Journal of Biomaterials Science, Polymer Edition

ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsp20

Synthesis and characterization of albumin

imprinted polymeric hydrogel membranes for
proteomic studies

Esra Feyzioğlu Demir, Emir Özçalışkan, Hayriye Karakaş, Murat Uygun, Deniz
Aktaş Uygun, Sinan Akgöl & Adil Denizli

To cite this article: Esra Feyzioğlu Demir, Emir Özçalışkan, Hayriye Karakaş, Murat Uygun,
Deniz Aktaş Uygun, Sinan Akgöl & Adil Denizli (2018) Synthesis and characterization of albumin
imprinted polymeric hydrogel membranes for proteomic studies, Journal of Biomaterials
Science, Polymer Edition, 29:18, 2218-2236, DOI: 10.1080/09205063.2018.1534423

To link to this article: https://doi.org/10.1080/09205063.2018.1534423

Published online: 05 Nov 2018.

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JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION
2018, VOL. 29, NO. 18, 2218–2236
https://doi.org/10.1080/09205063.2018.1534423

Synthesis and characterization of albumin imprinted

polymeric hydrogel membranes for proteomic studies
Esra Feyziog €

lu Demira, Emir Ozçalışkanb
, Hayriye Karakaşb, Murat Uygunc,d,
c,d
Deniz Aktaş Uygun , Sinan Akgo €l and Adil Denizlie
b

a
Vocational School of Health Services, Department of Medical Laboratory Techniques, Izmir
University of Economics, Izmir, Turkey; bFaculty of Science, Department of Biochemistry, Ege
University, Izmir, Turkey; cFaculty of Science and Arts, Chemistry Division, Adnan Menderes
University, Aydın, Turkey; dAdnan Menderes University, Nanotechnology Application and Research
Center, Aydın, Turkey; eDepartment of Chemistry, Hacettepe University Faculty of Science,
Ankara, Turkey

ABSTRACT ARTICLE HISTORY

In this presented study, a novel molecularly imprinted polymeric Received 14 September 2018
hydrogel membranes (PHMs) were developed to use for the albu- Accepted 5 October 2018
min depletion studies. For this, albumin imprinted poly(2-hydrox-
KEY WORDS
yethyl methacrylate-N-methacryloyl-(L)-phenylalanine methyl
Albumin depletion;
ester) polymeric hydrogel membranes [p(HEMA-MAP) PHMs] were polymeric hydrogel
synthesized by the photopolymerization technique, and then membrane; protein
characterized by SEM, EDX, FT-IR and swelling studies. imprinting
Synthesized PHMs had spherical structure and the MAP monomer
incorporation onto the PHMs was determined by EDX analysis by
using nitrogen stoichiometry. Also, the swelling ratio of the albu-
min imprinted p(HEMA-MAP) PHMs was determined as 215%. The
optimum albumin adsorption condition (adsorption capacity,
medium pH, adsorption rate, temperature, ionic strength) were
studied and the maximum albumin adsorption capacity was
found to be as 34.28 mg/g PHMs. Selectivity experiments were
also carried out with the presence of the competitive proteins
such as lysozyme and amylase, and the results demonstrated that
the albumin imprinted p(HEMA-MAP) PHMs showed high affinity
towards the BSA molecules than the competitive proteins of lyso-
zyme and amylase. Adsorbed albumin was desorbed from the
PHMs by 1.0 M of NaCl, and the reusability of the imprinted PHMs
was also demonstrated for five successive adsorption-desorption
cycles without any significant loss in the albumin adsorption cap-
acity. As an application, sodium-dodecyl sulfate polyacrylamide
gel electrophoresis was used to indicate the albumin depletion
efficiency of albumin imprinted p(HEMA-MAP) PHMs. This pre-
sented study showed that, these imprinted membranes are prom-
ising for proteomic studies and applications, and can be used for
the investigations for human diagnostics.

CONTACT Dr. Sinan Akgo€l sinanakgol@yahoo.co.uk Ege University, Faculty of Science, Department of
Biochemistry, Izmir, Turkey
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2219

