Organic–inorganic hybrid silica as supporting matrices for selective recognition of bovine hemoglobin via covalent immobilization

Research Article

Organic–inorganic hybrid silica assupporting matrices for selective recognitionof bovine hemoglobin via covalentimmobilization

The synthesis of poly-aminophenylboronic acid (APBA) imprinted hybrid silica-based

polymers for selective recognition of bovine hemoglobin (BHb) was described, where the

mesoporous hybrid silica supporting matrices were prepared by a mild sol–gel process

with tetraethoxysilane and 3-aminopropyltriethoxysilane as two precursors. Covalent

immobilization of BHb was adopted in order to create homogeneous recognition sites.

After removal of the template, the resulting imprinted polymers showed high binding

affinity toward BHb and the imprinting factor (a) reached 2.12. The specificity of the BHb

recognition was evaluated with competitive experiments, indicating the imprinted poly-

mers have a higher selectivity for the template BHb. The easy preparation protocol and

good protein recognition properties made the approach an attractive solution to depletion

of high-abundance protein from bovine blood.

Keywords: Covalent immobilization / Molecular imprinting / Organic–inorganichybrid / Protein / Sol–gelDOI 10.1002/jssc.200900295

1 Introduction

Molecular imprinting is a technique for preparing recogni-

tion sites of predetermined selectivity. The sites are tailor-

made in situ by copolymerization of functional monomers

and crosslinkers in the presence of template [1–3]. This

technique has been successfully applied to the fields of

chromatographic stationary phases [4–6], SPE [7–8], artificial

antibody mimics [9–10], catalysis [11–12], and biosensing

[13–15] over the past decades. Despite the attractive features

of this technique with specificity that has been largely

reserved for small molecules, imprinting biomacromole-

cules such as protein, saccharides, and viruses still

represents a great challenge due to their incompatibility of

these targets with organic solvents that are typically used for

imprinting. Furthermore, the large molecular size, the high

flexibility of conformation, and the complexity of surface

structures limit the biomacromolecular imprinting [3].

Generally, there are two distinct strategies to prepare

protein-template imprinted polymer. One is non-covalent

molecular imprinting, in which intermolecular interaction

such as hydrogen bonds, electrostatic interactions,

hydrophobic interaction, Van der Waals forces, etc. are

utilized to form functional monomer-template adducts in

solution [16]. Although non-covalent methods are easy to

perform and many kinds of functional monomers are

available, the homology generated binding sites in term of

affinity and selectivity is commonly low. This drawback can

be largely avoided by covalently immobilized approach, by

which more homogeneous recognition sites can be

obtained. Shiomi et al. [17] have first developed the covalent

imprinting for selective recognition of hemoglobin by

using sol–gel process. Bonini et al. [18] modified the

approach to prepare silica-based imprinted beads for human

serum albumin, which has been successfully applied to

removal of high-abundance template protein from human

serum. Besides, using a two-stage core-shell miniemulsion

polymerization, Tan et al. [19] have fabricated surface-

imprinted particles for specific recognition of BSA with

the immobilization of template protein molecules on poly-

meric supporting beads. Such template immobilization

strategy allows the imprinting of proteins that may not be

soluble in the polymerization mixture and can be potentially

employed as a generally applicable methodology for protein

imprinting.

3-Aminophenylboronic acid (APBA), as a popular

functional monomer, can be polymerized under the mild

Zian Lin1,2

Fan Yang1

Xiwen He1

Yukui Zhang1,3

1College of Chemistry, NankaiUniversity, Tianjin, P. R. China

2Ministry of Education KeyLaboratory of Analysis andDetection for Food Safety(Fuzhou University), Fuzhou,Fujian, P. R. China

3National ChromatographicResearch and Analysis Center,Dalian Institution of ChemicalPhysics, Chinese Academy ofSciences, Dalian, P. R. China

Received April 29, 2009Revised August 14, 2009Accepted August 14, 2009

Abbreviations: APBA, 3-aminophenylboronic acid; APTES,

3-aminopropyltriethoxysilane; APS, ammonium persulfate;

BHb, bovine hemoglobin; FT-IR, Fourier transform infrared;

