Facile Preparation of Glycoprotein-Imprinted 96-Well Microplates for

Dec 17, 2013 - A template glycoprotein was first immobilized by a boronic acid-modified microplate through boronate affinity binding, and then, a thin...
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Facile Preparation of Glycoprotein-Imprinted 96-Well Microplates for Enzyme-Linked Immunosorbent Assay by Boronate Affinity-Based Oriented Surface Imprinting Xiaodong Bi and Zhen Liu* State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, China S Supporting Information *

ABSTRACT: Molecularly imprinted polymers (MIPs), as inexpensive and stable substitutes of antibodies, have shown great promise in immunoassays. Glycoproteins are of significant diagnostic value. To facilitate the application of MIPs in clinical diagnostics, a general and facile imprinting method toward glycoproteins oriented for an enzyme-linked immunosorbent assay (ELISA) in the form of a 96-well microplate is essential but has not been fully explored yet. In this study, a new method called boronate affinity-based oriented surface imprinting was proposed for facile preparation of glycoprotein-imprinted microplates. A template glycoprotein was first immobilized by a boronic acid-modified microplate through boronate affinity binding, and then, a thin layer of polyaniline was formed to cover the microplate surface via in-water self-copolymerization. After the template was removed by an acidic solution, 3D cavities that can rebind the template were fabricated on the microplate surface. Using horseradish peroxidase (HRP) as a model target, the effects of imprinting conditions as well as the properties and performance of the prepared MIPs were investigated. α-Fetoprotein (AFP)-imprinted microplate was then prepared, and thereby, a MIP-based ELISA method was established. The prepared MIPs exhibited several highly favorable features, including excellent specificity, widely applicable binding pH, superb tolerance for interference, high binding strength, fast equilibrium kinetics, and reusability. The MIP-based ELISA method was finally applied to the analysis of AFP in human serum. The result was in good agreement with that by radioimmunoassay, showing a promising prospect of the proposed method in clinical diagnostics.

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diagnostics. The molecularly imprinted polymers (MIPs)coated 96-well microplate has been an important direction of the application of MIPs. Although efforts21−24 have been put forward in this direction, efficient and widely applicable approaches for the imprinting of glycoproteins are still much needed. Boronate affinity has gained increasing attention in recent years due to its unique reversible covalent binding with cis-diol containing compounds.25−27 Particularly, boronic acid-functionalized materials, such as monoliths,28−32 magnetic nanoparticles,33−35 and mesoporous silica,36 have been developed into important tools for selective extraction of glycoproteins. However, conventional boronate affinity materials are associated with some apparent drawbacks: (1) common boronic acids require alkaline pH for eventual binding while binding at lower pH requires particularly synthesized boronic acids29,31,37 or materials;32−34 (2) boronic acids alone provide broadspectrum selectivity and therefore nontarget cis-diol molecules may severely interfere with the binding of cis-diol containing targets; and (3) low binding strength (10−1−10−3 M for

olecular imprinting, as an efficient technology to create artificial receptors with antibody-like binding properties or enzyme-like activities, has been used in a variety of important applications such as chemical separation, molecular sensing, and catalysis.1−10 The imprinting of biological macromolecules especially proteins is important but rather challenging, because of conformational change during polymerization and slow mass transfer in the polymers.3,11 To solve these problems, a number of methods have been proposed, such as surface imprinting,12 epitope imprinting,13,14 microcontact imprinting,15 metal coordination,16 Pickering emulsion imprinting,17,18 and nanotechnology-based imprinting.19 However, these approaches are not generally applicable since they were not designed on the basis of common features of all proteins or a subclass of proteins. Glycoproteins are a large family of proteins that function significantly in many biological processes such as molecular recognition, cell signaling, immune response, and regulation of cellular development. As the expression of many glycoproteins is associated with the occurrence of diverse diseases, glycoproteins are of significant importance in clinical diagnositics.20 The 96-well microplate-based enzyme-linked immunosorbent assay (ELISA) has been a major platform for high throughput analysis of disease biomarkers in clinical © 2013 American Chemical Society

Received: November 18, 2013 Accepted: December 17, 2013 Published: December 17, 2013 959

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Figure 1. Schematic diagram of boronate affinity-based oriented surface imprinting of glycoproteins.