1. Introduction
As specific biomarkers, blood proteins have been preferred intensively for the diagno-
sis and the prognosis of various diseases. Unfortunately, the most of these proteins
are in low concentrations around nanogram or picogram levels for per milliliter sam-
ples [1,2]. Lower concentrations of these diagnostic proteins is very important for
early diagnosis, but the abundant proteins in blood (i.e. albumin, IgG) mask these
low level valuable biomarkers such as cytokines, troponins etc. [1,3,4]. Some of these
biomarkers are also important for prognosis. The complete removal or depletion of
these abundant proteins is very important, due to the low levels of the biomarker
proteins [1,5]. Albumin is the most abundant protein in blood (around 55% of total
blood proteins) [3,6]. It is acidic, very soluble and extremely robust protein: stable in
the wide pH range of 4.0–9.0, and stable for high temperatures till 60
C [7]. Due to
the high amount of the abounded protein in human blood, proteome analysis become
particularly difficult in serum, plasma cerebrospinal fluid, or synovial fluid samples
[8]. For this reason, these abundant proteins such as albumin and IgG must be
depleted from serum samples immediately before the analysis [1,4]. Several depletion
methods for removal of these high abundant proteins from body fluids have been
developed and various strategies have also been used [1,8]. Along these methods, dye
affinity methods (in which Cibacron Blue and Reactive Green are used as a dye-lig-
and) have been found a new and efficient strategy for albumin depletion [8,9]. In
recent years, molecular imprinting technology have been attracted great attention for
albumin depletion, due to its specificity and selectivity [6,10–12].
Molecular imprinting technology is one of the highly selective and productive tech-
niques used especially for the separation and recognition fields. Molecular imprinted
polymers have their own specific recognition sites for target proteins and can be pre-
pared easily in a short time by using a relatively cheaper way. These imprinted poly-
mers are highly stable and ready to use for molecular recognition [13–15]. Molecular
imprinting technique wisely consist by the polymerization of functional monomers
and a suitable cross-linker around the template molecule in order to create recogni-
tion sites and functional group orientation on the polymeric matrix [16]. Molecular
imprinted polymers (MIPs) can choose the template molecule based on the shape,
size, and functionality even through the other compound with similar structure in the
aqueous solution [17]. In recent years, molecular imprinting technique have been
attracted great attention due to the potential applications in many biotechnological
and medical fields such as biosensor [18–21], chromatographic separation, catalysis,
artificial antibody mimics [11, 17]. Various compounds have been also used as a tem-
plate molecule such as small metal ions [13,22], large proteins [11,23] and even bac-
terial cells [24,25].
Albumin is the most abundant protein in blood samples, and various depletion
techniques and support materials have been intensively used to deplete albumin. Bead
shaped spherical particles, monolithic polymers, thin films and membranes, and
nanoparticles with different functionalities have been applied for this purpose.
Albumin can deplete by using various techniques such as hydrophobic, hydrophilic,
metal chelate or dye affinity. Molecular imprinting is an innovative and efficient tech-
nique to create an artificial recognition region for target molecules. By this method,
2220

E. FEYZIOGLU DEMIR ET AL.

albumin can deplete efficiently and specifically by using the albumin imprinted
polymeric materials.
In this presented study, polymeric hydrogel membranes were chosen as a support
material for the albumin imprinting process. These membranes can be further used
for the preparation of the depletion cartridges for albumin from various biological
samples. For this purposes, albumin imprinted p(HEMA-MAP) PHMs were prepared
by UV-photopolymerization technique. These albumin imprinted PHMs were charac-
terized by using SEM, EDX, FT-IR and swelling studies. Albumin adsorption proper-
ties of the PHMs were also investigated under different adsorption condition such as
different initial albumin concentrations, pHs, adsorption times, temperatures and
ionic strengths. Selectivity of albumin toward to lysozyme and amylase was studied
and repeated usage of the albumin imprinted p(HEMA-MAP) PHMs was also
investigated.

2. Experimental
2.1. Materials
Bovine serum albumin (BSA), lysozyme, amylase, hydroxyethyl methacrylate (HEMA,
co-monomer), ethylene glycol dimethacrylate (EGDMA, crosslinker), 2,2-dimethoxy-2-
phenyl-acetophenone (DMPA, initiator), phenylalanine methyl ester and methacryloyl
chloride were supplied by Sigma Chemical Co (St. Louis, USA). All other chemicals were
of analytical grade.

2.2. Synthesis of N-methacryloyl-(L)-phenylalanine methyl ester

N-methacryloyl-(L)-phenylalanine methyl ester (MAP) was prepared according to
the previous procedures [26,27]. Briefly, 5.0 g of phenylalanine methyl ester and
0.2 g of sodium nitrite (NaNO2) were dissolved in potassium carbonate solution
(K2CO3, 5% (w/v)). Then, the mixture was transferred in a round-bottomed three-
necked flask with a dropping funnel. The reaction reservoir was cooled to 0
C by
using an ice-water bath and magnetically stirred under the nitrogen atmosphere.
Methacryloyl chloride (6.0 mL) was added drop wise into this reaction mixture.
Then, the reaction reservoir was transferred to room temperature for 2 h with
continuous stirring. Thereafter, pH of the mixture was adjusted to 3.0 and the
mixture was extracted by using chloroform. After the phase separation, the
organic phase was dried with magnesium sulphate, and the chloroform phase was
evaporated by using a rotary evaporator. The product (MAP) was washed with
NaOH and crystallized in ether–cyclohexane mixture. MAP was dissolved and
stored in ethanol.