MIP, molecularly imprinted polymer; NIP, non-imprintedpolymer; TEOS, tetraethoxysilane

Correspondence: Dr. Zian Lin, College of Chemistry, NankaiUniversity, No. 94 Weijin Road, Tianjin 300071, P. R. ChinaE-mail: [email protected]: 186-22-23494962

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2009, 32, 3980–39873980

aqueous conditions by chemical or electrochemical

initiation and is expected to interact with various sacchar-

ides and amino acid residues [20]. It has been usually

adopted for recognition studies. Bossi’s group [21] was the

first to report that this monomer is suitable for protein

imprinting. Rick and Chou [22] fabricated poly (APBA)-

based lysozyme (Lyz) and cytochrome c (Cyt C) imprinted

polymers on the gold surfaces of quartz crystal micro-

balance electrodes. Recently, Turner et al. [23] also

successfully imprinted thermal- and fluoro-alcohol-induced

a-lactoglobulin isoforms in poly (APBA)-thin films on

quartz crystal microbalance chips and the resulting poly-

mers showed better template selectivity than the corre-

sponding non-imprinted polymer (NIP).

Complexity reduction in samples is an important step in

facilitating access to the low abundant proteins of interest

for disease research and diagnostics. As one of the abundant

proteins, bovine hemoglobin (BHb) is considered as a

drawback, since it seriously masks and hampers the

detection of low abundant proteins. To selectively deplete

BHb, a novel method for the preparation of organic–

inorganic hybrid silica-based imprinted polymer was devel-

oped. The hybrid silica-based matrices fabricated by a mild

sol–gel process could create the uniform and small sizes of

the particles and offer high surface area for the immobili-

zation of template. The template was covalently immobi-

lized on glutaraldehyde-treated aminopropyl silica matrices

through amine bonds between amine groups of BHb

and aldehyde groups on the silica. The molecularly

imprinted polymers (MIPs) thus obtained were evaluated by

investigating the binding kinetics, the binding capacity,

the specificity and the recovery for template protein. In

addition, the feasibility for biological application was further

assessed by selective removal of template protein from

bovine blood.

2 Materials and methods

2.1 Materials and reagents

BSA (size 4.0� 4.0� 14.0 nm; Mw 67 kDa), lysozyme (Lyz;

size 3.0� 3.0� 4.5 nm; Mw 13.4 kDa) and BHb (size

5.5� 5.5� 7.0 nm; Mw 64.5 kDa) were purchased from

Shanghai Lanji (Shanghai, China). APBA was obtained

from Sigma (St. Louis, MO, USA). Tetraethoxysilane

(TEOS, 95%) and 3-aminopropyltriethoxysilane (APTES,

99%) were obtained from Acros Organics (Geel, Belgium),

which were used directly without further purification.

Ammonium persulfate (APS), MOPS, and CTAB were

obtained from institute of Tianjin Guangfu Chemicals

(Tianjin, China). BCA Protein Assay Kit was the product of

Pierce (Rockford, IL, USA). Other reagents were of

analytical grade or better. Deionized water was prepared

with a Milli-Q water purification system (Millipore, Milford,

MA, USA). Bovine blood was kindly gifted from Xiaochuan

Biotech (Tianjin, China).

2.2 Apparatus

The SEM images of the imprinted polymers were obtained

by a SS-550 scanning electron microscope (Shimadzu,

Japan). Fourier transform infrared (FT-IR) spectra

(4000–400 cm�1) in KBr were recorded using the AVATAR

360 FT-IR spectrophotometer (Nicolet, Waltham, MA,

USA). The data of adsorption were obtained by using UV-

2450 spectrophotometer (200–850 nm) (Shimadzu, Japan).

Pore-size distribution of the hybrid silica imprinted poly-

mers was measured by nitrogen adsorption method (Nowa

4000, Quantachrome, USA). Electrophoresis for proteins

was performed by using regular SDS-PAGE with 10%

polyacrylamide gel and 4% stacking gels (Bio-Rad, Hercules,

CA, USA).

