NH4OH.
3. Silanizing agent: Tetraethyl orthosilicate (TEOS).
4. Ultrasound bath.
5. Mechanical propeller shaker.
7. Buchner funnel.
9. Drying oven.
10. Fe3O4@SiO2 is finally obtained.
2.3 Functionalization 1. Pre-reaction mixture: Dissolve 250 mg of Fe3O4@SiO2 in 5 mL

of Fe3O4@SiO2 of toluene.
2. Functionalizing agent: 3-(Trimethoxysilyl)propyl methacrylate

(MPS).
3. Fe3O4@SiO2-C¼C is finally obtained.
2.4 Computer 1. Production of chemical structures: AVOGADRO® software.

Simulation to Choose 2. Density Functional Theory (DFT): ORCA package.
the Functional
3. Making the input files and submission in queues: either manu-
Monomer and/or ally or through programming code scripts.
Solvent
4. Complex calculations: Computer cluster.
5. Plotting energy graphs: Mathematical software.
6. Visualization of optimized structures and obtaining figures of
merit: Visual Molecular Dynamics (VMD).
2.5 Polymerization: 1. Pre-reaction mixture: Dissolve 1.0 mol L1 of the template,
Synthesis of MIP 4.0 mol L1 of functional monomer in 40 mL of solvent (H2O,
on Fe3O4@SiO2-C¼C methanol, ethanol, acetronitrile), and 200 mg of Fe3O4@SiO2-
C¼C.
2. Cross-linker: One can use ethylene glycol dimethacrylate
(EGDMA) as structural monomer (SM).
3. Radical initiator: One can use 2,20 -Azobis(2-methylpropioni-
trile) (AIBN).
5. Water bath with temperature control.
6. Buchner funnel.
8. Drying oven.
2.6 Extraction 1. Ideal washing solvent (H2O, methanol, ethanol, acetronitrile,

of Template from CH3COOH).
M-MIP Particles 2. Cellulose extraction cartridge.
3. Soxhlet extractor, an assembly of the system components is
depicted in Fig. 1.
4. UV–Vis spectrophotometer.
5. Buchner funnel.
7. Drying oven.
2.7 Sample 1. Sample dilution.

Application 2. Centrifuge.
Magnetic MIPs 89
Fig. 1 A schematic setup for washing MMIP
4. 0.45 μm RC filter.
5. Chromatograph fitted with a UV/visible detector.
2.8 Electrochemical 1. Galvanostat/potentiostat.

Measurements 2. Three-electrode cell of single compartment.
3. Reference electrode (RE): Ag/AgCl/KCl (3 mol L1).
4. Counter electrode (CE): Platinum coil.
5. Working electrode (WE): carbon graphite paste [4].
6. Solutions: Glycine, Britton-Robison buffer (H3BO3
0.04 mol L1, CH3COOH 0.04 mol L1, and H3PO4
0.04 mol L1).
7. Ultrasound bath.
3 Methods
3.1 Synthesis 1. Add 80 mL of H2O; the mixture should be kept under stirring
of Magnetic and N2 atmosphere for 30 min.
Nanoparticles 2. Slowly add 10 mL of NH4OH to the mixture; keep it under
mechanical stirring for 30 min. This yields Fe3O4.
Fig. 2 An illustrative scheme related to the synthesis of magnetite nanoparticles
3. Filter and wash the solution with H2O using vacuum pump and
neodymium magnet for better sedimentation. Dry the mixture
at 80 C overnight.
4. Weigh 300 mg of Fe3O4; add 4 mL of absolute ethanol and
4 mL of H2O to the 300 mg Fe3O4. The mixture should be
subjected to ultrasound bath for 15 min.
5. Add 5 mL of NH4OH and additional 2 mL of TEOS to the
mixture. Keep the mixture under mechanical agitation for 12 h.
This yields Fe3O4@SiO2.
6. Filter and wash the solution with ethanol using vacuum pump
and neodymium magnet for better sedimentation. Dry the
mixture at 80 C overnight.
7. Weigh 250 mg of Fe3O4@SiO2; add 5 mL of toluene and 5 mL
of MPS to the 250 mg Fe3O4@SiO2. Keep the mixture under
mechanical stirring for 12 h in N2 atmosphere. This yields
Fe3O4@SiO2-C¼C (Fig. 2).
8. Filter and wash the solution with ethanol using vacuum pump
and neodymium magnet for better sedimentation. Dry the
solution at 80 C overnight.
3.2 Molecular 1. Creation of structures and software used:

Modeling 2. Initially, one needs to construct the structures of the functional
monomers (FM) and template (T) which will be used for the
synthesis of the MIP. One does not need to construct the
structure of the solvent since the calculation is made based on
an implicit solvent (without using the structure of the solvent,
and using only a dielectric constant in the calculation). A free
software widely used for the construction of structures is AVO-
GADRO® (Fig. 3) [5].
Magnetic MIPs 91
Fig. 3 Development of the structure of 2-vinylpyridine
3. After that, one needs to simply save the structures constructed

in xyz format and convert them to the calculation and entry
submission files (the commands are typically characteristic of
software programs that come with the DFT method) (see Note
2).
4. Subsequently, the file containing the optimized structure of
FM and T is extracted and inserted into the VMD software
[6] to allow the visualization of the structure and the transla-
tion of the functional monomer to the template (or vice versa)
to form the FM-T complex. A recommended density functional
widely used in chemistry for DFT calculations is the hybrid
functional B3LYP [7].
5. After the calculation of T, FM, and FM-T complex, the “final
point energy” is extracted. The energy can be given in units of
Hartree, and one needs to convert it properly to kJ mol1.
Once this has been done, the sum of the energy of T is added to
that of FM (EFM + ET ¼ EFM-T). Next, the result obtained is
subtracted from the energy of the FM-T complex (EFM-T0 ):
ΔE ¼ EFM-T EFM-T’. After that, plots are constructed based
on the data of the complex relative to energy in kJ mol1 (see
Note 3).
3.3 Development 1. Synthesis by precipitation: This usually begins with a molar

and Characterization ratio of 1:4:100 for T:FM:SM, respectively.
of MMIPs 2. Mix T, FM, and 40 mL of solvent (H2O, methanol ethanol).
Fig. 4 An illustrative scheme related to the synthesis of MMIP, using acrylic acid as functional monomer,
EGDMA as structural monomer, and AIBN as radical initiator
3. The compounds in the mixture should be allowed to interact

with one another for 12 h at 25 C.
4. Thereafter, add 200 mg of Fe3O4@SiO2-C¼C nanoparticles to
the mixture and keep the system under constant mechanical
agitation for 2 h.
5. Add MS and 0.05 mmol of radical initiator to the system and
keep the mixture under constant stirring in N2 atmosphere.
The mixture should be subjected to heating at 60 C for 5 h.
6. Filter and wash the precipitation using vacuum pump and
neodymium magnet for better sedimentation. Dry the solution
at 80 C overnight (Fig. 4).
7. Wash the mixture in a Soxhlet system using the ideal washing
solvent (see Note 4).
8. Monitor the washing solvent by UV–Vis spectrophotometer
(or other analytical method) until the template is completely
removed.
9. Apply the same procedure without the addition of the template
for the synthesis of M-NIP (used as control material).
Magnetic MIPs 93
3.4 Characterization 1. Characterization can be performed by the following techni-

ques: Fourier transformed infra-red (FT-IR), scanning electron
microscopy (SEM), energy-dispersive X-ray spectroscopy
(EDS), and transmission electron microscopy (TEM). The
analysis can be conducted directly on solid samples (see Note
5).
2. The morphology of the material under investigation can also be
characterized by N2 adsorption/desorption, using the iso-
therm models of Brunauer, Emmett, and Teller (BET) to
obtain the surface area and the Barrett-Joyner-Halenda (BJH)
method to obtain the pore size.
3. The thermal stability and composition of the synthesized mate-
rials can be evaluated by thermogravimetry (TGA) and differ-
ential scanning calorimetry (TGDSC).
4. The characterization of the magnetite particles can be per-
formed by the vibrating sample magnetism (VSM) method.
3.5 Application 1. Add 5 mL of methanol (MeOH) in 500 mL of milk.

of MMIP as Dispersive 2. After that, sonicate the mixture for 10 min and remove 1.5 mL
Solid-Phase Extractor of the mixture and centrifuge it for 30 min at 15,100 g.
(DSPE) Subsequently, collect 250 μL of the supernatant, and dilute it
for the Determination with 50 mL of water.
of Ciprofloxacin 3. Add a previously prepared solution of ciprofloxacin at concen-
in Bovine Milk trations of 5.0, 10.0, and 50.0 μM.
4. Put 5 mg of MMIP (prepared as previously described, using
ciprofloxacin as template molecule) in tubes containing 1.0 mL
of the previously prepared solutions.
5. Keep the mixtures under agitation for 2 h using a rotary shaker;
this should be followed by the separation of the MMIP suspen-
sions using neodymium magnetic bar and filtering the solu-
tions with the aid of a 0.45 μm RC filter.
6. Analyze the material by High Performance Liquid Chromatog-
raphy with a UV/visible detector (HPLC/UV–Vis).
7. Apply the same procedure for MNIP.
3.6 Application 1. Disperse MMIP in a glycine solution, and sonicate it for 2 h.

of MMIP Particles 2. Add the water sample and shake the mixture for additional 2 h.
Previously Captured by
3. Pre-concentrate the MMIP on the surface of the magnetic
a Magnetic Sensor
sensor for 20 s.
for the Development
of an Electrochemical
4. Thereafter, transfer the magnetic sensor to the electrochemical
cell, and wait for 15 min before performing the voltammetric
Sensor
measurements (Fig. 5).
5. The following parameter conditions should be applied when
carrying out the voltammetry differential pulse analysis:
Fig. 5 An illustrative scheme related to the analytical procedure for capturing MMIP with a magnetic electrode
deposition potential of 0.20 V (for 60 s), potential range of

0.20 to 0.50 V, step potential of 4 mV, potential pulse of
0.12 V, and scan rate of 4 mV s1.
6. Once one notices the presence of a matrix effect, quantification
should be performed using the standard addition method.
7. Apply the same procedure for MNIP.
4 Notes
1. Magnetite nanoparticles can be obtained by different methods

of synthesis. The co-precipitation synthesis method is widely
used because of its outstanding advantages; this method is a
simple and relatively less costly way of synthesizing magnetic
particles in comparison with other chemical methods [8].
2. DFT (Density Functional Theory) is an ab initio computational
method of calculating electronic structure which is widely pop-
ular in chemistry and physics [7]. The technique can be applied
as an initial procedure for the development and optimization of
the synthesis of M-MIP. A wide range of software programs,
including GAMESS, ORCA, GAUSSIAN, VAS, among others,
are equipped with DFT [9–13]. Through the application of
DFT, one is able to identify which of the complexes has a more
favorable interaction based on the solvent chosen. For instance,
negative energies are associated with more favorable interac-
tions. One will note, however, that the complex with the
strongest interaction will not necessarily be the most appropri-
ate; this is because one needs to remove the template from the
MMIP for the cavity to be formed. Thus, it is essentially
important to choose complexes with intermediate energies.
3. It is at this moment that one specifies the maximum time of
calculation and number of functional processors, based on
Magnetic MIPs 95
other theoretical and computational parameters. Another

important point worth mentioning is that one needs to calcu-
late the structure of the analyte and the functional monomer
separately in the previously chosen solvent. By doing so, one
will be able to obtain the optimized structure and the “final
point energy” of each structure, which will be used in
subsequent steps. It is noteworthy that the use of more than
one FM may add interesting properties to the material, includ-
ing greater selectivity and specificity.
4. Each material can be developed based on the special features of
the template molecule; and the ratio of T:FM:SM may vary
according to the characteristics of the template molecule and
the objectives of the experiments. Similarly, the solvent used for
washing may be dependent on the characteristics of the tem-
plate molecule; for instance, characteristics such as molecule
solubility and pKa are determinants when it comes to the
choice of an ideal solvent. Also, in all methods applied, one
needs to perform a range of tests in order to achieve more
reliable results. By doing so, a regular scale will be developed
based on the best conditions for the polymeric material to
perform its functions effectively, including adsorption with
different MMIP masses, different adsorption times, variations
in solvents and pH, etc.
5. The application of a mean diameter of approximately
145–170 nm is found to be interesting; in this condition, one
obtains a good morphological symmetry, which ultimately
shows that the synthesis is effective and controlled. When the
magnetic nanoparticles are modified with TEOS and MPS, one
observes an increase of approximately 6.5 nm in the particles of
the material. The occurrence of MMIP polymerization in the
cluster of magnetite nanoparticles leads to the formation of a
polymeric layer with high porosity, which is regarded an essen-
tially important characteristic of this material.
Acknowledgments
The authors are grateful to FAPESP (#2014/50945–4 #2019/

10452-2, #2020/01493-4 and #2019/00677-7), CNPq (grants
#408050/2018-7 and #301728/2019-4), and CAPES for the
financial assistance granted in support of this work.
References
1. Pupin RR (2017) Nanomagnetic biomimetic immunosensors and immunoassays. Disserta-

polymers with restricted access (magnetic tion, State University of São Paulo
RAMIP) obtained by semicovalent and
non-covalent synthesis for application in
2. Khan S, Hussain S, Wong A, Foguel MV, Mor- 8. Nkurikiyimfura I, Wang Y, Safari B, Nshinga-
eira Gonçalves L, Pividori Gurgo MI, Taboada bigwi E (2020) Temperature-dependent mag-
Sotomayor M d P (2018) Synthesis and char- netic properties of magnetite nanoparticles
acterization of magnetic-molecularly imprinted synthesized via coprecipitation method. J
polymers for the HPLC-UV analysis of ame- Alloys Compd 846:156344. https://doi.org/
tryn. React Funct Polym 122:175–182. 10.1016/j.jallcom.2020.156344
https://doi.org/10.1016/j.reactfunctpolym. 9. Allouche A (2012) Software news and updates
2017.11.002 gabedit—A graphical user interface for compu-
3. Silva LM (2018) Synthesis, characterization tational chemistry softwares. J Comput Chem
and optimization of selective MIP-magnetic 32:174–182. https://doi.org/10.1002/jcc
to ciprofloxacin and evaluation of the viability 10. Frisch MJ, Trucks GW, Schlegel HB, Scuseria
of the simultaneous use of two functional GE, Robb MA, Cheeseman JR, Scalmani G,
monomers. State University of São Paulo Barone V, Petersson GA, Nakatsuji HX, Li
4. Ruiz-Córdova GA, Khan S, Gonçalves LM, MC, Marenich A, Bloino J, Janesko BG,
Pividori MI, Picasso G, Sotomayor MDPT Gomperts R, Mennucci B, Hratchian HP,
(2018) Electrochemical sensing using mag- Ortiz JV, Izmaylov AF (2016) Gaussian 09.
netic molecularly imprinted polymer particles Revis. A.02. Gaussian Inc., Wallingford CT
previously captured by a magneto-sensor. 11. Fabian MD, Shpiro B, Rabani E, Neuhauser D,
Talanta 181:19–23. https://doi.org/10. Baer R (2019) Stochastic density functional
1016/j.talanta.2017.12.085 theory. Wiley Interdiscip Rev Comput Mol Sci
5. Hanwell MD, Curtis DE, Lonie DC, 9:1–15. https://doi.org/10.1002/wcms.
Vandermeersch T, Zurek E, Hutchison GR 1412
(2012) Avogadro: An advanced semantic 12. Neese F (2012) The ORCA program system.
chemical editor, visualization, and analysis plat- Wiley Interdiscip Rev Comput Mol Sci
form. J Cheminform 4:17 2:73–78. https://doi.org/10.1002/wcms.81
6. Humphrey W, Dalke A, Schulten K (1996) 13. Schmidt MW, Baldridge KK, Boatz JA, Elbert
VMD – visual molecular dynamics. J Mol ST, Gordon MS, Jensen JH, Koseki S,
Graph 14:33–38 Matsunaga N, Nguyen KA, Su S, Windus TL,
7. Zhang IY, Wu J, Xu X (2010) Extending the Dupuis M, Montgomery JA (1993) General
reliability and applicability of B3LYP. Chem atomic and molecular electronic structure sys-
Commun 46:3057–3070. https://doi.org/ tem. J Comput Chem 14:1347–1363. https://
10.1039/c000677g doi.org/10.1002/jcc.540141112
Chapter 8
Water-Compatible Fluorescent Molecularly Imprinted

Polymers
Huiqi Zhang
Abstract
Preparation of molecularly imprinted polymers (MIPs) capable of directly and selectively recognizing small
organic analytes in aqueous samples (particularly in the undiluted complex biological samples) is described.
Such water-compatible MIPs can be readily obtained by the controlled grafting of appropriate hydrophilic
polymer brushes onto the MIP particle surfaces. Two types of synthetic approaches (i.e., “two-step
approach” and “one-step approach”) for preparing complex biological sample-compatible hydrophilic
fluorescent MIP nanoparticles and their applications for direct, selective, sensitive, and accurate optosensing
of an antibiotic (i.e., tetracycline (Tc)) in the undiluted pure bovine/porcine serums are presented.
Key words Molecularly imprinted polymers, Nanoparticles, Water-compatible, Controlled/“living”

radical precipitation polymerization, Hydrophilic polymer brushes, Fluorescent sensors, Complex
biological samples
1 Introduction
Molecularly imprinted polymers (MIPs) are artificial receptors with

tailor-made molecular recognition sites [1–6]. They have attracted
enormous interest in recent years because of their high molecular
recognition capability, outstanding stability, easy preparation, and
low cost. MIPs can be readily prepared by the versatile and straight-
forward molecular imprinting technique, which typically involves
the copolymerization of functional and cross-linking monomers in
the presence of a template molecule (i.e., the target analyte or its
fragment or analogue) and subsequent template removal from the
resulting cross-linked polymers. The imprinted binding sites com-
plementary to the template in size, shape, and position of the
functional groups are thus created in the MIPs, which allow them
to show substrate affinity and selectivity comparable to those of
biological receptors (e.g., antibody). These appealing physico-
chemical characteristics make MIPs highly promising substitutes
https://doi.org/10.1007/978-1-0716-1629-1_8,
97
98 Huiqi Zhang
for biological receptors in many molecular recognition-based appli-

cations such as separation/purification [2, 3, 5], biomimetic catal-
ysis [4], chemosensing [1, 5], and various biomedical purposes [6].
Despite significant progress made in the field of MIPs, most of
the presently developed MIPs targeting small organic templates can
only show good molecular recognition ability in organic solvents,
and they typically lose their specific template binding ability in
aqueous solutions [7–9]. This drawback largely limits the practical
application of MIPs in many bioanalytical areas involving aqueous
media such as food safety control, environmental monitoring, and
clinical diagnostics. Therefore, tremendous efforts have been
devoted to developing MIPs that can show good molecular recog-
nition ability toward small organic analytes in aqueous solutions
(i.e., water-compatible MIPs) in the past several decades [7–9].
Over the past decade, our group has been focusing on devel-
oping water-compatible MIPs with a ultimate goal to generate
MIPs that can eventually replace biological receptors in the bioa-
nalytical applications [6, 8–10]. So far, a series of facile, general, and
highly efficient approaches have been established in our lab for
preparing fully water-compatible MIP micro/nanoparticles by
their simple grafting of proper hydrophilic polymer brushes (the
resulting MIPs show largely enhanced surface hydrophilicity, which
can afford them significantly reduced hydrophobicity-induced non-
specific analyte bindings and efficient anti-fouling properties, thus
leading to their excellent molecular recognition ability in complex
biological aqueous samples) [6, 8–10]. To this end, we have devel-
oped a type of new polymerization technique named “controlled/
“living” radical precipitation polymerization (CRPP)’’ [10], which
combines the advantages of controlled/“living” radical polymeri-
zation (CRP) [11] and traditional precipitation polymerization
techniques [12]. CRPP can be easily realized by simply introducing
CRP mechanism into the traditional precipitation polymerization
system [10]. For example, atom transfer radical precipitation poly-
merization (ATRPP) and reversible addition-fragmentation chain-
transfer (RAFT) precipitation polymerization (RAFTPP) can be
readily implemented simply by replacing the free radical initiator
in the traditional precipitation polymerization system to atom
transfer radical polymerization (ATRP)-initiating system (e.g., a
combination of Cu(I) halide/ligand and an alkyl halide initiator)
and by adding a RAFT agent (e.g., a dithioester-containing species)
into the traditional precipitation polymerization system, respec-
tively. CRPP allows one-pot synthesis of well-defined spherical
polymer particles with surface-bound “living” initiating or chain-
transfer groups as well as other functional moieties. This character-
istic makes them easily functionalizable by using various surface-
initiated CRPs or coupling chemistries [8, 9].
Based on different synthetic routes, our developed approaches
for preparing water-compatible MIP particles can be mainly
Water-Compatible Molecularly Imprinted Polymers 99
Fig. 1 Schematic protocol for the preparation of QD-labeled hydrophilic fluorescent MIP nanoparticles with
surface-grafted PGMMA brushes via “two-step approach”. Reprinted with permission from [27]. Copyright
(2016) American Chemical Society
classified into “two-step approach” and “one-step approach”

[8, 9]. “Two-step approach” typically involves first the synthesis
of “living” MIP particles with surface-bound CRP-initiating or
chain transfer groups (either directly by CRPPs or by grafting
MIP layers onto the preformed “living” polymer/silica particles
via surface-initiated CRPs) and their subsequent grafting of hydro-
philic polymer shells (either via surface-initiated CRPs of hydro-
philic monomers [13] or via such approaches as RAFT coupling
chemistry [14] or thiol-epoxy coupling chemistry [15]). “One-step
approach” can directly afford hydrophilic MIP micro/nanoparti-
cles with hydrophilic polymer brushes by using hydrophilic macro-
molecular chain transfer agents (macro-CTAs) or macromolecular
initiating agents in the CRPP systems [16, 17]. In this chapter, we
will describe two routinely used methodologies (i.e., “two-step
approach” (Fig. 1) and “one-step approach” (Fig. 2)) in our lab
for preparing water-compatible MIPs with fluorescent labeling as
well as their application for direct and highly selective optosensing
of an antibiotic (i.e., tetracycline (Tc)) in complex biological sam-
ples (i.e., the undiluted bovine/porcine serums).
2 Materials
The following commercially available reagents (of the analytical

grade, Scheme 1a–f) were purified or modified prior to use as
shown below:
4-Vinylpyridine (4-VP), methacrylic acid (MAA), and ethylene
glycol dimethacrylate (EGDMA) were purified by distillation under
vacuum. Methanol was distilled. Acetonitrile was refluxed over
calcium hydride and distilled. N,N-Dimethylformamide (DMF)
100 Huiqi Zhang
Fig. 2 Schematic protocol for the synthesis of anthracene-labeled hydrophilic fluorescent MIP nanoparticles
with surface-grafted PHEMA brushes via “one-step approach” for direct and rapid drug optosensing in real,
undiluted biological samples. Reprinted from [22], Copyright (2015), with permission from Elsevier
was dried with anhydrous magnesium sulfate and then distilled

under vacuum. Copper(I) chloride (CuCl) [18] and
2-hydroxyethyl methacrylate (HEMA) [19] were purified accord-
ing to the literature methods. Azobisisobutyronitrile (AIBN) was
recrystallized from ethanol. Tetracycline (Tc) hydrochloride was
converted into its neutralized form following a literature
procedure [20].
The chemicals shown in Scheme 1g–l were synthesized in our
lab following the literature methods, including glyceryl mono-
methacrylate (GMMA) [21], (2-hydroxyethyl anthracene-9-car-
boxylate) methacrylate (AnHEMA) [22], tris
(2-(dimethylamino)ethyl)amine (Me6TREN) [23], cumyl dithio-
benzoate (CDB) [24], well-defined poly(HEMA) (PHEMA) with
a dithioester end-group (Mn,NMR ¼ 5140 g/mol) (i.e., PHEMA
macro-CTA) [22], BIBAPTES [25], an aqueous solution of CdTe
quantum dots (QDs) [26], and “living” CdTe QD-embedded SiO2
nanoparticles (CdTe QD-SiO2) with surface-bound alkyl bromide
groups (i.e., QD-SiO2-Br) [27].
The other chemicals used but not mentioned above (e.g.,
chloramphenicol (Chl, Scheme 1m), cefalexin monohydrate (Lex,
Scheme 1n), atenolol (Scheme 1o), and vancomycin hydrochloride
(Van, Scheme 1p)) are commercially available reagents (typically of
the analytical grade) and used directly. The standard fetal bovine
serum and porcine serum were stored at 20 C prior to use, and
the thawed serums were used directly.
Follow all waste disposal regulations when disposing of waste
materials.
Scheme 1 Chemical structures of some reagents used in this work
2.1 Synthesis 1. Pre-polymerization mixture: Dissolve 2 mmol of functional

of QD-Labeled monomer (4-VP) and an appropriate amount of template
Fluorescent MIP (Tc) in 150 mL of dried acetonitrile. The molar ratio of Tc to
Nanoparticles 4-VP may vary from 1/8 to 1/2. Pre-polymerization mixture
with Surface-Bound should be prepared prior to the polymerization and used
Alkyl Halide Groups immediately.
(i.e., ATRP-Initiating 2. Cross-linker: EGDMA.
Groups) (Briefly 3. Immobilized ATRP initiator: “Living” QD-SiO2-Br
QD-SiO2@MIP) via nanoparticles.
Surface-Initiated ATRP
4. Catalyst: CuCl/Me6TREN. The molar ratio of CuCl to Me6T-
(See Notes 1, 2, and 3)
REN may vary from 1/3 to 1.
5. Heating magnetic stirrer with temperature control (with
oil bath).
102 Huiqi Zhang
6. Template removal: Laboratory centrifuge/centrifuge tube

(PE, 50 mL)/methanol.
7. Laboratory vacuum oven.
2.2 Synthesis 1. Hydrophilic monomer: GMMA.

of QD-Labeled 2. Immobilized ATRP initiator: “Living” QD-SiO2@MIP
Fluorescent MIP nanoparticles.
Nanoparticles 3. Catalyst: a mixture of CuCl and CuBr2 together with a ligand
with Hydrophilic (2,20 -bipyridine). The molar ratio of CuBr2 to CuCl may vary
Poly(GMMA) (PGMMA) from 1/10 to 4/10, and that of CuCl+CuBr2 to 2,2-
Brushes (i.e., QD- 0
-bipyridine may vary from 1/4 to 1/2.
SiO2@MIP@PGMMA)
4. Sacrificial ATRP initiator: Ethyl 2-chloropropionate. The
via Surface-Initiated
added amount of the sacrificial ATRP initiator may vary, but
ATRP (See Notes 4 and
it is recommended to be around 1/4 of the used molar amount
5) of CuCl.
5. Solvent: A mixture of methanol and distilled water, typically in
a volume ratio of 1:1.
water bath).
7. Laboratory centrifuge.
2.3 Synthesis 1. Pre-polymerization mixture: Dissolve 0.834 mmol of func-

of Organic tional monomer (MAA) and appropriate amounts of fluores-
Fluorophore-Labeled cent monomer (AnHEMA) and template (Tc) in 60 mL of a
Fluorescent MIP mixture of dried acetonitrile and DMF (4:1 v/v). The molar
nanoparticles ratio of Tc to MAA to AnHEMA may vary from 1:1:0.1 to
with PHEMA Brushes 1:4:0.4. Pre-polymerization mixture should be prepared prior
(i.e., OF-MIP@PHEMA) to the polymerization and used immediately.
via Hydrophilic 2. Cross-linker: EGDMA.
Macro-CTA-Mediated 3. Initiator: AIBN.
RAFTPP (See Notes 1,
4. RAFT agents: CDB and hydrophilic PHEMA macro-CTA (Mn,
3, and 5)
NMR ¼ 5140 g/mol).

oil bath).
6. Template removal: Laboratory centrifuge/centrifuge tube
(PE, 50 mL)/a mixture of methanol and acetic acid (9:1 v/v).
3 Methods
3.1 The First Step 1. Add the functional monomer 4-VP (2 mmol), the template Tc
of “Two-step (0.5 mmol), and dried acetonitrile (150 mL) into a round-
Approach”: Synthesis bottom flask (250 mL). Magnetically stir the solution in the
of QD-SiO2@MIP (and dark at 25 C for 2 h to promote the formation of 4-VP/Tc
Its Non-imprinted or complex.
Control Polymer 2. Add the cross-linker EGDMA (6 mmol) into the solution.
(QD-SiO2@CP)) via 3. Degas the above solution by bubbling argon for 10 min in an
Surface-Initiated ATRP ice-water bath and then add CuCl (0.1 mmol) and Me6TREN
(See Notes 1, 2, and 3) (0.2 mmol) into the reaction system.
4. Degas the above mixture by bubbling argon for 10 min in an
ice-water bath and add “living” QD-SiO2–Br (200 mg) into
the reaction mixture.
5. Magnetically stir the reaction mixture at 60 C for 4 h under
argon atmosphere.
6. Collect the orange yellow QD-SiO2@MIP particles (with
bound Tc) by centrifuging the reaction mixture, thoroughly
wash the product with methanol to remove the template, and
then allow it to be dried at 40 C under vacuum to the constant
weight (yield: 211 mg).
7. Prepare and purify QD-SiO2@CP particles under the same
conditions as for QD-SiO2@MIP except in the absence of the
template (yield: 211 mg).
3.2 The Second Step 1. Add QD-SiO2@MIP or QD-SiO2@CP (100 mg), GMMA
of “Two-step (14.6 mmol), CuCl (0.146 mmol), CuBr2 (0.0438 mmol),
Approach”: Synthesis methanol (4 mL), and distilled water (4 mL) into a round-
of QD- bottom flask (25 mL). Degas the above mixture by bubbling
SiO2@MIP@PGMMA argon for 15 min in an ice-water bath.
and QD- 2. Add 2,20 -bipyridine (0.4085 mmol) into the above degassed
SiO2@CP@PGMMA via mixture.
Surface-Initiated ATRP 3. After degassing the above mixture by bubbling argon for
(See Notes 4 and 5) 15 min in an ice-water bath, add ethyl 2-chloropropionate
(0.036 mmol) into the reaction mixture.
4. Allow the polymerization to take place under argon atmo-
sphere at 25 C for 4 h with magnetic stirring.
5. Collect polymer particles by centrifuging the reaction mixtures,
thoroughly wash them with methanol, and allow them to be
dried at 40 C under vacuum to the constant weights, leading
to orange yellow QD-SiO2@MIP@PGMMA and
QD-SiO2@CP@PGMMA particles (their yields are 105 and
106 mg, respectively).
104 Huiqi Zhang
3.3 “One-step 1. Add MAA (0.834 mmol), Tc (0.834 mmol), AnHEMA

Approach”: Synthesis (0.0834 mmol), and a mixture of acetonitrile and DMF
of OF-MIP@PHEMA via (4:1 v/v, 60 mL) into a round-bottom flask (100 mL) succes-
Hydrophilic sively. Magnetically stir the mixture at ambient temperature for
Macro-CTA-Mediated 5 min to obtain a clear solution.
RAFTPP (See Notes 1, 2. Add EGDMA (2.501 mmol), AIBN (0.0566 mmol), CDB
3, and 5) (0.0552 mmol), and PHEMA macro-CTA (Mn,
NMR ¼ 5140 g/mol, 0.0342 mmol) into the above solution,
and degas the reaction mixture by bubbling argon for 30 min in
an ice-water bath.
3. Magnetically stir the reaction mixture in an oil bath at 60 C for
24 h under argon atmosphere.
4. Add 10 mL of methanol/acetic acid (9:1 v/v) into the poly-
merization mixture and allow the mixed sample to be kept at
room temperature for 30 min (to make the following centrifu-
gation easier).
5. Collect polymer particles by centrifugation, remove their tem-
plate by thoroughly washing them with methanol/acetic acid
(9:1 v/v) (until no template can be detectable in the centrifu-
gated supernatant), wash them with methanol thrice, and
finally allow the resulting polymer particles to be freeze-dried
under vacuum to the constant weight, leading to light pink
OF-MIP@PHEMA nanoparticles (yield: 432 mg).
6. Prepare and purify OF-CP@PHEMA nanoparticles (light pink
color) under the same conditions as for OF-MIP@PHEMA
nanoparticles except in the absence of the template (yield:
501 mg).
3.4 Calibration Perform spectrofluorometric titration experiments to establish the

Measurements calibration curves for the hydrophilic fluorescent MIP nanoparti-
with Fluorescent MIP cles according to the following procedures:
Nanoparticles 1. Measure the fluorescence intensities of the hydrophilic fluores-
with Hydrophilic cent MIP nanoparticles (1 and 0.5 mg/mL for QD-SiO2@-
Polymer Brushes MIP@PGMMA and OF-MIP@PHEMA, respectively) after
(or Briefly Hydrophilic their incubation with different concentrations of Tc in the
Fluorescent MIP complex biological sample (e.g., the undiluted pure bovine
Nanoparticles) (See serum) at 25 C for 2 h.
Note 6)
2. Plot the fluorescence quenching of the studied MIP nanopar-
ticles (i.e., (F0/F) 1) versus Tc concentration using the
Stern–Volmer equation:
F 0 =F ¼ 1 þ K SV C
where F0 and F are the fluorescence intensities in the absence and
presence of Tc, respectively, KSV is the Stern–Volmer quenching
constant, and C is the Tc concentration.
3. The linear calibration curves (plots of (F0/F) 1 versus C) can

thus be established for the hydrophilic fluorescent MIP nano-
particles within a certain Tc concentration ranges (0.5–50 and
0.5–20μM for QD-SiO2@MIP@PGMMA and
OF-MIP@PHEMA, respectively), which can be used for deriv-
ing the Tc concentration in other complex biological samples
by using these fluorescent MIP nanoparticles as the optosen-
sors (see Subheading 3.5).
3.5 Direct and Highly 1. Determine the fluorescence intensities (F0) of the hydrophilic
Selective Optosensing fluorescent MIP nanoparticles in the undiluted commercially
of Tc in Complex available bovine or porcine serum (no Tc was detectable in
Biological Samples these commercially available serums).
with the Hydrophilic 2. Determine the fluorescence intensities (F) of the hydrophilic
Fluorescent MIP fluorescent MIP nanoparticles after their incubation with the
Nanoparticles (See undiluted bovine or porcine serum samples that are spiked with
Note 6) different concentrations of Tc.
3. Derive the contents of Tc in the above spiked bovine or porcine
serum samples by comparing the determined F0/F values of
the samples with the calibration curves listed in Subheading
3.4.
4. Directly detect Tc concentrations in the undiluted bovine or
porcine serum samples in the presence of some interfering
species (e.g., Chl, Lex, Van, and atenolol) following the
above method.
4 Notes
1. The above-described hydrophilic fluorescent MIPs were

prepared with the antibiotic Tc as the template [22, 27]. How-
ever, water-compatible MIPs can also be prepared similarly for
a wide range of other templates such as herbicides (e.g.,
2,4-dichlorophenoxyacetic acid (2,4-D)) [28–31], food addi-
tives (e.g., folic acid (FA)) [32], and drugs (e.g., propranolol)
[15, 33]. Water-compatible MIPs of different compositions
can be readily obtained by employing other monomers, cross-
linkers, initiators, and solvents. The volume of the utilized
solvent in each case should be rationally tuned to obtain nar-
rowly dispersed MIP micro- or nanoparticles. Uniform spheri-
cal MIP particles with diameters ranging from about 50 nm
[22] to 3.6μm [14] may be obtained under different
conditions.
2. In the “two-step approach,” the core of the hydrophilic fluo-
rescent MIP particles is not limited to silica particles. It can be
any “living” polymer micro- or nanospheres prepared via
106 Huiqi Zhang
CRPPs [8–10]. Moreover, the hydrophilic polymer brush-

grafting method is also not limited to the surface-initiated
ATRP, it can be any kinds of surface-initiated CRPs (e.g.,
surface-initiated RAFT polymerization [13, 28, 30]) and vari-
ous efficient coupling reactions [14, 15, 31].
3. In the described examples, we use a polymerizable anthracene-
containing organic fluorescent monomer and quantum dots
(QDs) (i.e., green CdTe QDs) as the fluorescent labeling spe-
cies for the “one-step approach” and “two-step approach,”
respectively. Other fluorescent species can be used as well for
the preparation of water-compatible fluorescent MIP particles
[29, 31]. Moreover, dual fluorescent species can also be
incorporated into water-compatible MIP particles to prepare
complex biological sample-compatible ratiometric fluorescent
MIPs [29, 31].
4. In the second step of “two-step approach,” addition of CuBr2
is to make ATRP better controlled [18]. In addition, the
addition of a sacrificial ATRP initiator (i.e., ethyl
2-chloropropionate) into the surface-initiated ATRP system
has two functions [34]: (1) to improve the controllability of
ATRP; (2) to produce free polymers in the reaction solution,
whose molecular weights and molar-mass dispersities can be
used to represent those of the polymer brushes grafted onto the
MIP particles.
5. The hydrophilic polymer brushes are also not limited to
PGMMA and PHEMA. Other hydrophilic polymers such as
PNIPAAm and PEG can also be used [13, 14, 16, 31].
6. Application of hydrophilic fluorescent MIP particles for the
detection of Tc in the undiluted pure bovine and porcine
serums is described as an example. Analytical procedures are
generic and can be adapted to various analytes in other complex
biological media such as the undiluted pure milks [14, 17, 27,
29, 31, 33].
Acknowledgments
The author thanks the financial support from National Natural

Science Foundation of China (21574070, 22071121) and Project
supported by NCC Fund (NCC2020PY12).
References
1. Haupt K, Mosbach K (2000) Molecularly 2. Zhang H, Ye L, Mosbach K (2006)
imprinted polymers and their use in biomi- Non-covalent molecular imprinting with
metic sensors. Chem Rev 100:2495–2504 emphasis on its application in separation and
drug development. J Mol Recognit coupling chemistry. J Mater Chem B

19:248–259 7:2474–2483
3. Turiel E, Martı́n-Esteban A (2010) Molecu- 16. Pan G, Zhang Y, Ma Y, Li C, Zhang H (2011)
larly imprinted polymers for sample prepara- Efficient one-pot synthesis of water-compatible
tion: a review. Anal Chim Acta 668:87–99 molecularly imprinted polymer microspheres
4. Wulff G, Liu J (2012) Design of biomimetic by facile RAFT precipitation polymerization.
catalysts by molecular imprinting in synthetic Angew Chem Int Ed 50:11731–11734
polymers: the role of transition state stabiliza- 17. Ma Y, Pan G, Zhang Y, Guo X, Zhang H
tion. Acc Chem Res 45:239–247 (2013) Narrowly dispersed hydrophilic molec-
5. Chen L, Wang X, Lu W, Wu X, Li J (2016) ularly imprinted polymer nanoparticles for effi-
Molecular imprinting: perspectives and appli- cient molecular recognition in real aqueous
cations. Chem Soc Rev 45:2137–2211 samples including river water, milk, and bovine
6. Zhang H (2019) Molecularly imprinted nano- serum. Angew Chem Int Ed 52:1511–1514
particles for biomedical applications. Adv 18. Zhang H, Klumperman B, Ming W, Fischer H,
Mater 32:1806328 van der Linde R (2001) Effect of Cu (II) on the
7. Zhang H, Li C (2013) Recent advances in the kinetics of the homogeneous atom transfer rad-
development of water-compatible molecularly ical polymerization of methyl methacrylate.
imprinted polymers. Chin Polym Bull 1:13–25 Macromolecules 34:6169–6173
8. Zhang H (2014) Water-compatible molecu- 19. Beers KL, Boo S, Gaynor SG, Matyjaszewski K
larly imprinted polymers: promising synthetic (1999) Atom transfer radical polymerization of
substitutes for biological receptors. Polymer 2-hydroxyethyl methacrylate. Macromolecules
55:699–714 32:5772–5776
9. Zhang H (2018) Water-compatible molecu- 20. Schweitz L, Spégel P, Nilsson S (2000) Molec-
larly imprinted polymers. In: Kutner W, ularly imprinted microparticles for capillary
Sharma PS (eds) Molecularly imprinted poly- electrochromatographic enantiomer separation
mers for analytical chemistry applications. The of propranolol. Analyst 125:1899–1901
Royal Society of Chemistry, London 21. van Dijk-Wolthuis WNE, Franssen O,
10. Zhang H (2013) Controlled/“living” radical Talsma H, van Steenbergen MJ, Kettenes-van
precipitation polymerization: a versatile poly- den Bosch JJ, Hennink WE (1995) Synthesis,
merization technique for advanced functional characterization, and polymerization of glyci-
polymers. Eur Polym J 49:579–600 dyl methacrylate derivatized dextran. Macro-
molecules 28:6317–6322
11. Zhang Y, Zhang H (2008) Recent advances in
the preparation of molecularly imprinted poly- 22. Niu H, Yang Y, Zhang H (2015) Efficient
mers via controlled radical polymerization one-pot synthesis of hydrophilic and fluores-
techniques. Chin J React Polym 17:1–11 cent molecularly imprinted polymer nanoparti-
cles for direct drug quantification in real
12. Li GL, Möhwald H, Shchukin DG (2013) Pre- biological samples. Biosens Bioelectron
cipitation polymerization for fabrication of 74:440–446
complex core-shell hybrid particles and hollow
structures. Chem Soc Rev 42:3628–3646 23. Ciampolini M, Nardi N (1966) Five-
coordinated high-spin complexes of bivalent
13. Pan G, Zhang Y, Guo X, Li C, Zhang H (2010) cobalt, nickel, and copper with tris(2-dimethy-
An efficient approach to obtaining water- laminoethyl)amine. Inorg Chem 5:41–44
compatible and stimuli-responsive molecularly
imprinted polymers by the facile surface- 24. Le TP, Moad G, Rizzardo E, Thang SH (1998)
grafting of functional polymer brushes via Polymerization with living characteristics. PCT
RAFT polymerization. Biosens Bioelectron Int Appl WO 98/01478; Chem Abstr 128:
26:976–982 115390
14. Zhao M, Chen X, Zhang HT, Yan H, Zhang H 25. Unsal E, Elmas B, Çağlayan B, Tuncel M,
(2014) Well-defined hydrophilic molecularly Patir S, Tuncel A (2006) Preparation of an
imprinted polymer microspheres for efficient ion-exchange chromatographic support by a
molecular recognition in real biological sam- “grafting from” strategy based on atom trans-
ples by facile RAFT coupling chemistry. Bioma- fer radical polymerization. Anal Chem
cromolecules 15:1663–1675 78:5868–5875
15. Ma YJ, Gao J, Zheng C, Zhang H (2019) Well- 26. Yang Y, He X, Wang Y, Li W, Zhang Y (2014)
defined biological sample-compatible molecu- Epitope imprinted polymer coating CdTe
larly imprinted polymer microspheres by com- quantum dots for specific recognition and
bining RAFT polymerization and thiol-epoxy direct fluorescent quantification of the target
108 Huiqi Zhang
protein bovine serum albumin. Biosens Bioe- real biological sample. Chem Commun
lectron 54:266–272 50:2208–2210
27. Yang Y, Niu H, Zhang H (2016) Direct and 31. Hou Y, Zou Y, Zhou Y, Zhang H (2020)
highly selective drug optosensing in real, undi- Biological sample-compatible ratiometric fluo-
luted biological samples with quantum-dot- rescent molecularly imprinted polymer micro-
labeled hydrophilic molecularly imprinted spheres by RAFT coupling chemistry.
polymer microparticles. ACS Appl Mater Inter- Langmuir. 36:12403–12413. https://doi.
faces 8:15741–15749 org/10.1021/acs.langmuir.9b03851
28. Pan G, Ma Y, Zhang Y, Guo X, Li C, Zhang H 32. Yang Y, Wang Z, Niu H, Zhang H (2016)
(2011) Controlled synthesis of water- One-pot synthesis of quantum dot-labeled
compatible molecularly imprinted polymer hydrophilic molecularly imprinted polymer
microspheres with ultrathin hydrophilic poly- nanoparticles for direct optosensing of folic
mer shells via surface-initiated reversible acid in real, undiluted biological samples. Bio-
addition-fragmentation chain transfer poly- sens Bioelectron 86:580–587
merization. Soft Matter 7:8428–8439 33. Lu X, Zheng C, Zhang H (2019) Improve-
29. Xu S, Zou Y, Zhang H (2020) Well-defined ment of surface hydrophilicity and biological
hydrophilic “turn-on”-type ratiometric fluo- sample-compatibility of molecularly imprinted
rescent molecularly imprinted polymer micro- polymer microspheres by facile surface modifi-
spheres for direct and highly selective herbicide cation with α-cyclodextrin. Eur Polym J
optosensing in the undiluted pure milks. 115:12–21
Talanta 211:120711 34. Gorman CB, Petrie RJ, Genzer J (2008) Effect
30. Zhao M, Zhang C, Zhang Y, Guo X, Yan H, of substrate geometry on polymer molecular
Zhang H (2014) Efficient synthesis of narrowly weight and polydispersity during surface-
dispersed hydrophilic and magnetic molecu- initiated polymerization. Macromolecules
larly imprinted polymer microspheres with 41:4856–4865
excellent molecular recognition ability in a
Chapter 9
Generation of High-Affinity Aptamer-MIP Hybrid

Nanoparticles
Mark Sullivan, Rachel Hand, and Nicholas Turner
Abstract
Aptamers and molecularly imprinted polymers (MIPs) are two leading technologies used for the develop-
ment of protein biomimetics. By combining the two technologies, a new hybrid class of materials can be
created, which utilizes the interesting characteristics of both recognition materials, while negating several of
their drawbacks. This chapter describes the protocol for the synthesis of aptamer-MIP hybrid nanoparticles.
These materials exhibit exceptional affinity (into the nM range) and selectivity for their target template.
They can be developed for a wide range of targets, while exhibiting excellent robustness, solubility, and
flexibility in use. These are a new class of recognition materials with the potential for use as drug delivery
vectors, as well as use within sensing and recognition assays.
Key words Aptamers, Aptamer-MIP hybrid nanoparticles, Sensing, Recognition assays
1 Introduction
The molecular recognition of large biomolecules, such as nucleic

acids, viruses, and proteins, has become increasingly topical, espe-
cially with the aim of developing sensors for the detection of disease
markers [1]. Antibodies are well known for their use in analytical,
diagnostic, and therapeutic applications, due to their strong recog-
nition to target molecules [2, 3]. Although, they do have their own
disadvantages including cost, biological source, immunogenicity, as
well as poor shelf life and stability. As such a considerable research
field has emerged around the design and development of suitable
alternatives [4, 5].
One such antibody alternative that has demonstrated excep-
tional potential is the use of aptamers, small single-stranded RNA
or DNA oligonucleotides that are able to bind to target molecules
[6]. They have been shown to bind to a variety of molecular targets
such as small molecules, proteins, nucleic acids as well as cells,
tissues, and organisms [7, 8]. Aptamers bind to their specific target
https://doi.org/10.1007/978-1-0716-1629-1_9,
109
110 Mark Sullivan et al.
site through electrostatic interactions, hydrophobic interactions,

and their complementary shapes [9]. With molecular recognition
properties that rival that of antibodies, they have found use in
biotechnological and therapeutic applications [10, 11] as they can
offer advantages over antibodies because they are cheaper, readily
produced by chemical synthesis; possess desirable storage proper-
ties and elicit little or no immunogenicity in therapeutic
applications [6].
Another alternative method to antibodies is the use of molecu-
lar imprinting [12]. Molecularly imprinted polymers (MIPs) are
gaining in popularity in use due to their properties, such as high
affinity and selectivity, and resistance to extremes of temperature,
pressure, and pH variations [13, 14]. Molecular imprinting
involves the formation of selective sites in a polymer matrix, with
the memory of a template, that is able to mimic enzymes and
antibodies. The imprinting process usually involves the self-
assembly of functional monomers around a template, through
covalent or non-covalent bonds. The resultant template-monomer
complexes are subsequently copolymerized with a suitable cross-
linker. Removal of the template molecules from the polymer results
in the formation of specific binding cavities, complementary in
terms of structure and functionality to the template molecule
[15–17]. The development and enhancement of molecularly
imprinted polymeric nanoparticles (nanoMIPs) have opened new
perspectives in nanotechnology. NanoMIPs have attracted a lot of
attention due to their performance, meaning they have to potential
to transform traditional analytical methods in chemistry, biochem-
istry, environmental sciences, and biomedical fields [18, 19].
Integrating these two (aptamers and MIPs) antibody alterna-
tives into a hybrid system is a better option and represents a new
category of materials. This allows for the incorporation of the
robustness of the MIP with the recognition properties of the
aptamer [20]. By exploiting a modified polymerizable aptamer,
solid phase synthesis of aptamer-imprinted polymer hybrid nano-
materials capable of molecular recognition has been achieved. The
AptaMIP NPs have been shown to be resistant to digestion by
nuclease P1, and through thermal strain, which confirms the poly-
mer matrix acts as a shield for the incorporated sequence [20]. The
synthetic protocol described below allows for the rapid develop-
ment of these materials, with the ability to target a wide range of
templates and is flexible enough to use a full range of monomers.
The protocol below is described in terms of targeting protein
templates though other small template molecules, such as the
cocaine analog, benzoylecgonine (BEC), have also been reported
[20]. The same methodology is applied.
Aptamer-MIP Hybrid Nanoparticles 111
2 Materials
Prepare all solutions using deionized or Milli-Q water (subse-

quently referred to as “water” and analytical grade reagents
(>95%). Prepare and store all reagents at room temperature (unless
indicated otherwise).
2.1 Synthesis 1. 5-Iodo-20 -deoxyuridine

of Polymerizable Base 2. Pyridine (dry).
3. Dimethyoxytrityl chloride.
4. Sodium bicarbonate (NaHCO3).
5. Dichloromethane (DCM).
6. Sodium sulfate (Na2SO4).
7. Palladium acetate.
8. Dimethylformaide (DMF).
9. Tributylamine.
10. Methyl acrylate.
11. Celite®.
12. Magnesium sulfate (MgSO4).
13. N,N-Diisopropylethylamine (DIPEA).
14. 2-cyanoethyl N,N-diisopropylchlorophosphoramidite
15. 7 N ammonia in methanol.
2.2 Synthesis 1. Selected Aptamer sequence (see Note 1).

of Modified Aptamer 2. Synthesized base (~20–25 mg per coupling).
3. Phosphoramidite for A, G, C, T, and U (as required).
4. Methodology (instrument)-specific materials (solvents, wash-
ing solvents, solid-phases, etc.).
2.3 Preparation 1. Template. This is the target protein or selected epitope

of Template-Modified sequence and should be stored as recommended by the sup-
Solid-Phase Beads plier. The template should be brought up to room temperature
(RT) before synthesis procedure (see Notes 2 and 3).
2. Glass beads (average diameter 75–90 μm).
3. 3-Aminopropyltrimethyloxysilane (APTMS).
4. Succinimidyl iodoacetate (SIA).
5. Glutaraldehyde (GA) 7% (v/v) solution: glutaraldehyde in
0.01 M PBS (pH 7.2).
6. Anhydrous toluene.
7. Anhydrous acetonitrile.
8. Sodium hydroxide (NaOH).