2.3. Preparation of albumin imprinted p(HEMA-MAP) PHMs

The p(HEMA-MAP) polymeric hydrogel membranes (PHMs) were synthesized by
free radical UV-photo polymerization technique [28]. In order to prepare the albu-
min-MAP complex, 4.0 mg/mL of albumin solution (in 0.1 M pH 7.4 phosphate
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2221

buffer containing 40% ethanol) was mixed with MAP monomer solution [volume
ratio of albumin-MAP complex was 8:1 (v/v)]. This complex was further mixed for
3 h, in order to form a stable complex between albumin and MAP monomer. The
polymerization mixture (1 mL) of p(HEMA-MAP) PHMs was prepared by dissolving
225 mL of HEMA and 225 mL of albumin-MAP complex, with DMPA (as an initiator,
1 wt %) and EGDMA (as a crosslinker agent, 1.0 wt %). The mixture was incubated
for 5 min in a sonic bath and degassed by purging with nitrogen for about 5 min.
The mixture was then poured into cylindrical glass molds (10 mm of diameter) and
exposed to 365 nm of UV light with 1 mW/cm2 intensity for 10 min. After the poly-
merization, PHMs were cut into circular pieces (diameter: 0.35 cm) by using a perfor-
ator and then washed several times with distilled water, in order to remove the
reacted monomers, initiators, and other residuals. After the rinsing step, template
albumin molecules were removed from the synthesized molecular imprinted PHMs
by using 1.0 M of NaCl by orbital stirring for 24 h [6,29]. Then, PHMs were further
rinsed with distilled water. Non-imprinted PHMs were also prepared in the same
above mentioned method by using only HEMA.

2.4. Characterization studies

Albumin imprinted p(HEMA-MAP) PHMs systems were characterized by SEM, EDX
FT-IR, and swelling tests. The morphology and the overall structure of the PHMs
were investigated by a Scanning Electron Microscope (SEM) (Quanta 250 S FEG) after
sputtering with a homogeneous thin film of gold film. Energy Dispersive X-ray
(EDX) analysis was carried out simultaneously with SEM, in order to determine the
incorporated amount of MAP monomer in the PHMs backbone. The characteristic
functional groups of albumin imprinted p(HEMA-MAP) PHMs were analyzed by
Attenuated Total Reflection-Fourier Transform Infrared Spectrometer (ATR-FTIR,
Perkin Elmer). Swelling ratios of albumin imprinted p(HEMA-MAP) PHMs were also
determined. Briefly, dry PHMs were weighed first and then immersed in distilled
water at 25 ± 0.1
C. After a certain time intervals (t), PHMs were weighed by remov-
ing the excess surface solution, and then placed in the same solution, again. The per-
centage of swelling (S%) of the PHMs in distilled water was calculated from the
following equation Equation (1);

Mt M
S% ¼  100 (1)
M0

here Mt is the mass of the swollen PHMs at certain time (t) and Mo is the mass of
the dry PHMs at time of 0 [30].

2.5. Adsorption studies of albumin imprinted p(HEMA-MAP) PHMs

Albumin adsorption studies on the albumin imprinted p(HEMA-MAP) PHMs sys-
tems were carried out in a batch systems. The effects of time, pH of the medium, the
initial albumin concentration, temperature, and ionic strength on albumin adsorption
were also investigated. In order to investigate the effect of time, the adsorption
2222

E. FEYZIOGLU DEMIR ET AL.

experiments were performed with the time range of 5 and 180 min. Initial concen-
tration of albumin was changed between 0.1 mg/mL and 2.0 mg/mL, to demonstrate
the effect of initial albumin concentration on the adsorption efficiency. The pH of
solution was also changed between 5.0 and 8.0 and the experiments were also
studied in the temperature range of 4
C and 45
C, in order to show the tempera-
ture effect. The ionic strengths of the adsorption medium were also changed
between 0 and 0.5 M by using the NaCl solutions. All the adsorption experiments
were carried out by using 3 mL of albumin solution under the orbital stirring at
250 rpm. At the end of the adsorption equilibrium, the PHMs were removed from
the solutions and the adsorbed amount of albumin was calculated by measuring the
initial and final albumin concentrations in the adsorption medium by the method
of Bradford [31,32]. The Q values were calculated by following equation (Equation
(2)). It also should be noted here that, all adsorption curves are averages of at least
triplicated experiments.


