Talanta 252 (2023) 123819

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Disposable electropolymerized molecularly imprinted electrochemical

sensor for determination of breast cancer biomarker CA 15-3 in human
serum samples
Ana Elisa F. Oliveira a, *, Arnaldo C. Pereira a, Lucas F. Ferreira b
a
Departamento de Ciências Naturais, Universidade Federal de São João del-Rei, UFSJ, São João del-Rei, MG, CEP, 36307-352, Brazil
b
Laboratório de Eletroquímica e Nanotecnologia Aplicada, Instituto de Ciência e Tecnologia, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Rodovia MGT
367, Km 583, 5000, Alto da Jacuba, Diamantina, MG, 39100-000, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Initially, a printed electrode was fabricated in a paper substrate using carbon nanotube ink, graphite pencil and
Molecularly imprinted polymer silver nanoparticle ink. For that the electrode was modified with gold nanoparticles and a molecularly imprin­
Printed electrode tedpolymer (MIP) using CA 15-3 as target molecule. The Atomic Force Microscopy (AFM) images exhibited the
CA 15-3
change in the morphology after each electrode modification. The roughness increasedafter the electro­
Cancer biomarker
polymerization, and decreased after the extraction procedure. Next, slightly increased again associated to the
interaction of CA 15-3 and the imprinted sites. Finally, the Fourier Transform Infrared Spectroscopy (FTIR)
results suggested the extraction/rebinding of CA 15-3 in the MIP sensor and also indicated that the NIP sample do
not have specific cavities for the CA 15-3. In short, under optimized conditions, the CNE/AuNP is incubated with
CA 15-3 (40 U mL− 1) for 2 h at 4 ◦ C. Then the electropolymerization was carried out in the potential range of
− 0.2 to 1.0 V during 20 cycles at scan rate of 50 mV s− 1 using a solution containing 15 mM of oPD. After
electropolymerization, the sensor was washed with oxalic acid solution for 2 h, leading to the formation of
imprinted cavities. The rebinding process was subsequently constructed for 1 h at 4 ◦ C using CA 15-3 solution.
The reproducibility and interference studies showed that the sensor can be reproducible and specific for CA 15-3.
Then the sensor was applied in determination of CA 15-3 in samples of serum and saliva. The use in serum
presented good recovery, but the application in saliva was not satisfactory. Therefore, the sensor CNE/AuNP/MIP
could be used in the determination of CA 15-3 in serum samples.

1. Introduction in the fabrication of electropolymerized film is the ortho-phenylenedi­

The electrochemical route, also known as electropolymerization,
provides a simple, clean and efficient route for polymer synthesis. The
synthesized polymers do not have fragments of oxidants or catalysts, and
the chemical and physical properties of the polymer are dependent on
the electrochemical conditions employed during synthesis [1–3]. Elec­
tropolymerization is normally carried out in a three-electrode electro­
chemical cell, with an aqueous solution of monomer and supporting
electrolyte [1,2]. Several monomers can be used in the electro­
polymerization. The only requirement is that it must have an oxidation
potential and the possibility to be oxidized in solution [4]. Some ex­
amples are pyrrole, aniline, indole, methylene blue, EDOTh, amino­
phenol, dopamine and many others [5,6]. One important monomer used
amine (oPD).
The electropolymerization of oPD has received greater attention over
the last decades [7]. oPD was first electrodeposited from aqueous so­
lutions of phosphoric acid and sulfuric acid by D’Elia et al. and by
Hermas et al. [8,9]. The oPD contains free active functional amine
groups. The –NH2 may be oxidized during the electrochemical poly­
merization. The polymer form of oPD is known as poly (o-phenyl­
enediamine) (PoPD). The polymerization mechanism, structural
features and characteristics of oPD are not a well established yet [10,
11]. The application of oPD is also extended to the fabrication of MIP
sensors due the possibility of molecular imprinting target molecules
using electropolymerization. Several studies were conducted in this
area. Li et al. developed a sensor by electropolymerized the oPD onto a

* Corresponding author.
E-mail address: ana_elisa_oliveira@yahoo.com.br (A.E.F. Oliveira).