9. Needle and glass syringes suitable for anhydrous chemistry.
10. 250 mL sealable bottles, ideally with needle-through seals.
11. Sintered disc funnel.
12. 500 mL flasks.
13. Vacuum pump with cooled solvent trap. Ideally, this should be
electronically controllable.
2.4 Synthesis 1. Aptamer sequence. This is the aptamer sequence for the target
of AptaMIP NPs via protein or selected epitope and should be stored as recom-
the Template-Linked mended by the supplier. The aptamer should be brought up
Solid-Phase Method to RT before synthesis procedure (see Note 4).
2. N-Isopropylacylamide (NIPAM).
3. N,N0 -Methylene-bis-acrylamide (BIS).
4. Acrylic acid (AAc).
5. N-Tert-butylacylamide (TBAm).
6. N-(3-Aminopropyl)methacrylamide hydrochloride (NAMPA).
7. Ammonium persulfate (APS).
8. Tetramethylethylenediamine (TEMED).
9. Phosphate-buffered saline (PBS): phosphate buffer (0.01 M),
potassium chloride (0.00268 M), and sodium chloride
(0.140 M), pH 7.4.
10. 100-mL SPE cartridge fitted with a 20 μm porosity frit.
11. 100 mL flasks.
12. Aluminum foil.
3 Methods
The following protocol is split into six sections. The first two
describe the process of synthesizing the polymerizable base (Sub-
heading 3.1) and the subsequent aptamer (Subheading 3.2). Sub-
heading 3.3 describes using a whole protein as a template, while
Subheading 3.4 describes the use of an epitope as a target (see
Notes 2 and 3). After performing Subheadings 3.1 and 3.2, per-
form either one of Subheading 3.3 or 3.4 before continuing with
Subheading 3.5 and 3.6. The synthesis presented here is scalable
(see Note 5).
3.1 Synthesis This step describes the synthesis of a modified thymine (Fig. 1).
of Base Other bases are accessible to this chemistry.
1. To a solution of 5-Iodo-20 -deoxyuridine (1.5 g, 4.2 mmol) in
dry pyridine (50 mL) dimethoxytrityl chloride (1.9 g,
Fig. 1 Carboxy-dT-CE Phosphoramidite. An example polymerizable base. The

methoxyvinyl group is the site of polymerization but numerous other options
exist that can be attached through the 50 site using Heck or Sonogashira
reactions (see Note 7)
5.5 mmol) is added. After 4 h the reaction mixture is cooled

and neutralized with an NaHCO3 saturated aqueous solution
(200 mL), extracted with DCM, and dried over sodium sulfate.
The organic solution is evaporated under vacuum and the
residue is purified by chromatography (see Note 6).
2. Palladium acetate (35 mg, 0.16 mmol) and the compound
produced in step 1 (1.01 g, 1.54 mmol) is suspended in
DMF (3 mL) in a 10 mL microwave tube equipped with a
magnetic stirring bar.
3. Tributylamine (0.37 mL, 1.56 mmol) and methyl acrylate
(265 mg, 3.08 mmol) were then added to the microwave
tube and the suspension was stirred and degassed with argon
(nitrogen can also be used) for 10 min (see Note 7).
4. The tube is tightly sealed and the mixture irradiated in a
microwave for 10 min at 100 C. After the irradiation period,
the reaction vessel is cooled to room temperature before
opening.
5. The reaction is filtered through Celite®, rinsing with DCM
(50 mL). The organic phase was dried over magnesium sulfate
and evaporated under vacuum.
6. The crude product was purified by flash chromatography with

an eluent of 9:1 DCM: 7 N ammonia in methanol. The appro-
priate fractions were combined and evaporated to give off a
white product.
7. The compound produced in step 6 (696 mg, 1.13 mmol) is
dried via azeotroping with dry DCM (3 10 mL). The solid
was then redissolved in dry DCM (15 mL) and the solution
stirred under argon (nitrogen can also be used).
8. DIPEA (0.49 mL, 2.84 mmol) is added via syringe to the
solution, followed by the cautiously addition of 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (0.3 mL, 1.34 mmol)
to the mixture. The solution is left to stir under argon (nitro-
gen can also be used) until the reaction is complete and con-
firmed by TLC (approximately 3 h).
9. The reaction mixture is diluted with degassed DCM (50 mL),
and the solution is washed with degassed saturated sodium
bicarbonate solution (2 50 mL). The organic phases are
collected and dried over sodium sulfate, before being evapo-
rated under vacuum. The crude product is purified by flash
chromatography with an eluent of 9:1 DCM: 7 N ammonia in
methanol. The appropriate fractions are combined and evapo-
rated to produce an off white solid.
3.2 Sequence Numerous synthetic strategies exist for sequence synthesis so we

Synthesis have not included this step, in detail. Given that it is commonly
performed automatically using a programmable solid-phase synthe-
sizer, the authors recommended you follow the protocols depen-
dent on your specific instrument and use standard LC-MS
analytical techniques for confirmation of successful synthesis. It
must be noted here that the modification on the base that you
have made (Subheading 3.1) should not interfere with the oligo
synthesis chemistry so moieties need to be selected carefully. It is
recommended that ultra-mild reactions conditions are used.
The modified base can be used as a replacement for its regular
equivalent (e.g., a thymine can be replaced with a modified thy-
mine) wherever you choose along the sequence. Nominally, this
can be performed by component selection on the synthesizer (sep-
arate chemical channel). It is recommended that for best binding a
number of bases are replaced throughout the length of the
sequence to limit significant flexibility.
3.3 Preparation 1. Boil the glass beads in 4 M NaOH (0.8 mL of solution per
of Protein-Modified gram of glass beads) for 15 min in order to activate their
Solid-Phase Beads surface, and then rinse them thoroughly with deionized water
(eight times with 100 mL, for 30 g of beads), until the pH of
the water/bead solution is around 7 (make sure that the base
has been completely washed off). Rinse with acetone (twice
with 200 mL) and dry at 80 C for 3 h, and then seal in a dry
nitrogen-filled bottle with needle-through seal cap.
2. Prepare a solution of APTMS 3% (v/v) in anhydrous toluene,
using sealed vessel under nitrogen to maintain an anhydrous
nature. Using a needle and syringe technique, transfer the
required aliquot to the sealed bottle containing the dry beads.
Incubate the dry beads in the APTMS solution at 60 C for
24 h at RT in a sealed container, using 0.4 mL of solution per
gram of beads (see Note 8).
3. After incubation, decant the glass beads into a sintered disk
filter funnel, and thoroughly wash with 8 volumes (100 mL) of
acetone and 2 volumes (100 mL) of methanol. Dry the glass
beads under vacuum (see Note 9), and transfer them into an
oven at 150 C for 30 min.
4. Incubate the silanized beads in 7% (v/v) GA solution for 2 h
(using 0.5 mL of solution per gram of beads).
5. After incubation, place the beads into a sintered disk filter
funnel, and wash them with 8 volumes (100 mL) of deionized
water under vacuum (see Note 10).
6. Incubate the glass beads in a solution of the target protein
(0.5 mg/mL) in 10 mM PBS (pH 7.4) overnight. This is
performed in a 500 mL sealed bottle under nitrogen (see
Note 3).
7. Filter the glass beads and rinse them with deionized water
(8–10 volumes of 100 mL) in a sintered funnel under vacuum.
8. Dry the beads under vacuum and store them at 18 C under
nitrogen (see Note 11).
3.4 Preparation 1. Boil the glass beads in 4 M NaOH (0.8 mL of solution per
of Epitope-Modified gram of glass beads) for 15 min in order to activate their
Solid-Phase Beads surface, and then rinse them thoroughly with deionized water
(eight times with 100 mL, for 30 g of beads), until the pH of
the water/bead solution is around 7 (make sure that the base
has been completely washed off). Rinse with acetone (twice
with 200 mL) and dry at 80 C for 3 h, and then seal in a dry
nitrogen-filled bottle with needle-through seal cap.
2. Prepare a solution of APTMS 3% (v/v) in anhydrous toluene,
using sealed vessel under nitrogen to maintain an anhydrous
nature. Using a needle and syringe technique, transfer the
required aliquot to the sealed bottle containing the dry beads.
Incubate the dry beads in the APTMS solution at 60 C for
24 h at RT in a sealed container, using 0.4 mL of solution per
gram of beads (see Note 8).
3. After incubation, decant the glass beads into a sintered disk
filter funnel, and thoroughly wash with 8 volumes (100 mL) of
acetone and 2 volumes (100 mL) of methanol. Dry the glass

beads under vacuum (see Note 9), and transfer them into an
oven at 150 C for 30 min.
4. Incubate the silanized beads in succinimidyl iodoacetate (SIA)
solution in dry (anhydrous) acetonitrile for 2 h at RT (using
0.5 mL of solution per gram of beads).
5. After incubation, place the beads into a sintered disk filter
funnel and wash them with 8 volumes (100 mL) of deionized
water under vacuum (see Note 10).
6. Incubate the glass beads in a solution of the selected epitope
(0.2 mg/mL) (see Note 12) in 10 mM PBS (pH 8.3) enriched
with EDTA 5 mM (using 0.5 mL of solution per gram of
beads) overnight at RT in the dark (a foil-covered bottle will
suffice).
7. Filter the glass beads and rinse them with deionized water
(8–10 volumes of 100 mL) in a sintered funnel under vacuum.
8. Dry the beads under vacuum and store them at 18 C under
nitrogen (see Note 11).
3.5 Synthesis 1. Measure out 1.74 μmol of the aptamer and dissolve in 10 mL of
of AptaMIP NPs water. Degas the solution with a gentle stream of N2 or Ar for
10 min via a Pasteur pipette located in the bulk of the solution
(see Note 13).
2. While the solution is degassing, weigh 30 g of template-
derivatized glass beads into a 100 mL sealable bottle. Purge
N2 or Ar into this bottle for 5 min to ensure oxygen is removed.
3. Add the solution prepared in step 1 of this section to the glass
beads and incubate for 1 h at RT.
4. Weigh out 20 mg of NIPAM, 2.4 mg of NAMPA, and 1 mg of
BIS. Add 39 mL of water and dissolve the solids.
5. Weigh out 17 mg of TBAm in a separate vial and add 250 μL of
ethanol. Dissolve the monomer by vortexing and brief sonica-
tion if necessary, and then add this solution to the solution
prepared in the previous step and homogenize.
6. In a separate vial, dissolve 22 μL of AAc into 1 mL of water.
Then take 100 μL of this and add it to the solution prepared in
step 4 of this section. Gently swirl to homogenize the solution.
7. Adjust the total volume of the solution to 40 mL by adding
water and swirl.
8. Seal the flask, connect to a vacuum source, and sonicate under
vacuum using an ultrasonicator for 10 min. Then bubble the
solution with a gentle stream of N2 or Ar for 20 min via a
Pasteur pipette located in the bulk of the solution.
9. Weigh 15 mg of APS and dissolve in 250 μL of water and add

12.5 μL of TEMED.
10. Pour the degassed solution of monomers onto the glass beads
prepared in Subheading 3.2, step 3 and quickly add the solu-
tion prepared in the previous step (step 12) to the glass beads
dispersed in the monomer mixture.
11. Purge the headspace with N2 or Ar and then seal the vessel and
incubate for 1 h at RT. This should not be agitated, but instead
left to sit.
3.6 Selection 1. After incubation, transfer the whole content of the vessel (both
of High-Affinity beads and solution) into a 100 mL SPE cartridge fitted with a
AptaMIP NPs 20 μm porosity frit and male Luer fit.
2. By means of a plunger or vacuum, remove the solution, and
replace with freshwater (30 mL) at RT (see Note 14).
3. Remove low-affinity AptaMIP NPs by eluting the aforemen-
tioned solution at RT and replace it with 30 mL aliquots of
freshwater each time. Repeat this step eight times. Stir the
beads very gently every other washing step. This solution is
to be discarded, but it is recommended to keep it until the
process is fully completed.
4. Seal the bottom outlet of the SPE cartridge containing the
glass beads, e.g., using a female Luer cap.
5. Collect the high-affinity AptaMIP NP solution. Add 30 mL of
water pre-warmed to 60 C to the SPE cartridge containing the
glass beads, gently stir the beads, and then place the cartridge
into a water bath at 60 C. The top of the cartridge should be
covered with foil to avoid contamination.
6. After 20 min, collect the solution bearing the AptaMIP NPs by
means of a plunger or vacuum pump. Keep this solution in a
clean 250 mL flask.
7. Add a further 20 mL of water pre-warmed to 60 C and place
the SPE cartridge in the water bath at 60 C for 2 min, and then
collect the solution as in step 6 in the same flask.
8. Repeat step 7 until approximately 150 mL of solution has been
collected (see Note 15).
9. The AptaMIP NPs in solution should be stored at 4 C and
used readily. Before use solution should be gently agitated to
ensure all particles are in solution and to reduce sedimentation.
(see Note 16).
The whole sequence can be described in Fig. 2.
Fig. 2 Schematic representation of the solid-phase synthesis of aptaMIP NPs. I:

Synthesis of polymerizable aptamer sequence (using modified base). II:
Polymerizable sequence is incubated first with solid phase bearing the protein
template. III: Remaining classical monomers are added to the solid phase, and
the polymerization is initiated with the addition of APS and TEMED. IV: Low
affinity particles and unreacted monomers are washed at relative low
temperatures. The temperature is then increased and the high-affinity AptaMIP
hybrid NPs are eluted from the solid phase using water
4 Notes
1. Sequence of aptamer will need to be determined prior to

synthesis using standard SELEX process. Ideally, the location
for replacement base should be situated throughout the oligo.
2. The presented protocol in Subheading 3.3 was taken from a
protocol that described an imprint of a model enzyme trypsin.
Any protein can be used in this method as the monomer
selection is designed to be universal, although the
corresponding aptamer for the protein is needed.
3. The important points to note here are that the concentration
range is approximately that as described here (0.5 mg mL1),
and the template protein is brought to room temperature
before synthesis. The protein isoform will be in the conforma-
tion found under the conditions of the synthesis, where the
protein will incline to be fully hydrated given the aqueous
nature of the reaction conditions. While the presence of a
high amount of polymerization components can affect the
conformation of a protein template, the method presented
here has shown no structural changes to the protein structure.
This is due to the limited amount of reaction components
added to the reaction mixture.
4. The aptamer used needs to be modified enabling the aptamer

to be polymerizable. It is recommended that introducing C-5
alkene-modified 20 deoxyuridine residues into the DNA strand
will result in single to multiple anchoring points between the
aptamer and the polymer matrix.
5. Within reason, the process is scalable, although if making larger
volumes of material, ideally you should perform multiple
syntheses at this scale. The method has proven to be highly
reproducible given the simple conditions and methods of NP
selection used in the final steps. Going smaller than this will
depend on the selected glassware available. Although the
authors recommend that you do not scale down more than
2 to guarantee that you obtain a working volume of NPs.
6. The presented protocol in Subheading 3.1 was taken from a
protocol which described the synthesis using 5-Iodo-2-
0
-deoxyuridine, other 5-Iodo bases can be used in order to
produce other polymerizable bases.
7. In this protocol, methyl acrylate is used to produce a
polymerizable base with a carboxy functional group, other
polymerizable functional groups are available (e.g., N,N0 -
methylenebisacrylamide would be a good substitute [21]) for
use. The addition of a polymerizable functional group can be
achieved by either Heck coupling or Sonogashira coupling
reactions.
8. Make sure that the glass beads are completely dry and only
anhydrous toluene is used. Do not stir the beads with an orbital
shaker or by using a stirrer, just gently swirl them by hand,
instead. This is because any collisions between beads may
roughen their surface. This applies to all the steps involving
incubation of glass beads, unless otherwise stated.
9. The glass beads are stable for up to 1 month at room tempera-
ture, if they are stored dry and under nitrogen.
10. Before adding GA, check the pH of the bead solution and
adjust to 7.4, if needed. The formation of the Schiff base is
reversible, so it is recommended to not excessively wash.
Reduction with sodium cyanoborohydride would result in
the formation of a stable bond. The beads should not be stored
at this stage; move directly to the template conjugation step.
11. The beads with immobilized template can be stored dry (under
nitrogen) for up to 2 weeks at 18 C. Although, it is recom-
mended to use the beads as soon as possible.
12. The selected epitope should be modified with a terminal cyste-
ine (at the –COOH end). Epitopes should be selected by
(a) compatibility with the molecularly imprinting technology
(i.e., short epitope), (b) option to add a terminal cysteine for
the immobilization onto the solid phase, (c) availability for the
final NP-protein interaction (i.e., surface epitope), and
(d) significance of the sequence of the epitope. The haloacetyl
group of SIA reacts with the thiol group of the terminal cyste-
ine of the epitope, this results in the coupling of the epitope
onto the surface of the beads.
13. Do not use an excessive stream of N2 as it may cause evapora-
tion of water. The tip of stream should be central to the
solution and towards the bottom of the vessel, but not touch-
ing the glass.
14. It is imperative that the glass beads do not dry and should be
left with a water interface just above the glass beads.
15. Using a spatula, the beads should be stirred every time fresh
pre-warmed water is added. Leave the solution at RT for 4–6 h
for it to cool down, followed by storing the solution at 4 C.
The AptaMIP NPs stock solution can be stored 4 C for a few
months, subject to any potential bacterial/fungal contamina-
tion issues, which may arise during the storage due to handling
under non-sterile conditions. Sodium azide or similar preser-
vative could be considered for use, depending on the final use
of the sample. The sample should not be frozen.
16. Stability can be tested using enzymatic nucleic acid digestion
assay, or by performance after heating of NPs above 90 C.
References
1. Gui R, Jin H, Guo H, Wang Z (2018) Recent 7. Keefe AD, Pai S, Ellington A (2010) Aptamers
advances and future prospects in molecularly as therapeutics. Nat Rev Drug Discov
imprinted polymer-based electrochemical bio- 9:537–550
sensors. Biosens Bioelectron 100:56–70 8. Ruscito A, DeRosa MC (2016) Small-molecule
2. Hayden O, Lieberzeit PA, Blaas D, Dickert FL binding aptamers: selection strategies, charac-
(2006) Artificial antibodies for bioanalyte terization, and applications. Front Chem 4:14
detection—sensing viruses and proteins. Adv 9. Kulbachinskiy AV (2007) Methods for selec-
Funct Mater 16:1269–1278 tion of aptamers to protein targets. Biochemis-
3. Bedwell TS, Whitcombe MJ (2016) Analytical try (Mosc) 72:1505–1518
applications of MIPs in diagnostic assays: 10. Lakhin AV, Kazakov AA, Makarova AV, Pavlov
future perspectives. Anal Bioanal Chem YI, Efremova AS, Shram SI, Tarantul VZ
408:1735–1751 (2012) Isolation and characterization of high
4. Chames P, Van Regenmortal M, Weiss E, Baty affinity aptamers against DNA polymerase iota.
D (2009) Therapeutic antibodies: successes, Nucleic Acids Ther 22:49–57
limitations and hopes for the future. Br J Phar- 11. Missailidis S, Hardy A (2009) Aptamers as inhi-
macol 157:220–233 bitors of target proteins. Expert Opin Ther Pat
5. Ruigrok VJB, Levisson M, Eppink MHM, 19:1073–1082
Smidt H, Van Der Oost J (2011) Alternative 12. Turner NW, Jeans CW, Brain KR, Allender CJ,
affinity tools: more attractive than antibodies? Hlady V, Britt DW (2006) From 3D to 2D: a
Biochem J 436:1–13 review of the molecular imprinting of proteins.
6. Lakhin AV, Tarantul VZ, Gening LV (2013) Biotechnol Prog 22:1474–1489
Aptamers: problems. Solutions Prospect Acta 13. Mosbach K, Ramstron O (1996) The emerging
Nat 5:34–43 technique of molecular imprinting and its
future impact on biotechnology. Nat Biotech- phase synthesis of molecular imprinted poly-
nol 14:163–170 mer nanoarticles with a reusable template—
14. El-Sharif H, Hawkins DM, Stevenson D, “Plastic Antibodies”. Adv Funct Mater
Reddy SM (2014) Determination of protein 23:2821–2817
binding affinities within hydrogel-based molec- 18. Canfarotta F, Poma A, Guerreiro A, Piletsky SA
ularly imprinted polymers (HydroMIPs). Phys (2016) Solid-phase synthesis of molecularly
Chem Chem Phys 16:15483–15489 imprinted nanoparticles. Nat Protoc
15. Sullivan MV, Dennison SR, Archontis G, 11:443–455
Hayes JM, Reddy SM (2019) Towards rational 19. Poma A, Turner AP, Piletsky SA (2010)
design of selective Molecularly Imprinted Poly- Advances in the manufacture of MIP nanopar-
mers (MIPs) for proteins: computational and ticles. Trends Biotechnol 28:629–637
experimental studies of acrylamide based poly- 20. Poma A, Brahmbhatt H, Pendergraff HM,
ers for myoglobin. J Phys Chem B Watts JK, Turner NW (2014) Generation of
123:5432–5443 Novel hybrid aptamer-molecularly imprinted
16. Hawkins DM, Stevenson D, Reddy SM (2005) polymeric nanoparticles. Adv Mater
Investigation of protein imprinting in 27:750–758
hydrogel-based molecularly imprinted poly- 21. Allabush F, Mendes PM, Tucker JHR (2019)
mers (hydrogels). Anal Chim Acta 542:61–65 Acrylamide-dT: a polymerisable nucleoside for
17. Poma A, Guerreiro A, Whitcombe MJ, Piletska DNA Incorpation. RSC Adv 9:31511–31511
EV, Turner APF, Piletsky SA (2013) Solid-
Chapter 10
Molecularly Imprinted Solid-Phase Extraction Sorbents

for the Selective Extraction of Drugs from Human Urine
Hasan Basan
Abstract
Development of molecularly imprinted solid-phase extraction (MISPE) sorbents for the removal of drugs
from human urine is described. MISPE sorbents are synthesized through free radical polymerization
mechanism and non-covalent imprinting approach. After the completion of the polymerization, bulk
polymer is ground using an analytical mill and then wet-sieved. Later on, MISPE sorbent has been
dry-packed into solid-phase extraction cartridges after the extraction of template drug molecule. Eluates
from the cartridges are analyzed using a spectrophotometer or spectrofluorimeter for the determination of
target analyte by referring to the calibration curves.
Key words Molecularly imprinted polymers, Molecularly imprinted solid-phase extraction, Free-
radical polymerization, Non-covalent imprinting approach, Template extraction, Human urine, Spec-
trofluorimetry, Spectrophotometry
1 Introduction
Sample preparation is of prime importance because potential inter-

ferants should be removed in order to increase the sensitivity and
selectivity of the analytical methods [1]. The selectivity of solid-
phase extraction (SPE), which is one of the most widely used
sample preparation methods, depends upon the type of the adsor-
bent. Nowadays, sorbents in conventional SPE are replaced by
highly selective molecularly imprinted polymers (MIPs) and
classical solid-phase extraction is named as molecularly imprinted
solid-phase extraction (MISPE) [2]. MIPs are prepared by the
copolymerization of functional and crosslinking monomers in the
presence of a template or target molecule dissolved in a porogen.
After the removal of template or target molecule from the MIP
sorbent with extraction, mostly Soxhlet extraction, three-
dimensional cavities whose shape, size, and spatial arrangement of
the functional groups complementary to the template molecule are
https://doi.org/10.1007/978-1-0716-1629-1_10,
123
124 Hasan Basan
generated [3]. Due to higher affinity of the MIP sorbents towards

target molecules, MISPE has been widely used for the clean-up and
selective removal of the analytes from complex biological (e.g.,
human urine) and environmental samples [4], compared to con-
ventional SPE sorbents. In addition, analytes having low concen-
trations can be determined easily after a preconcentration step
using MISPE procedures. By doing so, lower concentrations of
analytes can be determined using very simple and cheap instru-
ments like spectrofluorimeters and spectrophotometers as well.
MISPE sorbents are usually prepared in monolithic format
(i.e., bulk form) by thermally (60–70 C) or UV light
(at 365 nm) initiated free radical polymerization mechanism.
After the completion of the polymeric reaction, the bulk polymer
is ground using an analytical mill and then wet-sieved to particle
size between 20 and 63 μm. However, the procedure of grinding
and sieving is cumbersome, and it causes a substantial loss of
imprinted polymer [5]. Some amount of seived particles are
dry-packed into the extraction cartridges having volume in between
1.5 and 3.0 mL. It should be pointed out that the packed amount
of sorbent and cartridge volume depend on the sample amount or
analyte concentration. In order to imprint sorbent to a high degree
(i.e., to obtain high imprinting factor) MIP formulation should be
carefully determined and optimized. The usual molar ratio of tem-
plate, functional monomer, and crosslinker is approximately
1:4:20. The porogen volume should be high enough to dissolve
ingredients of the polymeric mixture. Porogen should have low
polarity and should be an aprotic solvent such as chloroform,
toluene, and dichloromethane to enhance the intermolecular inter-
actions, in turn affinity, between template molecule and functional
monomer. Swelling degree of the MIPs should be carefully con-
trolled because swelling has a great negative influence on the rec-
ognition ability of the MIP sorbent, resulting in size and shape of
the imprinted sites of the MIP to change (losing its memory
effect) [6].
2 Materials
All chemicals are of analytical reagent grade and all of the solutions
are prepared using deionized or double-distilled water. Organic
solvents used in the experimental studies are of high purity since
solvent purity is very important in spectrophotometric and spectro-
fluorimetric methods as far as interferences and detection limit of
methods described are concerned (see Note 1).
2.1 Synthesis 1. MIP preparation mixture: Dissolve template and functional

of MISPE Sorbent monomer in approximately 1.0 mL of an organic porogen
(e.g., chloroform, CHL or acetonitrile, ACN) in a borosilicate
Molecularly Imprinted Solid-Phase Extraction Sorbents 125
test tube. Ideal molar ratios of template to functional monomer

is about 1:4.
2. Crosslinker: ethylene glycol dimethacrylate (EGDMA).
3. Initiator: N,N-azobisisobutyronitrile (AIBN).
4. Temperature controlled incubator or UV lamp (365 nm).
5. Analytical mill.
6. Analytical sieves 20 and 63 μm in size.
2.2 Soxhlet 1. Soxhlet extractor with cellulose extraction thimble.

Extraction 2. Extraction solvent mixture: methanol/acetic acid (9:1, v/v).
3. Spectrophotometer or spectrofluorimeter.
4. Vacuum oven or temperature controlled incubator.
2.3 Batch Rebinding 1. 5.0–75.0 mg dry MIP/NIP sorbent.

Studies 2. Binding solution: 4–5 mL of 5.0 μg/mL analyte (e.g., telmi-
sartan or indapamide) solution.
3. Magnetic stirrer.
4. Centrifuge.
5. Spectroflourimeter or spectrophotometer.
6. Standard solutions of the analyte: 0.14–1.5 μg/mL indapa-
mide and 0.04–1.4 μg/mL telmisartan.
7. Incubation solvents: dichloroethane (DCE), acetonitrile
(ACN), chloroform (CHL), and toluene (TOL).
8. Incubation time: 30–400 min.
9. Initial concentration of analyte: 1.0–15.0 μg/mL.
2.4 MISPE Protocol 1. Sorbent to be packed: 50.0 mg of MIP/NIP particles.

2. Cartridge and frits: 1.5 mL of SPE cartridge and two polyeth-
ylene frits.
3. Vacuum manifold.
4. Conditioning solvent: loading solvent.
5. Loading solvents: acetonitrile and pH 5.2 deionized water.
6. Washing solvent: acetonitrile.
7. Elution solvent or solvent mixture: methanol.
8. Regeneration solvent or solvent mixture: methanol.
2.5 Analysis 1. Blank human urine stored at 20 C.

of Human Urine 2. Centrifuge.
3. Nylon membrane (0.45 μm).
4. 1.0 M HCl or 1.0 M NaOH.
126 Hasan Basan
5. Methanol.
6. Volumetric flasks (10.0 or 100.0 mL).
7. Vacuum manifold with cartridge.
3 Methods
3.1 Synthesis 1. Dissolve 0.082 mmol of indapamide and 0.857 mmol of

of MISPE Sorbent 2-(trifluoromethyl) acrylic acid (TFMAA) in 1.0 mL of aceto-
nitrile in a borosilicate test tube and incubate for 15 min at
room temperature (see Note 2).
2. Add 3.0 mmol of EGDMA as a crosslinker and 7.0 mg of AIBN
as an initiator.
3. Polymerization process was conducted at 70 C in the incuba-
tor for 10 h (see Note 3).
4. After the bulk polymerization, tubes are crashed and bulk
polymer is ground with an analytical mill.
5. The particles are then wet-sieved using methanol to sizes
between 20 and 63 μm.
6. Place the particles into the extraction thimble and load thimble
into the Soxhlet extraction apparatus.
7. Put enough amount of extraction solvent mixture of metha-
nol/acetic acid (9:1, v/v) into the distillation flask.
8. Let the extraction procedure to last about 48 h until no more
indapamide is released from the sorbent. A spectrophotometer
is used to detect the released analyte.
9. MISPE sorbent is dried in the incubator at 50 C for 4 h and it
is ready to use.
10. Same procedures are applied for the non-imprinted polymer
(NIP). However, polymerization is performed in the absence
of template molecule.
3.2 Batch Rebinding 1. 30.0 mg of dry sorbent (MIP or NIP) is placed into a glass vial
Experiments containing about 4.0 mL of 5.0 μg/mL indapamide (IDPm)
solution and mixture is stirred for 3.0 h at room temperature.
2. The mixture is centrifuged at 1699 g for 30 min to separate
the supernatant and the particles.
3. The supernatant is analyzed using a UV–Vis spectrophotome-
ter (λ ¼ 285 nm) for the determination of free IDPm.
4. Amount of IDPm adsorbed by the MIP or NIP sorbents are
found by substracting free IDPm from the initial amount pres-
ent in the binding medium.
5. For the determination of optimum batch rebinding conditions,

type of incubation solvent (ACN, DCE, and TOL), incubation
time (30–480 min), amount of sorbent (5.0–75.0 mg), and
initial IDPm concentration (1.0–10.0 μg/mL) are tested (see
Note 4).
6. In order to determine degree of selectivity of the MISPE
sorbent, adsorption studies of indomethacine and telmisartan
are also conducted separately (see Note 5).
3.3 MISPE Protocol 1. 50.0 mg of MIP/NIP particles are dry-packed into 1.5 mL
SPE cartridge between two polyethylene frits and connected to
a vacuum manifold. MISPE procedure is represented shemati-
cally in Fig. 1 (see Note 6).
2. The sorbent inside cartridge is first conditioned with 2x0.5 mL
of loading solvent (ACN or pH 5.2 deionized water) (see Note
7).
3. 4 0.5 mL of 15.0 μg/mL IDPm solution prepared in ACN
or pH 5.2 deionized water is loaded into the cartridge.
4. Thereafter, the cartridge is washed wth 4 0.5 mL of ACN in
order to remove the interferants (see Note 8).
5. IDPm adsorbed into the sorbent is eluted from the cartridge
using 8 0.5 mL of MeOH.
6. Amount of the IDPm in the eluate is determined spectropho-
tometrically at 285 nm by referring to the calibration curve
ranging from 0.14 to 1.50 μg/mL.
7. Cartridges are regenerated using 6 0.6 mL of MeOH to be
used in the next analysis.
3.4 Analysis 1. Blank human urine samples are collected from a healthy volun-
of Human Urine teer and stored in a refrigerator at 20 C.
2. Human urine samples are centrifuged at 1699 g for 30 min
and then supernatant is filtered through 0.45 μm nylon mem-
branes and pH of the filtrate is adjusted to 5.2 with 1.0 M HCl.
3. Stock standard solution of IDPm is prepared in MeOH and
working standard solutions are obtained by diluting appropri-
ate amounts of stock soluton with blank human urine.
4. 4 0.5 mL of each standard solution is then passed through
MIP and NIP cartridges.
5. After applying washing and elution steps, matrix-matched cali-
bration curve in the range of 0.14–1.50 μg/mL is generated.
For the recovery and precision studies, 2.0 mL urine samples
spiked with 0.25–1.50 μg/mL IDPm solutions are employed.
128 Hasan Basan
Sample Sample
Conditioning loading washing Elution
Imprinted
cavities
Interfering
compounds
Analyte
Spectrophoto-
meter
Fig. 1 Schematic representation of MISPE-Spectrophotometric method
3.5 Analytical To show the suitability of the proposed MISPE spectrophotometric

Parameters method for the determination of IDPm in human urine, analytical
parameters including linearity, LOD, LOQ, within-day (repeatabil-
ity) and between-day (reproducibility) precisions, and accuracy are
validated.
1. For the validation of linearity, matrix-matched calibration
curve is obtained by spiking appropriate amounts of IDPm
into the blank human urine and a linear calibration graph is
obtained in the range of 0.14–1.50 μg/mL.
2. LOD and LOQ values are calculated using the standard devia-
tion value for the standard having the lowest concentration and
slope of the calibration graph.
3. The within-day precision is calculated by performing five
extraction of independently prepared urine samples spiked
with IDPm at different cocentrations over a day.
4. Between-day precision is assessed by studying five extractions
of independently prepared urine samples spiked with IDPm at
different concentrations in three different days.
5. Within-day and between-day precisions are determined by cal-
culating relative standard deviations in percent.
6. For recovery studies, urine samples prepared by spiking three
different concentrations of IDPm (0.25, 0.75, and 1.50 μg/
mL) are used.
4 Notes
1. The above-mentioned MISPE sorbent synthesis procedures

has been used for the imprinting and selective removal of
IDPm from human urine sample [7] and presented here as an
example.
2. So, by following the above-mentioned procedures, MISPE
sorbents having other functional monomers, crosslinkers, poly-
merization initiators. or porogens might be prepared for differ-
ent templates.
3. Similar experimental procedures have been followed and
MISPE sorbent for the telmisartan has also been generated
[6]. Main difference between two studies is the polymerization
mechanism in that photochemical UV polymerization method-
ology was used instead of thermal polymerization.
4. Before the application of the MISPE protocol, batch rebinding
studies should be performed in order to determine parameters
affecting analyte (or target compound) adsorption to the sor-
bent. This step helps optimization of MISPE protocol (e.g.,
solvent type, sorbent amount, pH, elution solvent).
5. Selectivity of MIP and NIP sorbents to IDPm is examined
using telmisartan and indomethacine as well. Indomethacine
is selected due to its structural similarity (structural analog) to
IDPm. Telmisartan has no structural similarity to the IDPm. It
has been concluded that among the three target compounds
IDPm is found to have highest affinity to the binding sites
present on MIP particles since it is the template molecule,
resulting in higher amount of adsorption.
6. Effectiveness of the imprinting process is determined by calcu-
lating value of imprinting factor. It is determined by ratio of
amount of IDPm adsorbed by MIP particles to the amount
adsorbed by NIP particles.
7. Before loading step, imprinted sites should be first activated
using loading solvent to maximize adsorbed amount of analyte.
8. A washing step is applied before the elution step. It helps to
appear selectivity of the MISPE sorbent by suppressing the
nonspecific interactions. Therefore, a special care must be
paid to the washing step in MISPE protocol.
References
1. Basan H, Yarımkaya S (2014) A novel solid- 2. Gorbani Y, Yılmaz H, Basan H (2017) Spectro-
phase extraction-spectrofluorimetric method fluorimetric determination of atenolol from
for the direct determination of atenolol in human urine using high-affinity molecularly
human urine. Luminescence 29:225–229 imprinted solid-phase extraction sorbent. Lumi-
nescence 32:1391–1397
130 Hasan Basan
3. Lavignac N, Allender CJ, Brain KR (2004) Cur- using molecularly imprinted polymers. Anl
rent status of molecularly imprinted polymers as Chim Acta 591:29–39
alternatives to antibodies in sorbent assays. Anal 6. Yılmaz H, Basan H (2015) Development of a
Chim Acta 510:139–145 molecularly imprinted solid-phase extraction
4. Lai JP, Chen F, Sun H, Fan L, Liu GL (2014) sorbent for the selective extraction of telmisartan
Molecularly imprinted microspheres fort he from human urine. J Sep Sci 38:1433–1439
anticancer drug aminoglutethimide: synthesis, 7. Yılmaz H, Basan H (2015) Preconcentration of
characterization, and solid-phase extraction indapamide from human urine using molecularly
applications in human urine samples. J Sep Sci imprinted solid-phase extraction. J Sep Sci
37:1170–1176 38:3090–3095
5. Baggiani C, Anfossi L, Giovannoli C (2007)
Solid-phase extraction of food contaminants
Chapter 11
Molecularly Imprinted Polymers Coupled

with Surface-Enhanced Raman Spectroscopy to Detect
Chemical Hazards in Foods
Marti Z. Hua, Shaolong Feng, and Xiaonan Lu
Abstract
Preparation of molecularly imprinted polymers coupled with surface-enhanced Raman spectroscopy
(MIP-SERS) sensor and its application in detecting chemical hazards in food matrices are described. Sample
cleaning is achieved by molecularly imprinted solid-phase extraction (MISPE), and target molecules are
detected by SERS. Procedures of MIP synthesis, MISPE preparation, SERS substrate preparation, spectral
collection, data analysis, and food analysis application are described.
Key words Molecularly imprinted polymers, Molecularly imprinted solid-phase extraction, Surface-
enhanced Raman spectroscopy, Chemical hazard detection, Food matrix, Sample preparation, Food
safety
1 Introduction
Detecting chemical hazards in food matrices face two major chal-

lenges, namely separating target molecules from the complex food
matrix and acquiring sufficient signal intensity from the trace
amount of the target. Molecularly imprinted polymers (MIPs) are
synthetic analogues to the antibody–antigen system [1] with many
advantages, such as high stability and low cost. Molecularly
imprinted solid-phase extraction (MISPE) technique utilizes
MIP’s specific affinity to target molecules, achieving the separation
and enrichment of target molecules from various food matrices [2–
4]. Surface-enhanced Raman spectroscopy (SERS) is a rapid, sensi-
tive, and label-free fingerprinting technique suitable to detect trace
levels of chemical residues [5]. Depositing relatively clean/pure
analyte onto the surface of SERS substrate (typically noble metal
nanoparticles) is the key to enhance the inherently weak Raman
scattering signal and reduce noise signals. Both challenges
https://doi.org/10.1007/978-1-0716-1629-1_11,
131
132 Marti Z. Hua et al.
mentioned above are addressed by coupling the two techniques,

MISPE and SERS.
Preparing an MIP-SERS sensor includes synthesizing MIPs,
packing MISPE, preparing SERS substrate, and building a standard
curve by collecting the enhanced Raman spectra and data analysis.
The actual food sample analysis using the developed MIP-SERS
sensor is simple, involving three steps. (1) Food sample
pre-treatment: target molecules are extracted from the complex
food matrix. (2) Clean-up by MISPE: co-extracted interfering
substances are removed, and the cleaned extract is collected and
enriched. (3) SERS detection: SERS spectra of the cleaned sample
are collected, and the concentration of target molecules is calcu-
lated by fitting the result to the standard curve. The MIP-enabled
separation techniques expand the application field of SERS to agri-
food testing as the application potential has long been limited to
analyzing pure substances or very simple matrix (e.g., environmen-
tal water samples). At the same time, SERS further proves the value
of MISPE which is the most visible commercial application of
MIPs [1].
2 Materials
2.1 Polymer 1. Functional monomer: 4-vinylpyridine (4-VP). Template:

Synthesis 2,4-dichlorophenoxyacetic acid (2,4-D). Crosslinker: ethylene
glycol dimethacrylate (EDMA). Radical initiator: 2,20 -Azobis
(2-methylpropionitrile) (AIBN).
2. Ultrapure water (18.2 MΩ·cm at 25 C) and HPLC grade
methanol for all synthesis steps. Deionized water and ACS
grade solvents for all other steps.
3. Equipment/apparatus: nitrogen gas (with tubing and attached
disposable glass Pasteur pipet), ultrasonic cleaner, magnetic
hotplate, water bath with magnetic stirring and temperature
control, ceramic pestle and mortar set, 200-mesh sieve, filter
paper sheet, and cotton twine (or cellulose extraction thimble),
Soxhlet extraction apparatus set, vacuum oven, UV–Vis spec-
trophotometer, or HPLC-DAD [optional].
2.2 Molecularly 1. Polypropylene solid-phase extraction cartridge (3 mL) and

Imprinted Solid-Phase compatible polytetrafluoroethylene (or polypropylene) frit.
Extraction (MISPE) 2. Equipment/apparatus: SPE Vacuum Manifold and accessories.
Vacuum source (vacuum water aspirator or vacuum pump).
2.3 Preparation 1. Oil bath with magnetic stirring and temperature control
of SERS Substrate (or use magnetic hotplate, thermometer, and
crystallizing dish).
MIPs Coupled with SERS to Detect Chemical Hazards in Foods 133
2. Three-neck round-bottom flask (250-mL). On the three necks:

a gas inlet adaptor for purging, a Liebig/Allihn condenser, and
a glass/PTFE stop cock. (The flask, adaptor, stop cock, and the
lower part of the condenser need to be washed with Aqua
regia.)
3. Centrifuge.
2.4 SERS Spectral 1. A gold-coated glass slide for sample deposition and nitrogen
Collection and Data gas for drying.
Analysis 2. A confocal micro-Raman spectroscopic system coupled with a
300-mW line shape 785 nm near-infrared laser. The system
includes a Raman spectrometer and a Leica microscope. The
spectrometer has an entrance aperture of 50 μm and a focal
length of 300 mm. A 1200-line mm1 grating is equipped as
well. The Rayleigh scattering light can be completely elimi-
nated by the filters. Only the Raman scattered light is collected
and dispersed by the spectrometer and recorded by a
578 385 pixel charge-coupled device (CCD) array detector
with a pixel size of 22 μm. A 50 objective (NA ¼ 0.75,
WD ¼ 0.37 mm) for spectrum collection.
3. Raman system controlling software.
4. Vancouver Raman Algorithm software for baseline
correction [6].
2.5 Detection 1. Pre-treatment: centrifuge, rotovap.

of Chemical Hazard 2. MISPE: SPE apparatus, vaccum source, nitrogen gas.
in Food Using
3. Listed in Subheading 2.4.
MIP-SERS sensor
3 Methods
3.1 Synthesis of 1. Into a 20-mL scintillation vial (see Note 1), add 4 mmol of
MIPs (and monomer (4-VP) and 1 mmol of template (2,4-D). Add 4 mL
Non-imprinted of methanol and 1 mL of water to dissolve the reagents with
Polymers [Optional]) magnetic stirring on a hotplate and/or sonication (see Note 2).
2. Dissolve 20 mmol of crosslinker (EDMA) and 0.3 mmol of
initiator (AIBN) into the 5 mL of solution, followed by mag-
netic stirring and nitrogen purging for 2 min. Screw the cap
onto the vial immediately after purging.
3. In the water bath, stir and heat the vial at 45 C for 4 h and then
at 60 C for 2 h or until the mixture is fully transformed to a
chunk of rigid polymer. Remove the vial from the bath and cool
to room temperature.
4. Wearing appropriate personal protection equipment (e.g.,

safety google, face shield, cut-resistant gloves), break the glass
vial, transfer the polymer into a mortar, and discard the glass
waste.
5. Wear a dustproof respirator (e.g., N95) and a safety goggle.
Crush the polymer carefully with the pestle (and remove the
stir bar). An aluminum foil skirt around the pestle can help to
reduce the dust being sent out. [Inhalation hazard caution!].
Pass the crushed fine polymer powder through a 200-mesh
sieve (see Note 3).
6. Fold a sheet of filter paper into a small packet and pack the
sieved powder of polymers. Ideally, the powder should evenly
spread the internal space of the packet, and 2–3 layers of paper
on both sides are needed to avoid leaking. Secure the closed
packet with cotton twine. An extraction thimble can also be
used with extra caution.
7. Load the packet/thimble into the chamber of a Soxhlet extrac-
tor and set up the entire Soxhlet extraction apparatus.
8. Add methanol with 15% (v/v) of acetic acid and some boiling
chips into the distillation round-bottom flask. The volume
should be 3–4 times of the main chamber volume and
1/3–1/2 of the flask volume. Remove the template from the
polymers by Soxhlet extraction for 24 h. Change the distilla-
tion solvent to pure methanol and extract for another 24 h.
9. Vacuum dry the polymers at 60 C for 4 h and collect the dried
polymer for further test and column packing.
10. [Optional] Preparation of NIPs follows the same protocol of
MIP synthesis without adding the template.
11. [Optional] Adsorption test for MIPs and NIPs can be done in a
system of 15 mg of polymers and 3 mL of template solution
(e.g., 2,4-D in 50% methanol). The polymer and template
solutions are mixed and shaken, followed by immediate centri-
fugation (e.g., 5 min at 22,000 g). The remaining concen-
tration of the template in the supernatant can be quantified
either by measuring the absorbance with a UV–Vis spectro-
photometer or by HPLC-DAD. The adsorption time varies in
the kinetic test, while the template concentration varies in the
static test. The specificity and adsorption capacity can be calcu-
lated to evaluate the performance of MIPs (see Note 4).
3.2 Preparation 1. Place a frit into a 3-mL polypropylene solid-phase extraction

of Molecularly cartridge. Pack 50 mg of MIPs and place another frit on top.
Imprinted Solid-Phase Make sure an even and tight packing of the MIPs.
Extraction (MISPE) 2. Set up the SPE manifold. Insert the packed SPE column to the
port of the manifold. Wash the column with 3 mL of water and
3 mL of methanol at a flow rate of ~2 mL per min. Repeat for

another two times.
3. Fill up the column with methanol and use parafilm to seal the
opening of the column upon use.
3.3 Synthesis 1. In a 250-mL three-neck round-bottom flask, dissolve 18 mg of

of SERS Substrate AgNO3 in 100 mL of water.
2. Immerse the flask in an oil bath. Place a condenser on the
middle neck, nitrogen purging with an adaptor on a side
neck. Place a glass/PTFE stop cork on the other side neck.
3. With constant stirring, water cooling, and nitrogen purging,
keep the oil bath at 140 C. When the solution starts to boil,
add 10 mL of freshly prepared 1% (w/w) sodium citrate solu-
tion via the third neck quickly. Reflux for 1 h with nitrogen
protection.
4. Cool the Ag colloid to room temperature overnight.
5. Without shaking the bottle, pour 100 mL of the colloid into
two 50-mL centrifuge tubes. Centrifuge at 3000 g for
40–60 min and remove the supernatant. Combine the remain-
ing part into one tube and add water up to 20 mL to resuspend
the nanoparticles. The resulted colloid is the SERS substrate
ready for use (see Note 5).
3.4 SERS Spectral 1. In a microcentrifuge tube, 100 μL of MISPE-cleaned sample or