Ci Cf  V
Q¼ (2)
weight of membrane ðg Þ

here, Q is the albumin adsorption capacity of PHMs systems (mg/g), Ci is the ini-
tial concentrations of albumin in the solution (mg/mL), Cf is the final concentra-
tion of albumin in the solution (mg/mL), V is the volume of the aqueous
phase (mL).

2.6. Selectivity of BSA imprinted p(HEMA-MAP) PHMs

The selectivity of BSA imprinted p(HEMA-MAP) PHMs was investigated towards the
competitive proteins of lysozyme (Mw ¼ 14.4 kDa, pI ¼ 11.0) and amylase (Mw ¼ 55.4
kD, pI ¼ 6.4). Adsorption experiments of the competitive molecules were carried out
by the same method for the albumin adsorption and concentrations were adjusted to
1 mg/mL. Absorbed albumin, lysozyme and amylase amounts were determined by
using the Bradford method.
Distribution coefficient (Kd) for lysozyme and amylase towards to albumin were
calculated by using the following equation (Equation (3)):
 
Kd ¼ ðCI_  Cf Þ=Cf x V=m (3)

here, Kd (mL/g) is a distribution coefficient for the competing proteins. Ci and Cf are
the initial and the final concentration of proteins (mg/mL), V is the volume of solu-
tion (mL), m is the weight of membrane (g).
The following equation was adopted to determine a selectivity coefficient (k) for
the adsorption of albumin onto the PHMs in the presence of a competing species of
lysozyme and amylase (Equation (4)):

k ¼ Kðtemplate proteinÞ =Kðcompeting proteinÞ (4)

 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2223

Figure 1. Schematically presentation of albumin imprinted p(HEMA-MAP) PHMs systems.

Relative selectivity coefficient was defined by the following equation

(Equation (5)):
k0 ¼ kimprinted =kcontrol (5)

here, kimprinted is albumin imprinted p(HEMA-MAP) PHMs in the presence of the

albumin-other proteins pairs, kcontrol is albumin non-imprinted PHMs in the presence
of the BSA-other proteins pairs [13].
2224

E. FEYZIOGLU DEMIR ET AL.

Figure 2. The SEM images of albumin imprinted p(HEMA-MAP) PHMs with different magnifications
(in scale of 500 mm, 100 mm, 40 mm and 10 mm).

2.7. Desorption and reusability of BSA imprinted p(HEMA-MAP) PHMs

Albumin desorption from the albumin imprinted p(HEMA-MAP) PHMs was carried
out by using 1.0 M of NaCl [6]. For this, albumin imprinted p(HEMA-MAP) PHMs
were placed in the desorption medium and stirred vigorously for 2 h at room tem-
perature. The amount of albumin in the desorption medium was determined spectro-
photometrically using the Bradford methods. Desorption ratio was calculated by
using the equation below. (Equation (6)):

Desorption Ratioð%Þ ¼ ½ðamount of desorbed albumin=amount of adsorbed albuminÞx100 (6)

Albumin adsorption-desorption cycle was repeated five times by using the same
PHMs systems, in order to determine the reusability of the PHMs systems, and the
experiments were carried out under the same conditions as described above. After
each adsorption-desorption cycle, the PHMs systems were washed with distilled water
and equilibrated with appropriate buffer solution.

2.8. SDS-PAGE imaging

The desorbed albumin from albumin imprinted p(HEMA-MAP) PHMs was analyzed
by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2225

Figure 3. EDX analysis of albumin imprinted p(HEMA-MAP) PHMs.

SDS-PAGE was performed according to Laemmli [33] using a Mini-Protean 3 electro-

phoresis cell (Bio-Rad Laboratories, Richmond, CA, USA), with the gel combination
of 12% separating gel in 1.5 M Tris–HCl (pH 8.8) and 4% acrylamide stacking gel in
1.0 M Tris–HCl (pH 6.8). Before loading, the initial, final and desorbed albumin sam-
ples were mixed with sample buffer (0.5% (w/v) bromophenol blue, 10% (w/v) SDS,
glycerol in the 0.5 M Tris-HCl, pH 6.8) separately. Tris–glycine buffer (pH 8.3) con-
taining 0.1% SDS was used as a running buffer, and running current was adjusted to
100 V 15 min for 4% stacking gel and 150 V 45 min for 12% separating gel. Then,
Coomassie Blue G-250 staining protocol was applied for the visualization of the pro-
tein bands.