https://doi.org/10.1016/j.talanta.2022.123819
Received 7 March 2022; Received in revised form 2 August 2022; Accepted 3 August 2022
Available online 10 August 2022
0039-9140/© 2022 Elsevier B.V. All rights reserved.
A.E.F. Oliveira et al.
glassy carbon electrode (GCE) for detection of ractopamine [12].
Al-Ammari et al. fabricated a MIP sensor using the oPD, zinc oxide
nanoparticle and graphene nanoplatelet into a GCE surface for detection
of 3-chlorophenol [13]. A MIP was constructed for butyrylcholinesterase
using the oPD and GCE by Ozcelikay et al. [14]. Many other reports were
published such as sensors for detection of sulfamethoxazole, troponin T
and atrazine [15–17].
The MIPs, also known as biomimetic receptors, are polymers with
specific molecular recognition for a specific molecule. Considering their
structure, the MIPs are commonly called plastic antibody by several
authors [18,19]. However, the MIPs have several advantages over the
antibodies, such as lower cost, easy preparation, better storage ability,
higher mechanical strength and greater durability to heat and pressure
[20–22]. The MIP synthesis involves polymerization using a functional
monomer in the presence of a template molecule. After polymerization,
the template was extract from the polymeric matrix, resulting in a
three-dimensional imprinted cavity, complementary to the template in
terms of size, shape and functional groups [18,22–24]. Therefore, the
MIPs enable the specific recognition of target analytes in samples.
Cancer Biomarkers are substances present in blood, body fluid and
tissue that provide information about the presence, progression or
remission of tumors [25]. Carbohydrate Antigen (CA 15-3) is a protein
produced by normal breast cells, product of the MUC-1 gene [26]. The
CA 15-3 is a tumor biomarker for breast cancer, since the level of CA
15-3 in human body increases as this protein is released by tumor cells
[27]. Therefore, the determination of CA 15-3 (limit value of 30 U mL− 1)
can be used to monitor the disease evolution and watch for breast cancer
recurrence [28–30]. The MIP sensor for determination of CA 15-3 is
based on the interactions between the polymeric film and the CA 15-3.
Some MIP sensors were fabricated in the last few years for determination
of CA 15-3 using different monomers and electropolymerization as the
synthesis technique [31–33]. It is important to emphasize, that to the
best of our knowledge, these are the only published papers concerning
the fabrication of electropolymerized MIP sensors for CA 15-3. That
suggests the novelty of this work.
In this work, a paper-based electrochemical sensor was fabricated,
modified with gold nanoparticle (AuNP) and molecularly imprinted
using o-phenylenediamine and CA 15-3. The AuNPs was utilized to
improve the electrochemical response and served as platform for CA 15-
3 incubation while MIP served as a recognition element. The imprinting
was conducted in two steps: (1) adsorption of CA 15-3 on the surface of
an electrode modified with AuNP and (2) electropolymerization of oPD
onto the adsorbed protein. The protein was further extracted, leaving
vacant sites for subsequent rebinding. The determination of the CA 15-3
was achieved using a redox probe, K4 [Fe(CN)6], responsible for the
electrochemical signal.
2. Experimental
2.1. Reagents
The MWCNT (purity99%, 6–13 nm diameter, 3.5–20 μm length)
were acquired from Nanocyl. Sodium tetrachloroaurate (III) dehydrate
(NaAuCl4), Ortho-phenylenediamine (oPD) and Sodium Dodecyl Sulfate
(SDS) were obtained from Sigma-Aldrich (Brazil). Potassium ferrocya­
nide (K4 [Fe(CN)6]), sulfuric acid (H2SO4) and ethylene glycol (EG)
were purchased from Neon (Brazil). Sodium phosphate dibasic hepta­
hydrate (Na2HPO4⋅7H2O), sodium phosphate monobasic monohydrate
(NaH2PO4⋅H2O), sodium citrate (Na3C6H5O7), nitric acid (HNO3), oxalic
acid (HO2CCO2H) and silver nitrate (AgNO3) acquired from Synth
(Brazil). Mineral spirit from Acrilex. Polyvinylpyrrolidone (PVP),
ethanol and methanol from Vetec.
Cancer antigen 15-3 (30 kU mL− 1) and normal saliva from single
human female donor from Lee Biosolutions. Cholesterol, uric acid, IgG
from human serum, dopamine and human serum from AB plasma (USA
sterile-filtered) from Sigma-Aldrich. Ascorbic acid obtained from Synth.
2
Talanta 252 (2023) 123819
Bovine serum albumin (BSA) molecular biology grade (20 mg mL− 1)
acquired from BioLabs. All solutions were prepared with water by Mil­
lipore Milli-Q system.
Pencil purchased from Faber-Castel (6 B), Compactor (Flat Tip Per­
manent Marker) and Molin (Gel Ink Rollerball Pen). Photo Paper Matte
A4 108 g/m2 from Spiral. Clear Sticker Paper A4 80 micra from USA
Folien. Self-Adhesive Photo Paper A4 180 g/m2 from Multilaser. The
stencil was cut using a cutter plotter MVSK800 - Visutec. Filter paper
from Unifil 80 g/m2.
2.2. Fabrication of the printed electrode
A scheme showing the steps involved in the fabrication of the printed
electrode using different conductive inks is shown in Fig. 1 (a).
The carbon nanotube ink and silver nanoparticle ink were prepared
according to the procedure described in previously work published by
our group [34,35]. In summary, the fabrication of the electrode includes
a mask with the sensor design was cut using a cutter plotter in clear
sticker paper. The stencil was applied in a photo paper matte, the inks
were printed and the mask removed. Finally, a mineral spirit layer was
applied on top of the sensor. This electrode will be called in the next
topics as Carbon Nanotube Electrode (CNE).
2.3. Development of the MIP sensor for determinations of CA 15-3
Fig. 1(b) shows an illustrating scheme of the steps on the construc­
tion of the MIP sensor and determination of CA 15-3 and Table 1 sum­
marizes the experimental conditions. Initially, the gold nanoparticles
(AuNPs) were synthesized, where the process and characterization was
already discussed. Then 30 μL of AuNPs was dropped on the previously
developed CNE and dried at 60 ◦ C for 10 min. Next, a CA 15-3 solution of
40 U mL− 1 was prepared (10 mM of Tris-HCl and 150 mM of NaCl at pH
7.5) and 30 μL was dropped onto the CNE/AuNP and incubated for 2 h at
4 ◦ C.
Then a 15 mM o-phenylenediamine (oPD) solution was prepared in
PBS (0.01 mol L− 1 PBS pH 7.5) and stored at 4 ◦ C in an amber flask to
avoid exposure to light. The electropolymerization was performed by
applying 20 consecutive voltammetric cycles between − 0.2 and 1.0 V in
the oPD solution at scan rate of 50 mV s− 1. The MIP was fabricated by
electropolymerizing the film on top of the CNE/AuNP/CA 15-3. While
the NIP sensor used as a control electrode was prepared using the same
procedure but without the CA 15-3 template (CNE/AuNP). After the
electropolymerization, the sensor was placed in PBS (0.01 mol L− 1 PBS
pH 7.5) and an additional 5 scans of cyclic voltammograms were applied
in order to remove polymerized monomer residues and to stabilize the
polymeric film. Then the sensor surface was gently washed with
deionized water and dried under N2 stream.
In order to remove the template molecule from the polymeric matrix,
an oxalic acid solution 10 mM in PBS (0.1 mol L− 1 pH 7.5) was incubated
in the electrode for 2 h at 4 ◦ C. Every 30 min the electrode was rinsed
with PBS (0.1 mol L− 1 pH 7.5) and the oxalic acid solution was changed.
Then to ensure the removal, the electrode was cyclic scanning between
0.8 and 1.2 V in PBS (0.1 mol L− 1 pH 7.5) for 10 cycles at scan rate of 50
mV s− 1. The resulting sensor was called: CNE/AuNP/MIP.
For the determination of CA 15-3, the MIP sensor was incubated with
30 μL of the sample containing CA 15-3. The incubation was performed
at 4 ◦ C for 1 h. The electrochemical measurements were carried out
using a redox probe of 1.0 mmol L− 1 potassium ferrocyanide (II) in PBS
(0.1 mol L− 1; pH 7.0) using Cyclic Voltammetry (CV). After each step in
the construction of the sensor, the electrode was washed using PBS (0.1
mol L− 1 pH 7.5) and dried with nitrogen blowing.
2.4. Optimization studies
The following electropolymerization conditions were optimized and
discussed: oPD (5, 10, 15 and 20 mM) and CA 15-3 (10, 20 and 30 U
A.E.F. Oliveira et al. Talanta 252 (2023) 123819

Fig. 1. (a) Carbon nanotube electrode fabrication using carbon nanotube and silver nanoparticle inks, and graphite pencil by handwriting technique; (b) Molecularly
imprinted carbon nanotube sensor modified with gold nanoparticle by electropolymerization of o-phenylenediamine for determination of cancer antigen 15-3. (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

amplitude of 5000 mV. The samples are placed in a circular metallic

Table 1
holder using silver tape. The Fourier Transform Infrared Spectroscopy
Summary of the steps involved in the construction of the MIP sensor for CA 15-3.
(FTIR) measurements were performed in the region 4000-400 cm− 1 by a
Step Solutions Conditions PerkinElmer FTIR-8300 using ATR mode.
Incubation of CA 15-3 CA 15-3 (40 U mL ) in − 1
30 μL onto the The electrochemical measurements were conducted in a Autolab
Tris-HCl/NaCl (10:150 mM electrode for 2 h at 4 ◦ C PGSTAT204 from Metrohm Autolab using NOVA 2.1 software. The
at pH 7.5) electrochemical response was evaluated using K4 [Fe(CN)6] in PBS (0.1
Electropolymerization of oPD (15 mM) 20 cycles between
oPD in PBS (0.1 mol L− 1 pH 7.5) − 0.2 and 1.0 V at 50
mol L− 1 pH 7.0) as redox probe.
mV s− 1
1
Stabilization of the PBS (0.1 mol L− pH 7.5) 5 cycles between − 0.2
2.6. Specificity and reproducibility
polymeric film and 1.0 V at 50 mV s− 1
Extraction (1) of CA 15-3 Oxalic Acid (10 mM) in 2 h at 4 ◦ C, changing
PBS (0.1 mol L− 1 pH 7.5) the solution every 30 For the specificity studies, several MIP sensors were fabricated and
min each one was incubated for 1 h at 4 ◦ C with a solution containing one
1
Extraction (2) of CA 15-3 PBS (0.1 mol L− pH 7.5) 10 cycles between 0.8 interfering substance and CA 15-3 of 20 U mL− 1. The interfering species
and 1.2 V at 50 mV s− 1
Rebinding of CA 15-3 CA 15-3 in Tris-HCl/NaCl 30 μL onto the
were cholesterol (0.19 mg mL− 1), dopamine (30 pg mL− 1), BSA (1.0 mg
(10:150 mM at pH 7.5) electrode for 1 h at 4 ◦ C mL− 1), uric acid (0.05 mg mL− 1), ascorbic acid (0.01 mg mL− 1) and IgG
(1.0 mg mL− 1). The concentration values were chosen according to the
reference normal concentrations founded in the human serum and
mL− 1) concentrations, number of cycles (10, 20 and 30 scans) and scan saliva, except the BSA that are not found in human serum, but it was
rate (25, 50 and 75 mV s− 1) of electropolymerization process, and CA used as an inexpensive analogue of Human Serum Albumin (HSA), most
15-3 extraction time (0:30, 1:00 and 1:30). For each study, one abundant protein in the blood.
parameter was varied, while the others were kept constant. In the op­ While the reproducibility was evaluated measuring the electro­
timizations studies, except the extraction time study, the sensor CNE/ chemical behavior of four electrodes fabricated identically in different
AuNP/MIP was incubated with CA 15-3 (20 U mL− 1) for 1 h at 4 ◦ C and conditions. Both studies were conducted using Differential Pulse Vol­
the electrochemical response was evaluated using chronoamperometry tammetry (DPV) and K4 [Fe(CN)6] in PBS (0.1 mol L− 1 pH 7.0).
in potential of 0.45 V for 60 s in 1 mmol L− 1 K4 [Fe(CN)6] and PBS (0.1
mol L− 1; pH 7.0).
2.7. Application in serum and saliva samples