Collection and Data standard solution in methanol is mixed with 50 μL of the
Analysis substrate.
2. Pipette 2 μL of the mixture onto a gold-coated glass slide and
dry with nitrogen.
3. Adjust the incident laser power to ~0.5 mW and record the
spectra with the 50 objective over a wavenumber range of
300–700 cm1 for 10 s. Four spectra for each sample should be
collected at different SERS active spots.
4. SERS spectra are normalized, and the baseline is corrected
using Vancouver Raman Algorithm software.
5. Establish a standard curve according to characteristic peaks
with spiked food samples (see Note 6).
3.5 Detection 1. Sample pre-treatment (see Note 7)

of Pesticide in Food (a) In a 50-mL centrifuge tube, mix 5 mL of skimmed milk
with Developed sample and 10 mL of water.
MIP-SERS Sensor (b) Quickly add 25 mL of acetonitrile (1% formic acid, v/v)
(Fig. 1) and shake the tube immediately for 10–15 s.
(c) Add a mixture of MgSO4 (6 g) and NaCl (1.5 g) and
shake the tube immediately for 10–15 s.
Fig. 1 Scheme of detecting pesticide in food with the MIP-SERS sensor
(d) Centrifuge the tube at 5000 g for 4 min. Collect the

upper layer (acetonitrile) of supernatant and evaporate to
almost dryness with a rotovap.
(e) Redissolve the residue with 2 mL of 50% methanol. This is
the “pre-treated sample.”
2. Clean-up via MISPE (see Note 8)
(a) Conditioning: open the sealing parafilm and turn on the
vacuum valve of the SPE manifold. Lower the methanol
level to the upper frit and condition the column with 2 mL
of 50% methanol.
(b) Loading: load the 2 mL of pre-treated sample.
(c) Washing: wash the column of 3 mL of 50% methanol.
(d) Eluting: elute the pesticide with 3 mL of methanol and
collect the eluate.
(e) Recovering: wash the column with 1 mL of 15% (v/v)
acetic acid in methanol and then 2 mL of methanol.
(f) Dry the eluate with nitrogen purging and redissolve with
100 μL of methanol. This is the cleaned sample for
further test.
3. Detection of target molecule with SERS
(a) Follow Subheading 3.4, Step 1–4.
(b) Fit the processed data into the standard curve to deter-
mine the concentration of pesticide in the cleaned sample.
(c) Calculate the original pesticide residue level in food in the
case of dilution or enrichment.
4 Notes
1. A regular 25-mL round-bottom flask can be used instead of the

vial. This may save the hassle to seek a vial holder (e.g., a screw
cap opener) and simplify the heating set up using a regular
two-prong clamp to hold the flask instead. However, in this
case, it is recommended to find flasks that are easy to break (i.e.,
making it disposable) for a few reasons. It can be challenging to
take the polymer out of the flask or break the chunk inside the
flask. At the same time, the polymerization residue on the wall
inside is hard to remove. Meanwhile, breaking high-quality
flasks is typically not too labor saving and economical.
2. At this volume, the polymerized chunk is typically rigid and
difficult to crush. Increasing the total volume of the solvent
makes the chunk easier to crush but may impair the perfor-
mance of MIPs.
3. A modified bulk polymerization method [7] is used in this
protocol to produce particles larger than 20 μm, considering
the MISPE steps. The typical pore size of the frit is 20 μm,
while precipitation and sol-gel polymerization methods typi-
cally produce submicron size polymers. The particle size of the
packed MIPs affects the binding/separation efficiency, flow
rate, pressure requirement, and volume of solvents for
MISPE. A 500-mesh (25 μm) sieve can be used with the
200-mesh (75 μm) sieve to further narrow the size distribution
of the polymers, which may improve the performance of the
packed SPE column. Wet sieving with methanol can be used
instead of dry sieving to reduce the risk of inhalation hazard
and product loss.
4. A guard column is recommended if the particle size distribu-
tion of MIPs is not determined (Nylon filters may also help to
prevent the MIPs remaining in the supernatant from entering
the HPLC system, but the potential binding between target
molecules to syringe filter may affect the result). Kinectic and
static adsorption tests may help to evaluate the performance of
MIPs compared with NIPs. However, excellent adsorption rate
and capacity to standard solutions (regardless the solvent) do
not sufficiently indicate the polymers’ performance when
packed as a MISPE column.
5. Pay attention to centrifugation speed as high centrifugal force
may compromise the Ag colloid stability, resulting in a compact
deposition/aggregation of Ag onto the wall (with metallic
shine) of the centrifuge tube. This washing step reduces the
concentration of citrate group and other substances as they
may mask the characteristic peak of target molecules in SERS
spectra [8]. Aggregation can be observed in the substrate
stored for more than 10 days, resulting in variation in SERS

spectra.
6. More complicated mathematical/chemometric models (e.g.,
PCA, PLSR, HCA [3, 4]) may be established if a simpler
standard curve is not available.
7. The pre-treatment method needs to be optimized for different
food matrices and target molecules. Solvent replacement by
rotovaping and redissolving may be skipped if a satisfying com-
promise between extraction and loading efficiency. For exam-
ple, too much co-extracted interfering substances are often
observed in a water-methanol extracting system for milk so
this system is not suitable for enriching/concentrating the
target molecules by evaporation. On the contrary, acetonitrile
precipitates proteins effectively, and the organic phase can be
almost completely separated from the aqueous phase and pre-
cipitates by the described protocol. However, acetonitrile at
room temperature is not suitable for loading samples but for
eluting the target out of the column.
8. During MISPE, try not to let the column dry by always adding
the next solvent/solution when the liquid level is getting close
to the upper frit. The only exception is the eluting step, where
the entire eluate should be collected, including the liquid in the
column. The flow rate is slower (1 mL/min) in loading and
eluting steps for a better recovery and is faster (1.5–2 mL/min)
for other steps to save time.
Acknowledgments
This work was supported by the fund awarded to X.L. by McGill

University new faculty start-up grant.
References
1. BelBruno JJ (2019) Molecularly imprinted poly- 176:123–129. https://doi.org/10.1016/j.

mers. Chem Rev 119(1):94–119. https://doi. foodchem.2014.12.051
org/10.1021/acs.chemrev.8b00171 4. Feng S, Gao F, Chen Z, Grant E, Kitts DD,
2. Feng S, Hu Y, Ma L, Lu X (2017) Development Wang S et al (2013) Determination of
of molecularly imprinted polymers-surface- α-tocopherol in vegetable oils using a molecu-
enhanced Raman spectroscopy/colorimetric larly imprinted polymers–surface-enhanced
dual sensor for determination of chlorpyrifos in Raman spectroscopic biosensor. J Agri Food
apple juice. Sens. Actuators B Chem Chem 61(44):10467–10475. https://doi.org/
241:750–757. https://doi.org/10.1016/j.snb. 10.1021/jf4038858
2016.10.131 5. Pilot R, Signorini R, Durante C, Orian L,
3. Hu Y, Feng S, Gao F, Li-Chan ECY, Grant E, Lu Bhamidipati M, Fabris L (2019) A review on
X (2015) Detection of melamine in milk using surface-enhanced Raman scattering. Biosens 9
molecularly imprinted polymers–surface (2):57. https://doi.org/10.3390/
enhanced Raman spectroscopy. Food Chem bios9020057
6. Zhao J, Lui H, McLean DI, Zeng H (2007) recognition element. Anal Chem 70
Automated autofluorescence background sub- (3):628–631. https://doi.org/10.1021/
traction algorithm for biomedical Raman spec- ac9711549
troscopy. Appl Spectrosc 61(11):1225–1232. 8. Hua MZ, Feng S, Wang S, Lu X (2018) Rapid
https://doi.org/10.1366/ detection and quantification of
000370207782597003 2,4-dichlorophenoxyacetic acid in milk using
7. Haupt K, Dzgoev A, Mosbach K (1998) Assay molecularly imprinted polymers–surface-
system for the herbicide enhanced Raman spectroscopy. Food Chem
2,4-dichlorophenoxyacetic acid using a molecu- 258:254–259. https://doi.org/10.1016/j.
larly imprinted polymer as an artificial foodchem.2018.03.075
Chapter 12
Molecularly Imprinted Polymer for a Smart Dispersive

Micro-Solid Phase Extraction Technique for Assessing Trace
Level Aflatoxins in Cultured Fish
G. D. Thilini Madurangika Jayasinghe and Antonio Moreda-Piñeiro
Abstract
Molecularly imprinted technology (MIT) consists of preparing materials exhibiting specific recognition
cavities to selective mimic the target analytes. The prepared materials promote selective interactions with
the targets and avoid interactions of concomitants from complex food, biological, clinical, and environ-
mental matrices. This chapter provides information on a recent development of a vortex-assisted micro-
solid phase extraction using a molecularly imprinted polymer (MIP) as an adsorbent for aflatoxins (AFs)
determination in cultured fish. MIP particles were synthesized by precipitation polymerization using
5,7–dimethoxycoumarin as a dummy template, methacrylic acid as a functional monomer, divinylbenzene
as a cross-linker, and 2,2-azobisisobutyronitrile as an initiator. Polymerization following the precipitation
method guarantees homogeneous particle size distribution and the integrity of the imprinted cavities. The
MIP microparticles were found to have 5 μm in diameter and a spherical shape. Important parameters such
as sample extract pH, adsorption stirring speed and time, desorption stirring speed and time, elution solvent
composition and volume, and polymer mass, were fully optimized. The pre-concentration method allows
therefore the assessment of four major AFs (B1, B2, G1, and G2) present in cultured fish at very low levels,
with pre-concentration factors from 15 to 50 depending of the volume of extract used for performing the
dispersive micro-solid phase extraction (D-μ-SPE).
Key words Molecularly imprinted polymers, Precipitation polymerization, Dispersive micro-solid

phase extraction, Aflatoxins, Cultured fish
1 Introduction
Before a successful analysis of complex samples, target extraction is

necessary to avoid interferences from sample’s matrix and also to
pre-concentrate (to enrich) targets since most of them are usually
present at very low levels. High degree of pre-concentration and
selectivity have been demonstrated to be reached when using
molecularly imprinted polymers (MIPs) as adsorbents for solid
phase extraction (SPE) procedures [1]. In addition to the
well-known on column (on-cartridge) SPE [2], batch procedures
https://doi.org/10.1007/978-1-0716-1629-1_12,
141
142 G. D. Thilini Madurangika Jayasinghe and Antonio Moreda-Piñeiro
(batch mode) have gained importance mainly since the introduc-

tion of magnetic adsorbents, some of them containing MIPs (mag-
netic MIP composites) [3, 4]. The batch mode is commonly
refereed as dispersive solid phase extraction (DSPE), and adsorbent
dispersion in the aqueous sample/extract or in the eluting solution
enhances analyte transfer [5]. DSPE procedures which use small
amounts of adsorbents (typically less than 50 mg) lead to dispersive
micro-solid phase extraction (D-μ-SPE) processes [6, 7], which
fundamentals are the same than those attributed to DSPE. Disper-
sion in DSPE and D-μ-SPE can be achieved by magnetic stirring
(use of magnetic adsorbents, such as magnetic MIPs) [4], or by
mechanical stirring, typically vortexing, or by ultrasound assistance
[5]. The dispersion of the adsorbent plays an essential role in
D-μ-SPE during the loading and elution stages, and a supplemental
energy source such as mechanical stirring (vortexing) and ultra-
sound sonication favours the adsorbent disaggregation and
increases the surface area of the adsorbent, and hence, retention
efficiency. Vortex stirring is a soft and low-cost shaking technique
which helps to enhance the mass transference in D-μ-SPE and gives
more repeatable results when comparing with ultrasound assistance
since ultrasound fluency is not the same in all places inside the
ultrasound water-bath [8]. The use of ultrasound probe avoids
this drawback, but the high and localized energy from the ultra-
sound probe tip can alter the targets. Vortex assistance avoids
analyte degradation and adsorbent aggregation although could
give lower extraction kinetics when comparing with ultrasound-
assisted processes [9, 10].
Despite there are several applications of magnetic adsorbents
(magnetic MIPs included) in DSPE and D-μ-SPE procedures, the
use of conventional MIPs for D-μ-SPE is scarce. This chapter
pretends to provide a snapshot of the benefits of an MIP-based
adsorbent for vortex-assisted D-μ-SPE when assessing aflatoxins
(AFs) in cultured fish. AFs, mainly B1, B2, G1, and G2, are highly
toxic compounds [11], and they can be present in animal feeds (fish
feed used in aquaculture facilities) and hence in cultured fish
[12]. The levels of AFs in the flesh of cultured fish are expected to
be very low and D-μ-SPE procedures involving high
pre-concentration factors and adsorbents highly selective such as
MIPs are an appealing choice.
2 Materials
Prepare all AFs (B1, B2, G1, G2) standard stock solutions
(1000 mg L1) in LC-MS grade methanol and store them at
20 C (AFM1 concentration was 508 mg L1). Prepare working
standard solutions just before analysis. All solvents such as
Dispersive MIPs for Micro-Solid Phase Extraction 143
methanol, acetonitrile, toluene, and formic acid must be of analyti-

cal grade. Reagents and samples are weighted in an analytical
balance.
Prepare other solutions such as potassium dihydrogen phos-
phate (0.1 M), and sodium hydroxide (0.1 M) using ultrapure
water (resistivity is 18 MΩ cm). Store all reagents/solutions accord-
ing to manufacturer’s recommendations. Follow all waste disposal
regulations for discarding AFs wastes and other waste materials.
2.1 MIP Synthesis 1. Pre-polymerization mixture: 5,7-dimethoxycoumarin (DMC)

as dummy template, methacrylic acid (MAA) as monomer, and
a mixture of 1:3 acetonitrile/toluene as a porogen.
2. Cross-linker: divinylbenzene (DVB).
3. Radical initiator: 2,20 azobisisobutyronitrile (AIBN).
4. Low profile roller placed inside a Boxcult temperature-
controlled chamber.
2.2 Template 1. Pressurized liquid extraction (PLE) device with 10 mL stainless

Removal from MIP steel cells, 60 mL of collection vial, and disposable cellulose
filters.
2. 8:2 Methanol/ultrapure water as washing solution.
3. Laboratory oven for drying.
2.3 Characterization 1. Fourier transform infrared spectrometer (FT-IR).

of MIP Beads 2. Field emission scanning electron microscope.
3. BET and porosity measurements.
2.4 AFs Extraction 1. Cultured fish species (gilt-head bream (Sparus aurata), Japa-
from Culture Fish Flesh nese sea bass (Lateolabrax japonicus), Brown trout (Salmo
trutta), and Turbot (Scophthalmus maximus).
2. Domestic blender for sample tissues homogenization.
3. 60/40 acetonitrile/0.1 M KH2PO4 (60:40) as extracting solu-
tion for AFs.
4. pH meter for pH adjustment of the extracting solution.
5. Sodium hydroxide for pH adjustment.
6. Ultrasound probe for AFs extraction.
2.5 Dispersive A schematic of the several stages of the dispersive micro-solid phase
Micro-Solid Phase extraction using MIP as a selective adsorbent is given in Fig. 1.
Extraction
1. 2 mL polypropylene Eppendorf tubes with 40 mg of synthe-
sized MIP.
2. Potassium dihydrogen phosphate (0.1 M KH2PO4, pH 6.0)
for MIP conditioning.
Fig. 1 Schematic of the vortex assisted D-μ-SPE procedure with MIP adsorbent
3. Vortex for MIP conditioning and for dispersing MIP beads

with the extract from the sample (loading stage) and with the
eluting solution (97.5:2.5 acetonitrile/formic acid) during the
elution stage.
4. Centrifuge for separating the MIP particles from supernatants
(conditioning solutions, extracts, and eluates).
5. PVDF syringe filters (4.0 mm diameter and 0.22 μm pore size)
and 2 mL disposable sterile syringes.
6. A metal block thermostat and nitrogen sample concentrator.
2.6 Liquid 1. LC/MS/MS with an electrospray ionization source, an HPLC

Chromatography- binary pump with an integrated vacuum degasser unit, and
Tandem Mass HPLC autosampler for chromatographic analysis.
Spectrometry 2. A reverse phase C-18 column (100 mm length, 4.6 mm i.d.,
Measurements 3.5 mm particle diameter) connected to a C-18 guard column
(4 mm length, 3.00 mm i.d) for chromatographic separation.
3. Column heater for controlling the column temperature.
4. 0.1% (v/v) formic acid in ultrapure water as mobile phase A,
and 0.1%(v/v) formic acid in methanol as mobile phase B.
5. Data processing software.
3 Methods
3.1 MIP Synthesis 1. Mix 0.0699 g (0.3392 mmol) of DMC (template) and 115 μL
and Characterization (1.356 mmol) MAA (monomer) in 25 mL of porogen (1:3
acetonitrile/toluene). Stir and let equilibrate overnight (see
Note 1).
2. Add 1.25 mL (6.98 mmol) DVB (cross-linker) and 0.1 g of
(0.607 mmol) of AIBN (initiator) and stir until total dissolu-
tion (see Note 2). The template/monomer/cross-linker molar
ratio is therefore set at 1:4:20.
3. Purge the mixture under nitrogen for 5 min and seal the
reaction tubes.
4. Place the tubes on the low-profile roller (33 rpm on its long
axis). Set the temperature of the temperature-controlled cham-
ber at 60 C (the roller is inside the chamber). The temperature
will be gradually increased from room temperature top 60 C in
2.0 h. Finally, let the polymerization reaction progress at 60 C
for 24 h.
5. Vacuum filter the synthesized material and wash with acetoni-
trile (20 mL, 3 times) and oven-dry at 40 C.
6. Prepare the NIP by following the same method as MIP but
without adding a template.
7. Remove the DMC template by PLE device (80 C, 103 bars,
and 60% flush volume for 20 min) with 8:2 methanol/ultra-
pure water (see Note 3). Let oven-dry the template-free MIPs
at 40 C for 12 h.
8. Confirm the template removal with LC-MS/MS.
9. Let oven-dry the template-free MIP at 40 C for 24 h and keep
it at room temperature until further use.
10. Characterize the synthesized material using SEM and evaluate
the shape and size of the MIP/NIP.
11. Characterize the synthesized material by FT-IR for evaluating
the presence of functional groups in MIP and NIP structures.
12. Characterize the synthesized material for pore volume (BET
measurements) and porosity.
3.2 AFs Extraction 1. Wash the samples with clean tap water and remove the gut,
from Fish by bones, head, fins, and scales. Collect the muscle and liver part
Ultrasound-Assisted of the fish separately.
Extraction (UAE) 2. Homogenize the samples using a domestic blender.
3. Mix 10 mL of 60:40 acetonitrile/0.1 M KH2PO4 (pH 6.0)
with 1.0 g of homogenized fish sample in a 30 mL
centrifuge tube.
4. Place the test tube in an ice-bath and insert the ultrasound tip
in the center of the tube (prevent the tip from touching the
bottom or walls of the tube). Set the ultrasound operating
conditions at 130 W (20 kHz frequency), 40% amplitude,
and continuous mode for 7.0 min.
5. Filter the mixture and separate the supernatant (final pH of the
extract within the 6.8–7.0 range).
6. Use the supernatant for dispersive micro-solid phase extraction
(D-μ-SPE).
3.3 Dispersive 1. Place 40 mg of MIP beads inside of the Eppendorf tubes.

Micro-Solid Extraction 2. Add 1.5 mL of fish extract (sample analysis), standard solutions
(D-μ-SPE) (calibration), fish extract spiked with 100 μg L1 for B1, B2,
G1, and G2, 20 μg L1 of M1 and 50 μg L1 of internal
standard (DMC) (optimization studies), or fish extract spiked
with several AFs concentrations and the same DMC concentra-
tion (standard addition calibration).
3. Subject the mixtures to vortex stirring at 206 g for 3 min,
and centrifuge (20,627 g, 10 min) for supernatant separa-
tion. Discard the supernatant.
4. Clean the isolated MIP beads by adding 1.5 mL of 0.1 M
KH2PO4 (pH 6.0) and stir (vortex, 206 g, 3 min). Centri-
fuge at 20,627 g for 10 min and discard the supernatant.
5. Add 0.5 mL of 97.5:2.5 acetonitrile/formic acid as an elution
solution and allow to vortex stirring at 367 g for 4.0 min.
6. Centrifuge again (20,627 g, 10 min) and collect the super-
natant (see Note 4).
7. Fully dry the eluate under N2 flow (50 C), redissolve the
residue with 100 μL of methanol, and filter the extract
(0.22 μm).
8. Add 1.0 mL of 0.1 M KH2PO4 (pH 6.0) in tube containing the
MIP particles, allow vortex stirring (206 g, 1.0 min), and
centrifuge (20,627, 10 min). Discard the washing solution.
The MIP particles will be conditioned for being reused.
3.4 Liquid 1. Perform working standards in methanol 2.5, 5, 10, 20, 50,
Chromatography- 100, and 200 μg L1 for B1, B2, G1, G2, and 2.5, 5, 10,
Tandem Mass 20, 30, 40, 50 μg L1 for M1. All standards must contain
Spectroscopy (LC-MS/ 50 μg L1 of internal standard (DMC). Use calibrations in
MS) methanol for D-μ-SPE optimization studies.
2. Perform the standard addition by spiking 1.5 mL of fish
extracts with 0.167, 0.333, 0.667, 1.33, 3.33, 6.67, and
13.3 μg L1 for B1, B2, G1, and G2, and with 0.167, 0.333,
0.667, 1.33, and 3.33 μg L1 for M1. Spike each fish extract
with DMC at 3.33 μg L1. Use standard addition for method
validation and sample analysis.
3. Use 0.1% formic acid in ultrapure water as mobile phase A, and
0.1% formic acid in methanol as mobile phase B with gradient
elution.
4. Set MS/MS parameters such as declustering potential (DP),
enhance potential (EP), collision energy (CE), and collision
cell exit potential (CXP) for each aflatoxin.
1. Optimize the several parameters such as the effect of the pH,

the effect of vortex speed and time for loading, the effect of
3.5 Optimization of desorption solvent, the effect of the desorption time and speed,
the D-μ-SPE the effect of MIP mass, and test the cross-reactivity and
Parameters imprinting effect of the synthesized MIP.
2. Study the effect of pH from pH 5.0 to pH 9.0, adding 0.1 M
formic acid to obtain the lower pHs and 0.1 M NaOH to
obtain higher pH values.
3. Illustrate the analytical recovery of each AF and select the
compromise pH which gives the highest analytical recovery.
These findings will show a neutral pH (pH 7.0) as optimum
value.
4. For efficiency target adsorption, investigate the influence of the
vortex time and speed in the range of 1–5 min and 23 g – 367
g, respectively. Initially fix the vortex speed at 367 g and
evaluate the vortex time. Select the best vortex time on the
highest analytical recovery obtained, and then evaluate the
effect of the vortex speed. This ensures that extraction effi-
ciency is decreasing when using vortex speeds higher than
206 g due to the back diffusion phenomenon reported in
several micro-extraction techniques.
5. The selection of the elution solvent is mainly based on the
solubility of the analyte. Test several proportions of acetonitrile
and formic acids such as 97.5/2.5, 95.0/5.0, 92.5/7.5, and
90.0/10.0 using a volume of 0.5 mL of eluting solution. Select
the optimum composition of the eluting solution (97.5:2.5
acetonitrile/formic acid).
6. Investigate the effect of the desorption (elution) time and
speed in the range of 1–5 min and 23 g – 367 g, respec-
tively. After selecting the best values (367 g, 4.0 min), opti-
mize the effect of MIP mass within the 10–60 mg range. Select
40 mg of MIP as the most suitable amount of adsorbent for
interaction with target molecules.
7. Check the imprinting properties and selectivity of the prepared
MIP by assessing the extraction efficiency (analytical recovery,
AR), the distribution ratio (D), and selectivity coefficients (S)
of the MIP and NIP adsorbents when pre-concentrating DMC
(template), the five AFs (B1, B2, G1, G2, and M1), and other
compounds such as vitamins and amino acids. Calculate the
extraction efficiency, distribution ratio, and selectivity coeffi-
cients by using the following equations (see Note 5).
A2
Extraction efficiency AR ¼ 100
AT
A2
Distribution ratio D ¼
A1
D DMC
Selectivity coefficient S DMC=X ¼
DX
where A1is the amount of analyte in aqueous solution at equilib-

rium; A2 is the amount of analyte enriched by MIP/NIP at equi-
librium, AT is the total amount of analyte used in the extraction,
DDMC is the distribution ratio of DMC (template), and DX is the
distribution ratio of tested compounds (AFs, vitamins, and amino
acids).
3.6 Analytical 1. Prepare several calibration curves in methanol using several

Performance analyte concentrations of 2.5, 5.0, 10, 20, 50, 100, and
200 μg L1 for B1, B2, G1, and G2 (2.5, 5.0, 10, 20, 30, 40,
and 50 μg L1 for M1) and with DMC (50 μg L1) as an
internal standard. To verify the existence of matrix effect, pre-
pare several standard addition calibrations by addition by spik-
ing 1.5 mL of fish extracts with 0.167, 0.333, 0.667, 1.33,
3.33, 6.67, and 13.3 μg L1 for B1, B2, G1, and G2, and with
0.167, 0.333, 0.667, 1.33, and 3.33 μg L1 for M1. Spike each
fish extract with DMC at 3.33 μg L1 (since the
pre-concentration factor of the D-μ-SPE is 15, these spiked
concentrations will be similar than those used for calibration
after subjecting the spiked fish extract to the D-μ-SPE proce-
dure). Compare statistically the mean slopes of the calibration
in methanol and the standard additions and verify the existence
of matrix effect (see Note 6).
2. Calculate the instrumental limit of detection (LOD) and limit
of quantification (LOQ) by taking into account the measure-
ment of the eleven blank samples after applying D-μ-SPE.
Follow the following equations for LOD and LOQ
calculations.
LOD ¼ 3 σ
LOQ ¼ 10 σ
where σ is the standard deviation of the measurement of
eleven blank samples.
3. Calculate the effective limit of detection (LOD) and limit of
quantification (LOQ) by taking into account the mean slope of
the standard addition (see equation below), the
pre-concentration factor achieved after D-μ-SPE, and the sam-
ple mass subjected to the ultrasound probe-assisted extraction
(see Note 7)
3σ
LOD ¼
m
10 σ
LOQ ¼
m
where σ is the standard deviation of the measurement of

eleven blank samples, and m is the mean slope of the standard
addition.
4. Evaluate the precision and accuracy of the method on the basis
of the relative standard deviation (RSD%) and analytical recov-
ery when performing intraday and inter-day assays. For intra-
day precision and analytical recovery, perform three standard
addition calibrations (D-μ-SPE implying a pre-concentration
factor of 15) on three different days. The first day perform
three replicates of each spiked concentration except the lowest
one, which is replicated seven times. The second day, perform
three replicates of each concentration levels, except an interme-
diate concentration, which must be replicated seven times.
Finally, the third day, perform three replicates of all spiked
fish samples, except those spiked with the highest concentra-
tion (seven replicates must be performed for this concentration
level). For inter-day precision and analytical recovery assays,
prepare seven standard additions on seven different days by
replicating each concentration level thrice.
5. To check the accuracy of the method, analyze a certified refer-
ence material (CRM) by the proposed method, and compare
the found values with those certified ones. Since there is no
commercially available CRM for AFs in fish flesh (liver), use
other CRMs to similar matrix with certified AFs content.
6. Study the reusability of the D-μ-SPE devices by subjecting
several aliquots of fish extract spiked with a known concentra-
tion of B1 (6.67 μg L1 before pre-concentration) to the
proposed D-μ-SPE. Perform the loading/elution cycle using
the same MIP mass (40 mg) in three different D-μ-SPE
devices. After performing each successive loading/elution
cycles, calculate the analytical recovery, and establish the num-
ber of loading/eluting cycle from which non-quantitative
values are obtained (analytical recovery lower than 80%).
7. Check the applicability of the developed D-μ-SPE method by
analyzing several cultured fish samples (flesh and liver).
4 Notes
1. MIP synthesis requires moderate amounts of the template

(target). High purity reagents that can be quite expensive will
lead to an expensive synthesis of the material or the need to
synthesize the molecule in the laboratory (also an expensive
and time-consuming procedure). In addition, there can be
other drawbacks if the targets used as templates are unstable
and highly toxic, such as AFs. In these cases, the target
molecule can be replaced by a compound (knowns as dummy

template) exhibiting a similar chemical structure and function-
alities than the analyte. This is the reason of choosing the DMC
as a template molecule (similar furanocoumarin structure as
AFs). A large porogen volume (25 mL) leads to MIP synthesis
by precipitation polymerization, which facilitates homoge-
neous and dispersed MIP particles and guarantees the integrity
of the generated imprinting cavities. First synthesis attempts
consisted of using a high acetonitrile proportion in the poro-
gen mixture, but the MIP obtained showed little adsorption
capacity through AFs. Then, a high proportion of toluene in
the porogen mixture was tested, and the obtained MIP parti-
cles were found to have available recognition cavities for the
targets dissolved in an organic extract from fish samples. The
MAA monomer is the most common monomer used for MIP
synthesis. This monomer fits well with the flat (planar) struc-
ture of the AF and DMC molecules, which was expected to
allow good template–monomer interactions.
2. DVB must be purified for removing polymerization inhibitors.
The purification process consists of passing a few milliliters of
the reagent through a previously prepared mini-column con-
taining neutral alumina. The mini-column is prepared as fol-
lows: a small portion of glass wool is placed inside a glass
Pasteur pipette and is pushed to the bottom of the pipette;
then approximately 0.5 g of alumina is introduced, and other
portion of glass wool is placed over the alumina adsorbent.
AIBN is purified by crystallization by dissolving the selected
amount of reagent in an small volume of methanol, and by
keeping the solution at 20 C for 1.0 h. Then, the methanol is
discarded, and the pure AIBN crystals are again dissolved in
methanol (the same volume than that used for crystalization)
previously heated at 50–60 C.
3. Template removal is a critical step when preparing MIPs. The
most conventional techniques for template removal are soaking
the MIP particles into organic solvents and speed-up the leach-
ing process by mechanical stirring and ultrasounds. Soxhlet
extraction is also a common technique for template removal.
These procedures are, however, time-consuming, and they
could require extreme pHs and temperatures that can lead to
distortion and even damage of the imprinting cavities. The first
attempt for DMC removal used Soxhlet extraction with
200 mL of acetonitrile/water/acetic acid (85:10:5), and two
12 h cycles (fresh washing solution in each cycle) was required
for template removal. As an alternative, we decided to use
pressurized liquid extraction (PLE), which is expected to
remove the template in shorter times, with a lower volume of
washing solution, and under moderate temperature a pH
condition. Parameters such as pressure, flushing volume, tem-

perature, and extraction time are the critical in PLE procedures
when removing the template since high pressures and high
temperatures for a long time can affect (damage) MIP beads.
The careful study of these variables led to an efficient method
for template removal, guaranteeing the integrity of MIP parti-
cles (integrity of the recognition cavities).
4. D-SPE and D-μ-SPE can be assisted ultrasound and mechanical
stirring by vortex during the loading and elution stages. The
supplemental energy from ultrasound and vortexing allows a
better interaction between adsorbent (MIP) particles and the
analytes in the extracts (loading step), and between the analyte
loaded in the adsorbent (MIP) particles and the eluting solu-
tion (eluting step). Vortexing offers certain advantages when
comparing to ultrasound assistance since it is a soft and
low-cost shaking technique and dispersion assistance is more
repeatable because of the ultrasound fluency (and hence the
dispersion) depends on the position inside the water-bath tank.
MIPs beads are lightweight particles and an additional centri-
fugation or filtration step is required before removing the
supernatants in order to avoid MIP particle losses.
5. Extraction efficiency of 40% was assessed for some investigated
compounds such as amino acids by using both MIP and NIP.
Since similar values were observed, the adsorption of these
compounds is nonspecific, and they cannot be attributed to
MIP recognition.
6. The matrix effect expressed as the percentage of the difference
between calibration and standard addition slopes was around
20% for some AFs (16% for B1, 23% for B2, and 19% for G2),
but it was very high for G1 (56%) and M1 (59%). ANOVA test
(95% confidence interval) showed that there was a significant
difference between the mean slopes of the calibration and
standard addition for all AFs.
7. The pre-concentration factor of the method is 15. The
pre-concentration factor could be improved when subjecting
volumes of fish extract higher than 1.5 mL to the D-μ-SPE
procedure. Results after applying the method to 3.0 mL
(pre-concentration factor of 30) and 5.0 mL
(pre-concentration factor of 50) of fish extract led to similar
AFs (B1) concentration in a fish flesh sample, independently of
the extract volume used for the analysis: 0.89 0.09,
1.05 0.11, and 0.76 0.08 μg kg1, for 1.5 mL (pre-con-
centration factor of 15), 3.0 mL (pre-concentration factor of
30), and 5.0 mL (pre-concentration factor of 50), respectively.
There were not found statistically significant differences among
these concentrations after applying an ANOVA test (95%
confidence interval). From these results, the D-μ-SPE can be

performed by loading up 5.0 mL of the fish extract without
accuracy losses, which implies a robust method and improved
LOD/LOQ.
Acknowledgments
This work was supported by the Direccion Xeral de I + D – Xunta

de Galicia Grupos de Referencia Competitiva (project number
ED431C2018/19), and Development of a Strategic Grouping in
Materials—AEMAT (grant ED431E2018/08).
References
1. Turiel E, Martı́n-Esteban A (2019) Molecu- green sample pre-treatments in food analysis.

larly imprinted polymers-based microextrac- Trends Anal Chem 118:1–18
tion techniques. Trends Anal Chem 8. Ojeda CB, Rojas FS (2018) Vortex-assisted
118:574–586 liquid–liquid microextraction (VALLME): the
2. Faraji M, Yamini Y, Gholami M (2019) Recent latest applications. Chromatographia
advances and trends in applications of solid- 81:89–103
phase extraction techniques in food and envi- 9. Adewuyi YG (2001) Sonochemistry: Environ-
ronmental analysis. Chromatographia mental science and engineering applications.
88:1207–1249 Ind Eng Chem Res 40:4681–4715
3. Capriotti AL, Cavaliere C, La Barbera G et al 10. Cai Q, Zhang L, Zhao P et al (2017) A joint
(2019) Recent applications of magnetic solid- experimental-computational investigation:
phase extraction for sample preparation. Chro- metal organic framework as a vortex assisted
matographia 82:1251–1274 dispersive micro-solid-phase extraction sorbent
4. Pichon V, Delaunay N, Combès A (2020) Sam- coupled with UPLC-MS/MS for the simulta-
ple preparation using molecularly imprinted neous determination of amphenicols and their
polymers. Anal Chem 92:16–33 metabolite in aquaculture water. Microchem J
5. Chisvert A, Cárdenas S, Lucena R (2019) Dis- 30:263–270
persive micro-solid phase extraction. Trends 11. IARC monographs on the evaluation of carci-
Anal Chem 112:226–233 nogenic risks to humans. Vol. 82 Traditional
6. Ghorbani M, Aghamohammadhassan M, herbal medicines, some mycotoxins, naphtha-
Chamsaz M et al (2019) Dispersive solid lene and styrene, (2002). World Health Orga-
phase microextraction. Trends Anal Chem nization International Agency for Research on
118:793–809 Cancer. IARC Press, Lyon, France
7. Moreda-Piñeiro J, Moreda-Piñeiro A (2019) 12. Dirican S (2015) A review of effects of aflatox-
Combined assisted extraction techniques as ins in aquaculture. Appl Res J 1:192–196
Chapter 13
Preparation of Monolithic Fibers in Fused Silica Capillary

Molds for Molecularly Imprinted Solid-Phase
Microextraction
Esther Turiel Trujillo and Myriam Dı́az-Álvarez
Abstract
In the last three decades, the use of molecularly imprinted polymers (MIPs) in sample preparation has
continuously increased due to the high selectivity that they provide to this critical step. Of particular interest
is the combination of molecular imprinting polymers and solid-phase microextraction (SPME) that allows
the development of rapid and environmental friendly analytical methods, with high sensitivity and selectiv-
ity. The protocol herein presented describes a very simple strategy for the direct preparation of monolithic
MIPs using silica capillaries as molds by the copolymerization of methacrylic acid and ethylene glycol
dimethacrylate in the presence of propazine as template. The main factors affecting the polymer synthesis
(e.g., porogen, monomer, cross-linker, polymerization mixture proportions, polymerization time, and fiber
thickness) are described in detail. The proposed strategy is easy to perform in any laboratory without special
equipment and allows precise control of the fiber thickness, overcoming this very common drawback in
MIP-based fiber preparation.
Key words Molecularly imprinted polymeric fibers, Solid-phase microextraction, Monoliths, Propa-
zine, Sample preparation
1 Introduction
1.1 Overview Solid-phase microextraction (SPME) was introduced by Pawliszyn

et al. in the 1990s [1] and was rapidly established as a robust and
simple sample preparation technique in most of analytical labora-
tories. The main advantage of SPME relies in the possibility of
performing sampling, analyte isolation, and enrichment in the
same step, thereby saving time and resources. In addition, a very
low consumption of organic solvents is required, making SPME
one of the most environmental friendly extraction techniques avail-
able nowadays. However, the main drawback is the limited variety
of the sorbents that are commercially available, which just covers
https://doi.org/10.1007/978-1-0716-1629-1_13,
153
154 Esther Turiel Trujillo and Myriam Dı́az-Álvarez
Fig. 1 Picture of a flexible fiber obtained by the proposed protocol
the scale of polarity of the fibers, thus leading to a lack of selectivity

of the extraction process.
Molecularly imprinted polymers (MIPs) are synthetic polymers
obtained by copolymerizing a monomer with a cross-linker in the
presence of a template molecule. After polymerization, the tem-
plate is removed leaving cavities in the polymeric matrix comple-
mentary in size, shape, and functionality to the template. Thus, the
obtained MIPs have high affinity for the template and related
compounds, allowing to perform selective extractions from com-
plex matrices [2].
In this sense, the combination of molecular imprinting tech-
nology and SPME, the so-called molecularly imprinted solid-phase
microextraction (MISPME), is nowadays one of the most advanced
technical applications of MIP technology in SPE. The different
MIP-based fiber preparation strategies described to date can be
grouped into two main categories: MIP-coated fibers and MIP
monoliths.
For MIP-coated fiber preparation, silica fibers are firstly acti-
vated by a silylation step, and subsequently placed in a small volume
of polymerization mixture for further polymerization. After poly-
merization, the fibers with the MIP coating on their surface are
pulled out of the semi-solid polymer and glued to a stainless-
steel tube.
Key parameters such as solvent and polymerization time affect
the fiber preparation reproducibility and the coating thickness,
which is critical in the step of pulling out the fragile fiber from
the semi-solid polymer [3–5]. Other strategies in MIP coating
preparation, such as multiple bulk copolymerization [6–9] and
surface reversible addition-fragmentation chain transfer polymeri-
zation (RAFT) [10], have been proposed with the aim of
controlling film thickness and surface morphology, as well as
enhancing the reproducibility in the preparation of MIP coatings.
Monolithic MIP Fibers for SPME 155
Another drawback of silica-coated fibers is their limited flexibil-

ity and fragility. To solve this issue, the preparation of metal-wire-
supported SPME fibers using different approaches has been
described. Some examples are the chemical bonding of the
imprinted coating to a previously modified metal wire [11, 12] or
the direct coating of conducting polymers (e.g., polypyrrole) onto a
metal support by electropolymerization [13–15]. However, in the
latter strategy high number of nonspecific interactions has been
observed; therefore, further development of this technique is
required and the use of other electropolymerizable monomers
should be considered.
The other category of MIP-based fibers comprises MIP fibers
(monoliths) that are directly synthesized using empty silica capil-
laries as molds [16, 17]. Monolithic MIPs are prepared via thermal
radical copolymerization of the polymerization mixture inside the
capillary, being silica etched away after polymerization. This proce-
dure involves easy steps that can be accomplished in any laboratory
equipped with basic instrumentation. The obtained fibers are flexi-
ble and can be slightly bent, thus reducing the risk of breakage
during handling (see Fig. 1).
This strategy allows a precise control of the fiber thickness,
which is limited to the inner diameter of the silica capillary, and
thus, the obtained fibers exhibit good preparation reproducibility.
In fact, fiber to fiber RSD values of extraction efficiencies as low as
7–15% [16], or even <6% [18] can be achieved.
It should also be noted that the monolithic MIP fibers, as well
as the silica-coated fibers, are thermally stable up to temperatures
around 250–300 C. Therefore, target analytes can be thermally
desorbed in the GC injection port or eluted with a suitable solvent
for further analysis. Subsequent separation and quantification are
usually carried out by GC coupled to MS, LC coupled to diode
array detection (DAD), or capillary electrophoresis (CE). Thus,
monolithic MIP fibers have been successfully applied in the SPME
of a large variety of compounds, including herbicides [16, 18–20],
disinfectants [21], insecticides [22], drugs [17, 23], personal care
products [24] industrial additives [25, 26], food preservatives [27],
and food sterols [28] in different highly complex matrices.
1.2 General Several important parameters should be considered in the prepara-

Considerations tion of SPME fibers based on monolithic MIPs, such as the solvent
in Monolithic Fiber used as porogen or the selected cross-linker and the polymerization
Preparation time among others. Some of these factors are directly related to the
success of the imprinting process, while others will affect the stabil-
ity of the obtained fiber. However, the effects of the different
parameters on the final fiber performance have to be considered
as a whole and not separately.
Optimization of MIP formulations: The composition of the
polymer, that is the type and amount of monomer, and the selected
cross-linker and porogen, must be carefully optimized, since they

will determine the final characteristics of the obtained material both
in terms of capacity, affinity, and selectivity for target analytes. For
instance, higher cross-linker ratios are usually preferred because
they use to provide good mechanical stability to the polymer,
which is essential both to maintain the integrity of SPME fibers
and to form a permanent macroporous structure [17]. Additionally,
the creation of macropores in the polymeric matrix is affected by
the nature and volume of the porogen (i.e., the pore volume
increases as the porogen volume up to a certain extent). A low
volume of solvent may produce gel-type polymer unable to form
suitable monoliths with the required macroporous structure. Nev-
ertheless, an excess of volume may lead to the obtainment of a
microgel powder, being it inadequate as well [17, 29].
Regarding the solvent nature, in non-covalent imprinting poly-
merization strategy non-polar aprotic solvents (e.g., toluene) are
preferred since they allow a better stabilization of the hydrogen
bonds responsible of the selectivity of the obtained fiber [29].
Fiber thickness: The fused silica capillaries that can be used as
molds are commercially available with different inner diameters.
Therefore, the final thickness of the fiber can be accurately con-
trolled by the proper selection of the capillary diameter. Likewise,
the amount of obtained sorbent will be also determined by the
chosen diameter. Moreover, thanks to the porous structure of the
obtained fibers, further rebinding processes will take place not only
on the fiber surface but also in its inner binding sites. For this
reason, a linear relationship between the obtained recoveries and
the volume of the fiber has been found [16].
Polymerization time: The polymerization time has an important
role in the degree of cross-linking. Too short polymerization times
may lead to a decrease of the SPME recoveries due to the lack of
definition of the binding sites. On the other hand, too long poly-
merization time produces highly cross-linked polymers with low
porosity reducing the extraction capability of the fiber [16].
Keeping all these comments in mind, polymerization protocols
must be optimized for each targeted template.
2 Materials
2.1 Polymerization 1. Template: propazine (PPZ) 99.9%, analytical standard.