3. Result and discussion

3.1. Characterization studies
In this presented study, a new generation albumin imprinted p(HEMA-MAP) PHMs
were prepared by using free radical photopolymerization technique. MAP, was pre-
ferred as a co-monomer, due to its hydrophobic phenylalanine groups to improve the
selectivity of the membrane towards albumin molecules. By incorporating the MAP
monomer onto PHMs, the membrane gained a hydrophobic character. Schematically
presentation for the preparation of albumin imprinted p(HEMA-MAP) PHMs sys-
tems is shown in Figure 1. These PHMs were characterized by using SEM, EDX, FT-
IR and swelling test.
As seen in Figure 2, albumin imprinted p(HEMA-MAP) PHMs had spherical pat-
terns within the whole structure, and all PHMs were adhered to each other. By this
way, the surface area of the PHMs was increased by presenting much more area to
functionalize [28].
The incorporated amount of MAP monomer into the PHMs was determined by
EDX analysis by using nitrogen stoichiometry and EDX spectrum of albumin
imprinted p(HEMA-MAP) is shown in Figure 3. As clearly seen here, synthesized
PHMs consisted of C, O and N atoms, and N only came from the MAP monomer by
2226

E. FEYZIOGLU DEMIR ET AL.

88 A
80

72

B
64
%Transmittance

56

48

40

32

24

16
A) MIP-p(HEMA-MAP) PHMs
8
B) NIP- p(HEMA) PHMs

4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600
Wavenumber (cm-1)

Figure 4. ATR-FTIR spectra of PHMs A) albumin imprinted p(HEMA-MAP) PHMs, B) non-imprinted

p(HEMA) PHMs.

incorporating onto polymeric backbone. Incorporated amount of the MAP monomer

was calculated as 0.707 mmol/g polymer by using the N stoichiometry.
The FTIR spectra of albumin imprinted p(HEMA-MAP) PHMs (4A) and non-
imprinted p(HEMA) PHMs (4B) are given in Figure 4. The FTIR spectrum of both
p(HEMA) and albumin imprinted p(HEMA-MAP) PHMs have the characteristic
stretching vibration band of hydrogen bonded alcohol, O–H, around 3385 cm1,
3391 cm1 respectively. The asymmetric and symmetric C-H stretching vibrations
bands are observed at 2952 cm1 and 2947 cm1 respectively. The bands at about
1706 cm1 and 1717 cm1 was assigned to carbonyl group of stretching vibration
bands, respectively. FTIR spectrum of albumin imprinted p(HEMA–MAP) PHMs has
also the characteristic amide I and amide II adsorption bands at 1480 cm1 and
1451 cm1, respectively; and C-H bending peaks around at 900 cm1 indicates the
incorporation of MAP monomer into the polymer structure [10,34,35].
The swelling behavior and swelling ratios of synthesized albumin imprinted
p(HEMA-MAP) PHMs and non-imprinted p(HEMA) PHMs were also investigated.
The swelling ratios of albumin imprinted p(HEMA-MAP) and non-imprinted
p(HEMA) PHMs were demonstrated in Figure 5. As seen in this figure, PHMs exhib-
ited rapid swelling character, and S% values of p(HEMA) PHMs was calculated about
236%. After the incorporation of MAP monomer into PHMs structure, the swelling
ratio of the PHMs decreased a little, and the S% values of albumin imprinted
p(HEMA-MAP) PHMs was also calculated as 215%. Due to the hydrophobic charac-
ter of MAP monomer, albumin imprinted p(HEMA-MAP) PHMs demonstrated
lower swelling ratios than that of p(HEMA) PHMs. It also should be mentioned here
that, PHMs demonstrated rapid swelling behaviors and whole PHMs reached the
equilibrium swelling degrees within 10 min.
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2227

Figure 5. Swelling isotherms of albumin imprinted p(HEMA-MAP) and non-imprinted p(PHEMA)

PHMs (T: 25
C).

3.2. Adsorption studies of albumin imprinted p(HEMA-MAP) PHMs

In this presented work, albumin imprinted p(HEMA-MAP) PHMs were preferred to
deplete the albumin from highly viscous and complex blood samples. Although there
were numerous studies, which used imprinting technology for albumin recognition
onto various support materials with different shapes and functionalities, imprinting
onto a membrane form is very rare. Also imprinting technology is not only preferred
as a depletion platform, but also used for detection of albumin and the other prepara-
tive purposes. Spherical bead shaped polymers have been used for the imprinting of
albumin. Pang et al. synthesized polyacrylamide gel beads, which had functional elec-
trostatic groups, and used for the molecular imprinting of albumin [36]. The
researchers prepared about 160 mm gel beads with porous structure. The synthesized
albumin imprinted gel beads demonstrated good ability to the albumin adsorption
with the separation factor of 4.71. Thin film polymers have been synthesized and
applied for the imprinting of albumin, but mainly used for the detection of albumin
from various samples. For example, a semi-covalent imprinting technology was
applied to albumin by Cieplak et al. [37]. Researcher used bis(2,20 -bithien-5-yl)me-
thane units for the construction of MIP backbone. These molecularly imprinted poly-
mers were prepared as a thin film form on an Au electrode to fabricate the
electrochemical chemosensor for albumin. These film shaped imprinted electrodes
showed good detection properties for albumin molecules with high affinity. A similar
study, which used albumin imprinted polyacrylamide hydrogel membrane grafted
non-woven polypropylene was carried out by Zhao et al. [38]. The synthesized
imprinted membranes showed good affinity to albumin molecules and demonstrated
improved selectivity towards competing proteins with the selectivity factor above 2.0.
A surface imprinted superparamagnetic polymer was prepared for albumin adsorption
by Gai et al. and its selectivity demonstrated by competitive adsorption tests [6].
These albumin imprinted magnetic polymers were successfully used for the adsorp-
tion of albumin from real samples. In this presented study, hydrogel membranes were
selected as the main polymeric structure imprinting for albumin. Using the MAP as a
functional monomer was also improved the possible specificity, which is very
2228