2.3 Preparation of organic–inorganic hybrid silica

matrices

The hybrid silica as supporting matrices was prepared as

described by Yan et al. [24] with some modification. The

mixed solution, consisting of 8.4 mL of TEOS, 8.85 mL of

APTES, 10.8 mL of anhydrous ethanol, 800 mg of CTAB,

and 1.6 mL of water, was stirred for 1 min, and then placed

in oven at 401C for 20 h. Subsequently, the supporting

matrices were washed three times with ethanol and

deionized water, respectively. After drying at room tempera-

ture, the obtained matrices were gently ground and sieved

(200 meshes) for next step.

2.4 Preparation of BHb-imprinted silica using immo-

bilized template

The aliquot of the supporting matrices (1.0 g) was incubated

with 100 mM phosphate buffer (PBS, pH 8.0) containing

5% glutaraldehyde at room temperature for 12 h in order to

introduce aldehyde groups. Then 1.0 mL, 5 mg/mL sodium

cyanoborohydride (NaCNBH3) was added in order to reduce

C==N to C–N, which could enhance the stability of

immobilized glutaraldehyde. The resulting matrices were

repeatedly washed with deionised water. Subsequently,

10 mL 2.5 mg/mL BHb solution containing 10 mM MOPS

(pH 5.5) and 0.1 M NaCl was admixed as the template with

incubation for 6 h at 41C in order to covalently bind the BHb

on the aldehyde groups. The residual aldehyde groups on

the surface of the supporting matrices were depleted by

adding 1 mL Tris-HCl (pH 8.0). Washes with deionized

water followed and finally 5 mL of 50 mM APBA water

solution was added to the resulting matrices. After 1 h

incubation, 5 mL of 25 mM APS was added in order to

initiate the polymerization reaction. The polymerization was

performed at room temperature for 2 h, after which the

polymers were washed again with deionized water for five

times. Finally, 5 mL of 0.5 M oxalic acid was added in order

to remove the template. This step was carried out at room

J. Sep. Sci. 2009, 32, 3980–3987 Other Techniques 3981


temperature for 24 h. The derivatization protocol was

checked at each step by FT-IR spectroscopy.

The NIPs were also prepared in the absence of template

using the same polymerization procedure as mentioned

above. The MIPs or NIPs needed to be conditioned with

10 mM PBS (pH 7.0) before use, in order to increase the pH

and remove the free of template in solution cleaved by the

oxalic acid.

2.5 Determination of immobilized BHb

The amount of immobilized BHb on the surface of the

supporting matrices was determined by BCA assay.

According to the manual of BCA assay, 0.01 g of the

imprinted polymer was immersed into 1.0 mL of 100 mM

NaOH for 5 h to cleave BHb completely. BHb standard

solutions were prepared with 100 mM NaOH in the

concentration range of 0–50 mg/mL. A volume of 100 mL of

each BHb standard and the cleaved BHb solution was mixed

with 900 mL of BCA reagent, respectively. After each mixture

was incubated at 371C for 30 min, the absorbance was

measured with a spectrophotometer at 562 nm, and the

content of immobilized BHb was calculated.

2.6 Determination of swelling ratio

The hybrid silica-based NIPs and MIPs that were initially

dispersed in deionized water were first isolated by

centrifugation at 14 000 rpm for 30 min. Measurement of

the polymers swollen weight (Ww) was made after the

supernatant was removed. Subsequently, the polymers were

dried at room temperature in vacuum box and weighed

again to obtain the dry weight (Wd). The swelling ratio of the

polymer was then calculated as follows:

SR ¼ ðWw �WdÞ=Wd ð1Þ

2.7 Binding capacity

To investigate the binding capacity, 30 mg of the polymers

were incubated with 3.0 mL of BHb solution at different

concentrations (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 2.0, and

2.4 mg/mL) for 5 h. Then the polymers were centrifuged at

14 000 rpm for 5 min and the concentration of BHb in the

supernatant was determined by using UV/Vis spectro-

photometer at the wavelength of 405 nm. The adsorption

capacity (Q, mg/g) was calculated according to the

difference of BHb concentration before and after adsorp-

tion, the volume of aqueous solution and the weight of the

imprinted polymers according to

Q ¼ ðC0 � CtÞV=m ð2Þ

where C0 is the initial BHb concentration (mg/mL), Ct the

BHb supernatant concentration (mg/mL), V the volume of

BHb solution (mL) and m is the weight of the imprinted

polymer (g).