2.5. Characterization techniques
The samples CNE, CNE/AuNP, CNE/AuNP/CA 15-3/MIP (before
extraction), CNE/AuNP/MIP (after extraction), CNE/AuNP/MIP/CA 15-
3, CNE/AuNP/NIP and CNE/AuNP/NIP/CA 15-3 were characterized
using different techniques. The Atomic Force Microscopy (AFM) was
performed using a Bruker Multimode 8 (MM8), scan size of 5.00 μm and
The sensor CNE/AuNP/MIP was used in the determination of CA 15-
3 in human serum samples and saliva samples. The serum was diluted in
the proportion of 1:7 (v/v) with PBS 0.1 mol L− 1 (pH 7.5), while the
saliva was used with no dilution. The sensors CNE/AuNP/MIP were
constructed as explained in the previously topics. The samples of serum
and saliva were spiked with CA 15-3 at different concentrations and the
resulting solution was incubated onto the MIP sensor. 30 μL was dripped

3
A.E.F. Oliveira et al.
onto each sensor and incubated for 1 h at 4 ◦ C.
3. Results and discussion
3.1. Fabrication of the MIP sensor for CA 15-3 determination
Several commercial methods have been developed for measuring the
CA 15-3. The most widely used rely on immunoassays, mainly through
Enzyme-linked immunosorbent assay (ELISA) test. That includes the
sandwich type that used two monoclonal antibodies and the competitive
type with one monoclonal antibody [36]. The approach signal trans­
duction in the immunoassays can be chemiluminescence, electro­
chemiluminescence, fluorescence, surface-enhanced Raman
spectroscopy (SERS) and electrochemical [36]. Most techniques include
complicated procedures, time-consuming, expensive and very depen­
dent on laboratory instrumentation [37]. Therefore, currently there is a
need for rapid and accurate detection of cancer biomarkers. The
manufacture of devices suitable for determining these biomarkers with
low costs, detection limits and time of analysis have become a potential
research area [38–40]. Regarding the determination of biomarkers,
electrochemical sensors have some advantages over conventional
techniques.
There are many advantages in using printed electrodes over con­
ventional electrodes. They can be fabricated on a large scale which will
allow a low-cost production. Since printed electrodes are disposable, the
cleaning and maintenance steps are eliminated. Consequently, printed
sensors are known as low-cost, miniaturized, disposableand highly
Fig. 2. (a) Schematic illustration of the fabrication of the MIP sensor for CA 15-3 detection; Cyclic voltammograms recorded during the electropolymerization of 15
mM of oPD on the CNE/AuNP in PBS (0.1 mol L− 1 pH 7.5) at 50 mV s− 1 for 20 cycles in (b) absence of (NIP) and (c) presence (MIP) of CA 15-3 (20 U mL− 1).
4
Talanta 252 (2023) 123819
sensitive devices. The conductive inks of carbon nanotube and silver
nanoparticle, and the graphite pencil, were effective in the fabrication of
the printed electrode. The paper-based electrode is an attractive alter­
native to conventional electrodes. It emerges as a simple, low cost and
flexible amperometric sensor.
The electropolymerization of o-phenylenediamine (oPD) and
following mechanism are discussed in the topics 1.1 and 1.2 of the
Supplementary Material. After the fabrication of the carbon nanotube
electrode (CNE), the construction of the MIP sensor for determination of
CA 15-3 is conducted in four main stages: (1) addition of AuNP layer, (2)
incubation of CA 15-3, (3) electropolymerization of oPD and (4) CA 15-3
extraction. The resulting sensor is called: CNE/AuNP/MIP. Fig. 2 illus­
trates the steps involved in the fabrication of the MIP and NIP sensor.
The first step is adsorption of AuNPs to the CNE. AuNP synthesized
was immobilized on the electrode surface by physical adsorption. The
AuNPs were used as platforms for the incubation of CA 15-3. Several
properties make the AuNP attractive for immobilization of bio­
molecules. The AuNPs can improve the electron transfer due their
excellent electrical conductivity [41,42]. Also, it increases the
surface-to-volume ratio, promoting the immobilization of a higher
amount of the subsequent layers and do not affect the activity or
structure of the biocomponent [41–45]. The bioconjugation of the AuNP
and CA 15-3 was performed by physical method, specifically adsorption
[46].
The next step was the electropolymerization of a oPD film on top of
the CNE/AuNP/CA 15-3. The MIP and NIP sensors were fabricated in the
same conditions, where the difference is the presence of CA 15-3 as the
A.E.F. Oliveira et al. Talanta 252 (2023) 123819