Mixture 2. Monomer: methacrylic acid (MAA) 99% and cross-linker eth-
ylene glycol dimethacrylate (EDMA) 98% are stabilized with
hydroquinone monomethyl ether (MEHQ), an inhibitor that
prevents unintended polymerization processes. Use an inhibi-
tor remover prior to polymerization (see Note 1). Store below
20 C.
3. Initiator: 2,20 -azobis(isobutyronitrile) (AIBN) 98.0%. Purify

AIBN by recrystallization from methanol (see Note 2) and
store protected from light in a refrigerator at temperature
below 10 C.
4. Porogen: toluene HPLC grade.
2.2 Preparation 1. Untreated fused silica capillaries with an internal diameter of

of Molecularly 0.53 mm (see Note 3).
Imprinted Fibers 2. PVC pump tubing with an outer diameter of 2.33 mm
(0.16 cc/min, color code: orange/yellow) (see Note 4).
3. Conventional binder clips.
4. Plastic syringes of 1 mL capacity and needles of 21G 100 ,
0.8 25 mm.
5. Conventional rubber eraser.
6. Laboratory oven.
7. Plastic screw cap container (60 mL).
8. Stir bar and magnetic stirrer.
9. Methanol, HPLC gradient grade.
10. Acetic acid glacial, 99.7%.
11. Water, HPLC grade.
12. Aqueous solution 3 M NH4HF2.
13. Nitrogen, 99.99%.
3 Methods
3.1 Preparation 1. Cut the silica capillaries into pieces of 30 cm.

of the Fused-Silica 2. Mark the position on the silica capillary where the windows will
Capillaries Used be later created. A schematic diagram of the capillary prepara-
as Molds tion protocol is depicted in Fig. 2.
3. Use the flame of a lighter to burn the protective layer from each
window for 2–3 s. Remove the ashes with an alcohol wipe (see
Note 5).
4. Cut a piece of 4 cm of the PVC pump tubing. Carefully
introduce one end of the silica capillary inside the tubing. It is
enough to introduce 3–4 mm.
5. Prepare sealing materials: cut the eraser in cubes of 5 mm side.
Use different eraser colors to distinguish between MIP and
NIP. Have binder clips on hand.
3.2 Preparation The same recipe but without template addition should be used to
of the Polymerization prepare the control NIP fibers.
Mixture
Fig. 2 Schematic diagram of the proposed fiber preparation protocol (PART 1). (1) Mark windows position.
(2) Burn windows protective layer and remove the ashes with an alcohol wipe. (3) With the aid of the tubing fill
the capillary with the polymerization mixture. (4) Close both ends firmly
1. Dissolve propazine (34.6 mg, 0.15 mmol) and MAA (51.9 μL,
0.60 mmol) in toluene (0.866 mL) using vortex.
2. Add EDMA (0.589 mL, 3 mmol) and AIBN (21.7 mg,
0.13 mmol) and vortex (see Note 6).
3. Remove the oxygen from the solution with a gentle N2 stream
during 5 min. Use the polymerization mixture immediately. If
not, store below 20 C (see Note 7).
3.3 Preparation 1. Use the syringe to introduce the polymerization mixture inside
of Molecularly the capillary with the aid of the PVC tubing (see Note 8).
Imprinted Fibers 2. Remove the needle from the PVC tubing and use the binder
clip to tighten the PVC tubing.
3. Place the filled capillaries in a laboratory oven at 65 C for
150 min (see Note 9).
4. Take the capillaries out of the oven and allow them to cool
down. Then use a cutter knife to cut the capillaries at the tick
marks made at the beginning of this procedure. A schematic
diagram can be seen in Fig. 3.
5. Remove the silica walls by immersing the fibers in a 3 M
aqueous solution of NH4HF2 for 12 h under agitation (see
Note 10).
6. Wash fibers gently with water to remove acid residues.
Fig. 3 Schematic diagram of the proposed fiber preparation protocol (PART 2). (1) Place the capillary into an
oven at 60 C during 150 min for polymerization. (2) Cut capillary at one edge of the windows. (3) Remove
silica walls of the four obtained fibers with NH4HF2 solution
7. Remove the template by immersing the fibers in a methanol:

acetic acid (1:1, v/v) solution for 2 h under stirring (see Note
11).
8. Remove the acetic acid by immersing the fibers in methanol for
30 min under stirring and store the fibers.
4 Notes
1. Ready to use prepacked columns are used to remove small

amount of inhibitors (use different prepacked columns for
each reagent). Consider the column capacity indicated in the
instructions (e.g., HQ/MEHQ: 3 L at 100 ppm) to calculate
the amount of reagent that can be percolated through the
column. Columns may be disposed when exhausted or reused
by filling the column with new packing material. Store the
purified reagent in the freezer. It is recommended to distribute
the purified reagent in small amber vials to be further defrosted
separately. In these conditions, reagents are properly main-
tained for months. Alternatively, monomers with low boiling
point as MAA can be purified from stabilizers by distillation
under reduced pressure.
2. AIBN 98% can be purified by recrystallization in methanol. Put
5 g of AIBN in an Erlenmeyer flask and add 200 mL of this
solvent. Use a magnetic stirrer and heat at low temperature

until AIBN gets completely dissolved. Allow the mixture to get
room temperature and separate the AIBN crystals by filtration
with a filter paper. Dry under vacuum at room temperature.
Alternatively, AIBN 99% recrystallized from methanol is avail-
able on the market and ready for use.
3. Untreated silica capillaries have different diameters and can be
selected depending on the desired use of the imprinted fibers.
For example, smaller diameters could be interesting for fibers
that will be desorbed in a GC chamber [30].
4. The purpose of the tubing is to help in the step of filling the
fused-silica capillary with the polymerization mixture. The sil-
ica capillary should be inserted few millimeters into the tubing,
consequently the diameter of the PVC pump tubing depends
on the diameter of the silica capillary. Another tubing of similar
nature may work.
5. The silica capillary is amber color. The window becomes trans-
parent when the protective layer has been correctly removed.
6. The proposed polymerization mixture is composed of propa-
zine, methacrylic acid, and ethylene glycol dimethacrylate
(1:4:20 molar ratio, respectively), which is a commonly used
formulation for non-covalent MIP preparations. Thanks to its
versatility, the non-covalent approach is the most widely used
for the preparation of MIPs.
7. The polymerization mixture should be protected from light
and immediately used; otherwise, it must be stored in the
freezer. The polymerization mixture can be stored in the
freezer for several months. Consider that templates with low
solubility in the mixture can precipitate when mixture freezes.
8. Insert the needle into the PVC tubing without piercing it. Press
gently the syringe plunger and stop when the mixture leaks
through the opposite end of the capillary. Then close immedi-
ately this edge with the eraser piece. It is important to avoid the
formation of bubbles during the filling process because, after
polymerization, those bubbles create voids that damage the
physical stability of the fiber.
9. AIBN, the most common initiator used in MIP preparation,
can be decomposed by photolysis (UV) or thermolysis at tem-
peratures above 60 C. The use of microwave irradiation for
the preparation of MIP monoliths considerably reduces the
polymerization time, due to the fast energy transfer and high
energy efficiency [25]. However, the use of microwave requires
solvents with specific dielectric properties (e.g., acetonitrile),
which may not be appropriate for MIP recognition of certain
templates. In the same way, the use of UV irradiation to initiate
polymerization is not adequate for solvents that present a
strong UV absorbance (e.g., toluene). For this reason, the use

of a conventional laboratory oven is considered the simplest
way to proceed.
10. A modified screw cap container can be used to treat several
fibers at the same time. Use a sharp object (i.e., a thick needle)
to drill holes in the plastic screw cap where the fibers will be
later placed. Leave some distance between the holes to work
comfortably. Fill the container with the corresponding solvent
and introduce the stir bar. Pierce a rubber piece with the
protected end of the fiber to support it on the drilled screw
cap. Use gentle agitation during the different treatments per-
formed to the fiber.
11. Although the NIP fibers do not contain the template, perform
the same washing steps for MIP and NIP fibers. In this way, the
fibers are exposed to identical conditions and thus reproducing
modifications that may occur in the polymeric matrix during
the treatments.
References
1. Pawliszyn J, Arthur CL (1990) Solid phase 8. Hu Y, Wang Y, Chen X et al (2010) A novel

microextraction with thermal desorption molecularly imprinted solid-phase microextrac-
using fused silica optical fibers. Anal Chem tion fiber coupled with high performance liq-
62:2145–2148 uid chromatography for analysis of trace
2. Turiel E, Martı́n-Esteban A (2009) Molecu- estrogens in fishery samples. Talanta
larly imprinted polymers for solid-phase micro- 80:2099–2105
extraction. J Sep Sci 32:3278–3284 9. Hu X, Pan J, Hu Y, Li G (2011) Preparation of
3. Koster EH, Crescenzi C, den Hoedt W et al molecularly imprinted polymer coatings with
(2001) Fibers coated with molecularly the multiple bulk copolymerization method
imprinted polymers for solid-phase microex- for solid-phase microextraction. J Appl Polym
traction. Anal Chem 73:3140–3145 Sci 120:1266–1277
4. Hu X, Hu Y, Li G (2007) Preparation and 10. Hu X, Fan Y, Zhang Y et al (2012) Molecularly
characterization of prometryn molecularly imprinted polymer coated solid-phase microex-
imprinted solid-phase microextraction fibers. traction fiber prepared by surface reversible
Anal Lett 40:645–660 addition-fragmentation chain transfer poly-
5. Hu X, Hu Y, Li G (2007) Development of merization for monitoring of Sudan dyes in
novel molecularly imprinted solid-phase micro- chilli tomato sauce and chilli pepper samples.
extraction fiber and its application for the Anal Chim Acta 731:40–48
determination of triazines in complicated sam- 11. Djozan D, Ebrahimi B, Mahkam M, Farajza-
ples coupled with high-performance liquid deh MA (2010) Evaluation of a new method
chromatography. J Chromatogr A 1147:1–9 for chemical coating of aluminum wire with
6. Hu X, Pan J, Hu Y et al (2008) Preparation and molecularly imprinted polymer layer. Applica-
evaluation of solid-phase microextraction fiber tion for the fabrication of triazines selective
based on molecularly imprinted polymers for solid-phase microextraction fiber. Anal Chim
trace analysis of tetracyclines in complicated Acta 674:40–48
samples. J Chromatogr A 1188:97–107 12. Prasad BB, Tiwari MP, Madhuri R, Sharma PS
7. Hu X, Pan J, Hu Y, Li G (2009) Preparation (2010) Enatioselective quantitative separation
and evaluation of propranolol molecularly of d- and l-thyroxine by molecularly imprinted
imprinted solid-phase microextraction fiber micro-solid phase extraction silver fiber cou-
for trace analysis of β-blockers in urine and pled with complementary molecularly
plasma samples. J Chromatogr A imprinted polymer-sensor. J Chromatogr A
1216:190–197 1217:4255–4266
13. Mazzotta E, Malitesta C, Dı́az-Álvarez M, 22. Hashemi-Moghaddam H, Jedi DJ (2015)

Martin-Esteban A (2012) Electrosynthesis of Solid-phase microextraction of chlorpyrifos in
molecularly imprinted polypyrrole for the anti- fruit samples by synthesised monolithic molec-
biotic levofloxacin. Thin Solid Films ularly imprinted polymer fibres. Int J Environ
520:1938–1943 Anal Chem 95:33–44
14. Dı́az-Álvarez M, Mazzotta E, Malitesta C, 23. Deng DL, Zhang JY, Chen C et al (2012)
Martı́n-Esteban A (2014) Evaluation of elec- Monolithic molecular imprinted polymer fiber
trochemically synthesized sulfadimethoxine- for recognition and solid phase microextraction
imprinted polymer for solid-phase microex- of ephedrine and pseudoephedrine in
traction of sulfonamides. J Mol Recognit biological samples prior to capillary electro-
27:415–420 phoresis analysis. J Chromatogr A
15. Liu X, Wang X, Tan F et al (2012) An electro- 1219:195–200
chemically enhanced solid-phase microextrac- 24. Dı́az-Álvarez M, Smith SP, Spivak DA, Martı́n-
tion approach based on molecularly imprinted Esteban A (2016) Preparation of molecularly
polypyrrole/multi-walled carbon nanotubes imprinted polymeric fibers using a single
composite coating for selective extraction of bifunctional monomer for the solid-phase
fluoroquinolones in aqueous samples. Anal microextraction of parabens from environmen-
Chim Acta 727:26–33 tal solid samples. J Sep Sci 39:552–558
16. Turiel E, Tadeo JL, Martin-Esteban A (2007) 25. Xu S, Zhang X, Sun Y, Yu D (2013)
Molecularly imprinted polymeric fibers for Microwave-assisted preparation of monolithic
solid-phase microextraction. Anal Chem molecularly imprinted polymeric fibers for
79:3099–3104 solid phase microextraction. Analyst
17. Djozan D, Baheri T (2007) Preparation and 138:2982–2987
evaluation of solid-phase microextraction fibers 26. Cai C, Zhang P, Deng J et al (2018) Ultrasen-
based on monolithic molecularly imprinted sitive determination of highly polar trimethyl
polymers for selective extraction of diacetyl- phosphate in environmental water by molecu-
morphine and analogous compounds. J Chro- larly imprinted polymeric fiber headspace solid-
matogr A 1166:16–23 phase microextraction. J Sep Sci
18. Zhang Z, Mei M, Huang Y et al (2017) Facile 41:1104–1111
preparation of a polydopamine-based monolith 27. Chen L, Huang X (2017) Preparation and
for multiple monolithic fiber solid-phase application of a poly (ionic liquid)-based
microextraction of triazine herbicides in envi- molecularly imprinted polymer for multiple
ronmental water samples. J Sep Sci monolithic fiber solid-phase microextraction
40:733–743 of phenolic acids in fruit juice and beer samples.
19. Djozan D, Ebrahimi B (2008) Preparation of Analyst 142:4039–4047
new solid phase micro extraction fiber on the 28. Kardani F, Mirzajani R, Ramezani Z (2018)
basis of atrazine-molecular imprinted polymer: Direct cholesterol and β-sitosterol analysis in
application for GC and GC/MS screening of food samples using monolithic molecularly-
triazine herbicides in water, rice and onion. imprinted solid-phase microextraction fibers
Anal Chim Acta 616:152–159 coupled with high performance liquid chroma-
20. Djozan D, Mahkam M, Ebrahimi B (2009) tography. J Iran Chem Soc 15:2877–2888
Preparation and binding study of solid-phase 29. Cormack PAG, Elorza AZ (2004) Molecularly
microextraction fiber on the basis of ametryn- imprinted polymers: synthesis and characterisa-
imprinted polymer. Application to the selective tion. J Chromatogr B Anal Technol Biomed
extraction of persistent triazine herbicides in Life Sci 804:173–182
tap water, rice, maize and onion. J Chromatogr 30. Rajabi Khorrami A, Narouenezhad E (2011)
A 1216:2211–2219 Synthesis of molecularly imprinted monolithic
21. Huang X, Zhang Y, Mei M, Yuan D (2014) fibers for solid-phase microextraction of acetal-
Preparation of monolithic fibers for the solid- dehyde from head-space of beverages stored in
phase microextraction of chlorophenols in PET bottles. Talanta 86:58–63
water samples. J Sep Sci 37:1185–1193
Chapter 14
Quick, Easy, Cheap, and Effective Method for the Synthesis

of Rugged Magnetic Molecularly Imprinted Stir-Bars
Patricia Regal, Mónica Dı́az-Bao, and Alberto Cepeda
Abstract
This contribution describes a fast and facile method for fabrication of robust magnetic stir-bars composed
of molecularly imprinted polymers (MIPs) combined with magnetite particles. This was achieved through a
prior optimization of the protocol presented here, in particular, the selection of cross-linker and porogen
suited for obtaining a durable monolithic magnetic stir-bar. In-house prepared magnetite nanoparticles
(Fe3O4) are used as magnetic core, coating them with molecularly imprinted polymers through a simple
process of bulk polymerization. Procedures for magnetite synthesis, preparation of polymerization mixture,
stir-bar synthesis, and analytical application are described.
Key words Molecularly imprinted polymers, Bulk polymerization, Magnetite, Stir-bar, Sorptive
extraction
1 Introduction
Since their discovery back to the early twentieth century, molecu-

larly imprinting polymers (MIPs) have become very popular sor-
bents in sample preparation due to their inherent selectivity,
robustness, low cost, and relatively straightforward preparation
[1]. Smart designs have been introduced progressively in molecu-
larly imprinting technology, including modifications of MIP struc-
ture, miniaturization, and hybridization of polymers with diverse
materials (magnetite, silica, glass), aimed at achieving better and
simpler extractive applications for real samples [2–4]. In this sense,
additional features such as magnetic properties can be incorporated
to the MIP sorbent, providing an attractive alternative to classic
clean-up protocols since the recovery of these polymers is per-
formed with the aid of an external magnetic field, avoiding tedious
steps such as sample filtration or centrifugation [2, 5]. Magnetic
nanoparticles are excellent candidates for the fabrication of new
functional materials, offering not only the possibility of quick
https://doi.org/10.1007/978-1-0716-1629-1_14,
163
164 Patricia Regal et al.
dispersion in sample solutions and readily recovery with the aid of

an external magnet, but also their ease of combination with other
materials such as MIPs [5].
Different approaches have been reported for the synthesis of
magnetic MIPs and MIP-based magnetic devices, including ferro-
magnetic nanoparticles coated with polymeric shells and stir-bars
with magnetic core, amongst others [5, 6]. A typical stir-bar for
sorptive extraction is composed of a polydimethylsiloxane film,
coated onto a glass jacket with an incorporated magnet core
[6, 7] Molecularly imprinting technology has introduced the out-
standing advantages of MIPs in stir-bar sorptive extraction. How-
ever, some significant challenges are still to be faced such as
template bleeding, low binding capacity, poor chemical and
mechanical MIP-coat stability, or template toxicity. Also, the depo-
sition of the MIP sorbent onto the magnetic core surface typically
requires pre-treatment or etching of those surfaces, creation of
superficial silanol groups, or the application of repeated coatings
to obtain a durable coat [6, 8, 9]. In this sense, monolith stir-bar
coatings from simple physical attachment methods represent a clear
advantage, but they have been reported to have poor structural
stability. Nonetheless, the method described here provided a quick,
easy, cheap, and effective alternative to fabricate robust magnetic
molecularly imprinted stir-bars (MMIP-SB) using only in-house
prepared materials. The MMIP-SB was prepared using methacrylic
acid as functional monomer, ethylene glycol dimethacrylate as
cross-linking monomer, toluene/methanol (90:10, v/v) as poro-
genic solvent and “chunks” of Fe3O4 magnetite as magnetic core.
Using MMIP-SBs, the extraction and clean-up procedures can be
carried out under stirring conditions by simply adding the sample
(milled or liquid) and required solvents to a laboratory flask con-
taining the stir-bar.
2 Materials
Check all solutions and reagents before their use and store accord-
ing to the manufacturer recommendations. Dispose all waste
according to applying regulations. Use ultrapure water (preferably
LC-grade) and analytical grade reagents. Indicated amounts of
reagents are for synthesizing one magnetic molecularly imprinted
stir-bar (MMIP-SB).
2.1 Synthesis – FeCl2 · 4H2O.

of Ferric Oxide (Fe3O4) – FeCl3 · 6H2O.
Nanoparticles
– Water.
– NH3 · H2O (25%).
Rapid Fabrication of Molecularly Imprinted Stir-Bars 165
– 250 mL round-bottom three-neck flask

– Round glass-coated stirring bars.
– Stainless steel magnet.
– Heating mantle for round bottom flasks with magnetic stirring
function (control of temperature and stirring independently).
– Mercury thermometer.
– PTFE thermometer inlet adapter with Viton gasket.
– Stainless steel support rod.
– Support rod clamp.
2.2 Polymeric – Template.

Mixture – Functional monomer: methacrylic acid (MAA).
– Cross-linker: ethylene glycol dimethacrylate (EGDMA).
– Radical initiator: 2,20 -azobis-(2-methyl-butyronitril) (AIMN).
– Porogen: toluene/methanol (90:10, v/v).
Prior use, MAA and EGDMA were freed from stabilizers by
distillation under reduced pressure. AIMN was recrystallized from
methanol.
2.3 Polymerization – Temperature controllable incubator.

Equipment and Other – Laboratory vortex mixer for tubes.
Material
– Magnetic stirrer.
– Analytical balance.
– Single-channel mechanical pipette and pipette tips.
– Polystyrene weighing dishes.
– 50 mL glass round bottom centrifuge tubes with phenolic screw
cap and rubber liner
– Tube rack for 50 mL centrifuge tubes.
– Round glass-coated stirring bars.
– Laboratory filter paper.
– Glass Petri dishes.
– Chemically inert glass 1.5 mL injection vials, crimp top.
– Aluminum vial caps, crimp closure type, and non-slit septa.
– Manual vial crimper.
– Fume hood.
– Methanol.
– Acetonitrile.
– Water.
2.4 Template – Toluene.

Removal, – Acetic acid.
Conditioning,
– Glass lab beaker 50 mL.
and Optimization
of the (MMIP-SB) – Glass lab bottle 50 mL; phenolic cap and PTFE liner.
– Magnetic stirrer.
– Soxhlet extractor.
3 Methods
The technique for obtaining a magnetic molecularly imprinted

polymeric stir-bar (MMIP-SB) comprises three major procedures.
The first step is the in-house synthesis of magnetic nanoparticles of
ferric oxide (Fe3O4 magnetite) by the co-precipitation method.
The second step is the preparation of the pre-polymerization mix-
ture. These two steps/reactions shall be performed in a fume hood.
The third step is the combination of all the above-mentioned
materials and their incubation to form an MMIP-SB by means of
bulk polymerization. After that, template removal is necessary,
followed by a proper optimization of different parameters (extrac-
tion protocol) affecting the selectivity and binding capacity of this
stir-bar for the selected target analyte and sample. Only brief
instructions for template removal and for optimization are
described here.
3.1 Synthesis 1. Assemble a 250 mL three-necked round bottom flask in a

of Magnetite by heating mantle with stirring function and position a mercury
Co-Precipitation thermometer inside the flask (lateral neck) using a PTFE ther-
mometer inlet adapter. Secure the flask with a support rod and
a clamp. See Fig. 1.
2. Place 0.01 mol of FeCl2 · 4H2O and 0.02 mol of FeCl3 · 6H2O
in 80 mL of water into the three-necked flask. Stir vigorously
(700–1000 rpm) with the aid of a glass-coated stir-bar and heat
until the temperature of the solution reaches 80 C.
3. Add 10 mL of NH3 · H2O (25%) to the mixture through the
lateral neck opposite to the thermometer and continue stirring
for 1 h at 80 C.
4. Turn off the heating and stirring; remove the stir-bar from the
flask with the aid of an external magnet.
5. Wait until the reaction temperature drops to room
temperature.
6. Isolate the obtained precipitate (magnetite) with the aid of an
external magnet and wash it with water several times until
neutral (see Note 1).
Fig. 1 Assembly of the equipment required for the synthesis of Fe3O4

nanoparticles by co-precipitation method
7. Dissolve the precipitate in 20 mL of water and remove it from

the three-necked flask by pouring it onto a glass Petri dish;
eliminate most of the water simply concentrating the nanopar-
ticles on the bottom of the dish with an external magnet and
discarding the water.
8. Let dry overnight in the Petri dish.
9. Keep the Fe3O4 magnetite (~3 g) in a dry place wrapped within
a filter-paper envelope (see Note 2) until use.
An assembly is depicted in Fig. 1.
3.2 Preparation 1. Dissolve 0.1 mmol of template in 1 mL of toluene/methanol

of Pre-Polymerization (90:10, v/v) into a 50 mL centrifuge tube with the aid of a
Mixture vortex mixer (see Note 3).
2. Add 0.4 mmol of MAA, cap the tube, vortex mix, and stir for
30 min with the aid of a small glass-coated stir-bar, placing the
capped tube in a rack on top of the magnetic stirrer
(200–400 rpm).
3. Add 2 mmol of EGDMA and 0.25 mmol of AIMN and vortex
mix until a clear solution is obtained.
4. Proceed immediately to the next step.
Fig. 2 Schematic of stir-bar fabrication using magnetite and bulk molecularly imprinted polymers (created
with BioRender.com)
3.3 Preparation 1. Place 0.5 mg of Fe3O4 magnetite pieces or “chunks” into a 1.5
of the Magnetic glass vial.
Molecularly Imprinted 2. Add freshly prepared pre-polymerization mixture up to ½ of
Stir-bar (MMIP-SB) the volume of the vial (see Note 4) and do not vortex or mix
mechanically but do it manually and softly instead.
3. Remove remaining air in the vial with a gentle flow of nitrogen.
4. Cap the vial immediately with an aluminum cap and crimp it
with a vial crimper.
5. Place the vial into the incubator, horizontally, laying down on
its side, and incubate the mixture at 60 C for 24 h for bulk
polymerization (see Note 5).
6. Remove the cap and break the glass vial carefully to release the
magnetic semi-cylindrical monolith (MMIP-SB).
A summary of stir-bar fabrication using magnetite and molec-
ularly imprinted polymers is depicted in Fig. 2.
3.4 MMIP-SB 1. Place the MMIP-SB in a 50 mL beaker containing 20 mL of

Template Removal methanol and stir for 30 min at ambient temperature; discard
and Optimization the methanol holding the MMIP-SB with the aid of an external
magnet.
2. Add 20 mL of acetonitrile and stir for 30 min at ambient

temperature; discard the acetonitrile.
3. Add 20 mL of water and stir for 30 min at ambient tempera-
ture; discard the water.
4. Carefully wrap the stir-bar in a thick filter paper envelope and
remove the template by Soxhlet extraction with methanol/
acetic acid (50:50, v/v) overnight (~ 12 h) (see Note 6).
5. Place the MMIP-SB in a 50 mL beaker and perform a final wash
under stirring at ambient temperature for 30 min, first with
methanol and secondly with acetonitrile.
6. Place the MMIP-SB in a 50 mL bottle and keep it wet by
covering it with methanol; cap the bottle to preserve the stir-
bar from drying until use.
7. Before using the stir-bar with real samples, perform an optimi-
zation of the extraction protocol using fixed amounts of analyte
and testing different solvents for loading, washing, and elution
(i.e., methanol, toluene, acetonitrile, and water); test also dif-
ferent stirring times for each extraction step (see Note 7).
4 Notes
1. The Fe3O4 magnetite can be considered neutral when the

ammonium odor is gone.
2. After drying, the magnetite nanoparticles will be compacted.
Do not loosen it or sieve it. Instead, use it as “chunks” of
magnetite to mix with the polymerization mixture in the vial.
3. Template amount may vary, but it is recommended to be in a
ratio of 1:4 with the functional monomer. For toxic analytes, a
dummy template is recommended. This protocol uses a
non-covalent approach, but other alternatives are possible
with small adaptations. Cross-linker and porogen solvent
should not be modified since the amounts and composition
presented here have been optimized this application. Pre-poly-
merization mixture should be prepared fresh before polymeri-
zation and used immediately for the synthesis of the stir-bar. It
is recommended to fabricate a non-imprinted polymer stir-bar
via the same procedure but without the template.
4. Do not fill the vial; always leave headspace and avoid reaching
the neck of the vial when lying.
5. In this protocol a temperature-controlled incubator is used for
thermal polymerization; alternative bulk polymerization strate-
gies may be used, for example, a water bath.
6. Alternatively, the template can be removed by washing the stir-
bar in a 50 mL beaker with 20 mL of a mixture of methanol and
acetic acid (50/50, v/v) for 12 h, refreshing the solvent every

30 min. The absence of template must be confirmed by chro-
matography, or any other alternative method suitable for the
selected template.
7. To optimize the extraction protocol using the obtained stir-
bars, like for any other MIP, it is necessary to carry out several
experiments, using different solvents. If possible, select sol-
vents that combine good recoveries with green aptitudes. Opti-
mize also loading, washing, and elution times in order to seek
the highest recoveries. The obtained MMIP-SB may require
relatively long adsorption times, from 15 min to even an hour
or more. For complex and/or fatty samples, a pre-clean of the
matrix prior to MMIP-SB extraction (precipitation, centrifuga-
tion) is recommended, in order to extend the life of the stir-bar.
References
1. Azizi A, Bottaro CS (2020) A critical review of 6. Hasan CK, Ghiasvand A, Lewis TW, Nesterenko
molecularly imprinted polymers for the analysis PN, Paull B (2020) Recent advances in stir-bar
of organic pollutants in environmental water sorptive extraction: coatings, technical improve-
samples. J Chromatogr A 1614:460603 ments, and applications. Anal Chim Acta
2. Turiel E, Martı́n-Esteban A (2010) Molecularly 1139:222–240
imprinted polymers for sample preparation: a 7. Kawaguchi M, Takatsu A, Ito R, Nakazawa H
review. Anal Chim Acta 668:87–99 (2013) Applications of stir-bar sorptive extrac-
3. Haupt K (2003) Peer reviewed: molecularly tion to food analysis. TrAC Trends Anal Chem
imprinted polymers: the next generation. Anal 45:280–293
Chem 75:376 A–383 A 8. Hu Y, Li J, Hu Y, Li G (2010) Development of
4. Hu Y, Pan J, Zhang K, Lian H, Li G (2013) selective and chemically stable coating for stir bar
Novel applications of molecularly-imprinted sorptive extraction by molecularly imprinted
polymers in sample preparation. TrAC Trends technique. Talanta 82:464–470
Anal Chem 43:37–52 9. Dı́az-Álvarez M, Turiel E, Martı́n-Esteban A
5. Keçili R, Büyüktiryaki S, Dolak İ, Hussain CM (2019) Molecularly imprinted polymer mono-
(2020) 5 - The use of magnetic nanoparticles in lith containing magnetic nanoparticles for the
sample preparation devices and tools. In: Mus- stir-bar sorptive extraction of thiabendazole
tansar Hussain C (ed) Handbook of nanomater- and carbendazim from orange samples. Anal
ials in analytical chemistry: modern trends in Chim Acta 1045:117–122
analysis. Elsevier, pp 75–95
Chapter 15
Determination of Neopterin as a Prognostic Indicator Using

Neopterin-Imprinted Cryogel Membranes
Okan Zenger, Burcu Eren, Pırıl Arısoy, Sibel Özdaş,
and Gözde Baydemir Peşint
Abstract
Neopterin (Neo) is thought of as a key biomarker for the diagnosis and prognosis of a wide variety of
diseases associated with cellular immune response. Therefore, it has become a vital need to be able to
specifically determine the Neo concentration in human serum. Molecularly imprinted cryogels have come
into prominence among other affinity systems by combining advantages of Molecular Imprinting Technol-
ogy (MIT) and cryogels. In this chapter, synthesis of novel Neopterin-imprinted cryogel membranes (Neo-
mip), characterization studies of synthesized materials, and their use in the determination of Neo in human
serum is described in detail. In addition, the evaluation of selective Neo adsorption properties of Neo-mip
against competitors (Pterin and Glucose) is discussed. Neo-mip will come into prominence as important
affinity materials for the selective Neo recognition in body fluids, prior to use in the health sector.
Key words Neopterin, Biomarker, Prognostic Indicator, Specific determination, Molecular imprint-
ing technique, Cryogel membranes
1 Introduction
Neopterin (Neo) is a catabolic product found in body fluids (i.e.,

serum, cerebrospinal fluid, urine) which is synthesized by mono-
cytes/macrophages upon the stimulation with the cytokine
interferon-gamma, and it is indicative of a pro-inflammatory
immune status. Neo serves as a key indicator of cellular immune
system activation [1]. Neo in serum is useful as a prognostic marker
for cancer patients, pro-inflammatory immune response in infec-
tions, cardiovascular, neurodegenerative, and autoimmune diseases
[2–6]. At this point, it is very important to be able to specifically
determine the Neo concentration in the diagnosis of diseases and in
the follow-up of treatment. Enzyme-Linked Immunosorbent Anal-
ysis (ELISA) [7], High-Pressure Liquid Chromatography (HPLC)
[8] and Nucleic Acid Testing (NAT) [9] are commonly used
https://doi.org/10.1007/978-1-0716-1629-1_15,
171
172 Okan Zenger et al.
techniques for the sensitive detection, purification, and quantifica-

tion of Neo. However, these methods are time and money consum-
ing and high skilled labor. Due to these disadvantages, the need for
new techniques that save time has increased [10].
Molecular Imprinting Technology (MIT) is a current approach
in which a target molecule is used as the template during the
synthesis of a polymeric system and recognition cavities are con-
structed for the specific detection, purification, determination,
depletion, etc. of the target molecule. In MIT, specificity of an
affinity gel system to the molecule of interest depends on the
physical, chemical, and geometrical characteristics of the template
molecule [11–13]. Molecularly imprinted polymers (mip) have
come into prominence because of their ease of availability, simple
preparation, reusability, as compared to traditional methods used in
detection, purification, and quantification processes of biomole-
cules [14–17]. mip can be prepared in different forms and used in
different applications such as microspheres, nanoparticles, and
monoliths [18–20]. Among these, cryogels are among the forms
that are frequently used in studies.
Cryogel membranes are synthesized via cryo-polymerization
process at temperatures below 0 C. Their interconnected macropor-
ous morphology provides ease of diffusion and helps molecules of
different sizes flow easily, allowing mass transfer of nano- and micro-
sized particles [21–23]. The interconnected three-dimensional pore
structure of cryogels provides simple of working with viscous liquids
such as blood. Advantages of molecular imprinting technique have
been combined with that of cryogels and a more favorable structure
has been obtained in molecularly imprinted cryogels [16].
In this chapter, synthesis of 2-hydroxyethyl methacrylate
(HEMA)-based Neopterin-imprinted cryogel membranes (Neo-
mip) via cryo-gelation process and the selective detection of Neo
directly in human serum are described in detail.
2 Materials
According to the protocol, purified ultrapure water (the conductiv-

ity is 18 MΩ-cm at room temperature) is used for all solutions.
Reagents are stored in accordance with the information provided in
the storage protocol. The amounts of reagents specified in the
protocol are for synthesizing Neo-mip with a dry weight of approx-
imately 0.125 g each.
Preparation of Neopterin-Imprinted Cryogel Membranes 173
2.1 Preparation of 1. Template molecule: Neopterin (Neo).

Neo-ImprintedCryogel 2. Monomers: 1-Vinylimidazole (VIM) and 2-Hydroxyethyl
Membranes (Neo-mip) methacrylate (HEMA).
3. Crosslinker: Methylene bisacrylamide (mBAAm).
4. Initiator and catalyst: Ammonium persulfate (APS) and N,N,
N0 ,N0 -tetramethylene diamine (TEMED).
5. Ultrapure water.
6. Freezer.
7. Magnetic stirrer and fish.
8. Petri dishes (id. 100 mm, glass).
9. Perforator.
10. Membrane holder (id. 25 mm).
2.2 Cleaning 1. Ethanol/water solution (70:30, v:v).

the Cryogel 2. 0.1 M HCl in water.
Membranes
and Template Removal
Studies 4. UV spectrophotometer (275 nm).
2.3 Characterization 1. Scanning Electron Microscopy (SEM).

of Neo-mip and NIP 2. Lyophilizer.
Cryogel Membranes
3. Electronic balance.
4. Filter paper.
2.4 Adsorption 1. Neo.

Studies 2. 0.05 M HCl in water.
2.5 Evaluation 1. Neo.

of Selective Adsorption 2. Pterin.
Properties of Neo-mip
3. Glucose.
for Neo
2.6 Desorption 1. 0.1 M HCl in water.

Studies 2. Peristaltic pump.
2.7 Recognition 1. Commercial human serum (sterile and filtered).

of Neo from 2. Phosphate-buffered saline (PBS) pH 7.4.
Human Serum
3. ELISA kit.
3 Methods
3.1 Preparation Neo-mip cryogel membranes with a monomer concentration of

of Neo-imprinted 16% (w:w) are produced. The production protocol is as follows.
Cryogel Membranes
1. Dissolve 1.5 mL of HEMA monomer in 4 mL of ultrapure
(Neo-mip) water (see Note 1).
2. Dissolve 275 mg of methylene bisacrylamide (MBAA; cross-
linker) in 5 mL water.
3. Mix the solutions prepared in steps 1 and 2 in a beaker (see
Notes 2 and 3.
4. Prepare Neo-VIM pre-complex by dissolving 0.1 mol of Neo
molecules and 50 μL of VIM functional monomers in 1 mL
water and store it in the refrigerator for 4 h to complete
complexation.
5. Add the pre-complex into the HEMA-MBAA polymerization
solution (see Note 3).
6. Add 100 μL APS (APS stock solution: 100 mg/mL in water)
and 30 μL TEMED into the final solution.
7. Mix the final solution for 5 min in ice (see Notes 2 and 3).
8. Pour the final polymer solution into the petri dishes
(id. 100 mm, glass) and cover is closed.
9. Keep the petri dishes in the freezer at 20 C for at least 24 h to
complete the polymerization process (see Note 4).
10. Take cryogel membranes from the freezer and allow them to
reach room temperature to form interconnected macroporous
structure.
11. Cut cryogel membranes into circular pieces (25 mm in diame-
ter) using a perforator (see Fig. 1) (see Note 5).
12. Place 5 cryogel membranes in a membrane holder.
13. As the control group, follow the same protocol for
non-imprinted cryogel membranes and use only VIM for
synthesis.
3.2 Cleaning 1. Wash the cryogel membranes with ethanol/water solution

the Cryogel (30:70, v/v) for 48 h at room temperature.
Membranes 2. Pass 5 mL of 0.1 M HCl through the Neo-MIP cryogel mem-
and Template Removal branes at a rate of 0.5 mL/min in a continuous system using a
Studies peristaltic pump.
3. Measure the elution solutions (after every elution cycle) using
UV spectrophotometer at 275 nm.
4. Repeat the elution steps until no Neo molecules are left (see
Fig. 2).
Fig. 1 Digital photos of Neo-mip cryogel membranes (thickness ¼ 2 mm,

radius ¼ 12.5 mm)
Fig. 2 Representative illustration of the complexation between Neo and 1-Vinylimidazole (VIM) molecules
5. Wash Neo-mip cryogel membranes, in which Neo was

removed and Neo-specific cavities are formed, with ethanol
and water (30:70, v:v) for 2 h at room temperature.
Fig. 3 SEM image of Neo-mip cryogel membranes
3.3 Characterization 1. Evaluate the morphology and stability of Neo-mip and NIP
of Neo-MIP and NIP cryogel membranes using the scanning electron microscopy
Cryogel Membranes (SEM).
(a) Initially, the cryogel membranes dry using lyophilizer in
order to dehydrate the membranes completely.
(b) Mount a cross-sectional fragment of the dried cryogel
membranes on an SEM sample mount and coat with gold.
(c) Mount the sample in an SEM.
(d) Scan the surface of the sample at the desired magnification
to study the cryogel membranes (see Fig. 3).
2. Dry an Neo-mip cryogel membrane using a lyophilizer to
evaluate swelling properties of Neo-mip.
(a) Submerge Neo-mip in water and release the cryogel mem-
brane at room temperature for 24 h.
(b) Weigh dried cryogel membrane (Wo) and swelling cryogel
membrane (Ws).
(c) Use the following formula to calculate the swelling ratio
(SR) of Neo-mip.
SR ð%Þ ¼ ½ðW s W o Þ=W o 100 ð1Þ

(d) Calculate the polymerization yield of Neo-mip using mt
and mdry values which are the total amounts of reagents
and the weight of dried Neo-mip in grams, respectively.

Polymerization yield ð%Þ ¼ mdry =mt 100 ð2Þ
(e) Determine the swelling properties and polymerization

yield of NIP using the same protocol and equations.
3.4 Adsorption 1. In a continuous system, pass 5 mL of Neo solution (0.05 M

Studies HCl in water, pH 7.4) through Neo-mip and NIP cryogel
membranes for 2 h with the flow rate of 0.5 mL/min at 25 C.
2. Use the following equation, calculate the adsorption capacity
(Q, mg/g):
Q ¼ ½ðC i C f Þ V =m dry ð3Þ
In Eq. [3], Ci is the equilibrium Neo concentration
(mg/mL) in adsorption solution before passing through
Neo-mip and NIP cryogel membranes.
Cf is the equilibrium Neo concentration (mg/mL) in
adsorption solution after passing through Neo-mip and NIPs
(see Fig. 4).
V and mdry refer to the volume of the adsorption solution
(mL) and the dry weight of Neo-mip (g), respectively.
3.5 Evaluation Selective Neo adsorption properties of Neo-mip were tested, as

of Selective Adsorption compared to that of Pterin and Glucose competitor molecules (see
Properties of Neo-mip Note 6).
for Neo 1. Two separate solutions of competitors and Neo (concentration
of each competitor and Neo should be 0.075 mg/mL in 5 mL
0.05 M HCl).
2. Interact the Neo-competitor solution with Neo-mip.
3. Determine the concentration of absorbed molecules using a
UV spectrophotometer at 275 nm.
4. Calculate the distribution coefficient (Dc, mL/g) using the
given equation:
Dc ¼ ½ðC i C f Þ=C f V =m dry ð4Þ

5. Calculate imprinting factor (IF) for Neo and competitors using
the following formula:
IF ¼ DcNeomip =DcNIPs ð5Þ
In Eq. [5], DcNeo-mip and DcNeo-mip are the distribution
coefficient of Neo-mip and NIPs.
6. Evaluate selectivity of Neo-mip for the template molecule
according to the selectivity coefficient (k) of Pterin and Glu-
cose. Use the following equation in order to determine k values
for each competitor.
k ¼ DcNeomip ðtemplateÞ=DcNeomip ðcompetitorÞ ð6Þ
Fig. 4 Schematic representation of selective adsorption study of Neo-mip cryogel membranes for Neo
The DcNeo-mip(template) is the maximum adsorption

capacity of the template.
The DcNeo-mip(competitor) is the maximum adsorption
capacity of each competitor molecule in Neo-mip.
3.6 Desorption 1. Treat with Neo-MIP and NIP cryogel membranes in 0.1 M
Studies HCl in water (V: 5 mL) in order to desorp adsorbed molecules,
in a continuous system for 2 h the flow rate of 0.5 mL/min
using a peristaltic pump.
2. Use UV spectrophotometer for the determination of the con-
centration of desorbed molecules in the desorption medium at
275 nm.
3. Following equation is used for the determination of desorption
ratio (DR).
Quantity of desorbed molecules

DR ð%Þ ¼ 100
Quantity of adsorbed molecules
4. Wash Neo-mip and NIPs with water after every
desorption step.
3.7 Recognition The selective Neo rebinding from human serum is performed.
of Neo from
1. Pour 5 mL of commercially supplied crude human serum in a
Human Serum
sterile glass test tube (Test tube A) (See Notes 7 and 8).
2. Test tube A spiked with Neo. Dissolve 0.05 mg of Neo in test
tube A to get 0.01 mg Neo/mL human serum (Test tube B).
3. Take 1 mL of Neo spiked human serum from test tube B and

pour in another test tube (Test tube C).
4. Add 4 mL of PBS buffer (10 mM, pH 7.4) in the test tube C for
1:5 (v:v) dilution.
5. Take 1 mL of Neo spiked human serum from test tube B and
pour in another test tube (Test tube D).
6. Add 9 mL of PBS buffer (10 mM, pH 7.4) in the test tube D
for 1:10 (v:v) dilution.
7. Pump 3 mL of serum sample in test tube A through Neo–mip
cryogel membrane column for 2 h at 25 C and adjusted flow
rate at 0.5 mL/min.
8. Take 500 μL of serum samples before and after step 7 and store
them at 4 C before measurement of Neo amounts.
9. Repeat steps 7 and 8 for serum samples in test tube C and D.
10. Calculate Neo rebinding amounts from human serum samples
obtained in steps 7–9, using the ELISA kit [24, 25] (see
Note 10).
11. Follow the ELISA Kit protocol, the absorbance is measured at
450 nm with a microplate reader (see Note 11).
4 Notes
1. Prepared solutions should be stored at 4 C until use, unless

different information is specified.
2. All polymerization work for the synthesis of cryogels must be
carried out at low temperature. Ice bath will be sufficient in this
study.
3. A magnetic stirrer should be used during the synthesis process,
in order to achieve a homogen polymerization reaction.
4. Petri dishes must be placed flat in the freezer.
5. Store all the cryogel membranes in ultrapure water at 4 C.
Before storage, washing is done with an ethanol/ water solu-
tion (30:70, v: v).
6. When preparing selectivity test solutions, two separate compe-
titors and Neo solutions should be 0.075 mg/mL in 5 mL
0.05 M HCl in water.
7. This serum is not heat inactivated.
8. Serum should be stored in sterile glass tubes (NaN3-free) away
from heat and light, under aseptic conditions at 20 C until
analysis.
9. Preparation of phosphate-buffered saline solution, pH 7.4:
Dissolve 7.19 g NaCl, 1.56 g KH2PO4, 6.74 g anhydrous
Na2HPO4, 8.45 g Na2HPO4.2H2O, and 17.01 g Na2H-

PO4.12H2O in 1 L of water. The final concentrations are
123 mM sodium chloride, 11.5 mM potassium phosphate,
and 47.5 mM sodium phosphate.
10. Serum Neo concentration is determined by sandwich ELISA
method using a commercial kit.
11. Serum Neo levels in the protocol are expressed in nmol/L.
Dilution is required according to the safe detection limit range
given in the kit protocol.
References
1. Kuzmits R, Ludwig H, Legenstein E, determination of neopterin. Microchim Acta

Szekeresz T, Kratzik C, Hofbauer J (1986) 180:1401–1409
Neopterin as tumour marker serum and uri- 11. Sellergren B (2001) Molecularly imprinted
nary neopterin concentrations in malignant polymers: man-made mimics of antibodies
diseases. J Clin Chem Clin Biochem and their applications in analytical chemistry,
24:119–124 1st edn. Elsevier, New York
2. Murr C, Widner B, Wirleitner B, Fuchs D 12. Martin-Esteban A (2013) Molecularly-
(2002) Neopterin as a marker for immune sys- imprinted polymers as a versatile, highly selec-
tem activation. Curr Drug Metab 3:175–187 tive tool in sample preparation. TrAC Trends
3. Haelst PLV, Liem A, Boven AJV et al (2003) Anal Chem 45:169–181
Usefulness of elevated neopterin and 13. Baydemir G, Denizli A (2015) Heparin
C-reactive protein levels in predicting cardio- removal from human plasma using molecular
vascular events in patients with non–Q-wave imprinted cryogels. Artif. Cells Nanomed Bio-
myocardial infarction. Am J Cardiol technol 43:403–412
92:1201–1203 14. Chen L, Xu S, Li J (2011) Recent advances in
4. Wirleitner B, Schroecksnadel K, Winkler C, molecular imprinting technology: current sta-
Fuchs D (2005) Neopterin in HIV-1 infection. tus, challenges and highlighted applications.
Mol Immunol 42:183–194 Chem Soc Rev 40:2922–2942
5. Chin GK, Adams CL, Carey BS et al (2008) 15. Cheong WJ, Yang SH, Ali F (2012) Molecular
The value of serum neopterin, interferon- imprinted polymers for separation science: a
gamma levels and interleukin-12B polymorph- review of reviews. J Sep Sci 36:609–628
isms in predicting acute renal allograft rejec- 16. Liu Y, Zhai J, Dong J, Zhao M (2015) Mag-
tion. Clin Exp Immunol 152:239–244 netic surface imprinted hydrogel nanoparticles
6. Sucher R, Schroecksnadel K, Weiss G et al for specific and reversible stabilization of pro-
(2010) Neopterin, a prognostic marker in teins. Mol Imp 3:47–54
human malignancies. Cancer Lett 287:13–22 17. Cetin K, Denizli A (2019) Microcryogels as
7. Parker DC, Mielke MM, Yu Q et al (2012) plastic antibodies for transferrin purification.
Plasma neopterin level as a marker of peripheral Process Biochem 79:174–184
immune activation in amnestic mild cognitive 18. Zheng C, Huang YP, Liu ZS (2013) Synthesis
impairment and Alzheimer’s disease. Int J Ger- and theoretical study of molecularly imprinted
iatr Psychiatry 28:149–154 monoliths for HPLC. Anal Bioanal Chem
8. Baydar T, Kemer O, Kilicarslan B, Cardak A, 405:2147–2161
Girgin G (2016) Detection of neopterin in tear 19. Yao CY, Li QQ, Lu YH (2011) Preparation of
samples. Pteridines 27(1–2):13–16 MIP microspheres by precipitation polymeriza-
9. Fuchs D, Milstien S, Kramer A, Reibnegger G, tion with 1-Phenyl-1-propanol as template.
Werner ER, Goedert JJ, Kaufman S, W€achter Adv Mater Res 415-417:1225–1230
H (1989) Urinary neopterin 20. Rahtuvanoğlu A, Akgönüllü S, Karacan S,
concentrations vs. total neopterins for clinical Denizli A (2020) Biomimetic nanoparticles
utility. Clin Chem 35:2305–2307 based surface Plasmon resonance biosensors
10. Sole RD, Scardino A, Lazzoi MR et al (2013) A for histamine detection in foods. ChemistrySe-
molecularly imprinted polymer for the lect 5:5683–5692
21. Lozinsky VI, Galaev IY, Plieva FM et al (2003) blood substitutes, and immobilization biotech-
Polymeric cryogels as promising materials of nology 41
biotechnological interest. Trends Biotechnol 24. Gieseg SP, Crone EM, Flavall EA, Amit Z
21:445–451 (2008) Potential to inhibit growth of athero-
22. Dainiak M, Plieva F, Galaev I, Hatti-Kaul R, sclerotic plaque development through modula-
Mattiasson B (2005) Cell chromatography: tion of macrophage neopterin/7,8-
separation of different microbial cells using dihydroneopterin synthesis. Br J Pharmacol
IMAC supermacroporous monolithic columns. 153:627–635
Biotechnol Prog 21(2):644–649 25. Ray KK, Morrow DA, Sabatine MS et al (2007)
23. Perçin I, Baydemir G, Ergün B, Denizli A Long-term prognostic value of Neopterin. Cir-
(2012) Macroporous PHEMA-based cryogel culation 115:3071–3078
discs for bilirubin removal. Artificial cells,
Chapter 16
Fluorescence Sensing with Molecularly Imprinted

Polymer-Capped Quantum Dots
Hanieh Montaseri, Heidi Abrahamse, and Patricia B. C. Forbes
Abstract
Procedures for the design of a fluorescence sensor based on molecularly imprinted polymer-capped
quantum dots (MIP@QDs) together with the synthesis of quantum dots and MIP@QDS are described.
Spherical and monodispersed nanoparticles are suitable for fluorescence sensing of an analyte such as
pharmaceuticals and polycyclic aromatic hydrocarbons (PAHs). In addition, excellent optical properties,
higher quantum yield, and photoluminescence efficiency as well as easy detection of emission spectra are
distinctive advantages of quantum dots as fluorescence sensors. Optimization of different variables and
analytical applications of the sensor are also presented, which are of value for fluorescence sensing.
Key words Molecularly imprinted polymer, Quantum dot, Surface imprinting polymerization, Sol-
gel approach, Functional monomer, Cross-linker, Fluorescence sensing
1 Introduction
Molecularly imprinted polymers (MIPs) are synthetic polymeric

materials possessing specific recognition sites complementary in
terms of shape, size, functionality, and spatial structure to the
template molecule [1]. MIPs have been used in chemical assays or
sensors and as natural receptors due to their low cost, long lifespan,
intrinsic robustness, and tailor-made recognition sites for target
analytes [1].
In molecular imprinting, functional monomers are located
around the template molecules by non-covalent interaction or
reversible covalent interaction, followed by polymerization and
template removal. Consequently, a molecular “memory” within
the imprinted polymer matrix is created, and then the target mole-
cule can be selectively distinguished based on the molecular “mem-
ory” [2]. In covalent molecularly imprinting, the functional
monomer is attached to the template by reversible covalent bonds
during polymerization [3] while in non-covalent interactions, the
https://doi.org/10.1007/978-1-0716-1629-1_16,
183
184 Hanieh Montaseri et al.
functional monomer associates with the template via intermolecular

interactions such as hydrogen bonding or ion-pairing [4].
Although conventional μm particle sized MIPs can be prepared
at low cost, they may possess some limitations such as incomplete
template removal due to the high cross-linking nature of MIPs, low
binding capacity (due to the low surface area to volume ratio) and
slow mass transfer which reduces the number of recognition sites
for rebinding of template molecule [5]. In order to circumvent this
issue, imprinting techniques have been combined with nanomater-
ials. Due to the small size of these particles, below 100 nm, surface
area to volume ratios is markedly improved having higher binding
capacity, sensitivity, and faster rebinding kinetics [6, 7]. Among
various types of luminescent nanomaterials, quantum dots (QDs)
have drawn a lot of attention for use in vivo or in vitro imaging,
toxin detection, and fluorescence immunoassays [8].
Generally, MIP-nanocrystals can be fabricated through the
following mechanisms:
1. Applying fluorescent monomers to prepare fluorescent MIPs
by using the conventional synthesis of MIPs (Fig. 1a).
2. Copolymerization of nanomaterials such as QDs and fluores-
cent dyes with functional monomers, cross-linkers, and a tem-
plate molecule (Fig. 1b).
3. Through the encapsulation approach which involves fixing of
nanomaterials and templates to pre-synthesized polymers via
chemical or physical techniques (Fig. 2).
Different techniques, which can be applied to synthesize an
MIP are summarized in Table 1. Each protocol possesses its own
benefits and limitations, which makes it suitable for various applica-
tions. Surface imprinting polymerization, for example, could be
used in diverse applications such as sensing, separation, and medical
applications [9].
2 Materials
All reagents are analytical grade and prepared in Millipore water

with a conductivity of <18 MΩ cm at 25 C. All glassware must be
cleaned with H2O2/H2SO4 mixture, detergents, Millipore water,
and acetone to remove any bound metals and contaminations
followed by drying in an oven. Prepare all precursors and solutions
before synthesis and use immediately. Disposing of wastes must be
followed based on disposal regulations in separated containers.
2.1 Quantum Dots 1. Precursor solutions: octadec-1-ene (ODE), oleic acid (OA),
trioctylphosphine oxide (TOPO), zinc oxide, cadmium oxide
(CdO), sulfur powder (S), selenium powder (Se) (see Note 1).
Molecularly Imprinted Polymer Capped Quantum Dot Fluorescence Sensors 185
Fig. 1 Formation process of (a) conventional MIPs through in situ polymerization and (b) luminescent MIP
nanocomposites (MIP-NCs) through copolymerization. Reproduced from [7] 2015 with permission from
Elsevier
2. Coil condenser must be cooled by water to avoid solvent

evaporation. An assembly is shown in Fig. 3.
3. 250 mL three-neck flask is fitted with a septum, refluxing
condenser, and thermometer.
4. High-quality nitrogen supply as an inert atmosphere is used.
Introduce the inert gas through the septum into the three-neck
flask.
5. Stirring heating mantle and magnetic bar.
Fig. 2 Preparation of quantum dots capped molecularly imprinted polymer

(MIP@QDs) nanospheres and the fluorescence quenching detection of analytes
based on specific recognition sites. The template, QDs, and copolymer were
dissolved in chloroform and then added into water. The MIP@QDs were prepared
via ultrasonication, evaporating of solvent and template removal via washing
with methanol. Reproduced from [15] 2012 with permission from American
Chemical Society
2.2 Water 1. Methanol (analytical grade), potassium hydroxide, thiol ligand

Solubilization (e.g., L-cysteine or glutathione), Millipore water.
of the QDs 2. Methanol, chloroform, and acetone for washing and
purification.
3. Magnetic stirrer, stir bar, and 250 mL beaker.
4. Laboratory centrifuge with at least 2520 g.
5. Centrifuge tubes.
2.3 Molecularly 1. Functional monomer: 3-amino-propyltriethoxysilane

Imprinted (APTES).
Polymer-Capped 2. Cross-linker: tetraethyl orthosilicate (TEOS).
Quantum Dots
3. Solvent: anhydrous ethanol (grade).
(MIP@QDs)
4. Catalyst: ammonia solution.
5. Template: for example, acetaminophen (AC).
6. Magnetic stirrer, stir bar, and 250 mL beaker.
7. Desiccator connected to a vacuum pump.
8. Fluorescence spectrofluorometer to record fluorescence emis-
sion spectra.
9. Quartz cuvette.
10. Ultrasonication water bath.
Table 1
Preparation of MIPs by different techniques [9–14]
MIP type Advantages Disadvantages

Bulk polymerization Simplicity, robustness, higher adsorption capacity, simple Irregular particles, loss of sample during crushing and sieving,
instrumentation lower MIP loading capacity, less recognition sites, high
consumption of the template, difficult to scale up, time-
consuming
Suspension Spherical particles, uniformly sized microspheres, reproducible, Surfactant polymers required, water is incompatible with
polymerization simple preparation, heterogeneous polymerization, excellent imprinting procedures
heat dispersion, suitable for industrial scale-up
Multi-step swelling Uniform and mono-dispersed spherical polymer particles Time-consuming and sophisticated procedures, difficult to
polymerization develop hydrophilic surface, requires aqueous emulsions
Precipitation Imprinted micro and nanospherical shapes, uniform size, higher High dilution factor, large amount of template, careful
polymerization active surface area, high yield, greater recognition binding optimization for monomer type and concentration,
ability compared to the conventional methods, no need for aggregation occurs
grinding and sieving
Surface Mono-dispersed particles, thin imprinted layers, simple, Complicated system, time consuming
polymerization inexpensive, diverse applications, easy to control MIP thickness
below 50 nm, fast response time, high selectivity towards the
template, more accessible sites, rapid binding kinetics and mass
transfer
In-situ Low cost, simple procedures, porous and permeable site, suitable Difficult optimization procedures for each template, large
polymerization for miniaturized devices, helpful for large scale, easy amount of cross-linker, poor mechanical stability, low
preparation, high-speed and high performance, high sensitivity, membrane permeability for separation purposes
selectivity and reproducibility, high yield, small amount of
template, suitable for large biopolymers and small molecules,
Molecularly Imprinted Polymer Capped Quantum Dot Fluorescence Sensors
rapid mass transport

187
2.4 Template 1. Laboratory centrifuge with at least 2520 g.

