Applied Surface Science 256 (2010) 3000–3005
Contents lists available at ScienceDirect
Applied Surface Science
journal homepage: www.elsevier.com/locate/apsusc
Protein imprinted polymer using acryloyl-b-cyclodextrin and acrylamide as
monomers
Wei Zhang a, Lei Qin a, Run-Run Chen a, Xi-Wen He a, Wen-You Li a,*, Yu-Kui Zhang a,b
a
b
Department of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 21 June 2009
Received in revised form 22 October 2009
Accepted 22 November 2009
Available online 3 December 2009
A novel protein imprinted polymer was prepared using acryloyl-b-cyclodextrin (b-CD) and acrylamide
as monomers on the surface of silica gel. The bovine hemoglobin was used as template and b-CD was
allowed to self-assemble with the template protein through hydrogen bonding and hydrophobic
interaction. Polymerization was carried out in the presence of acrylamide as an assistant monomer,
which resulted in a novel protein imprinted polymer. After removing the template, imprinted cavities
with the shape and spatial distribution of functional groups were formed. Bovine serum albumin (BSA)
cytochrome c (Cyt) and lysozyme (Lyz) were employed as non-template proteins to test the imprinting
effect and the specific binding of bovine hemoglobin to the polymer. The results of the adsorption
experiments indicated that such protein imprinted polymer, which was synthesized with b-CD and
acrylamide as monomers, could selectively recognize the template protein.
ß 2009 Elsevier B.V. All rights reserved.
Keywords:
Surface molecular imprinting
b-Cyclodextrin
Bovine hemoglobin
Recognition
1. Introduction
Molecularly imprinted polymer (MIP) was highly cross-linked
polymer prepared by copolymerizing functional monomers and
crosslinker in the presence of template molecule. After the
template molecule was removed from the polymer, the complementary binding sites with specific recognition ability were
created. The remarkable advantages of MIP compared to biosystem like antibodies are their reusability and low cost. To date,
MIP has been widely used in the fields of chromatographic
separation [1,2], as antibody mimetics and artificial receptor [3,4],
and catalysis [5].
The majority of the MIP has been prepared using small
molecules as template, however, imprinting against proteins
was still a challenge [6–13], which is primarily due to the
complexity of the protein structure and the variety of their
sequence. Hjerten’s group prepared polyacrylamide MIP with
protein as template and the results of the experiment showed that
the MIP could selectively recognize template protein [14,15].
Shiomi et al. [16] prepared protein imprinting polymer using
covalently immobilizing template protein on the surface of silica.
The results suggested that the imprinted cavities were successfully
created on the silica. Highly selective recognition of protein could
be achieved through a three-dimensional distribution of functional
* Corresponding author. Tel.: +86 22 2349 4962; fax: +86 22 2350 2458.
E-mail address: [email protected] (W.-Y. Li).
0169-4332/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsusc.2009.11.064
groups [17–19]. b-CD is an interesting molecule because it forms
inclusion compounds with hydrophobic guest through hydrophobic interaction and hydrogen bonding [20]. Hjerten’s group studied
different interactions between b-CD and the proteins and the
investigation showed that charged b-CD could affect the (capillary) electrophoretic separation of peptides and proteins [21]. The
b-CD as monomer was successfully employed to small molecules
[22–24]. Recently, Liu and coauthors [25] using b-CD as functional
monomer successfully prepared bilirubin imprinted polymer and
the MIP possessed high affinity and selectivity. Komiyama and
coauthors reported that two kinds of modified b-CD monomers
were applied to the imprinting toward amino acid derivatives and
oligopeptides, and the MIP could selectively recognize the
template molecules from mixture [26,27]. In our previous work
[28], a novel molecularly imprinted polymer selective for
tryptophan was fabricated with bonded b-CD and acrylamide
(AA), and the results indicated that MIP using bonded b-CD and AA
has higher imprinting effect to template molecule.