2.8 Binding kinetics

To evaluate the binding kinetics of the BHb-imprinted

polymers, the polymers of 30 mg were incubated with 3 mL

of 2.0 mg/mL BHb solution for different time. The

adsorption capacity was quantified as described in above

section.

2.9 Specificity of adsorption

To determine the adsorption specificity of the BHb-

imprinted polymers, 30 mg of the NIPs or BHb-MIPs was

placed in a centrifuge tube, where the different concentra-

tion of BSA or Lyz (1.0, 2.0, 4.0 mg/mL) was added to a fixed

initial concentration of BHb (2.0 mg/mL) and incubated for

5 h at room temperature. After centrifugation at 14 000 rpm

for 5 min, the concentration of BHb at the supernatants was

measured.

The specific recognition property of the MIPs is eval-

uated by imprinting factor (a), which is defined as the

following equation:

a¼QMIP=QNIP ð3Þ

where QMIP and QNIP are the adsorption amount of

template or analogues on MIPs and the corresponding

NIPs, respectively.

The selectivity factor (b) is expressed as the following

equation:

b ¼ atem=aana ð4Þ

where atem is imprinting factor of template molecule and

aana is imprinting factor of analogues.

2.10 Real sample analysis

To evaluate the feasibility of the imprinted polymers for

biological application, 50 mg of BHb-imprinted polymers

were immerged with 3 mL of bovine blood that had been

diluted 100-fold with 10 mM PBS (pH 7.0) and incubated for

5 h. The supernatant fluid was then collected by centrifuga-

tion and analyzed by SDS-PAGE assay.

3 Results and discussion

3.1 Preparation and characterization of hybrid silica-

based imprinted polymers

The general scheme for the preparation of APBA-based

imprinted hybrid silica-based polymers was illustrated in

Fig. 1, which involved the following steps: (i) preparation of

J. Sep. Sci. 2009, 32, 3980–39873982 Z . Lin et al.


hybrid silica matrices; (ii) covalent immobilization of

template protein; (iii) polymerization of APBA; and (iv)

removal of template.

In this work, a supramolecular template-based approach

combined with sol–gel chemistry was utilized for the

preparation of organic–inorganic hybrid silica matrices.

TEOS and APTES were used as two precursors, and the

surfactant CTAB was chosen as the supramolecular

template. The electrostatic interaction between them played

an important role in the formation of porous hybrid silica

matrices [24]. Furthermore, ethanol was added into the

reaction solution, not only to dissolve all sol–gel ingredients

homogeneously but also to retard the hydrolysis rate of

precursors. With an appropriate amount of water added, the

co-condensation of TEOS and APTES (with the optimized

molar ratio of 1:1) in the presence of CTAB occurred, and

the porous mesostructure of hybrid silica matrices with

active amine groups was obtained (see Fig. 2A). The

uniform and small sizes of the matrices could offer high

surface area for further immobilization of template.

Glutaraldehyde, commonly used bifunctional reagent

[17–19], was performed to couple the hybrid silica matrices

with BHb. After being reduced by NaCNBH3, the BHb was

immobilized on the surface of the silica matrices through a

covalent bond between e amine groups of lysines of the

template and aldehyde groups on the silica matrices.

APBA could be polymerized by APS to form relatively

short chains [25], which could flocculate in aggregates, and

deposit in a reasonably thin and ordered film on the surface

of protein-immobilized silica matrices [26]. Figure 2B

and C shows SEM images of the NIPs and MIPs after

polymerization of APBA, respectively. It can be clearly

observed that the MIPs have more homogeneous and

ordered small particles than the NIPs, suggesting the APBA

well interacted with BHb. Furthermore, the pore-size

distribution of the polymers was measured by nitrogen

adsorption method. The average pore diameter of the MIPs

and NIPs was 15.6 and 23 nm. The corresponding surface

areas were 132.3 m2/g for MIPs and 108.1 m2/g for NIPs,

respectively.

The SR values of the hybrid silica-based NIPs and MIPs

in water were determined based on the amount of water

uptake. It was found that the SR value for NIPs was 2.38,

which was lower than that obtained with MIPs, where the

SR value was 3.12. The exact reason for this was not known,

but it could possibly be due to the formation of binding

cavities on the surface of the MIPs, which enhanced water

penetration and results in higher water uptake and SR

value.