Removal from 2. Anhydrous ethanol (grade).
MIP@QDs
3. Centrifuge tubes.
3 Methods
3.1 Preparation 1. Prepare the Se precursor by dissolving 1.9 g of TOPO in 25 mL

of QDs of ODE and heating up to 80 C to form a clear solution. Add
0.3 g of Se powder and stir continuously to form the TOPSe
3.1.1 Preparation
solution (see Note 1).
of Precursors
2. Sulfur precursor: 1.9 g of TOPO in 30 mL of ODE, and 20 mL
of OA is heated to 80 C and when a clear solution is produced,
0.16 g of S powder is added.
3. Zinc precursor: 0.41 g of ZnO powder is dissolved in 30 mL of
ODE and 20 mL of OA.
3.1.2 Synthesis of QDs 1. Assemble reflux setup according to Fig. 3.

2. Turn on the heating stirrer mantle and set temperature to
~280 C.
3. Under inert atmosphere, 1.3 g of CdO is added into a solution
of 50 mL of ODE and 30 mL of OA and vigorously stirred the
solution to a temperature of ~280 C to fabricate a colorless
Cd-OA complex.
4. Inject TOPSe solution into the solution to allow for nucleation
and growth of core QDs which will result in an intense red
solution.
5. Allow the nucleation and growth of the core QDs to carry on
for about 15 min (Fig. 4a).
Fig. 3 Schematic reflux setup for the synthesis of QDs

Fig. 4 Synthesis of (a) QDs by the organometallic approach and (b) surface modification by thiol ligands.
Adopted from [16] 2018 Montaseri Thesis
6. Reduce the temperature to ~200 C by changing the setting of

heating mantle and then add zinc and sulfur precursors to
passivate epitaxially the core surface with a ZnS shell.
7. Take small aliquots of the sample at different time intervals and

record their fluorescence spectra to achieve a desired size.
8. Leave the QDs under the fume hood to cool them down and
then add methanol and leave for 15 min.
9. Discard the supernatant to completely remove unreacted
precursors.
10. Add acetone to the QDs and leave them overnight to precipi-
tate (OA–TOPO-capped QDs).
3.1.3 Water 1. Dissolve 3.0 g KOH with 40 mL of methanol then add 2.0 g
Solubilization of the QDs thiol ligand such as L-cysteine and glutathione (thiol–KOH
with Thiol Ligands methanolic solution.).
2. Discard acetone from the precipitate of the QDs achieved from
the previous procedure.
3. Disperse the purified OA–TOPO-capped QDs in chloroform.
4. Add thiol–KOH methanolic solution followed by the addition
of Millipore water to the QD solution and stir for 1 h (Fig. 4b).
5. Purify the QDs with acetone and chloroform via centrifugation
at 2520 g for 5 min and repeat centrifugation (see Note 2).
3.2 Synthesis 1. Dissolve 0.1 mmol AC in 20 mL anhydrous ethanol and add

of MIP@QDs 0.4 mmol of functional monomer (APTES). Stir and let equili-
brate for 1 h. Template to functional monomer ratios of 1:4
and higher are preferable for non-covalent imprinting (see Note
3).
2. Inject 5 mL of the QDs (see Note 4) followed by 100 μL of
cross-linker (TEOS) and 100 μL of catalyst (ammonia solu-
tion). Stir the mixture for 24 h.
3. Use anhydrous ethanol to purify the MIP@QDs via centrifuga-
tion at 2520 g for 5 min until the fluorescence intensity of
MIP-capped QDs remains constant.
4. Dry the resultant polymer under vacuum at room temperature
and store in a desiccator for further use.
5. Prepare non-imprinted polymers (NIP@QDs) in the same pro-
cedure but without the addition of template and keep them in a
desiccator at room temperature.
3.3 Optimization 1. Prepare different concentrations of MIP@QDs in 3 mL of

of Sensing Variables Millipore water and disperse by sonication for 10 min.
3.3.1 Concentration 2. Add a fixed concentration of a standard solution of the
of MIP@QDs template.
3. Measure fluorescence intensity of different concentrations of
MIP@QDs in the presence of the template.
4. Calculate a series of F0/F values. F0 and F are the fluorescence

intensity of MIP@QDs in the absence and presence of the
template, respectively (see Note 5).
3.3.2 Effect 1. Dissolve the optimum amount of the MIP@QDs in Millipore

of Incubation Time water, which can be found from the optimization of
concentration.
2. Add a fixed concentration of a standard solution of the
template.
3. Record fluorescence spectra every 2 min.
4. Calculate the ratio of F0/F. The values of F0 and F are the
fluorescence intensity of MIP@QDs in the absence and the
presence of the template, respectively (see Note 5).
3.4 Fluorescence 1. Dissolve the optimum concentration of the MIP@QDs in

Sensing Millipore water and incubate for the optimum time (Found
of the Template Using from Subheading 3.3).
MIP@QDs 2. Record the fluorescence spectrum of MIP@QDs in the pres-
in a Standard Solution ence of Millipore water as F0 value.
3. Inject known amounts of the template solution to the polymer
solution and consider incubation time as well.
4. Record F value as the fluorescence intensity of the polymer in
the presence of different concentrations of template (see Note
6).
5. Through the variation of template concentration, a series of
F0/F values are obtained.
6. In the system of MIP@QDs-template, fluorescence quenching
is observed which can be described according to Stern–Volmer
equation (Eq. 1).
F0
¼ 1 þ K sv ½Q ð1Þ
F
F0 and F are the steady-state fluorescence intensities in the
absence and presence of the quencher, respectively. [Q] is the
total analyte concentration and KSV represents the Stern–Vol-
mer quenching constant.
7. Calibration curve: Plot F0/F versus the concentration of tem-
plate in order to find the linear equation and to calculate limit
of detection (LOD) and limit of quantification (LOQ) as
follows:
LOD ¼ 3δ=m ð2Þ
LOQ ¼ 10δ=m ð3Þ
where m is the slope of the calibration curve and δ is the

standard deviation of the blank measurement (n ¼ 10) (see
Note 7).
3.5 Determination 1. Centrifuge samples at 2040 g for 5 min and then filter
of the Template through 110 mm pore size filter paper before analysis to eradi-
in Water Matrices cate suspended matter.
Using Fluorescence 2. Use standard addition to add different amounts of standard
Sensing of MIP@QDs solution of template to the water samples within the linear
range.
3. Carry out fluorescence measurements for each concentration
(F). Add real water sample to the solution of MIP@QDs and
measure fluorescence intensity (F0).
4. Use the linear equation, which can be found from Subheading
3.4 to find the concentration of the unknown in real water
samples.
4 Notes
1. The described procedure was applied for the synthesis of

CdSe/ZnS QDs. Regarding the synthesis of alloyed core/
shell QDs such as CdSeTe/ZnS QDs, premixed TOPTe solu-
tion is prepared with 0.48 g of Te and 1.93 g of TOPO in
25 mL of ODE and injected to the colorless Cd-OA complex
swiftly followed by the addition of TOPSe solution. Further-
more, core/shell/shell QDs, for example, CdSeTe/ZnSe/ZnS
QDs can be prepared as follows: prepare Zn and Se precursors
for the ZnSe shell as explained in Subheading 3.1. For the
passivation of the CdSeTe/ZnSe core/shell with a ZnS shell,
dissolve 0.407 g of zinc diethyldithiocarbamate (ZDC) in
30 mL of ODE and 20 mL of OA. This precursor is immedi-
ately added to the growth solution of CdSeTe/ZnSe QDs.
2. For the complex alloyed core/shell QDs or core/shell/shell
QDs, follow these steps for purification: step 1: acetone; step
2: chloroform; step 3: water: chloroform: acetone (1:1:2)
mixture; and step 4: chloroform followed by acetone.
3. In this procedure, MIP@QDs are prepared as an example via an
adapted Stöber method (sol-gel approach) and surface imprint-
ing technique to form non-covalent interactions between acet-
aminophen and the APTES [17]. However, different MIP
compositions comprised of various functional monomers,
cross-linkers, initiators, and solvents can be prepared. In the
case of expensive, template leakage, poorly available templates
or target analytes with low solubility, a structural analogue or a
dummy molecule can be used which has similar shape and
structure to the template. Regarding free radical polymeriza-

tions, use an initiator such as 2,20 -azobisisobutyronitrile
(AIBN) instead of catalyst [18]. Generally, the template can
participate in free radical polymerization if it would have any
polymerizable groups and is stable under polymerization con-
ditions; otherwise, other polymerization techniques such as
surface imprinting have to be adopted.
4. The appropriate concentration of the QDs for the synthesis of
MIP@QDs can be found by optimization of the QDs alone in
the presence of a fixed concentration of the template.
5. F0 value is the maximum fluorescence of the MIP@QDs in the
presence of Millipore water (in the absence of template). In this
regard, add Millipore water to the MIP@QDs and record the
fluorescence spectrum.
6. In order to employ the excitation mode for the fluorescence
measurements, record the absorption of the MIP@QDs with
UV/Vis spectroscopy and use the maximum absorption wave-
length as the excitation wavelength (λex). Emission wavelength
is adjusted over the range of (λex + 10)800 nm with excitation
and emission slit widths of 5 nm.
7. In order to calculate the standard deviation for LOD and LOQ
determination, the maximum fluorescence intensity of the
MIP@QDs alone is the F0 value while the maximum fluores-
cence intensity of the MIP@QDs in the presence of Millipore
water is considered as the F values (repeat it 10 times). So a
series of F0/F is achieved to calculate the standard deviation.
Ultimately, computational chemistry could assist in the selec-
tion of the templates, functional monomers and cross-linker to
optimize the binding affinity of the synthesized polymers with
target analytes. Different polymerization techniques can be applied
based on Table 1 in diverse applications such as sensing, separation,
biological and medical uses. However, a number of challenges still
remain, for example, low sensitivity of MIP-based fluorescent sys-
tems needs to be improved. It has been suggested that decreasing
the MIP-shell thickness and ratiometric fluorescence measurements
are promising approaches to improve sensitivity [19].
Acknowledgments
The authors sincerely thank the University of Pretoria, the Water

Research Commission (Grant K5/2438/1 and K5/2752), the
Photonics Initiative of South Africa (Grant PISA-15-DIR-06),
the University of Johannesburg, the National Laser Centre and
the National Research Foundation-South African Research Chairs
Initiative (NRF-SARChI) for their financial grant support (Grant
98337).
References
1. Chen L, Xu S, Li J (2011) Recent advances in analytical applications. Recent Research in
molecular imprinting technology: current sta- Polymerization. InTech, London, pp 47–67
tus, challenges and highlighted applications. 11. Pichon V, Chapuis-Hugon F (2008) Role of
Chem Soc Rev 40(5):2922–2942 molecularly imprinted polymers for selective
2. Fang C, Yi C, Wang Y, Cao Y, Liu X (2009) determination of environmental pollutants—a
Electrochemical sensor based on molecular review. Anal Chim Acta 622(1):48–61
imprinting by photo-sensitive polymers. Bio- 12. Yemis F, Alkan P, Yenigül B, Yenigül M (2013)
sens Bioelectron 24(10):3164–3169 Molecularly imprinted polymers and their syn-
3. Kempe M, Mosbach K (1995) Molecular thesis by different methods. Polym Polym
imprinting used for chiral separations. J Chro- Compos 21(3):145
matogr A 694(1):3–13 13. Beltran A, Borrull F, Marcé R, Cormack P
4. Yu C, Mosbach K (1997) Molecular imprinting (2010) Molecularly-imprinted polymers: use-
utilizing an amide functional group for hydro- ful sorbents for selective extractions. TrAC
gen bonding leading to highly efficient poly- Trends Anal Chem 29(11):1363–1375
mers. J Org Chem 62(12):4057–4064 14. Gokmen MT, Du Prez FE (2012) Porous poly-
5. Schirhagl R (2014) Bioapplications for molec- mer particles—a comprehensive guide to syn-
ularly imprinted polymers. Anal Chem 86 thesis, characterization, functionalization and
(1):250–261 applications. Prog Polym Sci 37(3):365–405
6. Figueiredo L, Erny GL, Santos L, Alves A 15. Zhao Y, Ma Y, Li H, Wang L (2012) Compos-
(2016) Applications of molecularly imprinted ite QDs@MIP nanospheres for specific recog-
polymers to the analysis and removal of per- nition and direct fluorescent quantification of
sonal care products: a review. Talanta pesticides in aqueous media. Anal Chem 84
146:754–765 (1):386–395
7. Ma Y, Xu S, Wang S, Wang L (2015) Lumines- 16. Montaseri H (2018) Quantum
cent molecularly-imprinted polymer nanocom- dot-molecularly imprinted polymer nanoma-
posites for sensitive detection. TrAC Trends terials for the fluorescence sensing of selected
Anal Chem 67:209–216 pharmaceutical and personal care products.
8. Rhyner MN, Smith AM, Gao X, Mao H, Thesis. University of Pretoria
Yang L, Nie S (2006) Quantum dots and mul- 17. Montaseri H, Forbes PB (2018) Molecularly
tifunctional nanoparticles: new contrast agents imprinted polymer-coated quantum dots for
for tumor imaging. Nanomedicine 1 fluorescence sensing of acetaminophen. Mater
(2):209–217 Today Commun 17:480–492
9. Yan H, Row KH (2006) Characteristic and 18. Nsibande SA, Forbes PB (2019) Development
synthetic approach of molecularly imprinted of a quantum dot molecularly imprinted poly-
polymer. Int J Mol Sci 7(5):155–178 mer sensor for fluorescence detection of atra-
10. Madikizela L, Tavengwa N, Pakade V (2018) zine. Luminescence 34(5):480–488
Molecularly imprinted polymers for pharma- 19. Chen L, Wang X, Lu W, Wu X, Li J (2016)
ceutical compounds: synthetic procedures and Molecular imprinting: perspectives and appli-
cations. Chem Soc Rev 45(8):2137–2211
Chapter 17
Dual-Fluorescent Nanoparticle Probes Consisting of a

Carbon Nanodot Core and a Molecularly Imprinted Polymer
Shell
Shan Jiang, Kornelia Gawlitza, and Knut Rurack
Abstract
Dual-fluorescent molecularly imprinted nanoparticles with a red-emissive carbon nanodot-doped silica core
and a chlorogenic acid-imprinted fluorescent polymer layer are prepared and their use in ratiometric
fluorometric analysis is described. Nanoparticle probes consisting of a shielded and stably emitting core
and a shell with embedded binding sites that indicates the presence of an analyte with a change in emission
allow for internally referenced measurements potentially accounting for detrimental influences from instru-
ment drifts, light source fluctuations or sensor materials-related inhomogeneities.
Key words Molecular imprinting, Fluorescence, Core-shell particles, Chlorogenic acid, Ratiometric
measurement
1 Introduction
Unlike conventional antibodies, molecularly imprinted polymers

(MIPs), being of nonbiological origin, are robust, resistant against
denaturing solvents and elevated temperatures, and can be repro-
ducibly prepared at low cost. Recent reports on “plastic antibodies”
mimicking natural antibodies with respect to both analytical per-
formance and application areas were met with excitement [1–
3]. Particularly, fluorescent MIPs (fMIPs) appear as one of the
most promising candidates for chemical sensing, thanks to the
high sensitivity, ease of operation, and format adaptability of fluo-
rescence [4–6].
However, known fMIP-based sensors for the quantification of
analytes almost exclusively refer to single-signal systems [7, 8]. Yet
especially in complex samples, single-signal measurements are often
affected by spatial or temporal changes in concentration or an
inhomogeneous distribution of the sensor materials, instrumental
fluctuations and environmental conditions [9], which make reliable
https://doi.org/10.1007/978-1-0716-1629-1_17,
195
196 Shan Jiang et al.
quantitative determination difficult. Compared with single-signal

output, ratiometric or dual-signal recording is independent of sen-
sor and/or probe concentration and provides an intrinsic correc-
tion to overcome the potentially detrimental effects mentioned
above, therefore allowing unequivocal quantification of analytes
[10–12]. A dual-fluorescent MIP system can be principally
designed as a core-shell platform with (1) a fluorescent core, the
fluorescence of which remains virtually unchanged, that is coated
with (2) an fMIP shell, the fluorescence of which responds to the
presence of an analyte that was initially imprinted as the template.
Herein, red-emissive carbon nanodots (R-CNDs), an emerging
class of fluorescent nanomaterials [13, 14], were enveloped with
silica (R-CSNs), serving as the fluorescent core of dual-fluorescent
MIPs (Fig. 1). After a two-step reversible addition–fragmentation
chain-transfer (RAFT) agent functionalization, an fMIP shell,
being imprinted with chlorogenic acid (CGA), a polyphenol
showing antioxidant, antibacterial, anti-inflammatory, anti-micro-
bial, and anti-hypertension activities [15], was grafted from the
surface of the core. The basic principle of molecular imprinting is
the creation of artificial binding cavities in a polymer network which
are complementary in size, shape, and spatial arrangement of func-
tionalities to the template. MIPs thus can provide strong affinity to
the template molecule with high selectivity. Obviously, CGA con-
tains multiple functionalities, e.g., a carboxylic acid group, a phenyl
ring and diol moieties, which can assist the formation of binding
cavities in an fMIP shell not only via covalent bonds, such as the
cyclic diester formed by the diol and boronic acid motifs, but also
noncovalent bonds, such as π-π stacking between phenyl rings.
Furthermore, based on previous studies [16, 17], deproto-
nated CGA was imprinted after reaction with tetrabutylammonium
hydroxide (TBA-OH) as the TBA salt with the urea-containing
fluorescent monomer (I). The fluorescence of I is usually enhanced
upon hydrogen bond-assisted complexation between the urea
motif and a carboxylate anion. Combination of the red emitting
CNDs with green emitting I allowed us to construct dual-
fluorescent CGA-MIP probes, in which use of a single excitation
wavelength (e.g., 410 nm) produces an analyte-insensitive fluores-
cence signal at ca. 680 nm, stemming from the R-CNDs, and a
CGA-sensitive fluorescence signal at ca. 510 nm, correlating to the
amount of CGA anion bound by I. With these features, dual-
fluorescent hybrid MIP probes can be generated against many
other bioactive compounds, eventually establishing fMIP-based
sensor platforms as a powerful approach to ratiometric (bio)-
chemical analysis.
Dual-Fluorescent Core-Shell Nanoparticle Probes 197
Fig. 1 (a) Schematic overview of the synthetic workflow for dual-fluorescent MIPs. (b) Hydrogen bond
formation between template and fluorescent monomer (I). (c) Structure of competitor caffeic acid (CA)
2 Materials
2.1 Synthesis 1. L-Glutathione reduced (98.0%).

of Red-Emissive 2. Formamide (99.5%).
Carbon Nanodots
3. Membrane filter (PVDF, 25 mm).
(R-CNDs)
4. Methanol and acetone (analytical grade, 99.5%), Milli-Q®
water (resistance 18 MΩ l cm at 25 C).
2.2 Synthesis 1. 4-Chloro-7-nitrobenzofurazan (99%).

of Fluorescent 2. NH3·H2O (32%).
Monomer (I)
3. 2,6-Di-tert-butyl-4-methylphenol (BHT) (>99%).
4. 2-Isocyanatoethyl methacrylate (98%).
5. Acetonitrile (MeCN) (HPLC grade, 99.95%), cyclohexane
and acetone (analytical grade, 99.5%), tetrahydrofuran
(THF) (anhydrous, 99.9%).
2.3 Synthesis of Red 1. Triton-X 100.

Carbon 2. Tetraethyl orthosilicate (TEOS) (>99%).
Nanodot-Doped Silica
3. Absolute ethanol and cyclohexane (analytical grade, 99.5%),
Nanoparticles
n-hexanol (99%).
(R-CSNs)
2.4 RAFT Agent 1. (3-Aminopropyl)triethoxysilane (APTES) (98%).

Functionalization 2. 4-Cyano-4-(thiobenzoylthio)pentanoic acid (CPDB) (97%).
3. Ethyl chloroformate (97%).
4. Triethylamine (99.7%).
5. Hydrochloric acid (37%).
6. Cyclohexane and petroleum ether (PE) (analytical grade,
99.5%), toluene (anhydrous, 99.8%).
2.5 MIP Synthesis 1. Chlorogenic acid (CGA) (97%).

2. Fluorescent monomer (I).
3. Tetrabutylammonium hydroxide solution (TBA-OH)
(1.0 mol L1 in methanol).
4. 4-Vinylphenylboronic acid (VBA) (97%).
5. Benzyl methacrylate (BMA) (96%).
6. Ethylene glycol dimethacrylate (EGDMA) (98%).
7. 2,20 Azobis(2,4 dimethyl)valeronitrile (ABDV).
8. Formic acid (>99%).
9. Chloroform (Anhydrous, 99.9%).
2.6 Equipment 1. Hydrothermal autoclave reactor (volume 30 mL).

2. Teflon-lined vessel (volume 30 mL).
3. Magnetic stirrer with heating function.
4. Column chromatography setup.
5. Beaker, round-bottom flask, glass vial, Eppendorf tube,
centrifuge tube.
6. Water bath sonicator.
7. Centrifuge with rotors.
2.7 Characterization 1. Caffeic acid (CA) (95%).

and Evaluation of MIP 2. Zetasizer with folded capillary cell and plastic disposal cuvette.
Binding Performance
3. Transmission electron microscope (TEM) with grid.
(Recommended)
4. Absorption and fluorescence spectrometers with stirring
function.
3 Methods (See Note 1)
3.1 Synthesis 1. Dissolve 4-chloro-7-nitrobenzofurazan (1 g, 5.01 mmol) in

of Fluorescent Probe 25 mL MeCN in a 50 mL round-bottom flask.
Monomer (I) (See Note 2. Add 3 mL NH3·H2O to the mixture and stir for 2 h at RT
1) (in this work, RT, i.e., room temperature refers to 22 1 C).
3. Filter out the insoluble solid of the reaction mixture with a filter
paper.
4. Evaporate MeCN with a rotary evaporator and purify the resi-
due by column chromatography (400 70 mm glass chroma-
tography column, acetone-cyclohexane from 3:1 to 1:1) on
silica gel to obtain 4-amino-7-nitrobenzofurazan in 0.54 g
(60%) as a brown powder. 1H NMR (400 MHz, CDCl3):
δ ¼ 8.46 (2H, d, J 8.0), 6.41 (2H, d, J 8.0).
5. Under Ar atmosphere, dissolve 4-amino-7-nitrobenzofurazan
(200 mg, 1.1 mmol) and 20 mg BHT in 10 mL anhydrous
THF in a 25 mL round-bottom flask.
6. Add 2-isocyanatoethyl methacrylate (257 mg, 1.7 mmol) to
the flask and stir the reaction mixture for 16 h at RT.
7. Evaporate MeCN with a rotary evaporator and purify the reac-
tion mixture by column chromatography (300 50 mm glass
chromatography column, acetone-cyclohexane from 5:1 to
2:1) on silica gel to yield I in 294.8 mg as a yellow powder.
1
H NMR (400 MHz, CDCl3): δ ¼ 8.49 (1H, d, J 8.5), 8.18
(1H, d, J 8.5), 8.13 (1H), 6.11 (1H, t), 5.65 (1H, t) 5.58 (1H,
t), 4.32 (2H, t, J 5.0), 3.63 (2H, q, J 5.0), 1.90 (3H, s). 13C
NMR (100 MHz, DMSO-d6): δ ¼ 166.02, 153.20, 144.86,
142.94, 136.33, 135.28, 127.04, 125.61, 108.93, 62.98,
37.97, 17.51. HRMS (ESI-): m/z [M-H] calcd. For
C13H13N5O6: 334.0793, found: 334.0790 [16].
3.2 Synthesis 1. Add 0.6 g glutathione and 20 mL formamide in a Teflon-lined

of R-CNDs vessel.
2. Tightly seal the prepared 3 wt% glutathione suspension in an
autoclave reactor.
3. Heat autoclave reactor at 160 C for 5 h in an oven.
4. Let the reactor cool down to RT.
5. Dilute the reaction mixture with 150 mL Milli-Q water.
6. Precipitate crude R-CNDs by the addition of 100 mL of
acetone.
7. Centrifuge the solution at 8900 g for 10 min.
8. Wash crude R-CNDs with 20 mL acetone once and with
methanol/acetone (10/90 vol. %) three times (10 min centri-
fugation at 8900 g for each washing step), respectively (see
Note 2).
9. Redisperse crude R-CNDs in 30 mL of methanol and remove

large particles of crude R-CNDs by filtration with a 0.22μm
membrane filter.
10. Evaporate methanol with a rotary evaporator.
11. Dry R-CNDs at RT in a vacuum oven overnight to obtain
0.1 mg R-CNDs as a black oily solid. (see Note 2).
3.3 Synthesis 1. Add 89 g Triton X-100, 385 mL cyclohexane, 80 mL

of R-CSNs n-hexanol, and 17 mL Milli-Q water to a 1000 mL beaker
and stir the emulsion (800 rpm) at RT for 15 min (see Note 3).
2. Dissolve 0.1 mg R-CNDs by adding 10 mL Milli-Q water and
transfer the solution to a glass vial.
3. Adjust the pH of R-CND solution to pH 2 by adding hydro-
chloric acid dropwise.
4. Add 2 mL of the R-CND solution to the beaker and stir the
emulsion (800 rpm) for another 5 min.
5. Add 5 mL TEOS to the beaker and stir the emulsion (800 rpm)
for another 30 min.
6. Initiate the hydrolysis of TEOS by adding 3 mL
NH3·H2O (32%) and stir the emulsion (500 rpm) at RT for
16 h (see Note 4).
7. To break the emulsion, add ca. 200 mL absolute ethanol to the
beaker and collect R-CSNs by centrifugation (8900 g for
5 min).
8. Wash R-CSNs twice with absolute ethanol and once with Milli-
Q water (ca. 250 mL, 5 min centrifugation at 8900 g, for
each washing step). Sonicate R-CSN suspension in every
washing step.
9. Dry R-CSNs at RT in a vacuum oven overnight to obtain a
white solid.
3.4 RAFT Agent 1. Add 1.0 g R-CSNs, 8 mL APTES, and 120 mL anhydrous
Functionalization toluene to a 250 mL round-bottom flask (see Note 5).
3.4.1 APTES 2. Sonicate the suspension for 20 min and further degas for
Functionalization 30 min.
3. Under Ar atmosphere, heat the suspension to reflux
(ca. 120 C) with stirring (500 rpm) for 16 h.
4. Let the reaction mixture cool down to RT.
5. Precipitate APTES-R-CSNs by adding 20 mL cyclohexane and
centrifuge for 5 min at 8900 g.
6. Wash APTES-R-CSNs three times with 80 mL toluene (5 min
centrifugation at 8900 g and 5 min sonication for each
washing step).
7. Dry APTES-R-CSNs at RT in a vacuum oven overnight to
obtain white solid.
3.4.2 4-Cyano- 1. Add 238 mg CPDB, 25 mL anhydrous THF, 82μL ethyl

4-(Phenylcarbonothioylthio) chloroformate, and 118μL triethylamine to a 100 mL round-
Pentanoic Acid (CPDB) bottom flask and flush the CPDB mixture with Ar while
Functionalization stirring.

2. Keep the mixture at 78 C for 40 min under an Ar
atmosphere.
3. In the meantime, dissolve 1.0 g APTES-R-CSNs in 15 mL
anhydrous THF, flush with Ar at 10 C.
4. Add cooled APTES-R-CSNs to the CPDB mixture under Ar
atmosphere and stir (500 rpm) for 24 h at RT.
5. Precipitate RAFT-R-CSNs by adding ca. 50 mL PE and centri-
fuge at 8900 g at 5 C for 5 min.
6. Wash RAFT-R-CSNs three times with 100 mL THF (5 min
centrifugation at 8900 g at 5 C and 3 min sonication for
each washing step).
7. Dry RAFT-R-CSNs at RT in a vacuum oven overnight to
obtain pink solid.
3.5 MIP Synthesis 1. Deprotonate the template, e.g., CGA (1.51 mg) with
(See Note 6) TBA-OH (4μL) in 600μL anhydrous CHCl3 (see Note 7).
2. For MIPs: Add as-prepared template (500μL CGA-TBA
CHCl3 solution), ABDV (3.5 mg), I (1.44 mg), VBA
(1.28 mg), BMA (5.8μL), and EGDMA (10.5μL) to the
degassed (by Ar purging for 10 min) RAFT-R-CSNs (50 mg)
in anhydrous CHCl3 (3 mL).
3. For NIP: Mix the same components mentioned in the previous
step but add 500 μL anhydrous CHCl3 to the reaction mixture
instead of CGA-TBA.
4. Degas both MIP and NIP mixtures for another 10 min in an ice
bath (ca. 4 C) and further stir at 50 C for 18 h and then at
70 C for another 2 h.
5. After reaction mixture has cooled to RT, add 10 mL PE and
collect CGA-MIP and NIP by centrifugation (5 min,
8900 g).
6. Wash both CGA-MIP and NIP three times with ca. 15 mL
anhydrous CHCl3 (5 min centrifugation at 8900 g and 5 min
sonication for each washing step).
7. Wash both CGA-MIP and NIP with a mixture of 80% metha-
nol, 15% formic acid, and 5% Milli-Q water (vol. %, in total
15 mL) for 1 h on a rotator.
8. Collect both CGA-MIP and NIP by 5 min centrifugation at
8900 g.
9. Repeat steps 6 and 7 three times.
10. Wash both CGA-MIP and NIP three times with ca. 15 mL
anhydrous CHCl3 (5 min centrifugation at 8900 g and 5 min
sonication for each washing step).
11. Dry CGA-MIP and NIP at RT in a vacuum oven overnight to
obtain a yellow solid.
3.6 Characterization 1. Determine hydrodynamic radius of nanoparticles with dynamic

and Evaluation light scattering (DLS).
of CGA-MIP Binding (a) Rinse disposable plastic cuvette twice with 2 mL Milli-Q
Performance water.
(Recommended) (b) Weigh 1.0 mg of R-CSNs, APTES-R-CSNs, and RAFT-
R-CSNs in glass vials.
3.6.1 Size Determination
(See Note 6) (c) Dissolve nanoparticles with 2 mL Milli-Q water to pre-
pare 0.5 mg mL1 suspensions.
(d) Sonicate as-prepared nanoparticle suspensions with water
bath sonicator for 10 min.
(e) Transfer 1.5 mL nanoparticle suspensions to a disposable
plastic cuvette.
(f) Perform at least three independent measurements per
sample. Measurement duration should be set according
to instrument recommendations, which depends on par-
ticle size and scattering characteristics.
2. Register transmission electron microscopy (TEM) images with
a (scanning) transmission electron microscope (Fig. 2).
3. Determine the diameter of nanoparticles from TEM images by
analyzing at least 50 nanoparticles with ImageJ software (Ver-
sion 1.52p, NIH, USA) [18]:
(a) Open a TEM image and draw a line over the scale bar,
select Analyze ! Set Scale in Set Scale window and enter
corresponding value into the “Known Distance” box.
(b) Change the “Unit of Measurement” box to nm.
Fig. 2 Representative TEM image of (a) R-CNDs, (b) R-CSNs, and (c) CGA-MIPs and (d) NIPs; scale bar 100 nm
(see Note 11)
(c) Draw an oval over single particle and select Analyze !

Measure to obtain the area (S) of the selected
nanoparticle. qffiffiffiffi
(d) Calculate the diameter (D) by equation: ¼ 4S π . The
mean value and SD were determined from at least
50 nanoparticles.
3.6.2 Zeta Potential 1. Dissolve R-CSNs, APTES-R-CSNs, and RAFT-R-CSNs with

Determination (See Note Milli-Q water to prepare 2 mL of 0.1 mg mL1 nanoparticle
8) suspensions. For R-CNDs, the concentration is ca. 10μg mL1.
2. Inject the nanoparticle suspension to a folded capillary cell until
the suspension covers both electrodes without any air bubble.
3. Measure Zeta potential three times and calculate the mean
value and SD.
3.6.3 APTES and CPDB 1. Weigh ca. 5 mg of R-CSNs, APTES-R-CSNs, and RAFT-R-
Determination CSNs, measure those samples with a thermogravimetric ana-
lyzer for thermogravimetric analysis (TGA) and evaluate the
data:
(a) Open the raw data and plot TGA curves.
(b) Calculate the final mass losses (%) of R-CSNs (msR-CSNs,
%), APTES-R-CSNs (msAPTES-R-CSNs, %) and RAFT-R-
CSNs (msRAFT-R-CSNs, %), respectively.
(c) Quantify the amount of APTES (cAPTES, mmol g1) and
RAFT (cRAFT, mmol g1) by following two equations:
ms msRCSNs
c APTES ¼ APTESRCSNs
MwAPTES
ms msAPTESRCSNs
c RAFT ¼ RAFTRCSNs
MwRAFT
2. Weigh ca. 5 mg of R-CSNs, APTES-R-CSNs, and RAFT-R-

CSNs, measure elemental analysis (EA) of the samples and
evaluate the data:
(a) Measure the elemental composition of nitrogen of
R-CSNs (NR-CSNs, %), nitrogen and sulfur of APTES-R-
CSNs (NAPTES-R-CSNs and SAPTES-R-CSNs, %) and nitrogen
and sulfur of RAFT-R-CSNs (NRAFT-R-CSNs and SRAFT-R-
CSNs, %), respectively.
(b) Quantify the amount of APTES (cAPTES, mmol g1) and

RAFT (cRAFT, mmol g1) by following two equations:
N APTESRCSNs N RCSNs
c APTES ¼
mNitrogen
S RAFTRCSNs S APTESRCSNs
c RAFT ¼
2 mSulfur
Fig. 3 Fluorescence titration (λex 410 nm) of (a) CGA-MIPs and (b) NIPs upon addition of 0–0.2 mM CGA-TBA in
anhydrous CHCl3 and (c) CGA-MIPs upon addition of 0–0.2 mM caffeic acid (CA) in anhydrous CHCl3. (d)
Sensing response of CGA-MIPs (red circles) and NIPs (blue triangles) towards CGA-TBA (IFfl ¼ 1.9) and of
CGA-MIPs to the closely related compound CA-TBA (black squares) (DFfl ¼ 3.0); [particles] ¼ 0.5 g L1,
anhydrous CHCl3
3.6.4 Evaluation of MIP 1. Weigh CGA-MIP (1.00 mg), NIP (1.00 mg), CGA (1.51 mg),
Binding Performance by and caffeic acid (CA, 0.77 mg) in four 3 mL brown glass vials
Fluorescence Rebinding (see Notes 7 and 9).
Test (Fig. 3) 2. Dissolve CGA-MIP and NIP with 2 mL anhydrous CHCl3,
CGA, and CA with 600μL anhydrous CHCl3, respectively.
3. Add TBA-OH (4μL) to CGA and CA solution, respectively.
4. Sonicate CGA-MIP, NIP, CGA-TBA, and CA-TBA with a
water bath sonicator for 5 min.
5. Measure UV-Vis absorption and fluorescence spectra of anhy-
drous CHCl3, CGA-MIP, and NIP suspensions.
6. Add 1–150μL CGA-TBA solution to CGA-MIP and NIP sus-
pension, respectively, and measure UV-Vis absorption and
fluorescence spectra.
7. Add 1–150μL CA-TBA solution to CGA-MIP suspension and

measure UV-Vis absorption and fluorescence spectra.
8. Start to stir CGA-MIP or NIP suspension for 2 min after the
addition of CGA-TBA or CA-TBA in CHCl3. Stop stirring and
measure fluorescence emission spectra from 420 to 750 nm
(slit 5 nm) with 410 nm excitation (slit 3 nm).
9. Evaluate the binding performance of CGA-MIP by both
imprinting factor (IFfl) and discrimination factor (DFfl),
which can be calculated by following three equations (see
Note 10):
ΔF r ¼ F r ðx Þ F r0

ΔF r:sat:=F
IFfl ¼
r0 CGAMIPþCGA
ΔF r:sat:=F
r0 NIPþCGA

ΔF r:sat:=F
DFfl ¼ CGAMIPþCGA
r0
ΔF r:sat:=F
r0 CGAMIPþCA
where Fr(x) refers to the relative fluorescence intensity II 510

680
of CGA-MIP or NIP at analyte (CGA or CA) concentration x.
Fr0 refers to the relative fluorescence intensity II 510 680
of
CGA-MIP or NIP when analyte (CGA or CA) is absent.
ΔFr. sat. refers to the relative fluorescence intensity change
of CGA-MIP or NIP when ΔFr of CGA-MIP is at its saturation
point.
4 Notes
1. Based on previous studies of the spectroscopic behavior of

fluorescent monomer I with carboxylate anions in CHCl3,
the determination of the binding constant (KD) of I to
CGA-TBA is conducted by fluorescence titration [16, 17]. A
relatively strong binding affinity of I to CGA-TBA, reflected by
a KD value such as 4.7 0.3 105 M 1 of the present host–
guest pair, is prerequisite for fixing the MIP recipe and synthe-
sizing MIPs based on the principle of stoichiometric imprint-
ing. A (very) low binding constant would either result in too
few complexes being present at equimolar concentrations of
I and CGA-TBA in the pre-polymerization mixture or in the
imprinting of unbound CGA-TBA when an excess is used to
engage all molecules I into a complex in the
pre-polymerization mixture. Moreover, pre-polymerization
studies on an MIP recipe are also crucial for the MIP synthesis.
Because, in simple fluorescence titrations, the concentrations of
I and CGA-TBA are generally kept at lower micromolar level,
to comply with absorption and fluorescence measurements in
conventional spectrometers and measurement configurations

(10 mm cells, 90 excitation-emission configuration), while in
the case of MIP synthesis, the concentrations used are usually
in the lower millimolar range. Thus, it is essential to investigate
if the spectroscopic behavior of I is roughly the same at dilute
(analytical) and polymerization (synthetic) conditions.
2. Cold methanol and acetone can reduce the loss of R-CNDs. If
not immediately used, R-CNDs should be stored at 20 C in
a tightly sealed brown glass vial [19].
3. Besides Triton-X 100, other surfactants, such as dioctyl sulfo-
succinate sodium salt (AOT) and polyoxyethylene-[5]-nonyl-
phenyl ether (NP-5), can be used in the synthesis of R-CSNs
as well.
4. The size of R-CSNs can be tuned by changing (a) the amount
of NH3·H2O (32%), e.g., the higher the amount of NH3·H2O,
the smaller the R-CSNs will be; (b) the molar ratio of both
water : surfactant and cosurfactant : surfactant, e.g., the higher
the molar ratio, the bigger the R-CSNs will be. However, the
size of silica nanoparticles synthesized by this water-in-oil
(W/O) microemulsion approach only lies in the 15–100 nm
range [20, 21].
5. After drying overnight, R-CSNs might form a bulk solid. For
full and even APTES functionalization, grind R-CSNs to pow-
der before dissolving in anhydrous toluene.
6. Before the synthesis of CGA-MIP, measurement of fluores-
cence, determination of particle size, Zeta potential, and quan-
tification of APTES and CPDB serve as quality control for all
synthesized particles in every step. Furthermore, checking
these parameters in different batches of R-CNDs, R-CSNs,
APTES-R-CSNs, RAFT-R-CSNs, and MIP/NIPs assures the
reproducibility.
7. Sonicate CGA and CA solution with water bath sonicator for
5 min because CGA and CA have relatively low solubility in
CHCl3. Vortex or gently shake CGA, CA, and MIP/NIP
solution or suspension for 10 s to ensure the homogeneity of
the sample.
8. The Zeta potential is the charge established on the surface of
materials when contacting with a liquid medium. It is thus an
interfacial property, given in millivolt unit. Over the last few
decades, Zeta potential measurements have become an impor-
tant characterization method for surface functionality or stabil-
ity of dispersed particles.
9. In fluorescence rebinding tests, CA is used as a competitor to
check if CGA-MIP can distinguish CGA and CA by comparing
the fluorescence increase in both CGA-MIP + CGA and
CGA-MIP + CA cases. It is always good to use a structurally

similar and application-related meaningful compound as com-
petitor to check the selectivity of MIPs.
10. In the current dual-fluorescent MIP system, the relative fluo-
rescence intensity Fr, obtained by normalizing the analyte-
sensitive signal If(510) of the MIP shell on the core’s reference
emission If(680), was used in the calculation of the performance
parameters IFfl and DFfl, instead of only the change in the
fluorescence intensity of the MIP (equivalent to If(510) here)
as is done in conventional single-signal fMIP studies.
11. In Fig. 2c, the MIP shell is hard to be visualized from the TEM
image, but it is proved by the size increase from R-CSNs to
CGA-MIPs.
Acknowledgments
This work has received funding from the European Union’s Hori-
zon 2020 research and innovation program under the Marie Skło-
dowska-Curie grant agreement No 721297.
References
1. Ahmad OS, Bedwell TS, Esen C et al (2019) conjugated O-acryloyl L-hydroxyproline. Bio-
Molecularly imprinted polymers in electro- sens Bioelectron 48:113–119
chemical and optical sensors. Trends Biotech- 8. Zhang X, Yang S, Jiang R et al (2018) Fluores-
nol 37:294–309 cent molecularly imprinted membranes as bio-
2. Turiel E, Martin-Esteban A (2010) Molecu- sensor for the detection of target protein. Sens
larly imprinted polymers for sample prepara- Actuators B-Chem 254:1078–1086
tion: a review. Anal Chim Acta 668:87–99 9. Guney O, Cebeci FC (2010) Molecularly
3. Mayes AG, Whitcombe MJ (2005) Synthetic imprinted fluorescent polymers as chemosen-
strategies for the generation of molecularly sors for the detection of mercury ions in aque-
imprinted organic polymers. Adv Drug Deliv ous media. J Appl Polym Sci 117:2373–2379
Rev 57:1742–1778 10. Chen L, Liu D, Peng J et al (2019) Ratiometric
4. Liu G, Huang X, Li L et al (2019) Recent fluorescence sensing of metal-organic frame-
advances and perspectives of molecularly works: tactics and perspectives. Coord Chem
imprinted polymer-based fluorescent sensors Rev 404:213113
in food and environment analysis. Nanomater- 11. Wu P, Hou X, Xu JJ et al (2016) Ratiometric
ials 9:1030 fluorescence, electrochemiluminescence, and
5. Rico-Yuste A, Carrasco S (2019) Molecularly photoelectrochemical chemo/biosensing
imprinted polymer-based hybrid materials for based on semiconductor quantum dots. Nano-
the development of optical sensors. Polymers scale 8:8427–8442
11:1173 12. Zhang R, Zhang Y, Deng X et al (2018) A
6. Wan W, Wagner S, Rurack K (2016) Fluores- novel dual-signal electrochemical sensor for
cent monomers: “bricks” that make a molecu- bisphenol A determination by coupling nano-
larly imprinted polymer “bright”. Anal Bioanal porous gold leaf and self-assembled cyclodex-
Chem 408:1753–1771 trin. Electrochim Acta 271:417–424
7. Inoue Y, Kuwahara A, Ohmori K et al (2013) 13. Baker SN, Baker GA (2010) Luminescent car-
Fluorescent molecularly imprinted polymer bon nanodots: emergent nanolights. Angew
thin films for specific protein detection Chem Int Ed 49:6726–6744
prepared with dansyl ethylenediamine-
14. Hola K, Zhang Y, Wang Y et al (2014) Carbon 18. ImageJ Basics. National Institutes of Health
dots-emerging light emitters for bioimaging, (NIH). https://imagej.nih.gov/ij/docs/
cancer therapy and optoelectronics. Nano pdfs/ImageJ.pdf. Accessed 15 Sep 2020
Today 9:590–603 19. Pan LL, Sun S, Zhang L et al (2016) Near-
15. Clifford MN, Jaganath IB, Ludwig IA et al infrared emissive carbon dots for two-photon
(2017) Chlorogenic acids and the acyl-quinic fluorescence bioimaging. Nanoscale
acids: discovery, biosynthesis, bioavailability 8:17350–17356
and bioactivity. Nat Prod Rep 34:1391–1421 20. Santra S, Bagwe RP, Dutta D et al (2005)
16. Wan W, Biyikal M, Wagner R et al (2013) Synthesis and characterization of fluorescent,
Fluorescent sensory microparticles that “light- radio-opaque, and paramagnetic silica nano-
up” consisting of a silica core and a molecularly particles for multimodal bioimaging applica-
imprinted polymer (MIP) shell. Angew Chem tions. Adv Mater 17:2165–2169
Int Ed 52:7023–7027 21. Bagwe RP, Yang CY, Hilliard LR et al (2004)
17. Shinde S, El-Schich Z, Malakpour A et al Optimization of dye-doped silica nanoparticles
(2015) Sialic acid-imprinted fluorescent core- prepared using a reverse microemulsion
shell particles for selective labeling of cell sur- method. Langmuir 20:8336–8342
face glycans. J Am Chem Soc
137:13908–13912
Chapter 18
Molecularly Imprinted Polymer-Based Quartz Crystal

Microbalance Sensor for the Clinical Detection of Insulin
Duygu Çimen, Nilay Bereli, Fatma Kartal, and Adil Denizli
Abstract
In this study, we reported the design of a quartz crystal microbalance (QCM) sensors for selective insulin
detection. In the first step, N-methacryloyl-(L) 3-histidine methyl ester (MAH) monomer was formed a
complex with insulin. Then, 2-hydroxyethyl methacrylate and ethylene glycol dimethacrylate were mixed
with MAH:insulin complex. Insulin-imprinted and non-imprinted QCM sensors were synthesized by
ultraviolet polymerization for the insulin detection. Insulin-imprinted QCM sensors was characterized by
the contact angle measurements, atomic force microscopy and ellipsometry. Limit of detection (LOD) was
found as 0.00158 ng/mL for the insulin-imprinted QCM sensors. Selectivity of insulin-imprinted and
non-imprinted QCM sensors was carried in the presence of glucagon and aprotinin. Insulin-imprinted
QCM sensor for insulin detection was also examined in the artificial plasma.
Key words Quartz crystal microbalance (QCM), Insulin, Sensor, Molecular imprinting method,
Clinical detection
1 Introduction
Insulin is an essential polypeptide hormone secreted by pancreatic

cells with a molecular weight of 5.8 kilodaltons (kDa). It is a small
protein with A and B polypeptide chains which are consisting of
21 amino acid residues and 30 amino acid residues crosslinked with
two disulfide bridges [1]. In addition to being the primary stabi-
lizer of carbohydrate metabolism, insulin also plays a role in fat and
protein metabolism associated with carbohydrate metabolism.
Additionally, insulin is an important regulator in glucose metabo-
lism as a hormone that is produced by beta cells located in the
pancreas and regulates glucose levels in the blood [2, 3]. The
amount of insulin secreted in response to glucose and blood glu-
cose increases proportionally. After the foods are digested by the
stomach, they are broken down by enzymes and converted into
glucose, then the glucose mixed with blood is balanced by the
https://doi.org/10.1007/978-1-0716-1629-1_18,
209
210 Duygu Çimen et al.
insulin hormone. The main task of insulin is to prevent excessive

glucose accumulation in the blood and if the secretion of this
hormone is inadequate and unable to fulfill its duty; it can lead to
many serious health problems, especially diabetes, kidney, and heart
disease. Insulin used as a biomarker is very important for the clinical
diagnosis of various types of diabetes, monitoring therapy, evalua-
tion of patients with insulinomas, doping control in athletes,
chronic pancreatitis, and breast cancer [4, 5]. Although there are
many determination methods such as enzyme-linked immunosor-
bent assay (ELISA), chromatography, and radioimmunoassay
(RIA) in the literature, most of these methods have a low selectivity
[6–8]. The constant need for fast and efficient new methods in the
fields of diagnosis, biotechnology has led researchers to conduct
better, more selective and sensitive analytical studies. In the litera-
ture, molecular imprinting methods in many areas such as chemical
sensors, catalysis, drug transport, biological antibodies, and recep-
tor systems create a considerable potential [9, 10]. Molecular
imprinting technique is frequently used in many fields such as
medicine, life, environmental sciences, and food. Stable synthetic
polymers with selective molecular recognition regions are prepared
by the molecular imprinting technique. Molecular imprinting tech-
nique is polymeric structures prepared by polymerizing the func-
tional monomer and crosslinker in the presence of a target molecule
that acts as a molecular mold. One of the application areas of
molecularly imprinted polymers is the use of sensors as recognition
elements. With molecular imprinting technique, selective and
unique artificial receptors belonging to a specific target molecule
can be designed. In the literature, there are studies for the selective
determination of biologically active molecules such as drug, pro-
tein, peptide, amino acid, and vitamin as molecular imprinting
polymer in recent years [11–14].
Mass-sensitive sensor devices work based on measuring the
changes of properties of piezoelectric or magnetoelastic material
via increased mass of biomolecules onto the sensor surface. Due to
mass is a common property of all ions, small molecules, proteins,
viruses, and cells. These sensors applicable to all target biological
structures without exception. Mass-sensitive sensors can work at
both liquid and gas phase. Mass-sensitive devices can use quartz
crystal microbalance, micro-cantilever, or magnetoelastic sensors.
Quartz crystal microbalance (QCM) sensors produce signal with
the changes in the resonance frequency of piezoelectric crystal
upon interaction with analyte. In the last decades, QCM-based
sensors have been increasingly utilized for label-free detection,
real-time analyses of various biological and chemical molecules
[15–18]. QCM sensors have also been applied for detection of
analytes of interest in medical diagnostics (cancer markers, antibo-
dies, proteins, hormones, drugs, vitamins. and allergy markers) and
environmental protection (phenols, aromatic hydrocarbons,
QCM Sensor for the Clinical Detection of Insulin 211
polychlorinated biphenyls, and pesticides). Here, we reported

kinetic studies of insulin-imprinted QCM sensor for the selective
detection of insulin in aqueous solution and artificial human
plasma [19].
2 Materials
2.1 Reagents All the water used for experiments in this study is purified using
reverse osmosis unit with a high flow cellulose acetate membrane
followed by organic/colloid removal and ion exchange packed-bed
system (R ¼ 18.2 MΩ/cm). All sample and buffer solutions are
prepared and stored at 25 C. Dispose of experimental waste mate-
rials in compliance with all waste disposal regulations. PBS buffer
was prepared by dissolving 0.01 M di-sodium hydrogen phosphate
(Na2HPO4) (Catalog No. 106586, Merck, Darmstadt, Germany),
0.002 M Potassium dihydrogen phosphate (KH2PO4) (Catalog
No. 104873, Merck, Darmstadt, Germany). The QCM sensors
was washed with ethyl alcohol/water solution (50%, v/v).
2.2 Equipment 1. Quartz crystal microbalance (QCM) (see Note 1).