E. FEYZIOGLU DEMIR ET AL.

Figure 6. A) The effect of the time on albumin adsorption onto the albumin imprinted p(HEMA-
MAP) PHMs (CBSA initial: 0.5 mg/ml; pH 7.4 containing 40% ethanol; Temperature: 25
C). B) The
effect of the initial albumin concentration on albumin adsorption (0.1 M phosphate buffer contain-
ing 40% ethanol, pH 7.4; Time: 120 min; Temperature: 25
C).

important for recognition and depletion of albumin from blood samples, efficiently.
Synthesized albumin imprinted PHMs were applied for the adsorption of albumin
and the optimal conditions for this process were also optimized.

3.2.1. Effect of time on adsorption

Figure 6(A) shows the time dependent albumin adsorption on the albumin imprinted
p(HEMA-MAP) PHMs. As seen here, albumin adsorption capacity was very fast at
the beginning (until 45 min), then increased gradually with time and reached a satur-
ation value within 120 min. This fast adsorption behavior at initial part of the adsorp-
tion time is probably due to the high geometric complementary and favorable
functional group orientation between albumin imprinted PHMs and the albu-
min molecules.

3.2.2. Effect of the initial albumin concentration on adsorption

The effect of initial albumin concentration on adsorption is given in Figure 6(B). As
seen here, albumin adsorption capacity of the PHMs increased with the increasing
albumin concentration up to 2 mg/mL. After the 1 mg/mL of albumin concentration,
albumin adsorption capacity of the PHMs reached a saturation value and maximum
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2229

Figure 7. (A) The effect of the pH on BSA adsorption (CBSA initial: 1mg/mL containing 40% ethanol;
Time: 120 min; Temperature: 25
C). (B). The effect of the temperature on BSA adsorption (CBSA
initial: 1mg/mL: 0.1 M phosphate buffer containing 40% ethanol, pH 7.4; Time: 120 min).

albumin adsorption capacity was found to be as 34.28 mg/g. The plain non-imprinted
p(HEMA) PHMs showed 4 times lower albumin adsorption capacity (about 9.16 mg/
g). These results demonstrated that, albumin adsorption process onto the albumin
imprinted p(HEMA-MAP) PHMs was considerably specific. Various researchers have
been used different support system for the adsorption of albumin by using diverse
techniques. For example, Gai et. al. [6] prepared surface-imprinted superparamagnetic
polymers for albumin separation, and the albumin adsorption capacity was deter-
mined as 80 mg/g. In another study, molecularly imprinted polymer beads were also
synthesized by using acrylamide and methacrylic acid (MAA) by Zhang et al., [39]
and the albumin adsorption capacity of MIP beads was found to be as 360 mg/g.
Wang et al., [14] were also prepared albumin imprinted polymer by using m-amino-
phenylboronic acid as a functional monomer, and the albumin adsorption capacity
was determined as 12 mg/g. Human serum albumin imprinted (pHEMAPA-HSA)
cryogels were also synthesized by Andaç et al. [10] for albumin depletion from
human serum and maximum adsorption capacity was found to be as 20 mg HSA/g.