To further determine the characteristics of the hybrid

silica-based MIPs, FT-IR spectra of the hybrid silica matri-

ces, modified glutaraldehyde, immobilized BHb, and poly-

merized APBA, together with the spectrum of APBA, were

compared in Fig. 3. The strong peaks near 1560 and

1407 cm1 were assigned as the –NH2 vibration (spectrum

(a)). The bands at 1720 cm�1 were C==O stretch (spectrum

(b)). This FT-IR spectrum suggests that the aldehyde groups

have been successfully grafted onto the surface of silica

matrices. The covalent immobilization of protein resulted in

the detection of signals typical of peptide bonds near

1650 cm�1 (spectrum (c)). The further polymerization of

APBA was confirmed by the vibration of phenylboronic

groups in the region from 1100 to 1200 cm�1, and boronate

contribution around 3200–3400 cm�1 (spectrum (e)).

In the covalent approach of protein imprinting, oxalic

acid is usually selected as eluent to remove the template,

because the formed C==N bond by immobilization of

template can be easily broken by oxalic acid [17, 18]. In this

work, 0.5 M oxalic acid was used and the eluate was detected

by UV–vis spectrophotometer at the wavelength of 405 nm.

It was observed (data not shown) that the absorbance of

BHb evidently decreased with the increase of elution time.

No template could be detected until the elution time reached

over 24 h. The result confirms the efficient removal of the

template form the MIPs.

3.2 Characterization of immobilized BHb

To determine the amount of immobilized BHb on hybrid

silica matrices, NaOH solution was used to cleave BHb from

supporting matrices, and then the supernatant was analyzed

by BCA assay. The result validated that the average 23.5 mg

of BHb was immobilized on 1 g of hybrid silica matrices.

The homogenous distribution of amine groups on the

surface of the hybrid silica matrices could largely reduce the

stereochemical hindrance, and thus increase the amount of

immobilized BHb.

3.3 Adsorption isotherms

To investigate the binding capacity for BHb of both NIPs

and MIPs, the adsorption isotherms were determined in the

range of 0.2–2.4 mg/mL initial concentration of BHb. As

shown in Fig. 4, it was observed that the adsorption amount

of BHb on MIPs and NIPs gradually increased with the

increase of BHb concentration from 0.2 to 1.6 mg/mL, and

came to equilibrium over 2.0 mg/mL. However, the

Figure 1. Schematic representation of protein imprinting basedon covalent immobilization.



obtained MIPs have a higher affinity for the template BHb

than the NIPs, and the a reached 2.12. The result can be

explained by the fact that the imprinting process in BHb-

imprinted polymers can form specific recognition cavities

that shows high binding affinity for BHb. In contrast, as for

NIPs, the non-specific adsorption has dominant effect due

to lack of recognition sites. Therefore, the binding amount

of BHb is low.

The adsorption behaviors of BHb-imprinted polymers

can be described with the Langmuir adsorption equation as

Ce=Qe ¼ Ce=Qmax þ 1=bQmax ð5Þ

where Ce is the equilibrium concentration of BHb

(mg/mL), Qe the adsorption capacity of BHb adsorbed per

unit weight of BHb-imprinted polymers at equilibrium

concentration (mg/g), Qmax the maximum adsorption

capacity (mg/g), and b is the adsorption equilibrium

constant (mL/mg).

In the BHb concentration range studied, the Langmuir

regression equation obtained is Ce/Qe 5 0.0229Ce10.0103

(r 5 0.9963). It was concluded that the Langmuir equation

fitted well for BHb adsorption under the concentration

range studied. The b and Qmax values could be calculated to

be 2.22 mL/mg and 43.6 mg/g. Compared with those using

silica beads as supporting matrices [17, 18], the hybrid silica-

based MIPs have a higher adsorption capacity.