2. UV light.
3. Contact angle.
4. Atomic Force microscope (AFM).
5. Ellipsometry.
2.3 Solutions 1. Use pure water as a cleaning solution.

2. Wash the clean H2SO4:H2O2 mix (hot or acidic piranha solu-
tion) on the QCM chip surface and use it as activation agent
(see Note 2).
3. Use allyl mercaptan (CH2 ¼ CHCH2SH) of 3 mM to form
thiol groups on the QCM chip surface.
4. Use ethyl alcohol (EtOH) as a washing solution for the QCM
chip surface.
5. Use 2-Hydroxyethyl methacrylate (HEMA) as co-monomer.
6. Use α,α0 -Azoisobutyronitrile (AIBN) as initiator for polymeri-
zation starting.
7. Use 0.1 M phosphate-buffered saline (PBS) of pH 7.4 as an
equilibrium buffer for the QCM sensor.
8. Prepare 0.5 M NaCl solution as a desorption agent.
3 Methods
3.1 Thiol Groups 1. Clean the QCM gold surface in acidic piranha solution (see
Modification of Note 2) and then dry.
QCM Chip 2. Drop 5 μL of allyl mercaptan (CH2 ¼ CHCH2SH) onto the
gold surface of QCM chip.
3. Incubate QCM chip overnight in a fume hood to introduce
thiol groups to the gold surface.
4. Clean QCM chip surface with ethyl alcohol:water (60%, v/v)
for removal of unbound allyl mercaptan molecules and dried.
3.2 Preparation of 1. Prepare the different monomer:template (MAH:insulin) ratios

QCM Sensor (1:1, 2:1, 4:1, 8:1 M ratio) complexes.
2. Mix the different monomer:template (MAH:insulin) com-
plexes with HEMA (1.21 μL) as co-monomer and EGDMA
(3.76 μL) as crosslinker (see Note 3).
3. Dissolve 4.0 mg AIBN in the polymer solution.
4. Mix the polymer solution at room temperature slowly on the
magnetic stirrer (see Note 4).
5. Drop 5 μL of polymer solution onto the thiol modified QCM
chip surface and coat a polymer mixture as homogenously by
using the spin coating method (see Note 5).
6. Initiate the polymerization under the UV light (100 Watt,
365 nm) for 45 min (see Note 6).
7. Clean insulin-imprinted QCM sensor with ethyl alcohol/water
solution (50%, v/v). Preparation of insulin-imprinted PHE-
MAH-based QCM sensor is depicted in Fig. 1.
3.3 Characterization 1. Insulin-imprinted and non-imprinted QCM chip surface

Studies should be characterized by FTIR-ATR, contact angle, atomic
force microscope, and ellipsometry analysis.
2. FTIR-ATR spectrophotometer in the wavenumber range of
700–4000 cm1 (Fig. 2a).
3. Perform a contact angle analysis for surface hydrophilic of
QCM chip.
4. Apply a sessile drop method to QCM chip surface for the
contact angle measurements and calculate average of contact
angle value (Fig. 2b, c).
5. Analyze surface deepness of insulin-imprinted QCM sensors by
atomic force microscopy. Take surface images and 3D images at
different scales. Display the sample field of 10 10 μm2 with a
resolution of 256 256 pixels and the 2 μm/s scanning rate
(Fig. 2d, e).
Fig. 1 Presentation of the insulin-imprinted PHEMAH-based QCM chip preparation. Reproduced from [19] with
permission of Elsevier
Fig. 2 FTIR-ATR, AFM studies, and ellipsometry images of the SPR chips in the surface morphology.
Reproduced from [19] with permission of Elsevier
Fig. 2 (continued)
6. Characterize insulin-imprinted QCM sensor average surface

thickness using a ellipsometer (Fig. 2f).
3.4 Kinetic Analysis 1. The imprinting factors at different molar ratios of MAH:insulin
were shown in Fig. 3.
2. Equilibrate insulin-imprinted QCM sensor at 0.1 M pH 7.4
PBS buffer solutions (see Note 7).
3. Apply insulin concentration in the linear range from 0.008 ng/
mL to 10.0 ng/mL to insulin-imprinted QCM sensors
(Fig. 4a).
4. Use insulin-imprinted QCM sensor for insulin kinetic analysis
with using QCM system (see Note 8).
5. Monitor signals in real time in the QCM system.
6. Utilize 0.5 M NaCl solution in the desorption step of QCM
sensor.
7. Clean QCM sensors with ethanol solution and pure water.
Fig. 3 The imprinting effect of insulin-imprinted and non-imprinted QCM sensors. Reproduced from [19] with
permission of Elsevier
Fig. 4 The real detection at different detection (A) and standard calibration (B) for insulin-imprinted QCM
sensors. Reproduced from [19] with permission of Elsevier
As seen in Fig. 4b, the equation of the linear graph taken from
the concentration range of 0.008–10 ng/mL is calculated as
y ¼ 0.3158 + 0.0439. The linearity of this equation is calculated
as R2 ¼ 0.9818. The limit of detection (LOD) and limit of quanti-
fication (LOQ) values are confirmed according to equations:
LOD: 3 S/m (1)

LOQ: 10 S/m (2)
where S is the standard deviation of the intercept and m is the

slope of the regression line. LOD and LOQ values were found as
0.00158 ng/mL and 0.0198 ng/mL, respectively. The detection
of limit values of other sensors studies is also given in Table 1 [19].
Table 1
The comparison of the prepared QCM sensor with literature
Method Linear range LOD Ref

Amperometry 0.1 nM–4000 nM 0.022 nM [20]
Amperometry 0.1–700 nM 0.04 nM [21]
Flow injection analysis 0.015–0.1 nM 0.0026 nM
Voltammetry 10–50 nM 2.8 nM [22]
Amperometry 50–500 nM 20 nM [23]
Flow injection analysis 100–1000 nM 50 nM [24]
Voltammetry 250 nM–1.6 nM 250 nM [25]
Electrochemical aptamer-based sensor 10 nM–200 nM 10 nM [26]
Electrochemical sensor 20 nM–1000 nM 5 nM [27]
Quartz crystal microbalance 0.00138–1.72 nM 0.00027 nM This work
3.5 Isotherm Models The isotherm model between insulin and QCM sensor interactions
was determined by carrying out association kinetic analysis, Scatch-
ard and Langmuir, Freundlich, and Langmuir-Freundlich adsorp-
tion isotherm models (Fig. 5). The isotherm model can be
calculated using the following equations [12, 13]:
Association kinetic analysis: dΔm/dt ¼ kaCΔmmax (kaC + kd)Δm (3)

Scatchard: Δmex/C ¼ KA(Δmmax Δmeq) (4)
Langmuir: Δm ¼ Δmmax C/KD + C (5)
Freundlich: Δm ¼ Δmmax C 1/n
(6)
Langmuir–Freundlich: Δm ¼ Δmmax C 1/n
/KD + C 1/n
(7)
1. C is insulin concentration (ng/mL).

2. Δmmax is the response measured by binding.
3. KD (mL/ng) and KA (ng/mL) are forward and reverse equi-
librium constants.
4. 1/n refers to Freundlich exponent; kd (1/s) and ka (ng/mL)
values refer to the forward and reverse kinetic rate constants.
5. The max, eq and ex, indicate maximum, equilibrium, and
experimental, respectively.
The calculated parameters for all models are shown in Table 2.
The best-fitted model to describe the interaction between the
QCM sensor and insulin molecule is the Langmuir model. Accord-
ing to these results, the binding of insulin-imprinted QCM sensor
is monolayer. The Δmmax value calculated by the Langmuir model
was close to the experimental Δmmax value.
Fig. 5 Adsorption models (Langmuir (a), Freundlich (b), and Langmuir-Freundlich (c)). Reproduced from [19]
with permission of Elsevier
Table 2
Isotherm and kinetic parameters
Association kinetics analysis Equilibrium analysis (Scathard)

ka (ng/mL s) 0.055 Δmmax (ng/cm2) 1.730
kd (1/s) 0.060 KA (ng/mL) 0.254
KA (ng/mL) 0.930 KD (mL/ng) 3940
KD (mL/ng) 1.075 R2 0.980
R2 0.926
Langmuir Freundlich Langmuir-Freundlich

Δmmax (ng/cm2) 0.460 Δmmax (ng/cm2) 4.088 Δmmax (ng/cm2) 0.139
KD (mL/ng) 0.275 1/n 0.462 1/n 0.462
2
KA (ng/mL) 3.636 R 0.849 KD (mL/ng) 1.145
2
R 0.999 KA (ng/mL) 0.873
R2 0.922
Table 3
The recoveries of insulin in artificial plasma samples (n ¼ 3)
Found Recovery
Added
(ng/mL) (%)
(ng/mL)
Artificial plasma QCM ELISA QCM ELISA
0.1 0.12 0.13 117.0 129.9
0.6 0.62 0.70 103.3 116.7
1.5 1.49 1.47 99.3 98.0
3.6 Detection of 1. Equilibrate insulin-imprinted QCM sensor at 0.1 M pH 7.4

Insulin from Artificial PBS buffer solutions (see Note 7).
Plasma 2. Spike the artificial plasma sample to insulin solutions with a
known concentration (0.1, 0.6, and 1.5 ng/mL) (Table 3).
3. Apply the insulin-spiked artificial plasma sample to insulin-
imprinted QCM sensors (Fig. 6) (see Note 9).
4. Monitor signals in real time in the QCM system.
5. Utilize 0.5 M NaCl solution in the desorption step of QCM
sensor.
6. Clean QCM sensors with ethanol solution and pure water.
3.7 Selectivity 1. Investigate the selectivity of insulin-imprinted and

non-imprinted QCM sensors with glucagon and aprotinin
molecules.
2. Apply 5.0 ng/mL insulin solutions to insulin-imprinted and
non-imprinted QCM sensors (Fig. 7).
3. Perform the protein interaction on the presence of 5.0 ng/mL
insulin and other proteins (aprotonin and glucagon) with
QCM sensors.
4. Show Δmmax value results that there decrease binding between
the insulin and other proteins (Table 4).
3.8 Reusability 1. Display the reusability of the QCM sensor by four equilibra-
tion–binding-regeneration cycles.
2. Use NaCl solution to remove the insulin-imprinted QCM chip
surface.
3. Repeat 0.25 ng/mL of insulin sample for four times with the
QCM sensors (Fig. 8a).
4. Under long-term storage conditions, QCM sensor was suc-
cessfully obtained reusability results for insulin detection.
Fig. 6 Real-time insulin detection in 1/5 diluted artificial plasma. Reproduced

from [19] with permission of Elsevier
Fig. 7 Selectivity studies of insulin-imprinted QCM sensors. Reproduced from

[19] with permission of Elsevier
Table 4
The selectivity coefficients for insulin-imprinted and non-imprinted QCM sensor
MIP NIP
Δm k Δm k k’
Insulin 0.387 – 0.046 – –
Glucagon 0.065 5.95 0.141 0.33 18.25
Aprotinin 0.055 7.04 0.105 0.44 16.06
Insulin + glucagon 0.308 1.26 0.121 0.38 3.31
Insulin + aprotinin 0.289 1.34 0.087 0.53 2.53
Insulin + glucagon + aprotinin 0.277 1.39 0.099 0.46 3.01
Fig. 8 Reproducibility studies (a) and short-term and long-term stability (b) of insulin-imprinted QCM sensors.
Reproduced from [19] with permission of Elsevier
5. After 90 days of storage of the QCM sensors, 70.3% of the

initial activity of the QCM sensors remained (Fig. 8b).
4 Notes
1. For the 5 MHz crystal in the QCM sensor, the sensitivity

should be 56.6 Hz cm2/μg.
2. Use H2O2 and H2SO4 as mixture ratio of 1:3 (v/v) for prepar-
ing piranha solutions.
3. The different monomer:template (MAH:insulin) ratios (1:1,
2:1, 4:1, 8:1 M ratio) complexes should be mixed in the rotator
for 1 h.
4. Mix all solution at room temperature slowly on the magnetic
stirrer.
5. Spread polymer solution on the gold surface of the QCM chip
to prepare the monolayer and homogeny surface.
6. Polymerization should be performed using UV light (100 W,
365 nm).
7. Equilibrate insulin-imprinted QCM chip with pH 7.4 PBS
buffer for 8 min, apply 0.008 ng/mL to 10.0 ng/mL insulin
samples on the QCM system for 35 min. Remove insulin with
0.5 M NaCl solution for 15 min.
8. Evaluate kinetic data with a QCM sensor software system.
9. All measurements should be made at room temperature.
References
1. Tiwari MP, Prasad BB (2014) An insulin mon- 13. Jalilzadeh M, Çimen D, Özgür E, Esen C,
itoring device based on hyphenation between Denizli A (2019) Designing and preparing
molecularly imprinted micro-solid phase imprinted surface plasmon resonance (SPR)
extraction and complementary molecularly nanosensor for detection of Zn(II)-ions. J
imprinted polymer-sensor. J Chromatogr A Macromol Sci A 56:877–886
1337:22–31 14. Çimen D, Bereli N, Günaydın S, Denizli A
2. Yu Y, Guo M, Yuan M, Liu W, Hu J (2016) (2020) Detection of cardiac troponin-I by
Nickel nanoparticle-modified electrode for optic biosensors with immobilized anti-cardiac
ultra-sensitive electrochemical detection of troponin-I monoclonal antibody. Talanta
insulin. Biosens Bioelectron 77:215–219 219:121259
3. Zhang B, Jiang Y, Kuang H, Yao C, Huang Q, 15. Cornelioa VE, Pedrosoa MM, Afonsoa AS,
Xu S, Tang D, Fu W (2008) Development of a Fernandesa JB, Silvaa MFGF, Fariaa RC,
spiral piezoelectric immunosensor based on Vieiraa PC (2015) New approach for natural
thiol self-assembled monolayers for the detec- products screening by real-time monitoring of
tion of insulin. J Immunol Methods 338:7–13 hemoglobin hydrolysis using quartz crystal
4. Gobi KV, Iwasaka H, Miura N (2007) Self- microbalance. Anal Chim Acta 862:86–93
assembled PEG monolayer based SPR immu- 16. Haupt K, Mosbach K (2000) Molecularly
nosensor for label-free detection of insulin. imprinted polymers and their use in biomi-
Biosens Bioelectron 22:1382–1389 metic sensors. Chem Rev 100:2495–2504
5. Xu M, Luo X, Davis JJ (2013) The label free 17. Şener G, Özgur E, Yilmaz E, Uzun L, Say R,
picomolar detection of insulin in blood serum. Denizli A (2010) Quartz crystal microbalance
Biosens Bioelectron 39:21–25 based nanosensor for lysozyme detection with
6. Ho JA, Zeng SC, Huang MR, Kuo HY (2006) lysozyme imprinted nanoparticles. Biosens
Development of liposomal immuno-sensor for Bioelectron 26:815–821
the measurement of insulin with femtomole 18. Wu AH, Syu MJ (2006) Synthesis of bilirubin
detection. Anal Chim Acta 556:127–132 imprinted polymer thin film for the continuous
7. Jaafarias M, Shams E, Amini MK (2011) Silica detection of bilirubin in an MIP/QCM/FIA
gel modified carbon paste electrode for electro- system. Biosens Bioelectron 21:2345–2353
chemical detection of insulin. Electrochim Acta 19. Kartal F, Çimen D, Bereli N, Denizli A (2019)
56:4390–4395 Molecularly imprinted polymer based quartz
8. Prasad BB, Madhuri R, Tiwari MP, Sharma PS crystal microbalance biosensor for the clinical
(2010) Imprinting molecular recognition sites detection of insulin. Mater Sci Eng C:730–737
on multiwalled carbon nanotubes surface for 20. Salimi A, Noorbakhash A, Sharifi E, Semnani A
electrochemical detection of insulin in real (2008) Highly sensitive sensor for picomolar
samples. Electrochim Acta 55:9146–9156 detection of insulin at physiological pH, using
9. Çimen D, Bereli N, Andaç M, Denizli A GC electrode modified with guanine and elec-
(2018) Molecularly imprinted cryogel columns trodeposited nickel oxide nanoparticles. Bio-
for Concanavalin A purification from jack bean sens Bioelectron 24:792–798
extract. Sep Sci Plus:1–10 21. Salimi A, Roushani M, Soltanian S, Hallaj R
10. Çimen D, Göktürk I, Yılmaz F (2016) (2007) Picomolar detection of insulin at
Removal of iron by chelation with molecularly renewable nickel powder-doped carbon com-
imprinted supermacroporous cryogel. Artif posite electrode. Anal Chem 79:7431–7438
Cell Nanomed B 44:1158–1166 22. Amini N, Gholivand MB, Shamsipur M (2014)
11. Çimen D, Denizli A (2020) Development of Electrocatalytic determination of traces of insu-
rapid, sensitive, and effective plasmonic nano- lin using a novel silica nanoparticles-Nafion
sensor for the detection of vitamins in infact modified glassy carbon electrode. J Electroanal
formula and milk samples. Photonic Sensors Chem 714:70–75
10:316–332 23. Ho ENM, Wan TSM, Wong ASY, Lam KKH,
12. Hüseynli S, Çimen D, Bereli N, Denizli A Stewart BD (2011) Doping control analysis of
(2019) Molecular imprinted based quartz crys- insulin and its analogues in equine urine by
tal microbalance nanosensors for mercury liquid chromatography-tandem mass spec-
detection. Global Chall 3:1800071 trometry. J Chromatogr A 1218:1139–1146
24. Salimi JA, Hallaj R (2012) Cobalt oxide 26. Gerasimov JY, Schaefer CS, Yang W, Grout RL,
nanostructure-modified glassy carbon elec- Lai RY (2013) Development of an electro-
trode as a highly sensitive flow injection amper- chemical insulin sensor based on the insulin-
ometric sensor for the picomolar detection of linked polymorphicregion. Biosens Bioelectron
insulin. J Solid State Electrochem 42:62–68
16:1239–1246 27. Guo C, Chen C, Luob Z, Chena L (2012)
25. Businova P, Prasek J, Chomoucka J, Electrochemical behavior and analytical detec-
Drbohlavova J, Pekarek J, Hrdy R, Hubalek J tion of insulin on pretreated nanocarbon black
(2012) Voltammetric sensor for direct insulin electrode surface. Anal Methods 4:1377–1382
detection. Proc Eng 47:1235–1238
Chapter 19
Preparing Selective Nanozymes by Molecular Imprinting

Yuqing Li, Xiaohan Zhang, and Juewen Liu
Abstract
Recently, many nanomaterials such as Fe3O4, CeO2, and gold nanoparticles have been reported to have
enzyme-like activities and they are called nanozymes. Although these nanozymes have oxidase or
peroxidase-like activities, they can catalyze the oxidation of many substrates and thus lack the specificity
expected for enzymes. The selectivity of nanozymes can be significantly enhanced up to 100-fold by coating
them with a molecularly imprinted polymer (MIP) layer. Since MIP creates specific binding pockets for the
imprinted substrate, the imprinted molecules can be enriched, selectively access the catalytic core, and be
oxidized, while other substrates are blocked from accessing the nanozyme surface. In this chapter, the
detailed protocol for the preparation of the MIP-coated Fe3O4 peroxidase-mimicking nanozymes is
described. In addition, some procedures needing special attention are described in detail, which will
facilitate the applications of MIP-coated nanozymes in analytical, biomedical, and environmental fields.
Key words Molecularly imprinted polymers, Nanozymes, Nanogels, Iron oxide, Catalysts
1 Introduction
1.1 Nanozymes Nanozymes are engineered nanomaterials with enzyme-mimicking

activities [1–4]. For example, iron oxide nanoparticles have
peroxidase-like activities, and they can oxidize a broad range of
substrates in the presence of H2O2 [5]. CeO2 nanoparticles were
reported to have oxidase, catalase, phosphatase, and nuclease mim-
icking activities [6–8], while gold nanoparticles are known for its
glucose oxidase-like activity [9]. Using nanoparticles as catalysts has
been executed for many years. What sets nanozymes apart from
typical inorganic catalysts is close to physiological reaction condi-
tions and the catalysis of biologically relevant reactions [4]. The low
cost and high stability of nanozymes make them attractive to
researchers.
1.2 Applications Nanozymes have been used for various applications mainly in three
of Nanozymes areas: analytical, biomedical, and environmental. For analytical
applications, they are used as labels for signal amplification, and a
https://doi.org/10.1007/978-1-0716-1629-1_19,
223
224 Yuqing Li et al.
classic example is to use iron oxide nanoparticles to replace horse-

radish peroxidase (HRP) for enzyme-linked immunosorbent assays
(ELISA). In addition, if a nanozyme can be selectively boosted or
inhibited by a molecule, this reaction may then be used to detect
that molecule as a promotor or inhibitor [10]. For example, F can
boost the oxidase-like activity of CeO2, which turned out to be
useful for F detection [11], and phosphite can accelerate the
oxidase-like activity of NiO nanoparticles [12]. For environmental
applications, nanozymes have been explored for the degradation of
organic molecules [13–15]. Nanozymes were also used as scaven-
gers for removing reactive oxygen species and killing cancer cells
[16, 17].
1.3 The Specificity Enzymes have a few fundamental features such as high catalytic
Problem in Nanozymes efficiency and high specificity. Improving the catalytic activity is
relatively simple, as it can often be made by using smaller nanopar-
ticles with appropriate surface modifications [10, 18]. However,
solving the specificity problem is more challenging. Unlike protein-
based enzymes, which possess three-dimensional binding pockets
for substrates and can selectively catalyze the conversion of its
substrate leaving other molecules intact, nanozymes do not have
such binding pockets, and they can hardly distinguish different
substrates. For example, glucose oxidase is selective for glucose,
while gold nanoparticles can also oxidize other reducing sugars
[19]. Improving specificity is critical for the development of the
nanozyme field since it can help nanozymes conceptually match
enzymes and also enhance applications.
To develop specific nanozymes, substrate binding pockets
might be constructed by attaching aptamers or antibodies
[20]. However, this method would defeat the cost and stability
advantages of nanozymes. Therefore, the method needs to match
the properties of nanozymes (i.e., robust and cost-effective).
1.4 Increasing Molecularly imprinted polymers (MIPs) are also known as plastic
Nanozyme Specificity antibodies [21], which are easy to prepare, stable, and can be mass
by Molecular produced with a low cost. We have grown an MIP hydrogel layer on
Imprinting a few nanozymes [22, 23], and also on a DNAzyme mimicking
peroxidase-like activity [24–26]. Under optimized conditions, the
selectivity of nearly 100-fold can be obtained for the imprinted
substrate. The improved selectivity partially came from the
improved activity of the imprinted nanozymes due to the enriched
substrate concentration and facilitated release of the product
[23]. In this chapter, we describe the detailed protocols of prepar-
ing MIP-coated nanozymes with peroxidase-like activities, specifi-
cally, the preparation of iron oxide nanozymes with an MIP layer.
Selective Nanozymes by Molecular Imprinting 225
2 Materials
2.1 Preparation 1. 0.2 M FeCl2 stock: freshly prepare 10 mL in Milli-Q water.

of Iron Oxide Protect the FeCl2 stock under nitrogen atmosphere to prevent
Nanoparticles oxidization.
(Fe3O4 NPs) 2. 0.1 M FeCl3 stock: freshly prepare 10 mL in Milli-Q water.
3. 0.2 M aqueous ammonia: freshly prepare 20 mL in Milli-Q
water.
4. A 50 mL 3-neck round bottle flask, with a magnetic stirring bar
placing on the bottom.
5. A thermometer: for heating and monitoring the temperature of
the solution.
6. A magnetic stirrer: for stirring during the synthesis of
Fe3O4 NPs.
7. An oil bath (1000 mL dimethicone).
8. Nitrogen gas: for degassing the solutions.
9. A syringe needle: for setting the nitrogen bubbles under the
solution.
2.2 Preparation 1. 20 mM HEPES buffer (pH 7.6): Dissolve 0.005 mol of

of MIP-Coated Iron HEPES (acid form) and 0.0049 mol of HEPES sodium salt
Oxide Nanoparticles (basic component) into approx. 450 mL of Milli-Q water.
(MIP-Fe3O4 NPs) Using 10% HCl or NaOH to adjust the pH to 7.6, and then
make up the volume to 500 mL with Milli-Q water.
2. 50 mM 3,30 ,5,50 -tetramethylbenzidine (TMB) stock: freshly
prepare 5 mL of 100 mM TMB in dimethyl sulfoxide
(DMSO), then dilute to 50 mM in 20 mM HEPES buffer.
3. Pre-polymerization mixture: Add 100 μL of 600 μg/mL
Fe3O4 NPs, 20 μL of 50 mM TMB, 210 μL of 0.2 M acrylam-
ide (AAm), 42 μL of 1 M N-isopropylacrylamide
(NIPAAm) (see Note 4), 160 μL of 0.1 M N,N´-methylenebi-
sacrylamide (MBAAm), 0.6 μL of N-[3-(dimethylamino)-
propyl]methacrylamide (DMPA), and 468 μL of 20 mM
HEPES buffer into a 1.7 mL microcentrifuge tube. The final
volume is 1 mL.
4. Nitrogen gas: for removing oxygen in the monomer solutions.
5. Polymerization initiator: 20 μL of 10% w/v ammonium per-
sulfate (APS), freshly prepared.
6. Polymerization catalyst: 0.6 μL of tetramethyl-ethylenediamine
(TEMED).
7. Surfactant: 20 μL of 60 mg/mL sodium dodecyl sulfate (SDS).
2.3 Template 1. Methanol with 20% v/v of acetic acid: mix 2 mL acetic acid
Removal with 8 mL methanol, for removing the imprinted
template TMB.
2. Methanol: for removing residual acetic acid, 10 mL.
3. Milli-Q water: for removing residual methanol, 10 mL.
4. UV-vis: for monitoring the removal of TMB at 652 nm.
5. Vacuum-centrifuge: set to 30 C for drying the nanoparticles.
2.4 Preparation 1. 20 mM acetate buffer (pH 4.0): Dissolve 0.6 g of acetic acid
of Substrate Solutions into approx. 450 mL of Milli-Q. Then titrate it to pH 4.0 with
1 M NaOH at the lab temperature of 22 C. Make up volume
to 500 mL with Milli-Q water.
2. 5 mM TMB stock: dilute 100 mM TMB stock (dissolved in
DMSO, see item 2 in Subheading 2.2) to 5 mM in 20 mM
acetate buffer.
3. 5 mM 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-
diammonium salt (ABTS) stock: freshly prepare 5 mL of
100 mM ABTS in Milli-Q water, then dilute it to 5 mM in
20 mM acetate buffer.
4. 5 mM dopamine stock: freshly prepare 5 mL of 100 mM
dopamine in Milli-Q water, then dilute it to 5 mM in 20 mM
acetate buffer.
2.5 Activity Assays 1. Instrument: Tecan Spark (microplate reader). Set the absorp-
tion wavelength to 652 nm for TMB and carry out activity
assays at room temperature (22 C).
2. TMB dilutions (from 0.05 to 1 mM): taking the preparation of
0.5 mM TMB as an example, mix 20 μL of 5 mM TMB with
20 μL of 50 mg/mL MIP-Fe3O4 NPs (containing 50 μg/mL
Fe3O4) and 140 μL acetate buffer in 1.7 mL microcentrifuge
tubes. The total volume is 180 μL.
3. Co-substrate: H2O2. Prepared 10 mM H2O2 stock in Milli-Q
water. Add 20 μL of 10 mM H2O2 into the above TMB
dilutions to normalize the final volume to 200 μL.
4. 96-well plate (transparent): for measuring UV-vis absorption
of TMB and calculate its oxidation amount. Transfer 100 μL of
the above TMB reaction solution to 96-well plate.
2.6 Selectivity Tests 1. Instrument: Tecan Spark (microplate reader). Set the absorp-
tion wavelength to 420 nm for ABTS, and 480 nm for
dopamine.
2. ABTS dilutions and dopamine dilutions (from 0.05 to 1 mM):
prepare in the same way as TMB.
3. Co-substrate: 20 μL of 10 mM H2O2.
4. 96-well plate (transparent): for measuring UV-vis absorption

of ABTS/dopamine and calculate their oxidation amounts.
Transfer 100 μL ABTS/dopamine reaction solution to
96-well plate.
3 Methods
3.1 Preparation 1. In a 50 mL 3-neck round bottle flask, add 1.5 mL of 0.2 M

of Iron Oxide FeCl2 and 4.0 mL of 0.1 M FeCl3. Stir for 10 min with a
Nanoparticles magnetic stirring bar, and at the same time degas it by nitrogen
gas. Set the nitrogen bubbles under the solution using a
needle.
2. Seal the flask with rubber caps and insert a thermometer under
the solution.
3. Place the flask in oil bath and heat it up until the temperature of
the solution reaches 80 C.
4. Drop-wisely add 15 mL of 0.2 M degassed aqueous ammonia
into the flask. Keep stirring at 80 C for 1 h with a gentle
nitrogen flow.
5. Gradually cool the black precipitate (i.e., Fe3O4 NPs) to room
temperature.
6. Add over 50 mL Milli-Q water into the mixture and sonicate;
keep stirring. Then let the Fe3O4 NPs to fully precipitate.
7. Pour the supernatant and repeat the wash process for five times
until the supernatant appears to clear.
3.2 Preparation In this part, the template (i.e., substrate to be imprinted) needs to
of MIP-Coated Iron be self-assembled with the functional monomers and crosslinkers
Oxide Nanoparticles first, which is called pre-polymerization. After the polymerization
(MIP-Fe3O4 NPs) and removal of the imprinted template, the binding pockets are
expected to be shaped chemically complementary to the template
due to the crosslinked polymer matrix (Fig. 1). In other words, the
geometry and chemical features of the substrate will be memorized
by the polymer, which leads to the specific substrate binding and
selective catalysis. Other non-imprinted substrates cannot effec-
tively access the nanozyme core.
1. In a 1.7 mL microcentrifuge tube, mix 100 μL of 600 μg/mL
Fe3O4 NPs (60 μg/mL) and 20 μL of 50 mM TMB (1 mM)
with 468 μL of 20 mM HEPES buffer (pH 7.6). The concen-
trations denoted in brackets are the final concentrations.
2. Add 210 μL of 0.2 M AAm (42 mM), 42 μL of 1 M NIPAAm
(42 mM), 160 μL of 0.1 M MBAAm (16 mM) and 0.6 μL
DMPA (see Note 1) to the above mixture to form a pre-
polymerization mixture. The final volume should be 1 mL.
Fig. 1 A scheme of preparation MIP-coated iron oxide. Reprinted with permission from Ref. [22]. Copyright
2017, American Chemical Society.
3. Purge nitrogen gas (bubbled into the solution) for 1 h to

completely remove the oxygen.
4. Incubate the tube at 4 C for 6 h, allowing the template TMB
to fully interact with the monomers.
5. Add 20 μL of 60 μg/mL SDS (1.2 mg), 10 μL of 10% w/v APS
(0.2 mg), and 0.3 μL of TEMED into the mixture to initiate
the polymerization (see Note 2). Gently convert the tube by
hand five times for mixing.
6. Place the tube on bench. Keep it unperturbed and wait for
nucleation and precipitation of the polymer. After 10–30 min,
when the products start to appear (white, tend to aggregate),
gently invert the tube for 3–5 times to fully disperse the pro-
ducts and then keep it still. Too much inversion or shaking
should be avoided at this stage to facilitate the nucleation (see
Note 3).
7. After waiting for another 10–30 min, when more white cloudy
products are formed, vortex the tube for 5 s to fully disperse the
aggregated product to milk-like dispersion. This step can help
keep the polymers in nanoscale.
8. Place the tube on bench overnight (room temperature), allow-
ing the polymerization to adequately process.
3.3 Template 1. Centrifuge the MIP-Fe3O4 NPs at 5000 rpm (2348 rcf) for
Removal 10 min, then remove the supernatant. Use a pipette to carefully
aspirate the supernatant rather than directly decanting the
sample.
2. Add 1 mL 20% acetic acid/methanol into the pellet, sonicate
the MIP-Fe3O4 NPs for 2 min to facilitate template removal.
Then centrifuge the tube at 5000 rpm (2348 rcf) for 10 min,
discard the supernatant using the pipette. Repeat this step for
three times to fully elute the template.
3. Add 1 mL methanol into the pellet, sonicate for 2 min to
remove the residual acetic acid. Then centrifuge the tube at
5000 rpm (2348 rcf) for 10 min and discard the supernatant.
Repeat this step for three times.
4. Add 1 mL Milli-Q water into the pellet, sonicate for 2 min to
remove the residual methanol in the sample. Then centrifuge
the tube at 5000 rpm (2348 rcf) for 10 min, and discard the
supernatant. Repeat this step two times.
5. Add 500 μL Milli-Q water into the tube and vigorously shake
or vortex the tube to fully disperse the MIP. After sonication
for 2 min, centrifuge at 5000 rpm (2348 rcf) for 10 min, and
collect the supernatant. Check the UV-vis absorption of the
supernatant at 652 nm to confirm that the absorbance is smal-
ler than 0.01 (indicating complete removal of the TMB tem-
plate). Normally, the absorbance should be below 0.01 after
the aforementioned washing steps.
6. Freezing the pellet for 2 h, and then dry it in vacuum-
centrifuge for 3 h (30 C).
3.4 Activity Assays 1. Dilute 5 mM TMB stock to various dilutions (from 0.05 to
1 mM) in 20 mM acetate buffer (see item 2 in Subheading
2.5). Add 20 μL of 50 mg/mL MIP-Fe3O4 NPs (containing
50 μg/mL Fe3O4) or 50 μg/mL free Fe3O4 NPs into the
diluted TMB, and normalize the final volume to 180 μL with
acetate buffer.
2. Add 20 μL of 10 mM H2O2 (final conc. 1 mM) into the above
solution. The total volume for catalytic reaction is 200 μL.
3. After 60 min incubation, transfer 100 μL reaction solution to
96-well plate for UV-vis measurement (652 nm) for TMB.
4. To measure the kinetics of the catalytic reaction, set the micro-
plate reader to kinetic mode. Before adding H2O2, place the
diluted TMB (concentrations from 0.05 to 1 mM) with 20 μL
of 50 mg/mL MIP-Fe3O4 NPs or 50 μg/mL free Fe3O4 NPs
mixture into 96-well plate for 5 min measurement of back-
ground catalysis. Normalize the volume to 180 μL as described
in step 1. Then, add 20 μL of 10 mM H2O2 into the well to
activate the reaction. Immediately start the kinetic scan (for
60 min) after adding H2O2.
5. Calculate the TMB conversion amount: use Beer-Lambert Law
A ¼ εcl, in which ε ¼ 39,000 M1 cm1 and l ¼ 1 cm.
6. According to the data obtained in step 3, use Michaelis–Men-
ten eq. (V ¼ Vmax[S]/(Km + [S])) and Vmax ¼ kcat[E] for cal-
culating V and kcat/Km, in which the [S], [E], Km, Vmax, and
V stand for substrate concentration, nanozyme concentration,
Michaelis constant, maximum oxidation rate and oxidation
rate, respectively.
3.5 Selectivity Tests 1. Dilute 5 mM ABTS and dopamine stock to various dilutions
(from 0.05 to 1 mM) in 20 mM acetate buffer, same as the
preparation of TMB dilutions (see item 2 in Subheading 2.5).
Add 20 μL of 50 mg/mL MIP-Fe3O4 NPs (containing 50 μg/
mL Fe3O4) or 50 μg/mL free Fe3O4 NPs into the diluted
ATBS/dopamine, and normalize the final volume to 180 μL
with acetate buffer.
2. Add 20 μL of 10 mM H2O2 (final conc. 1 mM), and the final
volume is 200 μL.
3. After 60 min incubation, transfer 100 μL solution to 96-well
plate for UV-vis measurement. Set 420 nm for ABTS and
480 nm for dopamine.
4. For measuring kinetic curve for the catalytic reaction, set the
instrument to kinetic mode. Before adding H2O2, place the
diluted ABTS/dopamine (concentrations from 0.05 to 1 mM)
with 20 μL of 50 mg/mL MIP-Fe3O4 NPs or 50 μg/mL free
Fe3O4 NPs mixture into 96-well plate for 5 min measurement
of background catalysis. The volume is 180 μL. Then, add
20 μL of 10 mM H2O2 into the plate to activate the reaction.
Immediately start the kinetic scan (for 60 min) after adding
H2O2.
5. Compare the product absorbance of TMB, ABTS, and dopa-
mine to calculate their overall conversion amount on MIP--
Fe3O4 NPs and free Fe3O4 NPs.
4 Notes
1. Although APS alone can be used as the initiator, it requires

high concentrations of monomers and the crosslinker (>1 M),
often resulting bulk hydrogels instead of nanoparticles. To
prepare the nanosized MIPs at room temperature, monomer
concentrations should be lowered. To achieve this, we used
APS and DMPA as the redox initiators because DMPA can
promote the polymerization rate and enhance the molecular
weight of the polyacrylamide [27]. Therefore, relatively low
monomer concentration (~100 mM) can result in polymer
precipitation, but it would not generate bulk gels. On the
other hand, since DMPA is positively charged, it can better
interact with the negatively charged TMB (but not the posi-
tively charged ABTS) and further improve the catalytic speci-
ficity and activity [22].
2. In MIP preparation step 5 in Subheading 3.2, since the sample
was already purged by nitrogen gas for 1 h, and the sample
volume was only 1 mL, special care needs to be paid when
adding SDS, APS, and TEMED. Try to minimize the time
when opening the cap and adding the reagents. Extended

exposure to air can increase the concentration of the dissolved
oxygen. If the oxygen content in solution raises too much, the
polymerization reaction might terminate in an early stage, and
the performance (selectivity) of the MIPs tends to be compro-
mised. Based on our experience, adding these solutions can be
finished in 1 min.
3. Special care needs to be paid for handing the microcentrifuge
tubes after adding the chemicals. For example, in step 6 of
Subheading 3.2, the tube should be kept in stationary first
without stirring or agitation. Any agitation will adversely influ-
ence nucleation, and consequently the polymer products are
most unlikely to be obtained. Only when some white products
start to appear, the tube should be gently agitated inverted,
until the aggregated products are fully dispersed. Then, wait
10–30 min for the further growth of the polymer in step 7 of
Subheading 3.2. When bulk products start to form (cloudy
aggregates), vortex the tube for 5 s to further disperse the
aggregated products. The well-prepared polymer solution is a
milk-like solution.
4. Special care needs to be paid when dealing with the acrylic
monomers due to their neurotoxicity, and the NIPAAm has a
very strong odor. Therefore, weighing them should be per-
formed in contained tubes with caps closed. We suggest weigh-
ing the empty tubes with caps (as W1) first. Then transfer a
small amount of the acrylic monomer into the tube in a fume
hood and weigh the tube (as W2). Therefore, the amount of
monomer is W2-W1. According to the intended concentra-
tion, calculate the volume of Milli-Q for preparing the mono-
mer solutions.
Acknowledgments
Funding for the Liu lab portion of the work reviewed here was
mainly from the Natural Sciences and Engineering Research Coun-
cil of Canada (NSERC).
References
1. Wei H, Wang E (2013) Nanomaterials with 3. Zhang R, Fan K, Yan X (2020) Nanozymes:
enzyme-like characteristics (nanozymes): next- created by learning from nature. Sci China Life
generation artificial enzymes. Chem Soc Rev Sci:1–18
42:6060–6093 4. Zhou Y, Liu B, Yang R, Liu J (2017) Filling in
2. Huang Y, Ren J, Qu X (2019) Nanozymes: the gaps between nanozymes and enzymes:
classification, catalytic mechanisms, activity challenges and opportunities. Bioconjug
regulation, and applications. Chem Rev Chem 28:2903–2909
119:4357–4412
5. Gao L, Zhuang J, Nie L, Zhang J, Zhang Y, Gu nanoparticles for targeting and visualizing
N et al (2007) Intrinsic peroxidase-like activity tumour tissues. Nat Nanotechnol 7:459–464
of ferromagnetic nanoparticles. Nat Nanotech- 17. Wang X, Hu Y, Wei H (2016) Nanozymes in
nol 2:577–583 bionanotechnology: from sensing to therapeu-
6. Xu C, Qu X (2014) Cerium oxide nanoparticle: tics and beyond. Inorg Chem Front 3:41–60
a remarkably versatile rare earth nanomaterial 18. Fan K, Wang H, Xi J, Liu Q, Meng X, Duan D
for biological applications. NPG Asia Mater 6: et al (2017) Optimization of Fe3O4 nanozyme
e90 activity via single amino acid modification
7. Kumar A, Das S, Munusamy P, Self W, Baer mimicking an enzyme active site. Chem Com-
DR, Sayle DC et al (2014) Behavior of nano- mun 53:424–427
ceria in biologically-relevant environments. 19. Lang NJ, Liu B, Liu J (2014) Characterization
Environ Sci Nano 1:516–532 of glucose oxidation by gold nanoparticles
8. Xu F, Lu Q, Huang P-JJ, Liu J (2019) Nano- using nanoceria. J Colloid Interface Sci
ceria as a DNase I mimicking nanozyme. Chem 428:78–83
Comm 55:13215–13218 20. Golub E, Albada HB, Liao W-C, Biniuri Y,
9. Comotti M, Della Pina C, Matarrese R, Rossi Willner I (2016) Nucleoapzymes: hemin/G-
M (2004) The catalytic activity of “naked” quadruplex DNAzyme–aptamer binding site
gold particles. Angew Chem Int conjugates with superior enzyme-like catalytic
43:5812–5815 functions. J Am Chem Soc 138:164–172
10. Liu B, Liu J (2017) Surface modification of 21. Volkert AA, Haes AJ (2014) Advancements in
nanozymes. Nano Res 10:1125–1148 nanosensors using plastic antibodies. Analyst
11. Liu B, Huang Z, Liu J (2016) Boosting the 139:21–31
oxidase mimicking activity of nanoceria by 22. Zhang Z, Zhang X, Liu B, Liu J (2017) Molec-
fluoride capping: rivaling protein enzymes and ular imprinting on inorganic nanozymes for
ultrasensitive F- detection. Nanoscale hundred-fold enzyme specificity. J Am Chem
8:13562–13567 Soc 139:5412–5419
12. Chang Y, Liu M, Liu J (2020) Highly selective 23. Zhang Z, Li Y, Zhang X, Liu J (2019) Molec-
fluorescent sensing of phosphite through ularly imprinted nanozymes with faster cata-
recovery of poisoned nickel oxide nanozyme. lytic activity and better specificity. Nanoscale
Anal Chem 92:3118–3124 11:4854–4863
13. Shen XT, Zhu LH, Wang N, Zhang T, Tang 24. Zhang Z, Liu B, Liu J (2016) Molecular
HQ (2014) Selective photocatalytic degrada- imprinting for substrate selectivity and
tion of nitrobenzene facilitated by molecular enhanced activity of enzyme mimics. Small
imprinting with a transition state analog. 13:1602730
Catal Today 225:164–170 25. Zhang Z, Liu J (2019) Molecular imprinting
14. Shen XT, Zhu LH, Huang CX, Tang HQ, Yu with functional DNA. Small 15:1805246
ZW, Deng F (2009) Inorganic molecular 26. Zhang Z, Liu J (2018) Intracellular delivery of
imprinted titanium dioxide photocatalyst: syn- a molecularly imprinted peroxidase mimicking
thesis, characterization and its application for DNAzyme for selective oxidation. Mater Horiz
efficient and selective degradation of phthalate 5:738–744
esters. J Mater Chem 19:4843–4851 27. Si K, Guo XQ, Qiu KY (1995) Initiation mech-
15. Zhang Z, Bragg LM, Servos MR, Liu J (2019) anism of radical polymerization using ammo-
Gold nanoparticles as dehydrogenase mimick- nium persulfate and polymerizable amine
ing nanozymes for estradiol degradation. Chin redox initiators. J Macromol Sci A
Chem Lett 30:1655–1658 32:1149–1159
16. Fan KL, Cao CQ, Pan YX, Lu D, Yang DL,
Feng J et al (2012) Magnetoferritin
Chapter 20
Enzyme-Mimics Molecularly Imprinted Polymers Based

on Metal Complexes: Electropolymerization
and Electrocatalytic Application
Elisabetta Mazzotta and Cosimino Malitesta
Abstract
The development of an electrosynthesized molecularly imprinted polymer (MIP) based on a metal complex
is here reported as an effective strategy for combining advantages coming from metal-ion coordination and
catalytic capabilities of metallic centers with ones deriving from electropolymerization. Metal ion coordina-
tion combines the flexibility of noncovalent imprinting approaches with the strength and specificity of
covalent ones representing an attractive binding mechanism in MIP design for the recognition of a vast
array of analytes. In addition, such a MIP possesses catalytic properties other than recognition capability,
which is not so common in MIP field. On the other hand, electropolymerization represents a highly
successful way of easily anchoring MIP-based sensing layers to the transducer surface. Procedures for
MIP electrosynthesis as well as for its analytical application in electrocatalytic sensing are described.
Key words Molecularly imprinted polymers, Electropolymerization, Metal-ion coordination, Elec-

trochemical sensors
1 Introduction
One of the most important issue in imprinting technology is the

development of materials having not only high selectivity but also
presenting fast desorption and rebinding kinetics under mild con-
ditions. The most common imprinting approaches are based on
covalent and noncovalent interactions, each of one presenting dif-
ferent levels of specificity and reversibility. Covalent bonding is
functional groups specific and directional but it is slown in rebind-
ing kinetics. On the other hand, the most common attractive
features of nonvalent interaction are its versatility, fast kinetics and
formation, and breakage under mild conditions. At the same time,
it suffers from less selectivity.
The use of metal coordination in the design of molecularly
imprinted polymers (MIPs) represents an attractive approach able
https://doi.org/10.1007/978-1-0716-1629-1_20,
233
234 Elisabetta Mazzotta and Cosimino Malitesta
to combine some of the most appealing features of other conven-

tional imprinting schemes. Due to its relatively high strength and
selectivity, it is well suited to molecular recognition. Moreover, its
formation and breakage can take place under mild condition also in
aqueous media not being affected by the solvent nature. Addition-
ally, the availability of a vast array of ligands allows one to control
and define the coordinating properties of metal centers towards
different substrates.
The use of metal coordination in imprinting procedures is
proposed since long with successful application in catalysis [1],
chemo- and enantioselective separation [2], and sensors [3], also
exploiting the incorporation of redox-active centers within the
imprinted cavities generating a polymer with enzyme-like
activity [4].
In this work, the protocol for preparing an electropolymerized
metal complex-based MIP is described. In particular, a
Co-porphyrin (Co(III)tetrakis(o-aminophenyl) porphyrin,
CoTAPP) is used as a monomer. The reported protocol reports
also the application of the prepared MIP to the electrochemical
detection of a toxic organohalide commonly used as pesticide,
2-(2,4-dichlorophenoxy) butyric acid (2,4-DB) exploiting the abil-
ity of cobalt porphyrin to catalyze, via reduction of metal center, its
reduction, which is a kinetically slow process requiring high over-
potentials thus running the risk of considerable interferences [3].
2 Materials
Prepare all solutions with ultrapure water (Millipore Milli-Q,

18.2 MΩ cm 1) before use. Use all analytical grade reagents and
solvents and store them according to the indication of the
manufacturer.
2.1 Electrode 1. Pt disc working electrode.