Synthetic protein imprinted polymer using acryloyl-b-CD and
AA as monomers was prepared in the present work. Introduction of
a large number of weak complementary interactions facilitated the
protein recognition [24,29]. So the acryloyl-b-CD and AA cooperated together were considered for the improvement of the
protein imprinted polymer performance. As shown in Fig. 1, the
large number of possible bonds can be created between the protein
and the functional monomers, and the spatial arrangement of the
complementary functional entities of the network, together with
the shape image correspond to the imprinted molecules.
W. Zhang et al. / Applied Surface Science 256 (2010) 3000–3005
3001
Fig. 1. The schematic representation for synthesis of the protein imprinted polymer.
Surface imprinting based on oriented immobilization of the
template on silica gel, which facilitates the mass transfer of
template protein, was employed in the present work. The bovine
hemoglobin (BHb) was used as the template protein and was
covalently immobilized on the surface of silica gel, and the MIP was
formed on the surface of silica gel. And after the template protein
was removed, complementary binding sites were thus created on
the surface of silica gel. A series of adsorption studies were
conducted and the results demonstrated that the MIP was capable
of selective recognition of the template protein.
2. Materials and methods
2.1. Apparatus
UV-2450 UV–vis spectrophotometer was from Shimadzu
(Kyoto, Japan). The surface morphology of the particles was
studied using a Quanta 200 scanning electron microscope (FEI,
Eindhoven, The Netherlands). Vario EL elemental analyzer
(Elementar, Hanau, Germany) was employed to investigate the
surface elemental composition of the particles. Fourier transform
infrared (FT-IR) spectra (4000–400 cm1) in KBr were recorded on
the AVATAR 360 FT-IR spectrophotometer (Nicolet, Waltham, MA,
USA).
2.2. Materials and reagents
Silica gel of ultra pure (40–60 mm, 15 nm, Acros Organics, Geel,
Belgium) was activated with acid before being silanized. Bovine
serum albumin (BSA, molecular weight (MW) 67 kDa, isoelectric
point (pI) 4.9), bovine hemoglobin (BHb, MW 66 kDa, pI 6.7),
cytochrome c (Cyt, MW 12.4 kDa, pI 10.2) and lysozyme (Lyz, MW
14.4 kDa, pI 11) used in this study were purchased from LanJi of
Shanghai Science and Technology Development Company (Shanghai, China). 3-Methylacryloxypropyl trimethoxysilane (WD-70)
and 3-aminopropyl trimethoxysilane (WD-56) were purchased
from Chemical Factory of Wuhan University (Wuhan, China). b-CD
was from Institute of Tianjin JingKe Fine Chemicals (Tianjin, China).
AA and N,N0 -methylenebisacrylamide (MBA) were purchased from
Chemistry Reagent Factory of Chinese QianJin (Tianjin, China).
W. Zhang et al. / Applied Surface Science 256 (2010) 3000–3005
3002
Potassium persulfate and m-nitrophenol were obtained from
Institute of Tianjin GuangFu Fine Chemicals (Tianjin, China).
2.3. Preparation of imprinted and non-imprinted polymers
To a three-necked round-bottomed flask, 5.0 g of activated
silica gel, 50 ml of toluene and 5 ml of silane coupling agents (VWD70:VWD-56 = 1:1) were added. The mixture was stirred and purged
with nitrogen 12 h at 70 8C. Then the silica gel was washed with
toluene, acetone and ether. The obtained silica gel (Fig. 1) was
dried at 70 8C for 6 h. And then the silica gel (5.0 g) prepared above
was soaked in 20 ml phosphate buffer solution (PBS, pH 6.2,
0.01 mol/l) containing 0.2 mol/l glutaraldehyde for 12 h. The
resulting silica gel (Fig. 1) was washed with doubly distilled water
repeatedly.