template. The electropolymerization was performed using cyclic vol­ (0.5 mol L− 1) and proteinase K (0.5 mol L− 1). As a result, the oxalic acid
tammetry from − 0.2 to 1.0 V for 20 scans at 50 mV s− 1. In order to avoid solution was chosen since it performed the highest difference in the
overoxidation, a lower range was used in the electropolymerization of responses before and after extraction [33].
MIP and NIP, compared to the previous study. Fig. 2(a) and (b) presents Unfortunately, due the quantity available of CA 15-3 in our labora­
the voltammograms obtained during the fabrication of the PoPD. tory, some studies had to be prioritized over others in this work. For
No remarkable difference is observed between the electrochemical example, the type of extraction solution was chosen according these
behavior of MIP and NIP. Both oxidation processes exhibit the absence previous reports. However, the extraction time was further optimized.
of a cathodic peak proving the irreversibility of the reaction and the Therefore, the extraction solution chosen was the oxalic acid. The pro­
current decreases after each scan, consistent with the formation of the cess consisted in disrupting the non-covalent bonds between the CA 15-3
electrically insulating PoPD film blocking the surface [32,47]. and the PoPD to allow the physically entrapped molecules to escape
The electropolymerization of NIP presents a maximum anodic cur­ from the polymeric network [48].
rent at about 0.53 V and the MIP at 0.39 V. This shift to more negative Also, to guarantee the efficient removal of CA 15-3 embedded in the
potential suggests that the MIP surface requires slightly less energy for polymeric matrix was carried out by overoxidation in PBS (0.1 mol L− 1
the oxidation of oPD. That can be associated to the attractive in­ pH 7.5). CV scanning was performed between 0.8 and 1.2 V for 10 cy­
teractions between the CA 15-3 and the polymer in the MIP. This peak is cles. The template can be removed from an MIP by film overoxidation as
representative of the oxidation of the amino group of the oPD mono­ reported by several authors [51–55]. The extraction creates cavities that
mers. However, the first peak in the electropolymerization of MIP ex­ are complementary in size, shape and functional groups to the CA 15-3
hibits a shoulder at 0.61 V, attributed to the oxidation of a dimer [32, molecules [33,48]. These cavities at the sensor will be occupied by the
48]. CA 15-3 protein in the electrochemical measurements. That will be
Also, the current peak indicates the occurrence of electro­ discussed in the next topic.
polymerization with a current 36% lower measured in MIP layers
compared to NIP. The current difference may be due to the presence of 3.2. Electrochemical behavior of the MIP sensor
the protein on the MIP layer, the non-electroactive CA 15-3, that results
in an additional barrier to diffusional monomer for its oxidation at the The determination of CA 15-3 was performed indirectly using the
electrode [32,49,50]. That suggested that the successful electro­ MIP sensor with a redox probe of K4 [Fe(CN)6]. The working principle of
polymerization of oPD entrapping CA 15-3 was achieved. the sensor is based on the binding between the CA 15-3 and the recog­
The final step in the construction of the MIP sensor is the CA 15-3 nition sites of the MIP. The current signal is proportional to the CA 15-3
extraction. The complete extraction of the entrapped molecule is concentration, since the binding MIP/CA 15-3 hider the electrode
crucial for the MIP sensor performance. This process can be difficult for transfer of the probe, decreasing the electrochemical response.
proteins due their size and complex structure. Therefore, an extensive Fig. 3 presented the voltammograms (a) and the peak current in­
research was conducted evaluating the different strategies used for the tensity (b) of CNE, CNE/AuNP, CNE/AuNP/CA 15-3/MIP (before
effective removal of molecule, especially proteins, from the imprinted extraction), CNE/AuNP/MIP (after extraction) and CNE/AuNP/MIP/CA
surface. The most common method is solvent extraction [33,48]. Some 15-3 (after rebinding of 20 U mL− 1 CA 15-3). The voltammograms shows
previous studies concerning the extraction of molecules from imprinted the not well-defined peaks of the redox pair [Fe(CN)6]4-/3-.
polymers are in Table 2. Using the bare CNE, the peak was approximately at 0.51 V. After
As observed, the target removal was performed using different addition of the AuNP is observed an increase of 58% in the peak current
technique, times and solutions. It is hard to find a pattern in this process. because the good electrical conductivity of the metallic nanoparticles.
Although, Pacheco et al. conducted an interest work optimizing the However, the oxidation potential shifts to a lower potential (− 0.11 V)
removal of CA 15-3 [33]. They tested several aqueous extraction solu­ due to the fast electron transfer that facilitates the oxidation process
tions: acetic acid/SDS (1% v/v), oxalic acid (0.5 mol L− 1), guanidine [50].
The formation of the PoPD film introduced additional barriers to the
electron transfer, resulting in a decrease of the current signal of 96% and
Table 2
Review of CNT inks synthesized in the past decade and their extraction method.
no observed peak. That confirms the formation of an insulation layer on
top of the CNE/AuNP. After the removal of CA 15-3, the response of the
Monomer Target Extraction Method Reference
sensor increases 8.5 times indicating the effective extraction of the
Pirrol α-amylase SDSa Solution Incubation overnight [23] template, that resulted in a less hindered electrode surface [32,56].
3-AMPb Tau PBSc pH 5.6 CVd; 10 scans; − 0.2 to [55]
After the rebinding of 20 U mL− 1 of CA 15-3, the redox peaks
protein 1.2 V
2-AMP CA 15-3e Oxalic Acid Incubation 12 h [27]
disappear and the current peak magnitude decreased 76%. The number
oPDf β-Amyloid Oxalic Acid Incubation 2 h [57] of cavities available for the redox probe to access the electrode surface
oPD BuChEg NaOH Shaking 300 rpm [42] decreases again, causing a lower redox signal. These results demonstrate
Solution overnight the possibility of the sensor to remove and rebind the CA 15-3 onto the
pPDAh Naloxone Methanol and Incubation 40 min [54]
MIP, thus offering accessible sensing cavities [14,29,57]. The NIP sensor
HCl Solution (change solution every
10 min) was used as reference system, to compare the MIP and NIP behavior in
TBi CA 15-3 NaOH CV; 200 scans; 0.0–0.4 V [25] the incubation of CA 15-3. As discussed, only the MIP surface contains
Solution (approximately 1 h) sensing cavities. While the NIP is formed of PoPD film without the CA
0.1 mol L− 1
15-3 template. That should affect the behavior of the sensor, since the
oPD CA 15-3 Trypsin (0.1 Incubation 90 min. CV; [26]
mol L− 1) 10 scans; − 0.3 to 0.3 V
MIP provides higher affinity with the CA 15-3 [29,32,49].
a
The determination of CA 15-3 was performed indirectly using the
SDS: Sodium Dodecyl Sulfate. MIP and NIP sensor. The voltammograms are presented in Fig. 4(c) and
b
AMP: Aminophenol.
c in Fig. 4(d) the peak current intensity, exhibiting the characteristic
PBS:Phosphate Buffer Saline.
d peaks of [Fe(CN)6]4-/3-. After incubation of CA 15-3, the MIP signal
CV: Cyclic Voltammetry.
e
CA 15-3: Carbohydrate Antigen 15-3. decreased 76%, while the NIP decreased 14%. The comparison of the
f
oPD: o-phenylenediamine. MIP and NIP indicates the MIP results in specific binding, while the NIP
g
BuChE:Butyrylcholinesterase. suggest nonspecific adsorption of CA 15-3 to the PoPD [14,29,57]. Also,
h
p-phenylenediamine. the behavior of CNE/AuNP/NIP/CA 15-3 is comparable with the
i
TB: Toludine Blue. CNE/AuNP/MIP, that suggests that both have a smaller amount of CA

5
A.E.F. Oliveira et al. Talanta 252 (2023) 123819

Fig. 3. Electrochemical behavior of MIP sensor: (a) Voltammograms obtained using electrode samples of CNE, CNE/AuNP, CNE/AuNP/CA 15-3/MIP (before
extraction), CNE/AuNP/MIP (after extraction) and CNE/AuNP/MIP/CA 15-3 (after rebinding of 20 U mL− 1 CA 15-3) in PBS (0.1 mol L− 1; pH 7.0) with 1 mmol L− 1 K4
[Fe(CN)6]; (b) Corresponding peak current intensity; Difference between MIP and NIP sensor: (c) Voltammograms obtained using CNE/AuNP/NIP, CNE/AuNP/NIP/
CA 15-3, CNE/AuNP/MIP and CNE/AuNP/MIP/CA 15-3 in PBS (0.1 mol L− 1 pH 7.0) using 1 mmol L− 1 of K4 [Fe(CN)6] and (d) Corresponding peak current intensity.