3.4 Adsorption kinetics

Figure 5 illustrated the adsorption kinetics of 2.0 mg/mL

BHb solution onto BHb-imprinted polymers. It could be

seen that the absorbance had a rapid increase in 2 h, and

then slowed down with the time extension. After 5 h, the

adsorption process almost reached equilibrium. Possible

reason was that BHb was easy to reach the surface

imprinting sites on the BHb-imprinted polymers at the

beginning. With the saturation of the sites, BHb began to

diffuse onto the surface of BHb-imprinted polymers non-

specifically.

Figure 2. (A–C) SEM images of hybrid silica matrices (A), NIPs (B) and MIPs (C).

Figure 3. FT-IR spectra of the (A) hybrid silica matrices, (B) aftermodification of glutaraldehyde, (C) after immobilization of BHb,(D) APBA, (E) poly-APBA MIPs.

Figure 4. Adsorption isotherms of BHb on the NIPs and MIPs.Amount of polymers: 30 mg; V 5 3.0 mL; CBHb 5 0.2–2.4 mg/mL;incubation time: 5 h; T 5 251C.



3.5 Adsorption specificity

The selectivity of BHb-imprinted polymers was evaluated by

using BSA and Lyz as competitive proteins. Same amounts

(30 mg) of the NIPs and MIPs were put into 2.0 mg/mL

BSA, Lyz, and BHb solution, respectively. Table 1 listed the

binding capacities of BSA, Lyz and BHb on the NIPs and

MIPs under equilibrium binding conditions. It was found

that the BHb-imprinted polymers did not show selectivity

for BSA and Lyz, where their corresponding a values were

only 1.12 and 1.03, respectively. Unlike the competitive

proteins, the BHb-imprinted polymers exhibited high

selectivity for BHb. The evidence indicates that the

imprinting process create a microenvironment based on

shape selection and position of functional groups that

recognizes the BHb-imprinted molecule.

To further illustrate the recognition specificity of the

BHb-imprinted polymers, NIPs and MIPs were subjected to

binary protein competitive assay, where a fixed concentra-

tion of the template and an increasing concentration of the

competitive protein were adopted. The results demonstrated

(Fig. 6A) that the binding amounts of BHb decreased on

NIPs and did not change significantly on MIPs when

increasing concentration of BSA from 1.0 to 4.0 mg/mL.

The shape effect of template can respond for the result.

Though BSA and BHb are both globular proteins and their

molecular weights are similar, BHb is a tetrameric protein

composed of pairs of two different polypeptides and has a

biconcave shape, and the size of BHb is about 65 A. Unlike

BHb, BSA consists of one polypeptide and has an ellipsoidal

shape, and the size of BSA is about 154 A, larger than BHb

[27]. Therefore, it is very difficult for BSA to enter and

occupy the recognition cavities tailored for BHb in a

competitive environment of protein adsorption. Oppositely,

non-specific adsorption and random diffusion play domi-

nant role in the NIPs environment, and thus the binding of

BHb is suppressed. Similar results were also obtained while

using Lyz as competitive proteins (Fig. 6B). Different from

the former, the adsorption of BHb has a slight decrease

when the Lyz concentration increased above 2.0 mg/mL.

Possible reason is that the imprinting cavities are usually

first occupied by smaller protein due to high diffusion

coefficients. Nevertheless, at later stages, BHb will gradually

displace the already adsorbed Lyz, since BHb has greater

affinity toward the imprinting sites [28]

Figure 5. Adsorption dynamics of BHb on the NIPs and MIPs.Amount of polymers: 30 mg; V 5 3.0 mL; CBHb 5 2.0 mg/mL;T 5 251C.

Table 1. Binding amounts of tested proteins on NIPs and BHb-

MIPs under equilibrium conditions (n 5 3)a)

Proteins Q (mg/g) a b

NIPs BHb-MIPs

BHb 14.3070.53 30.3471.42 2.12 –

BSA 10.1770.37 11.4070.47 1.12 1.89

Lyz 15.3170.58 15.8370.65 1.03 2.05

a) Experimental conditions: amount of polymers: 30 mg;

V 5 3.0 mL CBHb 5 CBSA 5 CLyz 5 2.0 mg/mL; T 5 251C.

Figure 6. (A, B) Competitive adsorption of BHb versus contras-tive proteins on the NIPs and MIPs. BHb1BSA; (B) BHb1Lyz;amount of polymers: 30 mg; V 5 3.0 mL; CBHb 5 2.0 mg/mL;T 5 251C.