To a 10 ml centrifuge tube, 80 mg of BHb, 5 ml of buffer (PBS, pH
7.0, 0.01 mol/l) and 1.0 g aldehyde-modified silica gel were added,
and the mixture was incubated 1 h at room temperature. Then
200 mg of acryloyl-b-CD (synthesized as described by the group of
Shun-ichi Nozakura and Makoto Komiyama [30,31]), 400 mg of AA
and 20 mg of MBA were added to this solution, which was
incubated 3 h under shaking for pre-polymerization. After the
mixture was purged with nitrogen to remove oxygen, the
polymerization was initiated by addition of 30 mg of potassium
persulfate and 40 mg of sodium hydrogen sulfite. Polymerization
was continued for 24 h and the obtained MIP (Fig. 1) was washed
successively with 10% (w/v) SDS-5% (v/v) HAc, 5% (w/v) oxalic acid,
ethanol, and doubly distilled water.
The non-imprinted polymer (NIP) was prepared using the same
procedure but without addition of template molecule.
2.4. Adsorption dynamics
In adsorption dynamics experiments, 50 mg of MIP or NIP was
suspended in 5 ml of an initial BHb concentration of 0.5 mg/ml. The
tube was incubated at room temperature with shaking. At different
time intervals, the amount of BHb adsorbed by MIP or NIP particles
was determined by UV–vis spectrophotometry. The amount of
protein adsorbed onto the polymer (Q) was determined according
to the following formula:
Q¼
ðC 0 C F ÞV
m
where C0 (mg/ml) and CF (mg/ml) represent the initial and final
protein solution concentration, respectively. V (ml) is the sample’s
volume and m (g) is the mass of the adsorbent polymer.
where QMIP and QNIP are the adsorption capacity of the template or
the analogue on MIP and NIP, respectively.
The selectivity factor (b) is defined as:
b¼
atem
aana
where atem is the imprinting factor towards the template molecule
and aana is the imprinting factor towards the analogue.
2.7. Competitive adsorption
Competitive adsorption of the template protein in respect to the
non-template protein was studied. The experiments were performed with a protein mixture made of a fixed concentration
(0.5 mg/ml) of the template protein BHb and a series of increasing
concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/ml) of the
competing protein BSA.
3. Results and discussion
3.1. Characterization studies
FT-IR spectra of activated silica gel, silica gel after silanization
treatment and aldehyde treatment silica gel were shown in Fig. 2.
The carbonyl groups of WD-70 were confirmed by the absorption
peak at 1726.06 cm1. FT-IR spectra provided information on bulk
composition of the particles rather than just the surface [11]. In
order to ascertain each modification, elemental analysis was
employed. The results were shown in Table 1. After the silanization
treatment, the nitrogen atomic content was increased from 0 to
2.22%, which suggested that amine groups were successfully
introduced onto the surfaces of the silica gel. After aldehyde
treatment, the carbon content was elevated from 6.32 to 6.71%.
After polymerization (MIP), it can be seen that the nitrogen content
was increased from 2.32 to 5.24%. The increase of nitrogen was
attributed to the formation of MIP on the surface of the silica gel.
As shown in Fig. 3, the activated silica gel exhibited irregular
shape (Fig. 3A) and its surface was smooth (Fig. 3B). After a layer of
polymer was formed on the surface of the silica gel, it exhibited a
rough surface (Fig. 3C and D). By comparing Fig. 3C with D, it can be
seen that there were no significant morphological differences
between MIP and NIP. The differences in protein adsorption
between MIP and NIP in the subsequent studies would be due to
2.5. Adsorption isotherm
In binding isotherm experiments, 50 mg of MIP was equilibrated with protein solution of varied initial concentrations in each
centrifuge tube. After 12 h, the saturated polymer was separated
by centrifugation, and the residual concentration of the protein
solution was measured by UV–vis spectrophotometry.