15-3 in their surface. The first due the absence of selective sites and the
last due the remaining CA 15-3 after the extraction process [32,49].
3.3. Characterization techniques
3.3.1. Atomic force microscopy
Atomic Force Microscope (AFM) was used to investigate the surface
topography of the imprinted polymer. The samples evaluated were CNE,
CNE/AuNP, CNE/AuNP/CA 15-3/MIP (before extraction), CNE/AuNP/
MIP (after extraction), CNE/AuNP/MIP/CA 15-3 (after rebind of 20 U
mL− 1 CA 15-3) and CNE/AuNP/NIP/CA 15-3. AFM images are shown in
Fig. 4. The AFM allows to measure the average roughness (Ra) and root
mean square (RMS), and consequently evaluate the surface of each layer
in the construction of the MIP sensor [58]. The Ra and RMS of CNE was
1.7 and 2.2 nm, showing a relatively flat and compact morphology. The
CNE exhibits the smallest roughness levels under AFM observation.
After the modification of CNE with the AuNP it is observed the
presence of randomly distributed worm-like structure. The deposition of
colloidal nanoparticles forms a diverse array of patters, such as worm­
like domains, isolated islands, continuous labyrinthine patterns and
polygonal networks. That is caused by the reduction of van der Waals
interactions due the solvent evaporation [59–61]. Also, the Ra and RMS
increase to 2.7 and 3.8 nm may be associated with the deposition of
AuNP.
The addition of the PoPD polymeric layer increased the Ra and RMS
of the overall materials to 9.8 and 12.8 nm. The surface of CNE/AuNP/
CA 15-3/MIP results in a much rougher surface, possibly related to the
successful deposition of the polymeric film. A compact almost globular-
like structure is observed with good coverage, resulting in a homoge­
neous and compact film of the surface [32,58,62,63]. This globular
structure can be associate with the protein CA 15-3.
The extraction of the CA 15-3 from the MIP, in CNE/AuNP/MIP
image, causes an important structural change as reveled by the image.
The topography becomes smoother and flatter, while the Ra and RMS
decreases to 2.5 and 3.4 nm, supporting the extraction procedure. The
process removes the CA 15-3 creating imprinted cavities, that leads to a
decrease in roughness [64–67]. That results indicates that the CA 15-3
are only partially embedded in the polymeric matrix, remaining traces
of the protein.
After rebinding, in CNE/AuNP/MIP/CA 15-3 image, the Ra and RMS
slightly increased to 3.9 and 5.4 nm. This increase may be related to the
interaction of CA 15-3 with the specific cavities formed during the
electropolymerization [29]. The amount of CA 15-3 attached seem to be
smaller than the MIP film before extraction, explaining the lower
roughness compared to CNE/AuNP/CA 15-3/MIP (9.8 and 12.8 nm, for
Ra and RMS respectively). That result is reasonable since the concen­
tration of CA 15-3 used in the electropolymerization is higher (40 U
mL− 1) than the used in the rebinding (20 U mL− 1). Also, it is unlike that