3.2.3. Effect of pH on adsorption

Albumin adsorption studies were carried out using different buffer solution such as
citrate buffer (pH:5.0) and phosphate buffers (pH range of 6.4–8.0) to investigate the
2230

E. FEYZIOGLU DEMIR ET AL.

Figure 8. (A) The effect of the ionic strength on BSA adsorption (CBSA initial: 1 mg/mL; 0.1 M
phosphate buffer containing 40% ethanol, pH 7.4; Time: 120 min; Temperature: 25
C).
(B) Adsorption–desorption cycle of BSA imprinted p(HEMA-MAP) PHMs (CBSA initial: 1 mg/mL;
0.1 M phosphate buffer containing 40% ethanol, pH 7.4; Time: 120 min; Desorption time: 120 min).

effect of pH on the albumin adsorption capacity. The pH profile of albumin

imprinted p(HEMA-MAP) PHMs was demonstrated in Figure 7(A). As seen here, the
maximum albumin adsorption was found as 41.05 mg/g PHMs at pH 7.4. The max-
imum adsorption capacity decreased significantly in excessive acidic and alkaline
pH regions.
It is very important to choose a appropriate polymerization solvent for the prepar-
ation of molecularly imprinted polymer to recognize the template molecule. In this
presented study, polymerization and the pre-complexation process were carried out at
pH 7.4. Thus, three dimensional structure of albumin improved the interactions
between the albumin and PHMs efficiently, because of the memory effect [10].

3.2.4. Effect of the temperature on adsorption

In order to investigate the effect of temperature, the albumin adsorption studies were
performed a range of temperature from 4 to 45
C. As given in Figure 7(B), the
adsorbed amount of albumin onto albumin imprinted p(HEMA-MAP) PHMs
increased from 12.32 to 44.95 mg/g with increasing temperature from 4 to 45
C.
These results indicate that, the main adsorption capacity of albumin on the albumin
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2231

Table 1. Langmuir and Freundlich adsorption isotherm constants.

Langmuir constants Freundlich constants
Experimental Shape of isotherm
Qex (mg/g) Qmax(mg/g) b(mL/mg) R2 Kf n R2 RL
34.28 68.97 0.694 0.9849 26.47 1.616 0.9017 0 < RL<1; favorable

imprinted p(HEMA-MAP) PHMs involve hydrophobic interactions with a higher per-

centage according to the other interactions, i.e. electrostatic interactions [40].

3.2.5. Effect of the ionic strength on adsorption

The effect of the ionic strength on albumin adsorption is given in Figure 8(A). As
shown in here, albumin adsorption capacity of albumin imprinted p(HEMA-MAP)
PHMs was decreased from 34.28 to 7.03 mg/g, as the NaCl concentration was
increased from 0.0 to 1.0 M. This decrease occurred with the increasing ionic strength
should be explained by; (i) the counter salt ions might interact with the albumin
molecules by the charge–charge interactions and mask the adsorption regions, and
(ii) the repulsive electrostatic forces between the albumin imprinted PHMs and
albumin molecules.

3.2.6. Adsorption isotherms

The Langmuir and Freundlich isotherms were used [41,42] for the modeling of equi-
librium data. The linear equation of Langmuir (Equation (7)) isotherm is represented
as follows:

Ce =Q ¼ ð1=Qmax bÞ þ ðCe =Qmax Þ (7)

where b is the Langmuir isotherm constant, Ce is the equilibrium albumin concentra-

tion, Q is the adsorption capacity, Qmax is the maximum adsorption capacity. The
other linear equations of Freundlich (Equation (8)) isotherms is also represented as
follows:

lnQ ¼ 1=nðlnCe Þ þ lnKf (8)

where Kf is the Freundlich constant, n is the Freundlich exponent and Ce is the equi-
librium albumin concentration. Langmuir isotherm can be expressed constitutively in
terms of a dimensionless constant separation factor or equilibrium parameter, RL that
is used to predict if an adsorption system is “favorable” or “unfavorable”. The separ-
ation factor, RL is defined by (Equation (9)):

RL ¼ 1=ð1 þ bC0 Þ (9)

where C0 is the initial albumin concentration (mg/L) and b is the Langmuir adsorp-
tion equilibrium constant (L/g). The value of RL indicates the shape of the isotherms
to be unfavorable (RL>1), linear (RL¼1), favorable (0 < RL<1), or irreversible (RL¼0)
[43]. And RL values at all initial concentrations of BSA were obtained in the range of
0-1, thus it was indicated the adsorption was favorable.
2232

E. FEYZIOGLU DEMIR ET AL.

Table 2. Kd, k and k’ values of lysozyme and amylase with respect to albumin.
Non-imprinted p(HEMA) PHMs Imprinted p(HEMA-MAP) PHMs
Protein Kd (mL/cm2) k Kd (mL/cm2 ) k k’
BSA 5.107 – 49.964 –
Lysozyme 5.363 0.952 37.330 1.338 1.406
Amylase 38.968 0.131 44.784 1.116 8.512

The Freundlich isotherm has been generally used to characterize the heterogeneous
adsorption systems and also reversible adsorption process, whereas Langmuir iso-
therm is usually provided information about the surface homogeneity [44–48].
A comparison of the experimental adsorption capacity and the theoretical values
are given in Table 1. According to the correlation values, the adsorption was compat-
ible with Langmuir model. Langmuir model is valid for monolayer sorption on to a
surface affinity with fixed number of identical sites. The ratio of Qmax gives the theor-
etical monolayer saturation of albumin molecules on the PHMs. The values of Qmax
is the maximum biosorption capacity when all sites are covered with albumin mole-
cules [28,45].