3.6 Reproducibility of BHb-imprinted polymers

The reproducibility of the bound template was evaluated by

repeated adsorption/desorption experiments. 10% w/v

acetic acid containing 10% SDS was used to desorb the

template and then the imprinted polymers were washed

with 10 mM PBS (pH 7.0) until the pH of the effluent

reached 7.0, which were used to absorb the template again.

Figure 7 showed that the adsorption capacity slightly

decreased after three cycle times. It is probable that the

poly-APBA is a thin film and do not strongly attach to the

surface of hybrid silica matrices. Thus, several incubation

steps give rise to the partial detachment of the matrices layer

and cause the loss of recognition properties of the BHb-

imprinted polymers. Despite the decrease of adsorption

capacity, it was still better than the NIPs for the recognition

property.

3.7 Specific removal of BHb from bovine blood

The practical applicability of the BHb-imprinted polymers

was demonstrated by selective removal of hemoglobin from

bovine whole blood. The high quantity of hemoglobin in

biological fluid is considered as a drawback, since it

seriously hampers the detection of low-abundance proteins

that are often marker of diseases. According to the

procedure as described in the experimental section, a

certain amount of bovine whole blood (1 mL) was drawn

and a 100-fold dilution with 10 mM PBS (pH 7.0) was

carried out. The supernatant fluid was then collected after

incubation for 5 h with the BHb-imprinted polymers. As

seen from Fig. 8A, the color of bovine blood was changed

from bright red to brown after incubation, implying that the

hemoglobin was partly absorbed by MIPs. Besides, the

supernatant fluid was further analyzed by SDS-PAGE and

the obtained electropherogram was illustrated in Fig. 8B,

including those for standard proteins (lanes 2–3), bovine

whole blood (lane 4) and bovine blood after removal of

hemoglobin (lane 5). The separation of bovine whole blood

without treatment (lane 4) revealed several major bands,

ranging in molecular weight from 14.4 to 67 kDa, which

might be attributed mainly to albumin and heme proteins,

etc. Only BHb band was faded obviously, leaving the other

bands nearly unchanged as seen from lane 5, suggesting

that the BHb-imprinted polymers have specificity for

hemoglobin. Interestingly, an extra thin band close to

26 kDa was found in lane 5. Possible reason is that the high-

abundance hemoglobin masks the unknown protein before

pretreatment. Nevertheless, the preliminary experiments

with a complex sample need further investigation where the

quantity of MIPs would be optimized to obtain an almost

complete depletion of hemoglobin from bovine blood.

Figure 7. Reproducibility of BHb-imprinted polymers. Amount ofpolymers: 30 mg; V 5 3.0 mL; CBHb 5 2.0 mg/mL; T 5 251C.

Figure 8. (A, B) Photograph of bovine blood treated with orwithout MIPs (A) and SDS-PAGE of competitive adsorption oftemplate protein (B) 1 – untreated bovine blood (100-folddilution); 2 – bovine blood treated with BHb-MIPs; 3 – deionizedwater. Lane (1) marker; (2) mixture of BSA, BHb and Lyz(each protein, 0.2 mg/mL); (3) BHb (0.2 mg/mL); (4) untreatedbovine blood (100-fold dilution); (5) bovine blood treated withBHb-MIPs.



4 Concluding remarks

In this study, a novel poly-APBA-imprinted hybrid silica-

based polymer has been synthesized based on covalent

immobilization of template BHb. The mesoporous hybrid

silica supporting matrices offer high surface area for the

immobilization of template, and thus higher binding capacity

for template can be obtained. The binding experiments

showed the prepared BHb-imprinted hybrid silica-based

polymers had specific recognition for template. Moreover,

the successful application in specific removal of BHb from

bovine blood suggested that the purposed method could be

expected to be an attractive solution for selective removal of

target protein from complex biological sample.

We are grateful to the National Basic Research Program ofChina (No. 2007CB914100), China Postdoctoral ScienceFoundation (20070420688) and Science Start Fund of FuzhouUniversity (0460022233) for financial supports.

The authors have declared no conflict of interest.

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