6
A.E.F. Oliveira et al.
Fig. 4. AFM phase images (right) and 3D-height images (left) of CNE, CNE/AuNP; CNE/AuNP/CA 15-3/MIP (before extraction), CNE/AuNP/MIP (after extraction),
CNE/AuNP/MIP/CA 15-3 (after rebinding) and CNE/AuNP/NIP/CA 15-3.
each binding site is reoccupied in the uptake process [68].
Finally, the image of CNE/AuNP/NIP/CA 15-3 presented a more
compact and smooth film compared to the CNE/AuNP/MIP/CA 15-3,
with Ra and RMS of 2.2 and 2.7 nm. Some areas in the image are
more flat than others and the entire film is not very homogeneous,
differently from the MIP sample. That can be associate with non-specific
adsorption of CA 15-3.
3.3.2. Fourier Transform Infrared Spectroscopy
The Fourrier Transform Infrared Spectroscopy (FTIR) was performed
to investigate the composition of each layer in the construction of the
MIP sensor and to prove the efficiency of the molecularly imprinted
polymer for CA 15-3 fabrication. Fig. 5 presents the infrared spectrum of
CNE, CNE/AuNP, CNE/AuNP/NIP, CNE/AuNP/NIP/CA 15-3, CNE/
AuNP/CA 15-3/MIP (before extraction), CNE/AuNP/MIP (after extrac­
tion) and CNE/AuNP/MIP/CA 15-3 (after rebinding of 20 U mL− 1 CA
15-3).
The CNE is fabricated by applying the carbon nanotube (CNT) ink in
a paper substrate, as described in the experimental section. The CNT ink
is fabricated by functionalizing covalently (HNO3:H2SO4) and non-
covalently (SDS) the CNT. The spectra of CNE exhibits the bands asso­
ciated with the sulfate groups presented in the SDS molecule: the SO2
asymmetric vibration (1217 cm− 1) and the SO2 symmetric vibration
(1078 cm− 1) [69–71]. The band at 3469 cm− 1 is related to the OH
asymmetric stretching probably associated to the hydroxyl groups of
physical adsorbed water molecules. The stretching vibration of CH2
(2908 and 2848 cm− 1) attributed to the carbon chain of SDS are also
seen. Finally, the band at 1475 cm− 1 is assigned to the C=C stretching of
CNT, suggesting the presence of carbon double bond (C=C) that con­
firms the sp2 structure of the CNTs [72,73].
The AuNP is fabricated using sodium citrate/sodium
7
Talanta 252 (2023) 123819
tetrachloroaurate. The CNE/AuNP spectra exhibits a band at 1710 cm− 1
associated with the C=O stretching due to carbonyl groups presented in
the sodium citrate. At 1230 cm− 1 and at 1076 cm− 1 is observed the C–O
stretching vibration, both associated with the sodium citrate [69,74].
However, some authors also associate the band at 1076 cm− 1 to the O–H
bending out of plane [75]. The band at 717 cm− 1 is assigned to the C–H
out of plane [76]. That suggested the formation of the citrate capped
gold nanoparticles. The bands associated with the CNT ink disappeared
in this spectrum, probably because the AuNP provides a good coverage
of the surface.
The CNE/AuNP/NIP spectra shows some peaks associated with
functional groups present within the polymeric matrices. The band at
3266 cm− 1 is assigned to the N–H stretching asymmetric vibrations. The
broad absorption band is characteristic of the polymer structure of
PoPD. The band at 1209 cm− 1 is assigned to the C–N stretching from the
secondary amine groups also presented in the PoPD structure [29,77,
78]. The band at 968 cm− 1 could be attributed to the out of plane C–H
deformation of 1,2,4 tri-substituted of benzene ring [79,80]. The bands
at 2912 and 2843 cm− 1 assigned to the stretching vibration of CH2 are
also observed. These bands can be associated with the carbon chain of
SDS, presented in the CNT ink, already observed in the CNE spectra.
Although, it can also be referring to the formation of PoPD film [29,73].
The spectra CNE/AuNP/NIP/CA 15-3 showed similar peaks
compared to the CNE/AuNP/NIP. That indicates that the incubation of
CA 15-3 did not modify or not modifiy enough the surface of the NIP
sensor to affect the infrared measurements. That is an interest result
since it suggests no strong specific binding of CA 15-3 and the PoPD film.
The spectra CNE/AuNP/CA 15-3/MIP, with the sample before
extraction of the target molecule, shows some peaks associated with the
PoPD film. The band at 3292 cm− 1 is assigned to the N–H stretching
asymmetric vibrations associated with the amine groups presented in
A.E.F. Oliveira et al.
Fig. 5. IR spectra of CNE, CNE/AuNP, CNE/AuNP/NIP, CNE/AuNP/NIP/CA
15-3, CNE/AuNP/CA 15-3/MIP (before extraction), CNE/AuNP/MIP (after
extraction) and CNE/AuNP/MIP/CA 15-3 (after rebinding of 20 U mL− 1 CA
15-3).
the PoPD structure. The band at 981 cm− 1 can be assigned to the out of
plane C–H deformation of 1,2,4 tri-substituted of benzene ring, resulted
from the polymerization of oPD. It is possible that the band at 1313 cm− 1
is assigned to the C–N stretching. The shift can be associated to the
presence of CA 15-3, that has the bond C–N in their structure. That also
explain the higher transmittance. The presence of CA 15-3, give rise to
new bands at 1610 cm− 1 and 777 cm− 1. The band at 1610 cm− 1 can be
associated with the C=O stretching due to carbonyl groups from amino
acid residues within the protein, while the bands at 777 cm− 1 can cor­
responds to the N–H wag of primary and secondary amines. These re­
sults indicate the CA 15-3 entrapped within the polymer, that evidences
the presence of the protein [29,81].
After extraction of CA 15-3, in the CNE/AuNP/MIP spectra, the
bands associated with their protein structure disappeared. The spectra
showed similar peaks of the CNE/AuNP/NIP, suggesting related
composition. The cyclic voltammetry studies indicate the presence of
some remaining CA 15-3, even after extraction. However, that amount
was not enough to change the infrared measurements. So the result
8
Talanta 252 (2023) 123819
exhibits only the peaks associated with the PoPD polymeric film. That
indicates the effective extraction of CA 15-3.
The next step is the rebinding of CA 15-3. As observed in the CNE/
AuNP/MIP/CA 15-3 spectra, the bands associated with the CA 15-3
protein structure is observed at 1606 cm− 1 and 772 cm− 1, assigned to
the C=O stretching and to the N–H wag of primary and secondary
amines. It is interest to observe the lower transmittance of these bands,
that can be related to a smaller amount of CA 15-3, compared to the
spectra before extraction. Overall, this result suggests the extraction/
rebinding of CA 15-3 in the MIP sensor and also indicates that the NIP
sample does not have specific cavities for the CA 15-3.
3.4. Experimental optimization for developing MIP sensor
As observed in the electrochemical studies, the voltammetric profile
in presence of K4 [Fe(CN)6] is not well-defined showing a distortion and
inclination due to resistive behavior. That behavior is also observed by
other authors when using a printed sensor with carbon nanotube ink.
Therefore, the following optimization studies, including the calibration
curve and application and biological samples were performed using
chronoamperometry. This method was chosen for detection of CA 15-3
since it usually offers better accuracy and sensitivity compared to cyclic
voltammetry [81,82]. The first optimization was the chro­
noamperometry potential applied during the measurements. The tem­
plate removal time was also optimized, whose experimental conditions
will be explained ahead.
3.4.1. Optimization of potential of chronoamperometry
Chronoamperometry is an electrochemical technique that measures
the current as a function of time at fixed potential [83]. In order to
optimize the potential applied during the chronoamperometry, five
potentials were selected in the range 0.35–0.55 V and amperometry
measurements were carried out for each potential. The goal was to
evaluate the effect of applied potential on the amperometric anodic
current due the redox reaction [84]. Therefore, the study was performed
using the bare CNE in presence and absence of K4 [Fe(CN)6]. The
chronoamperograms and resulting current variation (subtracting cur­
rent in presence/absence of redox probe) are presented in Figs. S1(a)
and S1(b), respectively.
The range of values was selected before and after the potential of the
oxidation peak of the potassium ferrocyanide using the CNE, as shown in
electrochemical behavior in Fig. S1. The blank measurement exhibits the
background current of the sensor, while the chronoamperograms in
presence of the electrochemical probe presents the current resulting
from the redox reaction. Therefore, the choice of the optimum applied
potential is essential to achieve the best electrochemical response of the
sensor [85,86].
As observed in the chronoamperograms, the current decreases
rapidly as a function of time and stabilizes after approximately 10 s, for
all the measurements. The initial decrease is associated with the
capacitive current due to the double layer charging [87]. Comparing the
responses, the applied potential of 0.45 V obtained the best signal and
higher current variation (Δi) in presence and absence of K4 [Fe(CN)6].
At this potential, the best electrochemical response was obtained due to