2.6. Specificity of adsorption
In order to determine the adsorption specificity of the MIP,
0.5 mg/ml of template BHb or 0.5 mg/ml of non-template proteins
including BSA, Lyz and Cyt was mixed with the MIP and the NIP,
respectively, and incubated for 12 h at room temperature. Then the
protein concentration of the supernatants was measured.
The specific recognition property of MIP is evaluated by
imprinting factor (a), which is defined as:
Q
a ¼ MIP
Q NIP
Fig. 2. FT-IR spectra of activated silica gel (1), silica gel after silanization treatment
(2), and aldehyde treatment silica gel (3). Peak identification: nOH: 3427.24 cm1
1
and 1644.86 cm1; nC5
; nSi–O–Si: 1197.36 cm1; nSi–O: 802.18 cm1
5O: 1726.06 cm
and 472.97 cm1.
W. Zhang et al. / Applied Surface Science 256 (2010) 3000–3005
3003
Table 1
Elemental composition of the support particles from elemental analysis.
Silica gel
Silanization treatment
Aldehyde treatment
MIP
C (%)
H (%)
N (%)
1.46
6.32
6.71
19.6
1.47
1.86
2.21
3.78
0
2.22
2.32
5.24
the molecular imprinting rather than morphological differences
[11].
3.2. Adsorption dynamics of polymers
Fig. 4 showed the dynamic adsorption process for the MIP and
NIP. The adsorption reached equilibrium at about 12 h. Compared
with the NIP, the MIP exhibited much higher capacity. At the early
time, a large number of imprinted cavities existed on the surface of
the support, so the template protein was easy to reach the specific
binding sites. When the recognition sites were filled up, the rate of
adsorption dropped significantly and adsorption process achieved
equilibrium. In the common adsorption process [32], the rate of
adsorption curve increased significantly at the beginning. However, in this rebinding process, the initial rate of adsorption was lower
than common binding process and adsorption reached equilibrium
in a longer time, which indicated that the b-CD played a vital role
in the process. The interactions between b-CD and protein involve
the hydrogen bonding and hydrophobic interaction, so the
prearrange sequence of the protein entered into the hydrophobic
moieties of the imprinted cavities and reached low energy spatial
Fig. 4. Adsorption dynamics of BHb on MIP (&) and NIP (~). Experimental
conditions: V = 5 ml; C = 0.5 mg/ml and the mass of polymer: 50 mg.
arrangement state, which needed a longer time. Therefore, the rate
of the rebinding process was much slower.
3.3. Adsorption isotherm
Binding isotherm was used to measure the concentration
dependence of the recognition behavior of the polymer. The
adsorbed amount of BHb increased with the increase of the initial
Fig. 3. The SEM microphotographs of silica gel (A), surface of silica gel (B), surface of MIP (C), and surface of NIP (D).
3004
W. Zhang et al. / Applied Surface Science 256 (2010) 3000–3005
Table 2
Imprinting factor (a) and selectivity factor (b) of the polymers.
Fig. 5. Adsorption isotherm of BHb on MIP. Experimental conditions: V = 5 ml;
C = 0.1–0.8 mg/ml; the mass of polymer: 50 mg and adsorption time: 12 h.
concentration of the BHb (Fig. 5). When the recognition sites were
saturated, the adsorption capacity reached a constant value. As
shown in Fig. 5, in the low concentration range of BHb, the amount
of BHb was not enough to saturate the specific binding sites.
Proteins
QMIP (mg/g)
QNIP (mg/g)
a
b
BHb
BSA
Lyz
Cyt
37.1
7.93
10.8
5.67
7.50
6.11
7.67
5.40
atem = 4.95
aana = 1.30
aana = 1.41
aana = 1.05
–
3.81
3.51
4.71
However, when the BHb concentration was increased, the specific
imprinted sites were filled with the BHb and the adsorption
process reached equilibrium.