Preparation 2. Water and ethanol for washing.
3. Microcloth and Nylon polishing pad and 1.0, 0.3, and 0.05
micron alumina powder for electrode polishing.
4. Ultrasonic bath.
5. One-compartment electrochemical cell.
6. Reference electrode: saturated calomel electrode (SCE)
7. Auxiliary electrode: platinum (Pt) wire.
8. Solution 0.5 M H2SO4.
9. Potentiostat/galvanostat.
Electropolymerization and Electrocatalytic Application of MIPs 235
2.2 Electropoly- 1. MIP polymerization mixture: 1 mM CoTAPP and 20 mM

merization 2,4-DB in 0.1 M tetrabutylammonium hexafluorophosphate
(TBAFP) in acetonitrile (see Note 1).
2. Not imprinted polymer (NIP) polymerization mixture: 1 mM
CoTAPP in 0.1 M TBAFP in acetonitrile.
3. One-compartment electrochemical cell.
4. Pt disc working electrode.
5. Reference electrode: Ag/Ag+ 0.1 M in acetonitrile.
6. Auxiliary electrode: Pt wire.
2.3 Washing 1. Acetonitrile.

for Template Removal 2. Methanol.
3. Magnetic stirrer and stir bar.
2.4 Electrochemical 1. One-compartment electrochemical cell connected to

(Voltammetric nitrogen line.
and Amperometric) 2. Working electrode: MIP- or NIP-modified Pt electrode.
Measurements 3. Reference electrode: Ag/Ag+ 0.1 M in acetonitrile.
4. Auxiliary electrode: Pt wire.
6. 2,4-DB at different concentrations (200μM–2 mM) in 0.1 M
TBAPF in acetonitrile.
7. 4-(2,4-dicholorophenoxy)propionic acid (2,4-DP) at different
concentrations (200μM–2 mM) in 0.1 M TBAPF in
acetonitrile.
8. Dichloroacetic acid (DAA) at different concentrations
(200μM–2 mM) in 0.1 M TBAPF in acetonitrile.
9. Phenoxyacetic acid (POAA) at different concentrations
(200μM–2 mM) in 0.1 M TBAPF in acetonitrile.
10. 4-chlorophenylacetic acid (PAA) at different concentrations
(200μM–2 mM) in 0.1 M TBAPF in acetonitrile
11. 4-chlorocathecol at different concentrations (200μM–2 mM)
in 0.1 M TBAPF in acetonitrile.
12. 2-chlorophenol at different concentrations (200μM–2 mM) in
0.1 M TBAPF in acetonitrile.
13. 0.1 M TBAPF in acetonitrile.
14. Magnetic stirrer and stir bar (for amperometric
measurements).
15. Volumetric pipette.
3 Methods
3.1 MIP and NIP 1. Clean the electrode surface before depositing MIP by electro-
Electropolymerization polymerization. To this aim, polish the Pt electrode surface
with 0.05 micron alumina powder on Microcloth polishing
pad: put small amount of alumina powder on polishing pad
and wet with distilled water. Hold the electrode vertically while
polishing and add distilled water if the pad gets dry. After
polishing, the surface electrode should be mirror shiny. If you
see scratches on it, you may need to go through 1.0, 0.3, and
0.05 micron alumina powder in a sequence, using the Nylon
polishing pad for 1.0 and 0.3 micron alumina.
2. Place the electrode in ultrasonic bath in ethanol/water 1:1
(V/V) for 10 min for removing alumina powder residues.
3. For electrochemical activation of the electrode, assemble the
three-electrode electrochemical cell comprised of the Pt work-
ing electrode, SCE reference electrode and Pt wire auxiliary
electrode. Cycle the potential between 0.2 and 1.2 V at a scan
rate of 100 mV/s in 0.5 M H2SO4 until a steady-state is
reached.
4. For MIP electropolymerization, assemble the three-electrode
electrochemical cell comprised of the cleaned/activated Pt
working electrode, Ag/Ag+ 0.1 M/acetonitrile reference elec-
trode and Pt wire auxiliary electrode. Cycle the potential
between 0.1 and 1.0 V at a scan rate of 200 mV/s in a
solution of 1 mM CoTAPP containing 20 mM 2,4-DB (MIP
electropolymerization mixture, Subheading 2.2, step 1) for
50 scans (see Note 2).
5. For NIP electropolymerization, use the same conditions
reported at step 4. in a monomer solution without adding
2,4-DB (NIP electropolymerization mixture, Subheading 2.2,
step 2).
6. After electropolymerization, rinse electrodes with acetonitrile,
dry, and keep them in the air at room temperature until use.
3.2 Calibration 1. Assemble the cell as described at Subheading 2.4.

Measurements 2. Place an exact volume of supporting electrolyte (0.1 M TBAFP
with Voltammetric in acetonitrile), measured using volumetric pipette, in the
MIP-Sensor measuring cell.
3. Turn on nitrogen flow and sparge solution for 5 min. Perform
calibration measurements under nitrogen flow at room
temperature.
4. Record a cyclic voltammetry in an electrolyte solution between
0.1 and 1.7 V at scan rate 100 mV/s.
Current (μA)
0
–2
–4 ip
–6
–1.8 –1.6 –1.4 –1.2 –1.0 –0.8 –0.6 –0.4 –0.2 0.0
Potential (V)
Fig. 1 Evaluation of ip as analytical signal for voltammetric calibration

measurements
5. Carry out calibration by adding known amounts of the analyte

stock solution and recording cyclic voltammetry as described at
step 4. after each addition.
6. Use the same procedure described at steps 1–5. both for MIP
and NIP electrodes.
7. After measurements, wash sensors with copious amounts of
acetonitrile, dry and keep them at room temperature in air
between measurements.
8. For quantitatively evaluate sensor response, measure peak cur-
rent ip for each tested analyte concentration, with peak poten-
tial located at about 1.5 V, as shown in Fig. 1.
9. Plot sensor response in coordinates 2,4-DB concentration ver-
sus ip and use linear regression for evaluating sensor linearity
range, sensitivity (i.e., slope of the regression curve) and limit
of detection, calculated as 3σ/a [5] (where σ is the standard
deviation of the linear regression and a is the slope of the
regression curve).
10. Perform at least three replicates on the same electrode for
evaluating sensor response repeatability and at least three mea-
surements on different electrodes (prepared under the same
conditions) for evaluating sensor response reproducibility. This
can be done evaluating the relative standard deviation asso-
ciated to slopes of compared calibration curves.
11. Compare MIP and NIP calibration curves (in the case of
unspecific response from NIP electrode) for evaluating the
imprinting factor, which can be expressed as the ratio between
MIP and NIP sensor sensitivity [6].
3.3 Calibration 1. Assemble the cell as described at Subheading 2.4.

Measurements 2. Place an exact volume of supporting electrolyte (0.1 M TBAFP
with Amperometric in acetonitrile), measured using volumetric pipette, in the mea-
MIP-Sensor suring cell and turn on stirring.
3. Turn on nitrogen flow and sparge solution for 5 min. Perform
calibration measurements under nitrogen flow at room
temperature.
4. Perform amperometric measurements by applying a fixed
potential of 1.8 V.
5. Start measurement in electrolyte solution and wait until stable
current is recorded. Then proceed by adding known amounts
of the analyte stock solution and wait until recorded current is
stable before adding the subsequent aliquot of analyte.
6. Use the same procedure described at steps 1–5. both for MIP
and NIP electrodes.
7. After measurements, wash sensors with copious amounts of
acetonitrile, dry, and keep them at room temperature in air
between measurements.
8. For quantitatively evaluate sensor response, measure reduction
current increase Δi after each analyte addition, as shown in
Fig. 2.
9. Plot sensor response in coordinates 2,4-DB concentration ver-
sus Δi and use linear regression for evaluating sensor linearity
range, sensitivity (i.e., slope of the regression curve) and limit
of detection, calculated as 3σ/a [5] (where σ is the standard
deviation of the linear regression and a is the slope of the
regression curve).
10. For evaluating MIP selectivity, use the same procedure
described at steps 1–5. for testing sensor response to other
interferences (2,4-DP, DAA, PAA, POAA, 4-chlorocathecol,
2-chlorophenol) (see Note 3). Interference ratio can be calcu-
lated as the ratio between MIP amperometric response to each
tested molecule and to 2,4-DB, both at a selected
concentration.
11. Perform at least three replicates on the same electrode for
evaluating sensor response repeatability and at least three mea-
surements on different electrodes (prepared under the same
conditions) for evaluating sensor response reproducibility. This
can be done evaluating the relative standard deviation asso-
ciated to slopes of compared calibration curves.
12. Compare MIP and NIP calibration curves (in the case of
unspecific response from NIP electrode) for evaluating the
imprinting factor, which can be expressed as the ratio between
MIP and NIP sensor sensitivity [6].
0
Δi
–5
Current (μA)
–10
–15
0 500 1000 1500 2000 2500 3000 3500

Time (s)
Fig. 2 Evaluation of Δi as analytical signal for amperometric calibration

measurements
4 Notes
1. The protocol here reported for electropolymerizing a MIP

based on CoTAPP for 2,4-DB can be used for the imprinting
of other organohalogen compounds whose electroreduction is
catalyzed by the cobalt center. Also, different cobalt complexes
and other metal complexes with electrocatalytic activity
towards organohalide reduction can be used, but it has to be
taken into account the need of having monomer with chemical
functionalities able to electropolymerize (e.g., pyrrole, aniline,
thiophene). If different monomers and templates are used,
experimental conditions for both electropolymerization and
calibration measurements should be modified accordingly.
2. Among experimental conditions influencing MIP-based elec-
trochemical performances, monomer:template ratio and film
thickness have a key role. A monomer:template ratio different
from 1:20 here proposed, commonly used in MIP electropo-
lymerization, is 1:4. In the case of electropolymerization by
cyclic voltammetry, film thickness can be controlled by varying
the number of scans.
3. For evaluating MIP selectivity, other molecules can be tested.
Selected molecules act as interference in the analyte detection
due to their co-existence in the real matrices and electroactivity
in the selected experimental conditions or to their high struc-
tural similarity (e.g., other chlorinated species).
References
1. Mohamed S, Balieu S, Petit E, Galas L, 4. Gu Y, Yan X, Li C, Zheng B, Li Y, Liu W,
Schapman D, Hardouin J, Baati R, Estour F Zhang Z, Yang M (2016) Biomimetic sensor
(2019) A versatile and recyclable molecularly based on molecularly imprinted polymer with
imprinted polymer as an oxidative catalyst of nitroreductase-like activity for metronidazole
sulfur derivatives: a new possible method for detection. Biosens Biolectron 77:393
mustard gas and V nerve agent decontamina- 5. Meier PC, Zund RE (2000) Statisitcal methods
tion. Chem Commun 55:13243 in analytical chemistry, 2nd edition, Wineford-
2. Oezcan AA, Say R, Denizli A, Ersoz A (2006) ner JD Ed., John Wiley & Sons: New York, NY
L-histidine imprinted synthetic receptor for bio- 6. Turco A, Corvaglia S, Mazzotta E (2015) Elec-
chromatography applications. Anal Chem trochemical sensor for sulfadimethoxine based
78:7253 on molecularly imprinted polypyrrole: study of
3. Mazzotta E, Malitesta C (2010) Electrochemical imprinting parameters. Biosens Bioelectron
detection of the toxic organohalide 2,4-DB 63:240
using a co-porphyrin based electrosynthesized
molecularly imprinted polymer. Sens Actuat B
148:186
Chapter 21
Using Molecular Dynamics in the Study of Molecularly

Imprinted Polymers
Gustaf D. Olsson, Jesper G. Wiklander, and Ian A. Nicholls
Abstract
Molecular dynamics (MD) simulations of prepolymerization mixtures can provide detailed insights
concerning the molecular-level mechanisms underlying the performance of molecularly imprinted polymers
(MIPs) and can be used for the in silico screening of candidate polymer systems. Here, we describe the use
of MD simulations of all-atom, all-component MIP prepolymerization mixtures and procedures for the
evaluation of the simulation data using the Amber simulation software suite.
Key words Computational design, Molecular dynamics, Molecular imprinted polymer, Prepolymer-
ization mixture
1 Introduction
The past two decades have witnessed the establishment of compu-

tational methods as valuable tools for the study of molecularly
imprinted polymers (MIPs) [1–6]. Computational strategies have
been used for both developing fundamental understanding of the
mechanisms underlying the molecular imprinting process and for
the design of imprinted materials. Among the range of computa-
tional approaches available, those based upon molecular dynamics
(MD) simulations have arguably proven the most valuable, as they
can provide molecular-level insights into the mechanisms underly-
ing imprinted polymer formation and even concerning polymer–
ligand recognition. MD studies of MIP prepolymerization mixtures
are currently the most widely used computational strategy [7–9], in
particular when applied to the simulation and evaluation of
all-atom, all-component MIP prepolymerization mixtures as devel-
oped and refined in our laboratory and by others [10–22]. The use
of such MD-based methodology is described here, with protocols
that rely heavily upon the Amber simulation software suite (see
Note 1) [23].
https://doi.org/10.1007/978-1-0716-1629-1_21,
241
242 Gustaf D. Olsson et al.
2 Materials
To set up and run MD simulations of MIP prepolymerization

mixtures, access to a computer is required. Most required software
will run on different operating systems (OSs). The process of
installation varies between operative systems. There are minimum
hardware requirements/recommendations for most software
though in general a multicore central processing unit (CPU) and
potential graphics processing unit (GPU) acceleration will increase
the speed of data production. Securing access to custom built
hardware for the intended purpose of planned simulations, either
locally or on shared resources, is often the best option for research
purposes, in particular for the simulation of larger systems.
The material presented is (unless otherwise specified) based on
the current versions of software, updated and patched, running on
macOS Mojave (10.14.6) or Ubuntu (18.04 LTS bionic) (see Note
2). The software/applications used (excluding choice of editor and
potential dependencies) in this protocol are the packages included
in Amber20/AmberTools20 (https://ambermd.org) as well as Che-
mAxon Marvin (https://chemaxon.com/products/marvin), Avo-
gadro (https://avogadro.cc/), Packmol (http://m3g.iqm.
unicamp.br/packmol/home.shtml), Grace/Xmgrace (https://
math.nyu.edu/aml/software/xmgrace.html), VMD (https://
www.ks.uiuc.edu/Research/vmd/), and UCSF Chimera (https://
www.cgl.ucsf.edu/chimera/).
3 Methods
3.1 System Design When considering performing simulations, some initial questions
are important. Firstly, should and can the system be simulated?
Secondly, what is the knowledge gap to be filled and thus, what
level of detail is required? Based on this, the simulations should be
designed and pursued to best fit the purposes. In the case of
simulations for prognostic purposes (e.g., finding the most suitable
monomer for a certain template or optimizing stoichiometries),
time-consuming all-atom, all-component MD simulations may
not be the optimal starting strategy. Instead, less demanding pro-
tocols such as smaller system/selected component MD screening,
docking, quantum mechanical methods, or chemometric analysis
can be more appropriate as initial steps. The need for experimental
validation in the absence of data for correlation with simulation
results must also be considered. In order to limit the scope of this
topic, the methodology presented is focused on diagnostic MD
studies of imprinted systems (i.e., correlating the performance of
MIPs with molecular events occurring in the corresponding pre-
polymerization mixtures).
Molecular Dynamics in Molecular Imprinting 243
Initially, some general consideration regarding system design is

appropriate. For diagnostic purposes, recreating the experimental
conditions is imperative. Thus, the components to be included
(template, solvent, functional and cross-linking monomers and
initiator) as well as their relative stoichiometries should reflect, as
close as possible, the prepolymerization mixtures studied. Approx-
imations and/or simplifications may be required; however, care
should be taken to ensure results that are still representative of
reality.
Once details regarding the system design are available, the next
step will be generating input structures with suitable parameters
followed by system equilibration, performing simulations (data
production) and finally processing and evaluation of the data
produced.
These steps will be addressed in a general manner focusing on
simulation of a random bulk liquid mixture/solution with compo-
nents of interest for MIP protocols.
3.2 Input Generation Generating all input required for MD simulations broadly involves
creation of molecular models, selecting and applying appropriate
parameters, and generating input/control files for the simulation
engine.
3.2.1 Virtual Molecular Having identified the components to be included in simulations,

Models virtual molecular models (VMMs) need to be created. A recom-
mendation is to start by documenting 2D structures of all compo-
nents which are to be simulated as these will be useful later on. As
ChemAxon provide licenses for academic and non-profit use, the
cross-platform MarvinSketch software is routinely used for these
purposes. There are several different file formats describing struc-
tural information which vary depending on how the structure was
determined, from which source information was obtained or which
software was used to create the file (see Note 3). Examples include
pdb, xyz, mol, mol2, xml, sy2, smiles, fasta, and more. For the
purpose of this protocol, the protein data bank file format (http://
www.wwpdb.org/documentation/file-format-content/for
mat33/v3.3.html), the default format for files hosted by the RCSB
PDB (https://www.rcsb.org/), will be used.
Some structures such as protein/peptides, DNA/RNA, and
carbohydrates may be available from (or can be constructed
using) online resources (see Note 4). Structures can also be gener-
ated locally using numerous different programs/applications. One
example is Avogadro [24], which is routinely used by the group and
will be discussed in the context of the example system presented.
Other tools, such as LEaP, which is included with instructions in
the Amber package, are also available.
The first step will be to generate VMMs of MIP components.
This example is built on acrylate MIPs and includes small organic
Fig. 1 The Avogadro GUI with some options/menus and the generated MAA structure displayed
molecules as monomers and solvent. Assuming that a template

belongs to this class of structures, the process is the same. If the
structures of the components are not available from online sources,
they must be constructed. For this example, Avogadro was used to
create methacrylic acid (MAA) (Fig. 1) (see Note 5). After generat-
ing/importing the structure, a conformer search can be used to
identify a low energy conformation. This is normally followed by a
geometry optimization and final energy minimization until conver-
gence criteria are met. Avogadro includes the Generalized Amber
Force Field (GAFF) [25], which can be used for these purposes.
This can be of interest as this example will make use of GAFF2
parameters later. After construction, optimization, and minimiza-
tion of MAA, the structure information is exported and saved as a
pdb file (Fig. 2). The generated pdb file will contain information
which may need to be modified or which is not used by Antecham-
ber/LEaP during later stages and can be removed. Files which will
be passed to Antechamber for parameterization and use with
GAFF2 can normally be used without modification. Regarding
files intended for use with parameters from other force fields
(FFs), the residue name (default being UNL, Fig. 2) normally
needs to be modified, atoms should be uniquely identifiable and
sometimes atom labels may need to be modified as well.
If the system to be simulated contains amino acid, nucleic acid
or carbohydrate sequences, acquiring experimentally derived struc-
tures from online sources (see Note 4), generating these using a
Fig. 2 The unmodified pdb file produced by Avogadro for MAA
Fig. 3 Using LEaP to generate a pdb file of a structure present in the available FFs
designated input generator or constructing these with LEaP is

recommended. The atom naming system used by Avogadro can
cause problems when applying parameters in LEaP as these are
not generated for the particular structure but rather applied. It is
important to pay attention to naming conventions. If a chosen
residue label exists in an FF to be used, problems will arise since
labels and structures will not match the FF information. In the same
manner, when creating VMMs where existing parameters are to be
applied, care must be taken to use accepted label convention to
ensure parameters are applied correctly.
There are several solvent models included with Amber. These
include different water models and a few common organic solvents
(see Note 6). As water will be used as solvent for this example, there
is no need to generate the structure using Avogadro. Appropriate
pdb files can be generated using LEaP when FFs contain the desired
structures. This process is shown in Fig. 3. The FF parameters
describing OPC water are sourced, parameters associated with the

OPC water model are assigned to the variable (“var”) and a pdb file
contains the information in “var” is written to disk. For reasons
explained later, this protocol will use a general water pdb file with
residue name “WAT” (as WAT or HOH standards used in the pdb
file format) containing three atoms named “O,” “H1,” and “H2”
(even though a single “O” would have sufficed). This was gener-
ated using Avogadro and manually edited, changing “UNL” to
“WAT,” adding 1 and 2 numbering to the respective hydrogens
and removing everything but the three lines containing coordinate
information. This file was saved as “solvent.pdb.” At this point, pdb
structure files for MAA (methacrylic_acid.pdb) and water (solvent.
pdb) should have been generated and available.
3.2.2 Generating GAFF Once VMMs are available, parameters will need to be generated for
Parameters those without existing parameters. Generating parameters can be
done in different ways though for this protocol, which will use
GAFF2 parameters for MAA, Antechamber and Parmchk2
(included with Amber) will be used. Parameters for the selected
solvent, water, are already available in an existing FF.
The command used to run Antechamber for the purpose of
generating input files for MAA is presented in Fig. 4 with a number
of options/flags included. It involves defining input/output (I/O)
filenames (i/o) and extensions (fi/fo), changing the VMMs
“UNL” residue label from Avogadro (Fig. 2) to MAA (rn, unique
three-letter abbreviations are required for residues/structures) and
setting the topology file name in the prep file to “methacrylic_acid”
(rf, the default otherwise being “molecule.res”). In the example,
the atom type is selected to GAFF2 (at) where GAFF is otherwise
the default. This choice is motivated by the fact that GAFF2 is now
the recommended alternative according to the Amber manual even
if it is not the default. The charge method is set to the AM1-BCC
(c) and the net charge (nc) for our neutral MAA set to zero (see
Note 7). One intermediary file produced by Antechamber is
“NEWPDB.PDB.” This pdb file will contain the changed labels
and using this file together with the newly produced prep and later
frcmod files will ensure naming of residues and atoms correlate
between files. Therefore, the “NEWPDB.PDB” file is routinely
renamed and retained, here to “methacrylic_acid_ac.pdb.” After
producing the prep file, Parmchk2 is executed to check the para-
meters produced (Fig. 4) where input file (i) and format (f) as
well as an output filename (o) are defined and the FF parameter
set is selected as GAFF2 (s 2, where GAFF is otherwise the
default). The two files, methacrylic_acid.prep and methacrylic_a-
cid.frcmod (Fig. 5), created here contain the information required
for MD simulation of MAA (see Note 8). For additional compo-
nents, the above process needs to be repeated.
Fig. 4 Commands used to run Antechamber and Parmchk2 to generate charge information and forcefield
modifications
3.2.3 Constructing the With VMMs and parameters available for all components to be used
Virtual Simulation System in the system under study in hand, assembling these into a virtual
simulation system (VSS) is the next step. A variety of methods and
tools are available (see Note 9). The method of choice for simula-
tion of MIP prepolymerization mixtures (and other systems)
adapted by the group is Packmol [26]. Using the example input
file for Packmol (Fig. 6), a simple cubic box of dimensions
30x30x30 Å, containing 100 MAA and 400 water molecules,
randomly distributed, can be created (see Note 10). The produced
VSS is written to “model-system.pdb” containing MAA and water.
In the pdb file, the correct abbreviations and renamed atoms
(Fig. 7) are included since the pdb file produced by Antechamber
was used to build the system. There is a lot of empty space in the
system (Fig. 7) when inspected visually. This is not optimal and the
packing can be optimized further; however, for this example this
will be resolved during equilibration. Note that leaving empty
space, thus creating inhomogeneous systems, may require extended
equilibration and can cause issues when running simulations using
GPU-accelerated calculations.
3.2.4 Applying With the VSS constructed the parameters, either generated or
Parameters available from FFs, need to be applied. For systems including
proteins/peptides, DNA/RNA, lipids and polysaccharides, there
are multiple FFs available in Amber and the manual provides
Fig. 5 Contents of the prep and frcmod files generated for MAA using Antechamber and Parmchk2
suggestions regarding their use. For proteins the recommendation

is ff14SB with ff19SB including improvements, DNA/RNA simu-
lations are recommended to be run with OL15/OL3, GLY-
CAM_06j is recommended for carbohydrates and lipid17 for
lipids. As discussed, several different water models are included
(see Note 6). GAFF is “a general force field, for organic molecule
like ligands” though the recommended version as of writing is
GAFF2. Parameters are also available for ions, organic solvents
and more.
For this example, GAFF2 parameters will be used for MAA,
combined with OPC parameters for water. The parameters are
applied using the program LEaP which is included in Amber in
two different versions xLEaP and tLEap. xLEaP (Fig. 8) is
launched with a graphical user interface (GUI) (see Note 11). For
Fig. 6 Example input file and selected output when using Packmol
this example, the text-based command line interface (CLI) alterna-

tive tLEaP will be used. A convenient way to organize LEaP
sessions is to write the commands into a file and then reading that
file into LEaP. This provides both a record of what has been done,
useful for identifying potential problems, as well as possible auto-
mation of processing repeated/multiple I/O. An example of such a
file is presented in Fig. 9.
First, parameters are loaded by “sourcing” FF files, for this
example the GAFF2 and OPC water FFs. After sourcing the FFs,
the parameter files and FF modifications produced using Ante-
chamber and Parmchk2, the prep (loadAmberPrep) and frcmod
(loadAmberParams) files are loaded. The order in which FFs and
parameter files are sourced is important, as these will be prioritized
in a “bottom-up” order, meaning that the last thing loaded will be
prioritized. Care must therefore be taken ensuring MAA para-
meters produced are sourced last as there may be deviations from
GAFF2 parameters that need to be prioritized. Different FFs define
Fig. 7 Extract from the Packmol generated pdb file (left), and the same pdb file visualized using UCSF Chimera
(right)
Fig. 8 The xLEaP GUI after loading the model-system.pdb

Fig. 9 LEaP input file used to prepare input files for simulations
Fig. 10 Part of the execution of the LEaP script in Fig. 9

Fig. 11 Using ParmEd to summarize a parameter/topology file
atom types/labels differently, and although there is little chance for

conflict in these instances, routinely checking the sourcing will save
time and prevent issues.
The system was assembled using Packmol with a general water
model pdb (residue name “WAT”). The intention, however, is to
use the OPC water model. As the default water model is TIP3P,
WAT needs to be defined as OPC (WAT ¼ OPC) (Fig. 9). LEaP
will then interpret all WAT residues as OPC water and apply the
correct atom names and parameters, instead of applying TIP3P
parameters or failing.
A variable is then defined, “sys,” and the “model-system.pdb”
file produced using Packmol is loaded as “sys” (sys ¼ loadPdb
model-system.pdb) (Fig. 9). A check is performed on “sys” to
inspect the system and its applied parameters (check sys). Box
dimensions are applied to the system using the van der Waals
volumes of components to apply system dimensions (setbox sys
vdw). Finally, input files for running the simulations are generated
(saveAmberParm sys model-system.parm7 sys model-system.rst7).
This includes an Amber topology file normally using suffixes such as
prmtop, parm7, or top and a coordinate file using suffices such as
inpcrd, restrt, rst7, or crd. Using appropriate suffixes is encouraged
and will help automatic identification of format when loading files.
However, in most cases it is not crucial as file types can be manually
defined. Amber now uses binary formats (NetCDF) for generated
trajectory data and restart files by default. This format can also be
used for the above-mentioned files. Finally, we exit the session

(quit).
Excerpts from the above process can be seen in Fig. 10, exclud-
ing some lines regarding loading and application of the parameters
(FFs). A warning is raised regarding the unperturbed charge of the
unit being non-zero and not integral (0.099900). Charges
derived using the AM1-BCC charge method (Fig. 5) rendered
the total charge of neutral MAA 0.000999. This may seem negli-
gible, but with 100 molecules in the system, the charge becomes
substantial. With 1000 molecules, the excess charge would be
almost 1 and a neutralizing charge would have to be added.
This problem can generally be resolved by deriving charges using
an appropriate quantum mechanical method (see Note 7). For the
purposes of this example, we will assume that the derived charges
and the observed deviation is acceptable. Included with Amber is
ParmEd (Fig. 11), a useful resource for extracting an overview of
the system and confirm that charges, composition, and other details
are correct (see Note 12).
3.3 Simulating The exact process of performing simulations will vary greatly
Constructed Systems depending on the system being simulated. Resource requirements
will depend on the size of the system, and with new multicore CPU
models and the possibility of performing GPU accelerated simula-
tions, users can perform simulations of larger and more demanding
systems locally. Getting access to shared computational resources is
another option. Running simulations requires defining input con-
trols and parameters; for this example, a series of input files are
generated and used as input for the simulation engine(s) of Amber
(SANDER/PMEMD, see Note 13). Following the outlined general
example for performing simulations of MIP prepolymerization
mixtures, an initial energy minimization of the system is performed
followed by steps to equilibrate the temperature and density of the
system before performing the final production phase simulations.
This is done using four different input scripts presented in Fig. 12
(see Note 14).
3.3.1 Energy The initial energy minimization is performed through a combina-

Minimization tion of steepest descent and conjugate gradient energy minimiza-
tion steps (Fig. 12, top-left “emin.in”). For the small example
system discussed, a short energy minimization of a total of
1000 cycles, switching from steepest descent to conjugate gradient
after 500 cycles is used. The number of cycles and settings may vary
strongly with the system/structure being minimized.
3.3.2 Temperature After initial energy minimization, the temperature of the system is
Equilibration equilibrated under conditions of constant number of particles and
volume (Fig. 12, top-right “eqTemp.in”). The simulations use
periodic boundary conditions to be representative of bulk solution
Fig. 12 Input files for energy minimization (top left), temperature equilibration (top right), pressure equilibration
(bottom left), and production phase (bottom right) calculations
conditions with constant volume and a 9 Å cutoff for long range

electrostatic interactions. Bonds to hydrogen are restrained using
the SHAKE algorithm allowing the time step of (0.002 ps). The
temperature is increased from 0 K to 293.15 K using Langevin
dynamics for temperature scaling with the collision frequency γ
given by gamma_ln. 50,000 MD steps are performed with a time
step of 0.002 ps for a simulation of 100 ps. Energy and coordinate
data is written to output every 1000 steps and to restart file every
2000 steps (used to restart/continue simulations if (in)voluntarily
terminated.
3.3.3 Pressure After temperature equilibration, the density of the system needs to
Equilibration be equilibrated by applying (equilibrating) pressure (Fig. 12,
bottom-left “eqPres.in”). This step is a continuation from the
temperature equilibration and information regarding coordinates
and velocities are read from the restart file from the preceding step.
Pressure equilibration is performed using isotropic position scaling
with a target pressure of 1 bar and a pressure relaxation time of 2 ps.
The temperature is kept constant at 293.15 K and constant pressure
periodic boundary conditions are used. Most other shown settings
are unchanged, this stage will be performed over 250 ps as density
equilibration normally is a slower process than temperature equili-
bration. For simulating bulk MIP prepolymerization mixtures, this
general procedure is normally enough to produce equilibrated and
stable systems (see Note 15). Some systems may need additional or
modified input and the number of MD steps (iterations) required
to achieve equilibrated systems are normally longer than in these
examples.
3.3.4 Data Production Following equilibration, the final MD simulation is started using
the production phase input file (Fig. 12, bottom-right “prod.in”).
This step is a continuation of the preceding step (pressure equili-
bration), reverting to constant volume periodic boundary condi-
tions with no pressure scaling. The assumption is that the system is
fully equilibrated and has arrived at a stable density. The data
production stage is divided into chunks of 1000 ps with the next
1000 ps block being a direct continuation of the previous chunk.
This produces output divided into files of manageable size. Addi-
tionally, if there is a problem breaking the simulation, at no point
will more than the last 1000 ps needs to be re-run. The resultant
trajectories can be manipulated post-production using Cpptraj to
join, split, compress, extract, and much more. The provided exam-
ples should constitute a working foundation to produce usable
input for simulations of MD prepolymerization mixtures (see
Note 16).
All of the calculations above can be executed either directly
from the CLI or by placing commands in a shell script (or job script
if using a workload management system such as Slurm (https://
slurm.schedmd.com/)). An example of such a shell script is pre-
sented in Fig. 13. Overwriting existing files (-O), on the first-line
input is presented to SANDER starting the energy minimization
using the emin input (i emin.in), writing output data to emin.out
(o emin.out). The model-system parameter/topology (p) and
input coordinate (c) files are specified and restart data is written to
emin.ncrst (r emin.ncrst). On the next line, the energy minimized
system is used as the starting point (c emin.ncrst) for the temper-
ature equilibration. This step also produces an MD trajectory file,
eqTemp.nc (x eqTemp.nc). The sequence progresses downwards
until the system is fully equilibrated, followed by the simulation
data production phase which can be extended for as long as needed
(see Note 17).
3.4 Processing and After production of simulation data, this needs to be processed and
Analysis of Data analyzed. The data output file contains some readable information
of potential interest; however, the bulk of the data will be contained
in the trajectory files which must be analyzed to extract information
regarding molecular interactions. This potentially involves some
processing of the output, for example, if wrapping (which is the
default and generally recommended setting) was not used, this may
be an important initial step if graphical representations of the
system are desired (see Note 18). Visual inspection is to be recom-
mended for MD simulations in order to quickly identify any major
Fig. 13 Shell script for running the complete set of calculations
problems with the simulation or system. This protocol will intro-

duce some frequently used methods and tools for analyzing Amber
MD simulations using the example system, equilibrated as
described with an additional 3 ns of production phase data
produced.
3.4.1 Minimization and The outcome of the minimization and equilibration steps should be
Equilibration Data Analysis evaluated before initiating longer production run simulations to
confirm that the systems have evolved to achieve stable temperature
and density. The validation of the equilibration phase will vary
depending on the system simulated. Membranes and/or amino
and nucleic acid sequences may require different equilibration pro-
cesses compared to simulation of bulk liquid mixtures containing
small organic molecules [27]. Evaluating the minimization/ equil-
ibration of MIP simulation systems can generally be achieved using
two scripts included with Amber, process_minout.perl and pro-
cess_mdout.perl. A number of new files should have been created
during equilibration, a series of “out,” “nc,” and “ncrst” files
(being data output, NetCDF trajectory, and restart files) as well as
a “logfile” and an “mdinfo” file with intermediary output from
simulations (Fig. 14). The files of interest are the emin, eqTemp,
and eqPres output (.out) files which are subsequently used as input
for the evaluation scripts, where process_minout.perl is used for the
energy minimization output and process_mdout.perl for the temper-
ature and pressure output. The evaluation scripts produce numer-
ous output files, and creating directories for this purpose is
generally a good idea. A suggested directory tree and extracts
from execution of the scripts are presented in Fig. 15. After execut-
ing the evaluation scripts, the energy of the system is found in the
file summary.ENERGY (Fig. 16) (see Note 19), and the tempera-
ture and density in summary.TEMP and summary.DENSITY,
respectively (Fig. 17).
3.4.2 Production Data Cpptraj is the main program used for processing coordinate trajec-
Analysis—Cpptraj tories and data files in the Amber package [28, 29]. It can be run
directly from the CLI, in interactive mode (not available for parallel
execution), or by supplying input files to be executed (see Note 20).
Fig. 14 Files present in the working directory after minimization, equilibration, and 3 ns production phase
simulation
Fig. 15 Suggested directory tree structure when using the evaluation scripts and part of the execution output
from evaluation of the energy minimization
As MIP prepolymerization simulations are split into series of 1 ns

chunks and often extend to 100 ns, input files are convenient for
routine analyses as this allows for easy rerunning, error checking
and provides a reasonable way to input the (up to) 100 files for
processing repeatedly with a single command. Figure 18 shows
how the input for Cpptraj can be structured, where the example
output coordinate trajectory files are being centered using the
autoimage function of Cpptraj. First, the parameter/topology
(parm7) file from LEaP is loaded (parm), followed by sequential
loading of the coordinate trajectory files produced during simula-
tion (trajin). After reading in the simulation data, the commands to
be executed are defined. In this case, autoimage is called to center
the simulation and wrap coordinates written to restart and trajec-
tory files back into a primary box. After this action is completed, a
new trajectory is written using the trajout command, defining the
name of the trajectory. This will also merge the three input trajec-
tory files into a single trajectory file. Figure 19 shows further
examples of command syntax for two routinely used analyses avail-
able in Cpptraj, the hydrogen bond and radial distribution function
(RDF) analyses (see Note 21). Hydrogen bond analysis is
Fig. 16 Processed data from the energy minimization, plotted as kCal/mol vs. iteration step using Grace
Fig. 17 Processed data from temperature (black, left y-axis) and pressure (red, right y-axis) equilibration,
plotted as K (left y-axis) and bar (right y-axis) vs. time (ps) using Grace
performed by calling the hbond function, defining donors and

acceptors using the abbreviations for molecules as defined in the
pdb and parm7 files, residue numbers, or other different masks
Fig. 18 Example input file for using the autoimage routine in Cpptraj
Fig. 19 Examples of commands to perform hydrogen bond and RDF analyses using Cpptraj
Fig. 20 Example output from hydrogen bond and RDF (inset) analyses
(available in the Amber documentation). The average hydrogen

bond information is written to file using the avgout option exclud-
ing intramolecular hydrogen bonds (nointramol), running the
analysis as defined (run) and then executing a lifetime analysis in
Fig. 21 The VMD GUI with some options/menus displayed and the unwrapped and wrapped trajectories loaded
Fig. 22 The Chimera GUI with some options/menus displayed and the wrapped trajectories loaded, showing
only MAA
the data present in dataset S1, saving the results in the lt_hbond.dat
file. For RDF analysis, the radial function is called with an output
filename, distance settings (0.2, 10), and the selections for which to
perform the analysis (:WAT,:MAA). The density is normalized from

the average volume of the system (volume) while ignoring intra-
molecular distances (nointramol), and finally integrating the RDF
(intrdf), writing the results to the same output file (can also be a
separate file). Further details regarding input options can be found
in the Amber and Cpptraj manuals. Example output from hydrogen
bond and RDF analyses are shown in Fig. 20. Here, hydrogen bond
analysis output specifies hydrogen acceptor and donor for each
interaction, the number and fraction of frames where these atoms
fulfill the hydrogen bond criteria (normally using the defaults) and
the average distance and angle. The RDF analysis output is in the
form of a three-column file containing the bin width (cumulative
distance/radius) in Ångströms, the RDF function, and the
integrated RDF function values. The first two columns are regularly
plotted producing graphs where the RDF reflects a local numerical
density of the analyzed “solvent” at that distance. For examples of
how this information is presented, consult any example of available
publications using these methods by the group [10–22]. In addi-
tion to Cpptraj, a number of other software packages are available
that can be useful for visualization and even for performing various
analyses. Of these, VMD [30] and Chimera [31] are routinely used
by us and others for these purposes and justify short presentations.
VMD is a software program that can be used to display and
analyze molecular assemblies, including output from MD simula-
tions. An example is shown in Fig. 21, visualizing the effect of
wrapping the system using Cpptraj as discussed above. VMD is a
good way to get a quick overview of a system and can be used to
produce graphics for publication as well as animations (see Note
22). Chimera is another program that can be used for interactive
visualization and analyses of molecular structures and assemblies,
trajectories, density maps, and more (Fig. 22). Chimera includes
several tools for preparing input files for simulations using Amber
and other simulation methods and software, and is frequently used
for visual inspection and production of high-quality graphic repre-
sentations (see Note 23).
4 Notes
1. The Amber20 software (http://ambermd.org/) is regularly

updated and well documented through an extensive manual.
There is access to multiple tutorials (http://ambermd.org/
tutorials/) and an active online community in the form of a
Mailing list (http://lists.ambermd.org/mailman/listinfo/
amber, http://archive.ambermd.org/). All-in-all making
Amber a good ingress point to MD simulations.
2. Including detailed installation instructions for the software

used is not realistic as it, for most software, would only apply
to individual versions of software, specific OSs and versions,
particular compilers, and so forth. Consult the individual doc-
umentation for available software regarding use and installation
instructions on the particular system intended to be used. Free
and open-source software will primary be recommended and
used. Do remember to cite the software you use as the survival
of many of the tools depend on it. Having access to a good
package manager is recommended. This will not automatically
solve all problems but will make installing software and depen-
dencies easier. On Ubuntu, the “aptitude” package manager is
included and ready for use. From a terminal, running “man
apt” or “man apt-get” will provide information on using these
tools. On macOS, Homebrew (https://brew.sh/) or MacPorts
(https://www.macports.org/) are good alternatives. There are
package managers for windows (https://chocolatey.org/,
https://docs.microsoft.com/en-us/windows/package-man
ager/winget/) though making use of the Windows Subsystem
for Linux (https://docs.microsoft.com/en-us/windows/wsl/
install-win10) would likely be a good choice compared to using
tools like Cygwin (https://www.cygwin.com/) or virtualiza-
tion software.
3. Software can be very particular regarding formats, being able to
convert file formats therefore is important. Open Babel: The
Open Source Chemistry Toolbox (http://openbabel.org/
wiki/Main_Page) is one tool designed for this purpose. There
are graphical user interphase front-ends available for different
operating systems such as iBabel for macOS (https://www.
macinchem.org/ibabel/version4/iBabel4_0.php).
4. There are several online resources hosting structures of pro-
teins, carbohydrates, nucleic acid sequences, and more
(https://www.rcsb.org/, https://www.ebi.ac.uk/pdbe/,
https://www.ccdc.cam.ac.uk/, http://csdb.glycoscience.ru/,
http://ndbserver.rutgers.edu/, https://dev.glycam.org/).
The quality of the structure information needs to be evaluated
before use and the structures checked for gaps/additions or
errors.
5. Avogadro (https://avogadro.cc/) contains several useful tools
for generating, analyzing and evaluating structures, building
proteins, crystal structures, generating input files for computa-
tional software including GAMESS (https://www.msg.chem.
iastate.edu/) and Gaussian (https://gaussian.com/) which can
be used for quantum mechanics-based structure refinement
and derivation of restrained electrostatic potential (RESP)
charges for MD simulations. There is a new version, Avogadro
2 (https://www.openchemistry.org/projects/avogadro2/),
which is a rewrite of the first-generation application and

libraries, that should be the more actively maintained version
though documentation is not clear regarding this.
6. Some of the available water models are TIP3P, TIP5P, SPC/E,
and OPC. Available organic solvents are methanol, chloro-
form, N-methylacetamide, and urea (or urea-water mixes).
There are online resources for obtaining/deriving parameters
for additional components not covered by the FFs included
with Amber, for example, the “AMBER parameter database”
(http://research.bmh.manchester.ac.uk/bryce/amber). When
available, established and tested parameters are recommended.
LEaP can directly be used to add solvent and ions, eliminating
the need for obtaining solvent (and ion) VMMs. This proce-
dure will not be covered here as bulk liquid mixtures of MIP
simulation systems are preferably constructed in another
manner.
7. A range of charge methods are available, of these the
AM1-BCC method is fast and executed directly on the system.
The AM1-BCC charge method sometimes produces
non-integer charges, which can become a problem when work-
ing with larger systems where the excess charge accumulates to
values approaching full charges. In such cases, deriving the
charges from quantum mechanical calculations of optimized
structures is recommended, something which is also routinely
done. For example, Avogadro can produce input, for example,
Gaussian or GAMESS. From optimized structures, charges can
be derived/calculated and applied using Antechamber.
8. For details regarding the different file formats used by Amber,
see https://ambermd.org/FileFormats.php.
9. Online resources such as CHARMM -GUI (http://www.
charmm-gui.org/) can be used to produce both random and
more organized systems. As with Packmol, control of the dis-
tribution and placement of components in the system is possi-
ble. LEaP can also be used to solvate or insert/remove
structures.
10. The Packmol website (http://m3g.iqm.unicamp.br/packmol/
home.shtml) contains excellent examples and a detailed man-
ual regarding installation, I/O, and general usage. There are
also additional tools such as the solvate.tcl script included when
installing from source instead of using the Packmol bundled
with Packmol-memgen included in Amber (used for producing
membrane embedded starting structures for simulations with
Amber).
11. The three-button mouse emulation on macOS and XQuartz
does not work properly. To make any greater use of the func-
tions available within the GUI using the mouse or editing any
text fields inside of the GUI, you need to attach a PC USB

connected mouse and keyboard. There is, for example, no way
to zoom while in system/unit edit mode using only the track-
pad or Apple magic BT mouse. If you have a retina display, the
font size and weight are both small. This can be changed
though is somewhat “non-trivial.” xLEaP can be launched
from the command line with additional arguments: “xleap -fn
‘*courier-bold-r*150*’ to change the font temporarily. For a
deeper insight into fonts and xLEaP, you can read https://
www.oreilly.com/library/view/x-window-system/
9780937175149/Chapter05.html. If you use the GUI system
editor launched through the “edit” command and manage to
select atoms/residues, there is no way to deselect. You cannot
use any of the “close window” buttons (red Xs) under macOS.
If you close the editor using the close button (red X), you close
the entire program and lose all unsaved changes.
12. There are many additional tools and functions in ParmEd,
consult the documentation for further examples (https://
parmed.github.io/ParmEd/html/index.html). Cpptraj also
has tools for checking structure validity and other details,
consult the documentation for details (https://amber-md.
github.io/cpptraj/CPPTRAJ.xhtml).
13. The Simulated Annealing with NMR-Derived Energy
Restraints (SANDER) engine is the base simulation engine of
the Amber package, available in the non-proprietary version of
Amber (AmberTools). The Particle Mesh Ewald Molecular
Dynamics (PMEMD ) engine is included in the proprietary
part of Amber. It is a version of the simulation engine opti-
mized with respect to the performance of the most frequently
used sander methods, including periodic boundary simula-
tions, using particle mesh Ewald summation for treating
long-range interactions. Both of these can be compiled for
parallel calculations, and there are particular versions for local
enhanced sampling calculations using sander.
14. The VIM editor is used to edit files in this protocol. Having
access to and understanding a text editor is important. A
potential problem when switching editors and/or operating
systems is the newline (\n) character, which is treated differ-
ently under Windows/dos and Linux. Something as trivial as a
“no end of line” notification can cause problems processing
and executing input files. It is generally not a bad idea to
terminate all input files with a blank line (this is even required
by some software).
15. Regarding SHAKE, the method to restrain bonds to hydrogen.
The time step needs to be maximum 0.001 ps if not using
SHAKE and should be shorter if higher temperatures are used.
Also, it is important to note that the initial density of the

system is highly important, particularly if using GPU calcula-
tions which are less forgiving for inhomogeneous systems.
There are also different requirements if performing calcula-
tions in serial or parallel and the output run information,
such as errors, may differ. If you have issues equilibrating
the system, a general recommendation is to use a different
barostat.
16. There are several default options which have not been dis-
cussed in the examples, and some options “auto selects”
dependencies which are not presented either. For an in-depth
insight into available options/settings, the manual should be
consulted. These input files are also dependent on the system
under investigation.
17. If compiled in parallel, sander.MPI will produce output at a
faster rate than the serial version sander. If a license for the
proprietary pmemd has been purchased, the parallel version will
decrease the time consumption for simulations and the parallel
pmemd.MPI even further. If these simulations are run in paral-
lel, particularly on shared resources, additional input is needed
to control CPU allocation and distribution. Normally, compu-
tational centers use some form of job control system to
handle this.
18. Unless you are running a larger system for long periods of time
where post-production wrapping may cause problems. The use
of wrapping or not is highly system dependent. For MIP pre-
polymerization mixtures, iwrap can normally be used unless
including larger amino or nucleic acid constructs. The iwrap
command does not image the system cleverly but rather just
based on the first atom or similar. For post-simulation imaging
using CPPTRAJ autoimage, there are a lot more possibilities
for creating a nicely centered and correctly wrapped trajectory.
19. For this, plotting software such as Grace (https://plasma-gate.
weizmann.ac.il/Grace/) or gnuplot (http://www.gnuplot.
info/) can be used directly from the command line for a
quick inspection.
20. 20.Cpptraj is available on GitHub (https://github.com/
Amber-MD/cpptraj) and can be installed independently of
Amber, being actively developed to support data from other
simulation engines.
21. As the simulation example input files (Fig. 12) are using some
recommended default options, such as iwrap ¼ 0, the analyses
of trajectory data need to take this into account. The rdf
analyses are by default imaging the system during analyses,
there are really only fringe cases where the option noimage
would be of any use. This means that regardless of whether
the trajectory has been “wrapped” during production (setting