the fact that it reached the maximum current variation value. This is in
agreement with the oxidation peak observed in Fig. 4 for Fe(CN)4− 6 /Fe
(CN)3−
6 . So the following studies were conducted applying a fixed po­
tential of 0.45 V for 60 s during the electrochemical measurements.
3.4.2. Template removal time
As discussed previously, the removal of CA 15-3 was performed using
an oxalic acid solution. This step is crucial in the construction of the MIP
sensor since it creates the specific sites for interaction with the CA 15-3
[88]. The extraction time was optimized by washing the CNE/AuNP/CA
15-3/MIP sensors with oxalic acid for different times (0:30–3:00). The
current was measured using chronoamperometry after extraction in
A.E.F. Oliveira et al.
presence of K4 [Fe(CN)6]. The current obtained from the chro­
noamperograms are in Fig. S2(a).
The increase in the current is observed from 0:30 to 2:00, then it
stabilized. Based on the electrochemical results, the enhancement of the
extraction time leads to higher CA 15-3 extraction. That is indicated
since the removal of CA 15-3 results in a less hindered electrode surface,
causing an increase of the current. After 2 h, the response increasing
with time do not modifies significantly. Hence, the time of 2:00 was
chosen for the extraction of CA 15-3 from the MIP sensor [63,88,89]. In
order the guarantee that the oxalic acid solution only extracts the CA
15-3 and does not modify the polymer film itself, samples of the sensors
CNE/AuNP/CA 15-3/MIP and CNE/AuNP/NIP were washed with oxalic
acid solution for 2 h and the results were compared. The resulting
amperograms are exhibits in Fig. S2(b).
In the NIP sensor, after extraction step the current only slight in­
creases, probably due the removal of unbounded PoPD, increasing the
electrochemical response. That result is important since it suggests that
the solution did not affect highly the polymeric film [64]. In the
meantime, after extraction the MIP sensor shows a higher current
indicating the efficiency of the extraction procedure with oxalic acid.
3.5. Optimization of electropolymerization process
In order to develop a sensitive PoPD polymeric film, to be used as
electrochemical sensor, it is crucial to optimize the electro­
polymerization process. The oPD film is strongly influenced by the
electropolymerization conditions such as monomer and template con­
centration, number of cycles and scan rate. Hence, those experimental
conditions were optimized to improve the MIP sensor performance
resulting in higher sensitivity.
The studies were performed using chronoamperometry (0.45 V for
60 s) and the sensor CNE/AuNP/MIP. The chronoamperograms were
obtained before and after incubation with CA 15-3 (20 U mL− 1). The
parameter exhibiting the higher Δi was chosen as the optimum value
since it resulted in higher interaction between CA 15-3 and recognition
sites of the MIP.
3.5.1. Monomer and template concentration
Usually, the monomer concentration is proportional to the thickness
of polymeric film and the amount of imprinted templates [90,91]. When
the thickness is too thick the film traps the molecules deeply in the
polymeric matrix, while too thin limits the amount of the imprinted sites
[92]. To determine the effect of monomer concentration on the response
of MIP sensor to CA 15-3, the polymeric films were grown in solutions of
oPD at different concentrations (5–25 mM). Fig. S3(a) shows the current
variation values as function of the monomer (oPD) concentration, before
and after incubation.
The results indicate the current is proportional to the thickness of
recognition layer and consequently the amount of cavities in the elec­
tropolymerized layer. Initially the current increases with increasing
concentration of oPD. That is due to the enhancing of the sensor sensi­
tivity due the higher amount of recognition sites [91]. Then the oxida­
tion current variation decreased since the excess o-PD monomer forms
non-conductive membrane without recognition sites [93]. That in­
dicates the maximum interaction between the MIP and CA 5–13 was
achieved at 15 mM. Therefore, the best sensor response and conse­
quently the optimum monomer concentration chosen was 15 mM [94,
95].
The CA 15-3 concentration was also optimized. The template con­
centration affects directly the amount of recognition sites. This can
ensure that the CA 15-3 is incorporated into the surface of the polymeric
matrix during the electropolymerization, to be further extracted leaving
sites complimentary to itself [90]. Different concentrations of CA 15-3
were tested for the optimum template concentration in presence of
constant oPD. The Δi obtained before and after incubation of CA 15-3
(20 U mL− 1) is presented in Fig. S3(b).
9
Talanta 252 (2023) 123819
As observed, at 40 U mL− 1 exhibited the best electrochemical
response. Before and after this value the current variation decreased. A
smaller concentration of oPD produced a less sensitive sensor, probable
because the smaller number of recognition sites in the polymeric matrix
[94]. While a higher concentration may result in CA 15-3 forming
complex with all the monomers decreasing the possibility of
cross-linking and consequently entrapment of the template molecule
[93]. Therefore, at 40 U mL− 1 the amount of CA 15–3 may have reached
the maximum, and consequently the amount of recognition sites.
3.5.2. Number of cycles and scan rate of electropolymerization
A lower number of cycles may not be enough to ensure that the
template is adequately embedded in the polymeric matrix, after
removing of CA 15-3. While higher cycles lead to more extensive elec­
tropolymerization, and consequently the formation of thicker PoPD film
with less accessible imprinted sites [90]. Therefore, the number of cycles
is important in the fabrication of well-defined recognition sites. In order
to study this behavior, the films were grown at different scan rates
(25–75 mV s− 1). Fig. S4(a) shows the current variation values as func­
tion of number of cycles, before and after incubation of CA 15-3.
The amount of imprinted sites increases with increase of cycles, and
then decreases after 20 cycles since a higher number of cycles may result
in too thick films that give poor site accessibility and low binding ca­
pacity [50,63,92]. Hence, 20 cycles can be defined as the optimum value
for analytical performance.
Next, the scan rate was optimized. Lower scan rates form thicker
films becoming difficult to remove the molecular template entrapped in
the polymeric film. While higher scan rates result in relatively porous
and thin films [91]. Hence, the optimized scan rate was found to be 50
mV s− 1, as observed in Fig. S4(b).
3.6. Calibration curve of the MIP sensor for CA 15-3
The performance of the proposed sensor CNE/AuNP/MIP was eval­
uated by assaying the CA 15-3. The electrochemical measurements were
performed by incubating different samples of the sensor with solutions
of increasing concentrations of CA 15-3 (5–35 U mL− 1). In order to
conduct the studies, the CA 15-3 solution was dropped on the sensor and
incubated for 1 h at 4 ◦ C, followed by measurements using chro­
noamperometry (0.45 V for 60 s) in presence of the redox probeK4 [Fe
(CN)6]. The results are shown in Fig. 6(a).
The chronoamperograms shows a decrease in the current with in­
crease in the CA 15-3 concentration. That occurs since the use of CNE/
AuNP/MIP to determine CA 15-3 is based on the interaction between the
CA 15-3 and the recognition sites of the MIP. The binding of MIP and CA
15-3 hider the electrode transfers of the redox probe, decreasing the
electrochemical response. As observed, the current signal is proportional
to the CA 15-3 concentration resulting in the calibration curve. At 35 U
mL− 1 the curve achieves the limit of linearity, after the concentration is
not directly proportional to the concentration of CA 15-3.
Fig. 6(b) exhibits the current obtained in the chronoamperograms in
function of CA 15-3 concentration. The calibration curve exhibited the
linear regression equation of i [μA] = − 0.013936 [U mL− 1] + 5.74714,
with a linear range of 5–35 U mL− 1 and coefficient of determination (r2)
of 0.9981. The developed sensor provided a sensitivity of 0.013936 μA/
U mL− 1. The limits of detection (LOD) and quantification (LOQ) repre­
sents the lowest amount of CA 15-3 that can be detected and quantita­
tively determined, respectively. The LOD and LOQ were calculated as
three and ten times the standard deviation of a blank solution divided by
the sensitivity. The values found were 1.16 U mL− 1 (LOD) and 3.87 U
mL− 1 (LOQ).
The linear range and LOD published in previous reports using MIP
sensors for determination of CA 15-3 and the results in this work were
compared in Table 3. As discussed, at the best of our knowledge, these
are the only published papers in this area. All the works developed a MIP
sensor by modification of commercial screen-printed electrode.
A.E.F. Oliveira et al. Talanta 252 (2023) 123819