The binding behavior of the polymer adapted the Langmuir
model equation [12] as Ce/Q = Ce/Qmax + 1/(bQmax), where Ce is the
equilibrium concentration of BHb (mg/ml), Q (mg/g) is the
adsorption capacity of BHb at equilibrium concentration, Qmax is
the theoretical maximum adsorption capacity (mg/g), and b is the
Langmuir adsorption equilibrium constant (ml/mg). The Qmax
values can be calculated to be 62.8 mg/g from the slope of the
Langmuir model equation (Fig. 6).
3.4. Adsorption specificity
In this experiment, BSA, Lyz and Cyt were used as non-template
proteins to further investigate the specificity of the BHb–MIP
(Fig. 7 and Table 2).
It can be seen from Fig. 7 that the BHb–MIP exhibited high
adsorption selectivity for template BHb and the results of the
adsorption were summarized in Table 2. The selectivity factor
(b) to BSA, Lyz and Cyt was 3.81, 3.51 and 4.71, respectively,
which indicated that the adsorption capacity of the template
BHb was much higher than those of the non-template proteins.
Because many specific recognition sites respect to template
protein were generated on the surface of the support, the
template protein was strongly bound to the polymer in the
rebinding process. Since the recognition sites of the imprinting
cavities were not complementary to BSA, Lyz and Cyt, the BHb–
MIP adsorbed less capacity of the non-template proteins than
that of the template BHb. In contrast, the NIP adsorbed template
much less than that of MIP since NIP had not generated specific
recognition sites due to the absence of template protein.
Therefore, the non-specific adsorption was the primary factor
for the adsorption of NIP.
Fig. 6. Adsorption isotherm of BHb on MIP, linearized according to the Langmuir
model.
Fig. 7. Selective adsorption of BHb, BSA, Lyz or Cyt on the MIP and NIP. Experimental
conditions: V = 5 ml; CBHb = 0.5 mg/ml; CBSA = 0.5 mg/ml; CLyz = 0.5 mg/ml;
CCyt = 0.5 mg/ml; the mass of polymer: 50 mg and adsorption time: 12 h.
Fig. 8. Effect of the competitive protein BSA on the rebinding of template protein
BHb on the MIP. Rebinding was done with a fixed concentration of BHb and
increasing concentration of BSA and adsorption time: 12 h.
W. Zhang et al. / Applied Surface Science 256 (2010) 3000–3005
3.5. Competitive adsorption
Competitive binding of the template protein was studied
(Fig. 8). In order to carry out this experiment, a series of binary
protein solution of BHb–BSA were prepared. The concentration of
the template protein was fixed at 0.5 mg/ml. The competing
protein concentration varied from 0.5 to 3.0 mg/ml.
Fig. 8 showed that the adsorption of the template protein BHb
has been little affected by the increase of the ratio of CBSA/CBHb. The
MIP recognized proteins by the synergistic effects of shape
complementarity and multiple weak interactions (e.g. hydrophobic interaction and hydrogen bond interaction) provided by the
functional monomers. Though the molecular volumes of BHb and
BSA are quite similar, the recognition sites of the imprinting
cavities were not complementary to the BSA. Consequently, the
competing protein had little effect on the adsorption of the
template protein.
4. Conclusions
In this study, synthetic protein imprinted polymer was
prepared using acryloyl-b-CD and AA as monomers. BSA, Lyz
and Cyt were chosen as non-template proteins to test the
imprinting effect and the specific binding of BHb to the polymer.
The selectivity factor (b) to BSA, Lyz and Cyt was 3.81, 3.51 and
4.71 after reaching adsorption equilibrium. The results of the
adsorption experiments indicated that such protein imprinted
polymer could selectively recognize the template. Easy preparation of the MIP and good protein recognition properties may make
this method attractive and broadly applicable in the field of
biomedical materials.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 20875049), the National Basic Research
3005
Program of China (973 Program) (Nos. 2007CB914100 and
2006CB705703).
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