3.3. Selectivity of albumin imprinted p(HEMA-MAP) PHMs

The selectivity of template molecule is an important parameter for MIP systems and
the molecular recognition depends on specific interaction between template molecule
and monomers. For this reason, lysozyme (Mw¼ 14400 Da, pI 11.0) and amylase
(Mw '¼ 55400 Da, pI ¼ 6.4) were used as competitive proteins. Kd, k and k values of
lysozyme and amylase were given in Table 2. The relative selectivity coefficient (k) is
the indicator for the adsorption affinity of imprinted sites [49]. The relative selectivity
coefficients of albumin imprinted p(HEMA-MAP) PHMs for albumin/lysozyme and
albumin/amylase were 1.41 and 8.51 times greater than that of NIP p(HEMA) PHMs,
respectively. Molecularly imprinted polymers can recognize its template molecules
efficiently, due to the specific adsorption interactions, adsorption orientations, shape
and size compatibility between the target molecule and functional groups of the sup-
port material, which are act as an adsorption region. Additionally, strength of this
recognition interaction determines the selectivity and the specificity. As mentioned
here, albumin imprinted PHMs demonstrated improved selectivity and specificity
towards the albumin molecules in comparison with the competitor molecules of lyso-
zyme and amylase.

3.4. Desorption and repeated use

After the adsorption experiments, albumin imprinted p(HEMA-MAP) PHMs were
treated with 1 M of NaCl solutions for desorption of adsorbed albumin, and the
desorption ratio was found to be as around 88.71%. Adsorption-desorption cycle was
repeated five times using the same albumin imprinted p(HEMA-MAP) PHMs so as
to show the reusability of albumin imprinted p(HEMA-MAP) PHMs.
Adsorption–desorption cycle of albumin imprinted p(HEMA-MAP) PHMs was given
in Figure 8(B). As seen here, at the end of the fifth cycle, adsorption capacity of
 JOURNAL OF BIOMATERIALS SCIENCE, POLYMER EDITION 2233

Figure 9. SDS-PAGE imaging of desorbed BSA (Line 1: maker; Line 2: initial BSA solution; Line 3:
desorbed BSA solution and Line 4: denatured BSA solution).

albumin imprinted p(HEMA-MAP) PHMs decreased only about 21%. It can be con-
cluded from these results that, albumin imprinted p(HEMA-MAP) PHMs can be
used repeatedly without any significant loss in the adsorption capacity.

3.5. SDS-PAGE imaging

SDS-PAGE was used to show the efficiency of albumin imprinted p(HEMA-MAP)
PHMs for albumin depletion. SDS-PAGE analysis of albumin samples before and
after treatment with albumin imprinted p(HEMA-MAP) PHMs were given in Figure
9. As seen in this figure, Line 1 is the molecular weight marker (kDa). Line 2
contains the initial albumin solution before treatment with albumin imprinted
p(HEMA-MAP) PHMs. Line 3 involves desorbed albumin from albumin imprinted
p(HEMA-MAP) PHMs. Line 4 also contains denatured albumin solution with heat-
ing. As clearly concluded from this figure that, albumin imprinted p(HEMA-MAP)
PHMs can be used for the albumin depletion studies efficiently. As also seen here,
desorbed albumin preserved its original structure and thus, can be used for the fur-
ther preparative applications.

4. Conclusions
In this presented study, a new generation molecular imprinted polymeric hydrogel
membranes were synthesized via UV-photo polymerization technique, and used for
the adsorption of albumin. These PHMs had original particulate morphology with
spherical structures. FTIR and EDX results showed that, MAP monomers successfully
2234

E. FEYZIOGLU DEMIR ET AL.

incorporated into the PHMs backbone. The maximum albumin depletion capacity of
the PHMs was found to be as 34.28 mg/g. According to the selectivity studies, albu-
min imprinted p(HEMA-MAP) PHMs demonstrated high specificity to albumin than
the competitive proteins of lysozyme and amylase. This selectivity of PHMs resulted
from the unique molecular imprinted technology, which creating the special albumin
recognition sites inside the polymeric structure. Moreover, albumin imprinted
p(HEMA-MAP) PHMs could be used several time without any significant loss in its
albumin adsorption capacity.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This research was financially supported by The Scientific and Technological Research Council
of Turkey, Undergraduate Domestic Research Project Support Program (2209-A).

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