Fig. 6. (a) Chronoamperograms obtained in different CA 15-3 concentrations (5–45 U mL− 1) in PBS (0.1 mol L− 1
pH 7.0) using 1 mmol L− 1
of K4 [Fe(CN)6]; (b)
Calibration curve.

days, solutions, materials and equipment [52]. The repeatability was

Table 3 also considerate since each electrode was measured 3 times. The chro­
Comparative study of electrochemical MIP sensors for determination of CA 15-3.
noamperograms are exhibited in Fig. 7(c) and the resulting current
Immunosensor Linear Range Limit of Detection Reference variation before and after CA 15-3 incubation in Fig. 7(d).
Au-SPE/MIP 0.25–10 U mL− 1 0.05 U mL− 1 [32] The relative standard deviation (RSD) of the current response for CA
Au-SPE/AOT/MIP 0.10–100 U mL− 1 0.10 U mL− 1 [31] 15-3 detection was 2.77%, indicating the precision of the MIP sensor.
Au-SPE/MIP 5–50 U mL− 1 1.50 U mL− 1 [33] These observations suggest that the electrode fabrication and modifi­
CNE/AuNP/MIP 5 - 35 U mL− 1 1.16 U mL− 1 This Work
cation have appreciable reproducibility [96].
a
Au-SPE: Au-screen printed electrodes.
b
DPA/SA: Dopamine/4-Styrenesulfonic Acid Sodium Salt.
c
phenylenediamine. 3.8. Application in serum and saliva samples
d
AOT/GLT: 8-amino-1-octanethiol/Glutaraldehyde.
e
TB: Toluidine Blue. The analysis of human serum and saliva samples were tested in order
f
2-AMP: 2-aminophenol.
to evaluated the possibility to detect the CA 15-3. First, these samples
were spiked with different concentrations of CA 15-3. Then the sensors
Therefore, this may be the first research developing a MIP sensor for CA CNE/AuNP/MIP were incubated with these solutions for 2 h at 4 ◦ C. For
15-3 fabricating the base printed electrode. Although there is not much each concentration, three sensors were fabricated and the chro­
to compare, the linear range and LOD obtained are very similar to the noamperometry measurements performed in triplicate [29].
work of Pacheco et al. and a little higher than the work of Gomes et al. The normal level of CA 15-3 in human serum is 30 U mL− 1 [29]. As
and Ribeiro et al. [31–33]. That suggests that the proposed discussed in the topic of the calibration curve, the linear range of the
CNE/AuNP/MIP sensor could be an effective alternative in the deter­ sensor CNE/AuNP/MIP was 5–35 U mL− 1 and the LOQ of 3.87 U mL− 1.
mination of CA 15-3 in biological sample. But first, reproducibility and Hence, it is important to dilute the human serum sample at 1:7 (v/v) for
interference studies were evaluated. the determination of CA 15-3 using the proposed sensor. The analysis
was conducted using chronoamperometry (0.45 v for 60 s).
3.7. Specificity and reproducibility studies The serum samples were prepared and spiked with different con­
centrations of CA 15-3 (28, 56 and 84 U mL− 1). The values chosen were
Initially, the specificity of the CNE/AuNP/MIP was evaluated in the below and above the normal level (30 U mL− 1). Those samples were
presence of interfering species such as ascorbic acid (AA), uric acid (UA), diluted at 1:7 in PBS (0.1 mol L− 1 pH 7.5) and incubated in the sensor.
BSA, cholesterol (CLR), dopamine (DPM) and human IgG. Each inter­ The resulting concentration of CA 15-3 after dilution was 4, 8 and 12 U
fering specie was added individually with CA 15-3 (20 U mL− 1) onto the mL− 1.
sensor and incubated. The chronoamperograms (0.45 V for 60 s) were Figure S5(a) presents the current obtained for each MIP sensor and
obtained in presence of the redox probeK4 [Fe(CN)6]. Fig. 7(a) shows the Table 4 exhibited the recovery studies of the proposed sensor in the
chronoamperograms and Fig. 7(b) the current variation before and after diluted human serum samples. As observed, the relative standard devi­
CA 15-3. ation (RSD) was between 0.7 and 3.6%, and the average recovery be­
Some interfering species caused slight increases in current response tween 101.8 and 104.3%. There is no specific regulation concerning the
(BSA: 6% and IgG: 5%), while the addition of cholesterol, dopamine, MIP electrochemical sensors. However, several previous studies
uric acid and ascorbic acid did not lead to significant effects. The current considerate appropriated the range between 80 and 120% for recovery
variation for all the samples was under 6% of the response without in­ values [97,98]. Hence, these results suggest the accuracy of the data
terferences (blank). These results indicate that the CNE/AuNP/MIP can produced by the sensor CNE/AuNP/MIP, indicating their suitability to
be specific for CA 15-3. Therefore, proposed sensor may be suitable for detect CA 15-3 in serum samples.
application in biological samples. Then the electrochemical studies were conducted in saliva samples.
Then the reproducibility of CNE/AuNP/MIP was tested by measuring The normal level of CA 15-3 in saliva samples is not well-stablished yet
the current response of four sensors constructed identically in different in the literature. Although, according to Farahani et al. for healthy

10
A.E.F. Oliveira et al. Talanta 252 (2023) 123819

Fig. 7. Specificity of the sensor CNE/AuNP/MIP incubated with CA 15-3 (20 U mL− 1) and interfering species (ascorbic acid 0.01 mg mL− 1, uric acid 0.05 mg mL− 1,
BSA 1.0 mg mL− 1, cholesterol 0.19 mg mL− 1, dopamine 30 pg mL− 1, and IgG 1.0 mg mL− 1): (a) chronoamperograms and (b) analytical response. Error bar =
standard deviation (n = 3); Reproducibility of sensor CNE/AuNP/MIP incubated with CA 15-3 (20 U mL− 1): (c) chronoamperograms and (d) analytical response.
Error bar = standard deviation (n = 3).

The concentration of biomarkers within the saliva is usually much

Table 4
lower compared to those that can be detected within the human serum.
Recovery studies of the proposed immunosensors in diluted human serum
These low concentrations can limit the applicability of CA 15-3 detec­
samples.
tion [101]. For example, the LOQ of the sensor is 3.87 U mL− 1, value
Sample Added/μM Found/μM RSD % Recovery %
above the normal level (2.71 U mL− 1) according to Laidi et al. [100].
1 4.00 4.32 3.6 108.0 Hence, the use of the sensor CNE/AuNP/MIP may not be appropriated
2 4.00 4.18 104.5 for determination in saliva sample.
3 4.00 4.02 100.5
However, since the normal level is not completely defined, the sensor
Average 4.00 4.17 104.3
will be evaluated in the determination of CA 15-3 in saliva. In order to
4 8.00 8.11 1.3 101.4
evaluate these low concentration of CA 15-3 presented in saliva, the
5 8.00 8.25 103.1
6 8.00 8.05 101.6 samples were not previously diluted. The saliva samples were prepared
Average 8.00 8.13 102.0 and spiked with different concentrations of CA 15-3 (4, 8 and 10 U
7 12.00 12.31 0.7 102.6
mL− 1). Next, the samples were incubated in different sensors. Fig. S5(b)
8 12.00 12.14 101.2 shows the current obtained for each MIP sensor and Table 5 presents the
9 12.00 12.21 101.8 recovery studies of the sensor in the saliva.
Average 12.00 12.22 101.8 As obtained, the average recovery was between 61.7 and 75.8% and
the RSD between 7.0 and 19.8%. That results do not show good recovery
values and accuracy. The inherent viscosity variability in human sam­
woman the average concentration is 4.5 U mL− 1, while for breast cancer
ples are the most common disadvantages of using saliva to detect bio­
patients 9.2 U mL− 1 [99]. Another study was conducted by Laidi et al.
markers [101,102]. Saliva is viscous and can not be used with the
where it was found the concentration of 2.71 U mL− 1 (healthy woman)
proposed sensor with dilution due the low analyte concentrations. That
and 4.77 U mL− 1 (cancer patients) [100].

11
A.E.F. Oliveira et al. Talanta 252 (2023) 123819

Table 5 Data availability

Recovery studies of the proposed immunosensors in human saliva samples.
Sample Added/μM Found/Мm RSD % Recovery % Data will be made available on request.
1 4.00 2.98 19.8 74.5
2 4.00 2.01 50.3 Acknowledgements
3 4.00 2.41 60.3
Average 4.00 2.47 61.7 We thank UFSJ, FAPEMIG, CAPES, CNPQ and Rede Mineira de
4 8.00 5.94 7.0 74.3 Química for the continuous support of our research. We thank professor
5 8.00 6.53 81.6 Thalita Chiaramonte and Filipe Santos Souza for the AFM measure­
6 8.00 5.71 71.4
ments. We also thank professor Lucas Franco Ferreira from Universidade
Average 8.00 6.06 75.8
Federal dos Vales do Jequitinhonha e Mucuri for the help and charac­
7 10.00 5.89 11.7 58.9 terizations. The research reported in this work was supportedby Con­
8 10.00 7.43 74.3
9 10.00 6.55 65.5
selho Nacional de Desenvolvimento Científico e Tecnológico - CNPq -
Average 10.00 6.62 66.2 INCT DATREM [465571/2014-0], Conselho Nacional de Desenvolvi­
mento Científico e Tecnológico - CNPq [406381/2018-6; 303802/2019-
7] and Fundação de Amparo à Pesquisa do Estado de Minas Gerais [CEX
must have affected the incubation of CA 15-3 on the imprinted sites of - APQ-02375-18]. A sincere thank you to Beatriz Boari Bonsolhos for her
the MIP, explaining the low recovery values. In conclusion, the sensor diligent proofreading of this paper.
CNE/AuNP/MIP is not suitable to detect CA 15-3 in saliva samples.
Appendix A. Supplementary data
4. Conclusions
Supplementary data to this article can be found online at https://doi.
The carbon nanotube electrode (CNE) was developed using hand­ org/10.1016/j.talanta.2022.123819.
writing technique, that allowed the use of a simple fabrication process.
The paper-based carbon nanotube sensor emerged as an attractive
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