iwrap ¼ 1) or afterwards (CPPTRAJ autoimage), running
radial will by default image coordinate unless forced not
to. However, depending on the system, the choice to image
or not can affect hydrogen bond (hbond) analyses. For the MIP
simulations, it is very likely that there will be an effect.
Wrapping/imaging the system during or after production will
likely produce reasonable results. For the purpose of analyzing
MIP prepolymerization simulations, the keyword image
should likely almost always be included, in particular if
performing analyses on a small number of template structures
and a dilute solution of multiple monomer (as is common)
where the effect of not correctly imaging coordinates will be
noticeable.
22. There are extensive resources available for installing and
learning to use VMD on the program website (https://www.
ks.uiuc.edu/Research/vmd/) as well as other resources and
plugins.
23. Chimera is developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of California,
San Francisco (supported by NIGMS P41-GM103311). At
the developer’s website (http://www.rbvi.ucsf.edu/chimera),
descriptive tutorials are available including several examples of
usage. There is also a very active user mailing list where support
can be found.
References
1. Nicholls IA, Olsson GD, Karlsson BCG, molecularly imprinted polymer for a pharma-
Suriyanarayanan S, Wiklander JG (2018) The- ceutical impurity in supercritical CO2: rational
oretical and computational strategies in molec- design using computational approach. J Clean
ularly imprinted polymer development. In: Prod 168:1025–1031. https://doi.org/10.
Kutner W, Sharma PS (eds) Molecularly 1016/j.jclepro.2017.09.026
imprinted polymers for analytical chemistry 5. O’Mahony J, Karlsson BCG, Mizaikoff B,
applications, Polymer chemistry series, vol 28. Nicholls IA (2007) Correlated theoretical,
The Royal Society of Chemistry, Cambridge, spectroscopic and X-ray crystallographic stud-
pp 197–226 ies of a non-covalent molecularly imprinted
2. Cowen T, Busato M, Karim K, Piletsky SA polymerisation system. Analyst
(2016) In silico synthesis of synthetic recep- 132:1161–1168. https://doi.org/10.1039/
tors: a polymerization algorithm. Macromol b706258c
Rapid Commun 37:2011–2016. https://doi. 6. O’Mahony J, Moloney M, McCormack M,
org/10.1002/marc.201600515 Nicholls IA, Mizaikoff B, Danaher M (2013)
3. Bates F, Busato M, Piletska E, Whitcombe MJ, Design and implementation of an imprinted
Karim K, Guerreiro A, del Valle M, Giorgetti A, material for the extraction of the endocrine
Piletsky S (2017) Computational design of disruptor bisphenol A from milk. J Chroma-
molecularly imprinted polymer for direct togr B 931:164–169. https://doi.org/10.
detection of melamine in milk. Sep Sci Technol 1016/j.jchromb.2013.05.025
52:1441–1453. https://doi.org/10.1080/ 7. Yañez F, Chianella I, Piletsky SA, Concheiro A,
01496395.2017.1287197 Alvarez-Lorenzo C (2010) Computational
4. Viveiros R, Karim K, Piletsky SA, Heggie W, modeling and molecular imprinting for the
Casimiro T (2017) Development of a development of acrylic polymers with high
affinity for bile salts. Anal Chim Acta not enough: principal component analysis of
659:178–185. https://doi.org/10.1016/j. the influence of template complexation in
aca.2009.11.054 pre-polymerization mixtures on imprinted
8. Tsyrulneva I, Zaporozhets O, Piletska E, polymer recognition and morphology. Int J
Piletsky S (2014) Molecular modelling and Mol Sci 15:20572–20584. https://doi.org/
synthesis of a polymer for the extraction of 10.3390/ijms151120572
amiloride and triamterene from human urine. 17. Cleland D, Olsson GD, Karlsson BCG,
Anal Methods 6:3429–3435. https://doi.org/ Nicholls IA, McCluskey A (2014) Molecular
10.1039/c4ay00318g dynamics approaches to the design and synthe-
9. Wren SP, Piletsky SA, Karim K, Gascoine P, sis of PCB targeting molecularly imprinted
Lacey R, Sun T, Grattan KTV (2015) Compu- polymers: interference to monomer–template
tational design and fabrication of optical fibre interactions in imprinting of 1,2,3-
fluorescent chemical probes for the detection trichlorobenzene. Org Biomol Chem
of cocaine. J Lightwave Technol 12:844–853. https://doi.org/10.1039/
33:2572–2579. https://doi.org/10.1109/Jlt. c3ob42399a
2015.2389036 18. Olsson GD, Karlsson BCG, Schillinger E,
10. Golker K, Olsson GD, Nicholls IA (2017) The Sellergren B, Nicholls IA (2013) Theoretical
influence of a methyl substituent on molecu- studies of 17-β-estradiol-imprinted Prepoly-
larly imprinted polymer morphology and rec- merization mixtures: insights concerning the
ognition - acrylic acid versus methacrylic acid. roles of cross-linking and functional monomers
Eur Polym J 92:137–149. https://doi.org/10. in template complexation and polymerization.
1016/j.eurpolymj.2017.04.043 Ind Eng Chem Res 52:13965–13970. https://
11. Shoravi S, Olsson GD, Karlsson BCG, doi.org/10.1021/ie401115f
Bexborn F, Abghoui Y, Hussain J, Wiklander 19. Golker K, Karlsson BCG, Olsson GD, Roseng-
JG, Nicholls IA (2016) In silico screening of ren AM, Nicholls IA (2013) Influence of com-
molecular imprinting prepolymerization sys- position and morphology on template
tems: oseltamivir selective polymers through recognition in molecularly imprinted polymers.
full-system molecular dynamics-based studies. Macromolecules 46:1408–1414. https://doi.
Org Biomol Chem 14:4210–4219. https:// org/10.1021/ma3024238
doi.org/10.1039/c6ob00305b 20. Schillinger E, Möder M, Olsson GD, Nicholls
12. Golker K, Nicholls IA (2016) The effect of IA, Sellergren B (2012) An artificial estrogen
crosslinking density on molecularly imprinted receptor through combinatorial imprinting.
polymer morphology and recognition. Eur Chem Eur J 18:14773–14783. https://doi.
Polym J 75:423–430. https://doi.org/10. org/10.1002/chem.201201428
1016/j.eurpolymj.2016.01.008 21. Olsson GD, Karlsson BCG, Shoravi S, Wiklan-
13. Olsson GD, Niedergall K, Bach M, Karlsson der JG, Nicholls IA (2012) Mechanisms under-
BCG, Tovar G, Nicholls IA (2015) Simulation lying molecularly imprinted polymer molecular
of imprinted emulsion prepolymerization mix- memory and the role of crosslinker: resolving
tures. Polym J 47:827–830. https://doi.org/ debate on the nature of template recognition in
10.1038/pj.2015.63 phenylalanine anilide imprinted polymers. J
14. Golker K, Karlsson BCG, Wiklander JG, Mol Recognit 25:69–73. https://doi.org/10.
Rosengren AM, Nicholls IA (2015) Hydrogen 1002/jmr.2147
bond diversity in the pre-polymerization stage 22. Karlsson BCG, O’Mahony J, Karlsson JG,
contributes to morphology and MIP-template Bengtsson H, Eriksson LA, Nicholls IA
recognition - MAA versus MMA. Eur Polym J (2009) Structure and dynamics of mono-
66:558–568. https://doi.org/10.1016/j. mertemplate complexation: an explanation
eurpolymj.2015.03.018 for molecularly imprinted polymer recognition
15. Shoravi S, Olsson GD, Karlsson BCG, Nicholls site heterogeneity. J Am Chem Soc
IA (2014) On the influence of crosslinker on 131:13297–13304. https://doi.org/10.
template complexation in molecularly 1021/ja902087t
imprinted polymers: a computational study of 23. Case DA, Belfon K, Ben-Shalom IY, Brozell
prepolymerization mixture events with correla- SR, Cerutti DS, Cheatham TE III, Cruzeiro
tions to template-polymer recognition behav- VWD, Darden TA, Duke RE, Giambasu G,
ior and NMR spectroscopic studies. Int J Mol Gilson MK, Gohlke H, Goetz AW, Harris R,
Sci 15:10622–10634. https://doi.org/10. Izadi S, Izmailov SA, Kasavajhala K,
3390/ijms150610622 Kovalenko A, Krasny R, Kurtzman T, Lee TS,
16. Golker K, Karlsson BCG, Rosengren AM, LeGrand S, Li P, Lin C, Liu J, Luchko T,
Nicholls IA (2014) A functional monomer is Luo R, Man V, Merz KM, Miao Y,
Mikhailovskii O, Monard G, Nguyen H, 27. Roe DR, Brooks BR (2020) A protocol for
Onufriev A, Pan F, Pantano S, Qi R, Roe DR, preparing explicitly solvated systems for stable
Roitberg A, Sagui C, Schott-Verdugo S, molecular dynamics simulations. J Chem Phys
Shen J, Simmerling CL, Skrynnikov NR, 153:054123. https://doi.org/10.1063/5.
Smith J, Swails J, Walker RC, Wang J, 0013849
Wilson L, Wolf RM, Wu X, Xiong Y, Xue Y, 28. Roe DR, Cheatham TE 3rd (2018) Paralleliza-
York DM, Kollman PA (2020) AMBER 2020. tion of CPPTRAJ enables large scale analysis of
University of California, San Francisco molecular dynamics trajectory data. J Comput
24. Hanwell MD, Curtis DE, Lonie DC, Chem 39:2110–2117. https://doi.org/10.
Vandermeersch T, Zurek E, Hutchison GR 1002/jcc.25382
(2012) Avogadro: an advanced semantic chem- 29. Roe DR, Cheatham TE 3rd (2013) PTRAJ and
ical editor, visualization, and analysis platform. CPPTRAJ: software for processing and analysis
J Cheminform 4:17. https://doi.org/10. of molecular dynamics trajectory data. J Chem
1186/1758-2946-4-17 Theory Comput 9:3084–3095. https://doi.
25. Wang J, Wolf RM, Caldwell JW, Kollman PA, org/10.1021/ct400341p
Case DA (2004) Development and testing of a 30. Humphrey W, Dalke A, Schulten K (1996)
general amber force field. J Comput Chem VMD: visual molecular dynamics. J Mol
25:1157–1174. https://doi.org/10.1002/ Graph 14:33–38. https://doi.org/10.1016/
jcc.20035 0263-7855(96)00018-5
26. Martinez L, Andrade R, Birgin EG, Martinez 31. Pettersen EF, Goddard TD, Huang CC,
JM (2009) PACKMOL: a package for building Couch GS, Greenblatt DM, Meng EC, Ferrin
initial configurations for molecular dynamics TE (2004) UCSF chimera—a visualization sys-
simulations. J Comput Chem 30:2157–2164. tem for exploratory research and analysis. J
https://doi.org/10.1002/jcc.21224 Comput Chem 25:1605–1612. https://doi.
org/10.1002/jcc.20084
Chapter 22
The Search for Peptide Epitopes for Molecular Imprinting

Through Bioinformatics
Alessandra Maria Bossi and Laura Pasquardini
Abstract
Epitope imprinting is an effective strategy to prepare molecularly imprinted polymers (MIPs) for protein
recognition. Indeed, the idea to use as a template just a fragment of the protein of interest, called the
epitope, instead of the whole protein, presents some key advantages for the imprinting process, in particu-
lar: cutting the costs for MIP production and avoiding protein unfolding during the imprinting process, so
to ultimately improve the quality of the stamped binding sites. How to select an epitope for the imprinting
is the strategic question. Here, the bioinformatics tools to search for suitable epitopes for the imprinting
process and rational tools to select the most suitable epitope are briefly introduced along with protocols for
their practical use.
Key words Molecularly imprinted polymers, Protein imprinting, Epitope imprinting, Epitope pre-
diction, Protein database, Bioinformatics
1 Introduction
The technique of molecular imprinting permits to prepare synthetic

polymeric receptors suitable to bind a target analyte [1, 2]. The
recognition properties of the formed molecularly imprinted poly-
mers (MIPs) are entailed during the very polymerization process
that is based on a template-assisted synthesis in which the given
molecular target is used as a template.
Merits of the MIP materials are the high affinity and selectivity
for their targets that rival monoclonal antibodies; moreover, MIPs
outpass antibodies for their physical properties that are typically
alike those of the polymeric materials. In fact, MIPs present toler-
ance to solvents, to extreme pHs, and to temperatures, undergo
sterilization, show polymer-like strain properties, processability and
integrability to electronics and to devices.
For templates being small molecules, the imprinting process is
well established and the recognition abilities of the prepared MIP
https://doi.org/10.1007/978-1-0716-1629-1_22,
269
270 Alessandra Maria Bossi and Laura Pasquardini
materials are generally exquisite. More challenging is to imprint

larger molecular targets, such as proteins [3] or supramolecular
assemblies, including cells, bacteria, and viruses [4–6]. Indeed, it
has been discussed how the size of a template matters for the
imprinting process [7].
Issues with proteinaceous templates arise both from their poly-
peptide nature and from their three-dimensional
(3D) conformation, or folding [8]. In fact, the composition in
amino acids of the protein’s backbone and its rearrangement into
a defined 3D conformation, ultimately impart to the protein pecu-
liar physicochemical characteristics (e.g., hydrophobicity, negative
or positive charge, hydrophilicity) that ultimately permit to exert its
particular function.
In the perspective of using a protein as a template in the
imprinting process, the composition and fold of the protein dictate
both its solubility in the polymerization conditions and, most
importantly, dictate the chemical and physical conditions for
keeping that protein folded in its 3D shape during the MIP synthe-
sis process [9]. Indeed, the recognition by an MIP of a folded
protein is assured for imprinted binding sites that possess shape
and chemical functionalities complementary to that particular fold
[10]. Finally, an additional problem when choosing a protein as a
template is the cost of the purified protein that makes the MIP
material expensive, scarcely mass producible and ultimately not
competitive for the market.
So far, the best solution to the issues of imprinting a protein is
taken straight from observing how nature achieves molecular rec-
ognition. Antibodies and receptors bind their target protein
through a binding area that is complementary just to a small
fragment, or peptide, of the whole protein. These fragments, called
epitopes, are exposed at the protein’s surface and are immunogenic.
Epitopes can be linear sequences, or can be conformational, also
called discontinuous, which means that the epitope arises from the
spatial organization of the protein backbone in the folded state, so
that few amino acids or short peptide sequences, belonging to distal
portions of the protein sequence, are clustered together in the
folded protein to form a functional area.
Copying nature means that MIPs to recognize a protein can be
prepared by choosing a fragment of a protein as a template to
imprint, in an approach that is defined epitope imprinting [11–
13]. Epitope templates are small oligopeptides, typically having
molecular weights in the range 500–3000 Da. Chemically, epitopes
are apt to withstand many different polymerization conditions
without being altered or degraded [14]. Additionally, epitope tem-
plates can be easily obtained by artificial synthesis, even for rare
proteins, significantly cutting the costs of the final MIP
material [14].
Epitope Imprinting through Bioinformatics 271
There are various strategies to imprint an epitope. The easiest

one is to imprint the free epitope, solvated together with the
monomers and the crosslinker, in a method referred as bulk epitope
imprinting [15, 16]. As an alternative, the epitope can be immobi-
lized by its chemical coupling to a solid support: this strategy has
the inherent advantage to permitting the choice of the orientation
of the template, either exposing the N- or C-terminal, thus pro-
ducing directional imprinted binding site [17–20]. Finally, when in
need of mimicking the spatial arrangement and the exposition of
the epitope in the folded protein, strategies are devised to introduce
conformational constrains in the epitope, such as synthesizing a
template peptide that contains the epitope sequence flanked by
cysteines, so to form a cyclic peptide epitope [21], or to provide
the epitope sequence with both cysteines to form the cycle and an
amino acidic tag suitable for its immobilization to a support, or
alternatively to induce in the epitope the formation of a particular
secondary structure, mimicking the folded parental protein, such as
using a coiled peptide as a template [22].
The effectiveness of the epitope imprinting approach is largely
sustained by the many examples proposed in the literature, which
counts 94 papers on the subject with just the keyword “epitope
imprinting” (source: Scopus at September 2020). Moreover, there
is a growing presence on the market of companies dedicated to
prepare custom-made MIP materials [23–25].
To successfully perform the epitope imprinting, not only it is
vital to select the most appropriate epitope imprinting strategy, but
the choice of the epitope template in the given protein target is of
fundamental importance.
Over the Subheading 3, we will discuss the bioinformatics tools
available to choose the epitope to imprint and give the operational
sequences to access the resources.
2 Materials
Access to bioinformatics free web resources.
3 Methods
3.1 Selection of When to use it: to select a known immunogenic epitope of the target
Immunogenic Peptide protein.
Epitopes Brief description of the available resources: free bioinformatics
tools and repositories of curated data relevant to immune reactions
and specific pathogens.
The Immune Epitope Database and Analysis Resource (IEDB)
is a freely available resource that contains an extensive collection of
experimentally measured immune epitopes and a suite of tools for
Fig. 1 Homepage of the Immune Epitope Database (IEDB). From www.iedb.org
predicting and analyzing epitopes [26]. The database is funded by

the National Institute of Allergy and Infectious Diseases (NIAID).
Data are curated from peer-reviewed scientific literature and from
data submitted by researchers. As of September 2020, over 21,400
references have been curated, and the database contains over
787,000 peptide epitopes and over 1,880,000 B cell, T cell,
MHC binding, and MHC ligand elution assays (positive and
negative).
How to use:
1. Go to http://www.iedb.org/
2. The site opens on the Home Page (Fig. 1).
3. Select the Epitope’s structure among linear, discontinuous, or
non-peptidic.
4. Select the Host organism in which the epitope should be
found.
5. Select the Assay through which the epitope has been identified.
6. Select the Disease, either a specific disease or the class of the
disease.
7. Start the Search.
Fig. 2 Example of a results webpage of IEDB. From www.iedb.org

8. The results are presented in a Table, as in the example of Fig. 2.

Each record is completed with the epitope sequence, the anti-
gen for the epitope, the organism, the references, and the
assays.
9. Select Details to find more information about an epitope.
3.2 Rational When to use it: to select a unique linear peptide sequence from the
Selection of Linear target protein, without discriminating for the localization of such
Peptide Epitopes unique peptide, being this found either at the protein surface or
buried inside the protein’s core. It is meant for analytical shotgun
proteomic approaches, in which the detection and quantification of
the protein will imply its digestion into peptides.
Brief description of the available resources: free web-curated
repositories of proteomics information and portals for peptide
manipulation and sequences alignment and comparison [27].
l Free databases storing the known protein sequences, such as the
UniProt Knowledgebase (UniProtKB) [28] and free sequences
manipulation tools, that provide the sources to find unique
peptide sequences within the targeted protein.
In particular, UniProtKB is the central hub for the collec-
tion of protein sequences and functional information on pro-
teins, with accurate, consistent, and rich annotation [28]. Each
UniProtKB entry contains the amino acid sequence, protein
name or description, taxonomic data and citation information,
the biological ontologies, classifications and cross-references,
and indications of the quality of annotation in the form of
evidence attribution of experimental and computational data.
l Tools for in silico handling protein sequences, such as ExPASy,
that stays for Expert Protein Analysis System, that is the SIB
Bioinformatics Resource Portal and provides access to scientific
databases and software tools (i.e., resources) for sequences
manipulation, such as tools for in silico enzymatic cleavage of
the protein sequences [29].
l Tools to align in silico peptide and protein sequences, such as the
Basic Local Alignment Search Tool (BLAST) [30], provided by
the National Center for Biotechnology Information, is a tool
that finds regions of local similarity between sequences and can
be used to infer functional and evolutionary relationships
between sequences. The sequence similarities are expressed in
number, thus comparison between the scores enable to easily
rank the similarity. Here, BLAST is used to find “signature”
unique epitopes of a protein.
How to use:
The workflow for the identification of the unique epitope is
discussed in [31], the key steps are:
l Find the protein’s sequence.

l Cut the protein in silico into its constituent peptides.
l Retain peptides with significant length (8–15 amino acids),
while discard shorter peptides, whose sequences are statistically
not uniques, and remove peptides longer than 16 amino acids,
that might coil up in secondary structures.
l Align each peptide to the whole protein sequences stored in the
database. The best peptide epitope is the one that aligned
towards the whole protein database provides the best match
(highest score) for the very parental protein, while having the
lowest E-value (number of distinct alignments with a score
equivalent to or better than S, but expected to occur in a
database search by chance) [32].
l Scores definitions:
Max(imum) Score: it is the highest alignment score of a set
of aligned segments from the same subject (database) sequence.
The score is calculated from the sum of the match rewards and
the mismatch, gap opens and extends penalties independently
for each segment. This normally gives the same sorting order as
the E Value.
Tot(al) Score: it is the sum of alignment scores of all seg-
ments from the same subject sequence. This sorting order may
help promote the position of mRNA matches to genomic
sequences where there are multiple exons. The Total Score is
useful for distinguishing hits to functional multi-exon genes
from those to the corresponding intronless retro-transposed
pseudogenes.
E(xpect) Value: it is the number of alignments expected by
chance with a particular score or better. The expect value is the
default sorting metric and normally gives the same sorting order
as Max Score.
Per(centage) Coverage: it is the percent of the query length
that is included in the aligned segments. This is calculated over
all segments as with the Tot Score.
To find the identification number (ID) and the amino acid
sequence of the target protein:
1. Go to the website http://www.uniprot.org/
2. Type the name of the protein and start search.
3. Browse the list of proteins, choose the protein of interest.
4. Click on the Entry corresponding to the correct protein ID.
5. Scroll the Display left panel to find for Sequences.
6. Click Sequences to access to the protein sequence.
7. Copy the sequence in FASTA canonical format.
In silico breaking into peptides of the sequence of the target

protein:
8. Go to the website http://web.expasy.org/peptide_cutter/
9. Paste the ID of the protein or the FASTA sequence in the
appropriate box.
10. Define the criteria for cleavage (enzymes vs. chemicals) by
selecting “Only the following selection of enzyme and chemi-
cals”; trypsin is suggested.
11. Select the option “Table of sites, sorted sequentially by amino
acid number”.
12. Select the option “Enzymes and chemicals cleaving exactly”
insert the number 1 “times”.
13. Click “Perform” to run the process to in silico cleave the
protein.
14. Obtain the list of tryptic peptides that displays the following
information: position of cleavage site; name of cleaving
enzyme; resulting peptide sequence; peptide length [aa]; pep-
tide mass [Da].
To select which peptides are potential candidates for the role of
“signature” epitope:
15. Discard from the list all the peptides that are shorter than
8 amino acids (too short to be unique).
16. Discard from the list all the peptides that are longer than
16 amino acids (too long are expensive to be synthesized,
often difficult to solubilize in aqueous environment, tend to
coil in secondary structure).
17. Prepare a table listing solely these peptides encompassing the
range of 8–15 amino acids; keep data on peptide sequence;
peptide length; peptide mass.
Rational method to define the degree of uniqueness of a
peptide:
18. Go to the site http://blast.ncbi.nlm.nih.gov/Blast.cgi
19. Select “protein blast”.
20. Feed each peptide from the previous list, one at a time in
the box.
21. Choose the parameters: * Database: non-redundant protein
sequences (nr); * Organism: e.g., Homo sapiens; * Algorithm:
blastp (protein–protein BLAST).
22. Run BLAST.
23. For each blasted peptide, the result is a table with the following
parameters: Description; Maximum Score; Total Score; Query
cover; E-value; Percentage of Identity; Accession number.
24. Check if the description matches the identity of the target

protein; if not, the blasted peptide is not a candidate for the
imprinting.
25. Retain the scores solely for the blasted peptides whose descrip-
tion matches the identity of the target protein.
26. Prepare a Table to list the results, equipped with the following
columns: Sequence of the blasted peptide; Total Score and the
E-value for each peptide blasted.
27. Blast one by one all the peptides reported in the Table at
point 17.
28. Compare the Maximum scores, the Total scores, and the
E-values of the basted peptides.
29. Choose the peptide with the highest scores and the lowest
E-value.
30. When two peptides display the same scores, the suggested
criteria for the selection of the epitope is to discriminate the
epitopes on the basis of their physicochemical parameters,
selecting the one that has characteristics fully compatible with
the MIP polymerization conditions.
To get the physicochemical parameters of the peptides:
31. Go to the website http://web.expasy.org/protparam/
32. Enter the sequence of the selected peptide in the box.
33. Run to estimate the parameters: molecular weight, isoelectric
point, number of negatively and positively charged residues,
GRAVY index.
34. Get as results the requested values.
35. Run each selected peptide candidate.
3.3 Selection of When to use it: it is a strategy that helps to define the epitope’s
Structured Epitopes localization in the protein and to define the orientation of the
epitope in the protein 3D structure. Information about the locali-
zation of the peptide in the protein and the orientation of the
epitope on the protein 3D structure is a prerequisite for a successful
imprint. It enables to perform epitope imprinting, not just with
linear peptides, but with directional peptides (e.g., circular pep-
tides, or immobilized through C- or N-terminal), properly mim-
icking the peptide exposure and orientation in the folded protein
and ultimately permitting a gain in MIP selectivity [21, 22].
Brief description of the available resources: free accessible protein
tools that enable the 3D view of proteins and the localization of
secondary structures in the protein conformation.
Tools for the 3D views of proteins, such as the interactive 3D
structure viewer iCn3D, permit to define the epitope localization in
the protein structure. The iCn3D (“I see in 3D”) is a WebGL-
Fig. 3 Example of a 3D view of a protein structure. From https://www.ebi.ac.uk/

pdbe/entry/pdb/1cx8 UniProt database
Fig. 4 Example of 3D view of protein with dual color code for secondary
structures. From https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html
based viewer for interactive viewing of three-dimensional macro-

molecular structures and chemicals on the web, that does not
require to install a separate application. It can be accessed from
the “molecular graphic” that appears on the structure summary
page for any record in the Molecular Modeling Database
(MMDB) [33].
Fig. 5 Example of zoom to focus particular residues in the protein structure

(https://www.ncbi.nlm.nih.gov/Structure/icn3d/full.html; alternatives, e.g., www.
pymol.org)
Fig. 6 Example of secondary structures, such as localization of turns onto the sequence of a protein. From
www.uniprot.org
The localization information is integrated with other data, such

as databases that store experimental evidences of protein–protein
interactions areas, literature metadata, and predicted information
gathered by any available in protein–protein interaction databases
(see for example: http://string-db.org; https://www.ebi.ac.uk/
intact/) so to ultimately finalize the criteria for the selection of a
one final epitope sequence.
How to use:
To localize the epitope in the 3D conformation of the protein:
1. Go to the website http://www.uniprot.org/
2. Insert the name of the target protein and run the search.
3. Get as a result the target protein sequence identification
number (ID).
4. Scroll down the result webpage to the section Structure.
5. In the section Structure, select the pdb of the protein that links
to the https://www.ebi.ac.uk/pdbe/entry/pdb
6. Select Structure analysis.
7. Go to 3D visualization, by typing https://www.ebi.ac.uk/
pdbe/entry/pdb/ insert the ID of the target protein /analy
sis) and browse the 3D structure of the protein to localize the
peptides of interest (example in Fig. 3).
Click the amino acids composing the peptide of interest to
get information.
As an alternative:
8. Go to https://www.ncbi.nlm.nih.gov/Structure/MMDB/
mmdb.shtml
9. In the page 3D macromolecular structures, select iCn3D.
10. Open iCn3D.
11. Insert the ID of the protein of interest.
12. Click on Select 3D to define domains and helix (red)/strand
(green) (Fig. 4).
13. Browse the structure to highlight particular amino acids
(Fig. 5).
To map the functions and particular secondary structures of the
epitopes along the protein sequence:
14. Go to https://www.uniprot.org/uniprot/ insert the ID of
the target protein /protvista
15. Select Feature Viewer.
16. Select the structure of interest, e.g., turn (Fig. 6).
Click on the structure of interest and get information on
length and sources.
Prepare the structured epitope for imprinting:
17. Impart orientation to a linear epitope by classical immobiliza-
tion chemistries [34] that allow to couple the epitope to a
supporting material [35].
18. Impart orientation to a structured epitope by mimicking its 3D
exposure, by cyclizing the epitope, synthesize the epitope
peptide by flanking the N- and the C-terminus with a

cysteine [21].
19. Impart orientation to a structured epitope by mimicking its 3D
exposure, by cyclizing the epitope, by flanking the N- and the
C-terminus with a cysteine followed by amino acids suitable for
its immobilization to a support, e.g., His-tag [35, 36].
20. Mimic the secondary structure of the epitope by computational
analysis of the protein of interest and selection of self-coiling
peptide modules, able to fold into a stable piece of secondary
structure [22].
4 Conclusions
The giant leap forward made in sharing data concerning macro-

molecules and in building and curating freely accessible repositories
of information about proteins, including protein structural data
and protein functional data, antigenicity, and interaction sites,
offers a set of invaluable tools for the quest of protein imprinting.
Indeed, the strategy to imprint a protein’s fragment, i.e., the epi-
tope, can rely on a number of different bioinformatics approaches
to find the most appropriate peptide to imprint.
Immunogenic epitopes, that are accessible peptides present at
the protein’s surface, can be searched by web resources dedicated to
the storage of immunological information.
Unique peptide sequences, whether located internally or at the
surface of the protein, can be identified by means of proteomic
tools, such as those dedicated to sequences comparison. Herein,
the protocols for the use of sequence comparison tools are
explained with the aim of searching sequences that are signatures
for the intended protein target, so to find the best possible epitope
to imprint. In this case, the epitope will be a sequence that will not
necessarily possess a functional role, instead it will be selected for its
particular sequence, which is ideally not shared by any other pro-
teins in the database, thus guaranteeing the MIP-binding sites will
perform a unique recognition towards the target protein.
Finally, the search for an epitope might go beyond just the level
of amino acidic sequence and take into account its placement in the
folded protein. Oriented peptides and structured epitopes can be
identified by browsing databases dedicated to the storage of struc-
tural information about proteins. Tools that permit to localize
secondary structure portions and turns on the protein fold find
use to localize the putative epitope on the protein 3D coil and are
helpful to decide whether the epitope to imprint shall have a
particular orientation, or a structure, so to yield to the successful
stamping of binding cavities in the formed MIP material.
The crescendo in the level of complexity of the epitopes corre-

lates with the final use of the MIP materials. Intended in vivo
biomedical applications of MIPs necessarily imply the necessity of
homogeneous and stringent recognition of a protein in a particular
fold and the formed MIP-binding sites.
References
1. Wulff G, Sarhan A (1972) Use of polymers films. Biomacromolecules 8:2781–2787.

with enzyme-analogous structures for the res- https://doi.org/10.1021/bm7004774
olution of racemates. Angew Chem Int Ed 11. Rachkov A, Minoura N (2000) Recognition of
11:341–342 oxytocin and oxytocin-related peptides in
2. Arshady R, Mosbach K (1981) Synthesis of aqueous media using a molecularly imprinted
substrate-selective polymers by host-guest polymer synthesized by the epitope approach. J
polymerization. Die Makromol Chem Chromatogr A 889:111–118. https://doi.
182:687–692. https://doi.org/10.1002/ org/10.1016/s0021-9673(00)00568-9
macp.1981.021820240 12. Rachkov A, Minoura N (2001) Towards
3. Bossi A, Bonini F, Turner APF et al (2007) molecularly imprinted polymers selective to
Molecularly imprinted polymers for the recog- peptides and proteins. The epitope approach.
nition of proteins: the state of the art. Biosens Biochim Biophys Acta 1544:255–266.
Bioelectron 22:1131–1137. https://doi.org/ https://doi.org/10.1016/S0167-4838(00)
10.1016/j.bios.2006.06.023 00226-0
4. Piletsky S, Canfarotta F, Poma A et al (2020) 13. Nishino H, Huang CS, Shea KJ (2006) Selec-
Molecularly imprinted polymers for cell recog- tive protein capture by epitope imprinting.
nition. Trends Biotechnol 38:368–387. Angew Chem Int Ed 45:2392–2396. https://
https://doi.org/10.1016/j.tibtech.2019.10. doi.org/10.1002/anie.200503760
002 14. Yang K, Li S, Liu L et al (2019) Epitope
5. Haupt K, Medina Rangel PX, Tse Sum Bui B imprinting technology: progress, applications,
(2020) Molecularly imprinted polymers: anti- and perspectives toward artificial antibodies.
body mimics for bioimaging and therapy. Adv Mater 31:1902048. https://doi.org/10.
Chem Rev 120(17):9554–9582. https://doi. 1002/adma.201902048
org/10.1021/acs.chemrev.0c00428 15. Emgenbroich M, Borrelli C, Shinde S et al
6. Gast M, Sobek H, Mizaikoff B (2019) (2008) A phosphotyrosine-imprinted polymer
Advances in imprinting strategies for selective receptor for the recognition of tyrosine phos-
virus recognition a review. Trends Anal Chem phorylated peptides. Chem Eur J
114:218–232. https://doi.org/10.1016/j. 14:9516–9529. https://doi.org/10.1002/
trac.2019.03.010 chem.200801046
7. Li S, Cao S, Whitcombe MJ et al (2014) Size 16. Bertolla M, Cenci L, Anesi A et al (2017)
matters: Challenges in imprinting macromole- Solvent-responsive molecularly imprinted
cules. Progr Polym Sci 39:145–163. https:// nanogels for targeted protein analysis in
doi.org/10.1016/j.progpolymsci.2013.10. MALDI-TOF mass spectrometry. ACS Appl
002 Mater Inter 9:6908–6915. https://doi.org/
8. Kryscio DR, Peppas NA (2012) Critical review 10.1021/acsami.6b16291
and perspective of macromolecularly imprinted 17. Xing R, Wang S, Bie Z et al (2017) Preparation
polymers. Acta Biomater 8:461–473. https:// of molecularly imprinted polymers specific to
doi.org/10.1016/j.actbio.2011.11.005 glycoproteins, glycans and monosaccharides via
9. Kryscio DR, Fleming MQ, Peppas NA (2012) boronate affinity controllable–oriented surface
Conformational studies of common protein imprinting. Nat Protoc 12:964–987. https://
templates in macromolecularly imprinted poly- doi.org/10.1038/nprot.2017.015
mers. Biomed Microdevices 14:679–687. 18. Li S, Yang K, Liu J et al (2015) Surface-
https://doi.org/10.1007/s10544-012-9648- imprinted nanoparticles prepared with a his-
9655 tag-anchored epitope as the template. Anal
10. Turner NW, Liu X, Piletsky SA et al (2007) Chem 87:4617–4620. https://doi.org/10.
Recognition of conformational changes in 1021/ac5047246
β-lactoglobulin by molecularly imprinted thin
19. Moczko E, Guerreiro A, Caceres C et al (2019) 29. Artimo P, Jonnalagedda M, Arnold K et al

Epitope approach in molecular imprinting of (2012) ExPASy: SIB bioinformatics resource
antibodies. J Chromatogr B 1124:1–6. portal. Nucleic Acids Res 40(W1):
https://doi.org/10.1016/j.jchromb.2019. W597–W603. https://doi.org/10.1093/
05.024 nar/gks400
20. Medina Rangel PX, Laclef S, Xu J et al (2019) 30. Altschul SF, Gish W, Miller W et al (1990)
Solid-phase synthesis of molecularly imprinted Basic local alignment search tool. J Mol Biol
polymer nanolabels: affinity tools for cellular 215:403–410. https://doi.org/10.1016/
bioimaging of glycans. Sci Rep 9:3923. S0022-2836(05)80360-2
https://doi.org/10.1038/s41598-019- 31. Bossi AM, Sharma PS, Montana L et al (2012)
40348-5 Fingerprint-imprinted polymer: rational selec-
21. Cenci L, Guella G, Andreetto E et al (2016) tion of peptide epitope templates for the deter-
Guided folding takes a start from the molecular mination of proteins by molecularly imprinted
imprinting of structured epitopes. Nanoscale polymers. Anal Chem 84:4036–4041. https://
8:15665–15670. https://doi.org/10.1039/ doi.org/10.1021/ac203422r
C6NR03467E 32. Henikoff S (1996) Scores for sequence
22. Chou C-Y, Lin C-Y, Wu C-H, Tai D-F (2020) searches and alignments. Curr Opin Struct
Sensing HIV protease and its inhibitor using Biol 6:353–360. https://doi.org/10.1016/
“helical epitope”—imprinted polymers. Sen- s0959-440x(96)80055-8
sors (Switzerland) 20:3592. https://doi.org/ 33. Wang J, Youkharibache P, Zhang D et al
10.3390/s20123592 (2020) iCn3D, a web-based 3D viewer for
23. MIP Diagnostics Limited. https://www.mip- sharing 1D/2D/3D representations of biomo-
dx.com/. Accessed 25 Sept 2020 lecular structures. Bioinformatics 36:131–135.
24. AFFINISEP. https://www.affinisep.com/. https://doi.org/10.1093/bioinformatics/
Accessed 25 Sept 2020 btz502
25. MIP Technologies. http://www. 34. Boutureira O, Bernardes GJL (2015) Advances
miptechnologies.com/. Accessed 25 Sept in chemical protein modification. Chem Rev
2020 115:2174–2195. https://doi.org/10.1021/
26. Fleri W, Paul S, Dhanda SK et al (2017) The cr500399p
immune epitope database and analysis resource 35. Xu J, Medina-Rangel PX, Haupt K et al (2017)
in epitope discovery and synthetic vaccine Guide to the preparation of molecularly
design. Front Immunol 8:278. https://doi. imprinted polymer nanoparticles for protein
org/10.3389/fimmu.2017.00278 recognition by solid-phase synthesis. Meth
27. Xu D (2012) Protein databases on the internet. Enzymol 590:115–141. https://doi.org/10.
Curr Protoc Mol Biol Chapter 19:Unit 19.4. 1016/bs.mie.2017.02.004
https://doi.org/10.1002/0471142727. 36. Xu J, Merlier F, Avalle B et al (2019) Molecu-
mb1904s97 larly imprinted polymer nanoparticles as poten-
28. The UniProt Consortium (2019) UniProt: a tial synthetic antibodies for immunoprotection
worldwide hub of protein knowledge. Nucleic against HIV. ACS Appl Mater Interf
Acids Res 47:D506–D515. https://doi.org/ 11:9824–9831. https://doi.org/10.1021/
10.1093/nar/gky1049 acsami.8b22732
INDEX
A Epitope imprinting...................................... 270, 271, 275
Aflatoxins .............................................................. 141–151 F

Aptamer-MIP Hybrid Nanoparticles .................. 109–120
Aptamers............................ 109–112, 116, 118, 119, 224 Fluorescence ......................... 47, 48, 104, 105, 184, 186,
189–193, 195, 196, 198, 204–207
B Fluorescence sensing............................................ 191, 192
Food matrix ................................................. 131, 132, 138
Bacteria ............................................................. 43–50, 270 Food safety ...................................................................... 98
Bioinformatics ...................................................... 269–282 Free-radical polymerization ................................. 124, 193
Biomarker ............................................................... 71, 210
Functional monomer (FM) ......................... 2, 3, 5, 9, 10,
Bovine serum albumin (BSA).................... 54, 55, 57, 58, 19–21, 23, 30, 35–38, 56, 58, 73, 74, 88, 90–92,
60–63, 67, 71–82 95, 101–103, 110, 124, 125, 129, 132, 164, 165,
Bulk polymerization.............................86, 126, 137, 166, 169, 183, 184, 186, 189, 192, 193, 210, 227
168, 169, 187
G
C
Green technology............................................................ 20
Catalysts ................................ 73, 76, 101, 102, 186, 189,
193, 223, 225 H
Chemical hazard detection .................................. 131–138
Chitosan....................................................... 44, 45, 47, 48 High-abundant proteins ................................................. 71
Chlorogenic acid .................................................. 196, 198 Human urine ........................................................ 123–129
Clinical detection ................................................. 209–220 Hydrophilic groups ...................................................54, 60
Complex biological samples ..........................99, 104, 105 Hydrophilic polymer brushes ..........................98, 99, 106
Computational design .................................................. 241
I
Controlled/“living” radical precipitation
polymerization..................................................... 98 Insulin ................................................................... 209–221
Core-shell particles........................................................ 195 Iron oxide ..................................... 85, 223–225, 227, 228
Cross-linker ........................ 2, 3, 5, 8, 26, 35–39, 46, 48,
73, 88, 101–103, 143, 144, 154, 156, 165, 169 L
Cryogel membrane .............................................. 171–179
Liquid chromatography (LC)...................... 1, 3, 6, 7, 54,
Cryogels.........................72, 73, 75, 76, 78, 82, 172, 179
57, 62, 64, 66, 68, 93, 144, 155, 171
Cultured fish......................................................... 141–152
M
D
Macromolecules exclusion.............................................. 63
Depletion .......................................................... 71–82, 172
Magnetic molecular imprinted polymer ........................ 85
Dispersive micro-solid phase extraction
Magnetic nanoparticles ........................43, 85, 89, 90, 95,
(D-μ-SPE)................................................. 141–151
163, 166
Double-layer imprinted polymer..............................71–82
Magnetite .................................... 85, 86, 90, 93–95, 163,
Drug Delivery ....................................... 19, 20, 29, 35–36
164, 166–169
E Membranes ..........................4, 23–25, 27–30, 33, 34, 36,
38, 125, 127, 173, 174, 176, 187, 197, 200, 211,
Electrochemical sensors ................................................ 216 256, 263
Electropolymerization .................................155, 233–239 Metal-ion coordination................................................. 233
https://doi.org/10.1007/978-1-0716-1629-1,
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer
Nature 2021
285
MOLECULARLY IMPRINTED POLYMERS: METHODS AND PROTOCOLS
286 Index
Microspheres ............................................. 1, 10, 172, 187 Purification devices ...................................................39, 40
Molecular dynamics (MD) ...........................88, 241–244,
254–257, 261, 262, 264 Q
Molecular imprinting ........................ 9, 19, 22, 110, 154, Q-NMR ....................................................... 12, 14, 15, 17
172, 183, 196, 210, 223–231, 241, 269–282
Quantum dots (QDs) .......................................... 183–193
Molecular imprinting methods .................................... 210 Quartz crystal microbalance (QCM) .................. 209–220
Molecular imprinting technique (MIT) ................ 19, 20,
24, 40, 72, 97, 172, 210 R
Molecularly imprinted polymer (MIP) .......................5, 6,
9–14, 16, 19–40, 44, 53, 54, 56–58, 60–62, 67, Restricted access media (RAM)........................................ 2
72, 85–95, 97–106, 110, 123–127, 129, 131, Restricted access molecularly imprinted polymer
134, 141–151, 154–157, 160, 161, 163, 164, (RAMIPs) ..................... 54–56, 59, 60, 62–64, 67
168, 170, 172, 183–193, 195–207, 209–220,
S
224, 229, 230, 233–237, 239, 241–266,
269–271, 275, 277, 278, 282 Sample preparation ........................... 53, 54, 64, 80, 123,
Molecularly imprinted polymeric fibers ....................... 153 153, 163
Molecularly imprinted solid-phase extraction Sensing.......................................... 19, 183–193, 195, 204
(MSPE) ...........................123, 131, 132, 134, 135 Sensor ................................... 20, 93, 109, 132, 133, 135,
Monoliths ......................9, 154–156, 160, 164, 168, 172 136, 183, 195, 196, 209–220, 234, 237
Sol-gel approach............................................................ 192
N Solid-phase extraction (SPE).................2, 30, 31, 37, 57,
Nanogels ........................................................................ 223 60, 62, 64, 67, 86, 112, 117, 123–129, 132–134,
Nanoparticles............................. 9–17, 43, 60, 85–87, 90, 136, 137, 141–151, 154
92, 94, 95, 98–102, 104, 105, 110, 131, 135, Solid-phase microextraction (SPME) ................. 153–161
164, 165, 167, 169, 172, 195–207, 223–227, 230 Sorptive extraction ........................................................ 164
Nanospheres ......................................................... 105, 186 Specific recognition.............................................. 183, 186
Nanozymes ........................................................... 223–231 Spectrophotometry .............................................. 124, 126
Neopterin ............................................................. 171–180 Stir-bar ................................ 56, 134, 157, 161, 163–170,
186, 235
P Supercritical CO2 ............................................................ 19
Surface imprinting..........................................23, 192, 193
Pickering emulsion....................................................43–49 Surface imprinting polymerization .............................. 184
Precipitation polymerization ................. 9, 11, 13–17, 67, Surface-enhanced Raman spectroscopy (SERS). 131–138
98, 150, 187
Pre-polymerization mixture....................... 101, 102, 143, T
166–169, 205, 225, 227
Prognostic indicator............................................. 171–180 Template extraction ..................................................11, 12
Propazine ..................................................... 156, 158, 160 Two-step swelling and polymerisation ........................ 1–8
Propranolol............................................................. 15, 105
W
Protein database ............................................................ 275
Protein exclusion................................... 55, 57–58, 62–63 Water-compatible .................................... 98, 99, 105, 106
Protein imprinting ........................................................ 278

Molecularly Imprinted Polymers 2021

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Molecularly Imprinted Polymers 2021

Uploaded by

Copyright:

Available Formats

Methods in

Molecular Biology 2359

Antonio Martín-Esteban Editor

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Madrid, Spain Antonio Martı́n-Esteban

1 Synthesis of Molecularly Imprinted Polymers by Two-Step Swelling

13 Preparation of Monolithic Fibers in Fused Silica Capillary Molds

HEIDI ABRAHAMSE • Faculty of Health Sciences, Laser Research Centre, University of

GUSTAF D. OLSSON • Bioorganic & Biophysical Chemistry Laboratory, Department of

Synthesis of Molecularly Imprinted Polymers by Two-Step

There are some methods to prepare microsphere MIPs [1–

functional monomer could be interfered since water is used as a

Fig. 1 Preparation of MIPs and RAM-MIPs by two-step swelling and polymerization

13]. RAM-MIPs for bisphenol A (BPA)-d16 were prepared and

2.2 Preparation Uniform-sized, polystyrene seed particles utilized as the shape

3. Add 0.95 mmol/L styrene with stirring at 350 rpm and at

2.3 Preparation 1. Prepare the uniform-sized, polystyrene seed particles as in Sub-

2.4 Preparation 1. Prepare the uniform-sized, polystyrene seed particles as in Sub-

The prepared MIPs and RAM-MIPs, respectively, were packed into

3.1 Separation 1. HPLC conditions: column, MIP for (S)-nilvadipine

Fig. 2 Chromatograms of a racemic mixture of nilvadipine enantiomers (a) and

2. Obtain chromatograms of a racemic mixture of nilvadipine

3.2 Simultaneous 1. Pretreatment conditions: column, RAM-MIP for BPA-d16

5. Obtain chromatograms of river water sample by a column-

1. Sodium chloride was added to adjust the ionic strength of the

polystyrene seed particle to dibutyl phthalate was changed to

Molecularly Imprinted Polymeric Nanoparticles

Molecular imprinting is a simple and effective method of imparting

suspension polymerization [6], multi-step swelling [7], and precip-

2.1 Polymerization 1. Monomers: Methacrylic acid as functional monomer (FM) and

3. Template (T): ()-Propranolol (PNL). PNL is commercially

2.2 Template 1. Methanol:acetic acid mixture, 9:1 by volume. Analytical grade

2.3 Quantitative 1. 400 or 600 MHz NMR machine.

Fig. 1 Setup for polymerization and nitrogen gas purging

4. Polymerize the reaction mixtures for 24 h in a water bath or an

6. If a peak is observed at 340 nm, then PNL is likely to still be

3.3 Quantitative 1H 1. Transfer 0.50 mL of the MIP or NIP pre-polymerization feed

Fig. 2 An example of a 1H NMR spectrum used for q-NMR

components post-polymerization is a measure of the polymer

1. The column can be prepared using a disposable syringe (50-mL

7. The concentration of the rebinding stock solution may vary. It

15. The pre-polymerization solution can be stored in the refrigera-

The authors acknowledge the technical assistance of Monica Ros-

MIP Synthesis and Processing Using Supercritical Fluids

In the past few decades, the interest in Molecular Imprinting Poly-

removal of the template molecule from the polymer is performed

Fig. 1 Phase diagram of carbon dioxide (adapted from [6])

solvent processes [31]. The extraction is ten-fold more efficient

Fig. 3 MIPs synthesized in scCO2 for different applications [20]

Fig. 4 Physical appearance of the synthesized MIP powder under scCO2

covalent, non-covalent, semi-covalent (by covalent and

porogenic solvent, but can also be tuned by the CO2 pressure

[19, 20]. A deep knowledge on the methodology is well described

ScCO2 operates at high pressures (above 200 bar), thus it is man-

3. Magnetic stirrer and Teflon-coated magnetic stirring bar.

12. Functional monomer(s), e.g., Subheading 3.5.1 itaconic acid

2.3 ScCO2-Assisted Materials required for the impregnation of compounds in the

Fig. 8 High-pressure cell used for membrane preparation [42]

8. Solvent, e.g., Subheading 3.5.3: dimethylformamide (DMF).

Thermostated water bath

Materials required to assess the affinity and selectivity of the

SR ð%Þ ¼ ½ðW s W 0 Þ=W 0 100 ð1Þ

Q ¼ ½ðC i C f Þ V =m dry ð4Þ

Q d ¼ ½ðC i C f Þ=C f V =m dry ð5Þ