binding with sugars and glycoproteins in free solution).38 Recently, we have established a universal and straightforward approach for the imprinting of glycoproteins, called photolithographic boronate affinity molecular imprinting.39 The prepared MIPs exhibited several highly attractive features: (1) excellent specificity (the cross-reactivity to interfering proteins was less than 9%), (2) dramatically improved binding strength (enhanced by 6 orders of magnitude as compared with binding in free solution), (3) widely applicable binding pH (from 5.0 to 9.0), and (4) superb tolerance for interference (tolerant for the interference of competing sugars at 1 million time higher concentration). Due to these highly beneficial merits, MIPs prepared by this approach can be promising substitutes for real antibodies for practical applications. However, because this method is based on UV-initiated polymerization, suitable substrates are limited to only an open surface that can well accept UV radiation. Particularly, when this approach is used to fabricate MIP-coated 96-well microplates, it is hard to ensure that the prepolymer solution in each well receives evenly distributed UV radiation and, meanwhile, no MIP can be formed onto the well wall. To facilitate the boronate affinitybased imprinting method for wider applications, a new imprinting approach is very necessary. To overcome the above issue, in this study, we propose a new approach, called boronate affinity-based oriented surface imprinting, for the imprinting of glycoproteins with an emphasis on ELISA application. Figure 1 illustrates the principle of the proposed approach. First, a 96-well microplate is functionalized with a common boronic acid such as 4formylphenylboronic acid at the well surface, including the bottom and the wall. Then, a target glycoprotein is immobilized onto the well surface by virtue of boronate affinity of the boronic acid. After that, a hydrophilic coating with appropriate thickness formed by in-water self-copolymerization of aniline is deposited onto the well surface. After the template is removed by disrupting the boronate affinity binding with an acidic solution, 3D cavities that are complementary to the molecular shape of the template are formed, which can rebind with the target. Using horseradish peroxidase (HRP) as a representative target, we first investigated the effects of imprinting conditions and characterized the properties and performance of the prepared MIP. Then, we further prepared α-fetoprotein (AFP)imprinted microplate and developed a MIP-based ELISA for the analysis of AFP. The prepared MIPs inherited all favorable features of the previous boronate affinity-based molecular imprinting method. Also, they exhibited faster equilibrium kinetics as compared with real antibodies and were reusable for multiple times. The MIP-based ELISA method was finally

applied to determine the concentration of AFP in human serum. The result was in good agreement with that by radioimmunoassay.

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EXPERIMENTAL SECTION Reagents and Materials. AFP and normal human serum was purchased from Shuangliu Zhenglong Biochemical Products Lab (Sichuan, China). HRP-labeled mouse monoclonal anti-AFP antibody, 3,3,5′,5′-tetramethylbenzidine dihydrochloride (TMB) substrate solution (A, B two components), and enzyme-labeled antibody diluent were obtained from Zhongkai Keyue Biotech (Beijing, China). Ovalbumin (OVA) from chicken egg and albumin from bovine serum (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMannose was purchased from Alfa Aesar (Tianjin, China). Glucose, 4-formylphenylboronic acid, sodium cyanoborohydride, and ammonium persulfate (APS) were from J&K Scientific (Beijing, China). 3-Aminopropyltriethoxysilane (APTES) was purchased from Aladdin Reagent (Shanghai, China). Aniline, HRP, H2SO4 (98%), HNO3 (63%), HCl (36%), glacial acetic acid (HAC), anhydrous methanol, NaH2PO4, Na2HPO4, and Tween 20 were of analytical grade and purchased from Nanjing Reagent Company. Ultrapure water used in all experiments was purified by a Milli-Q water purification system (Millipore, Billerica, MA, USA). Instruments. A synergy Mx microplate reader from BioTek (Winooski, VT, USA) was used for the preparation condition optimization, characterization, and ELISA assay. Polystyrene (PS) 96-well microplates (LOT No.165305) from Thermo Fisher Scientific (San Jose, CA, USA) were used for all experiments. Modification of Phenylboronic Acid on the Inner Surface of a 96-Well Microplate. The amino group was first introduced onto the surface of the microplate wells as reported previously.40 Briefly, the wells were filled with a 3:1 (v/v) H2SO4 (98%)/HNO3 (63%) mixture (250 μL/well) and kept at room temperature for 30 min. After being washed with water to achieve a neutral pH, the wells were filled with 5% aqueous APTES solution, pH 6.9 (250 μL/well), and slightly shaken at room temperature for 2 h and then dried by air at 62 °C for another 2 h. Then, the microplate was washed with anhydrous methanol and dried by air. After the wells were filled with 10 mg/mL 4-formylphenylboronic acid dissolved in anhydrous methanol (150 μL/well), the microplate was sealed and slightly shaken at room temperature for 12 h. Subsequently, each well was supplemented with 100 μL of 10 mg/mL sodium cyanoborohydride dissolved in anhydrous methanol, and then, the microplate was sealed again and gently shaken at 960

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phosphate buffer (pH 5.0, 6.0, 7.4, or 8.5) were employed as the samples. Each well was filled with 50 μL of the sample under investigation and incubated for 30 min at room temperature. After washing with 200 mM phosphate buffer containing 0.05% Tween 20 (pH 7.4) for 2−3 times, 200 μL of TMB solution was added and the absorbance at 650 nm was recorded immediately after 5 min of incubation. Interference Experiment. A 100 ng/mL HRP sample dissolved in 200 mM phosphate buffer (pH 7.4) was taken as a control sample, and the interfering substance (OVA, BSA, glucose, mannose) was then spiked in it at a concentration of 1 mg/mL. Each well was filled with 50 μL of the sample being tested and incubated for 30 min at room temperature. After washing with 200 mM phosphate buffer containing 0.05% Tween 20 (pH 7.4) for 2−3 times, 200 μL of TMB solution was added and the absorbance at 650 nm was recorded immediately after 5 min of incubation. Binding Equilibrium. 100 ng/mL HRP dissolved in 200 mM phosphate buffer (pH 7.4) was employed as the sample. Each well was filled with 50 μL of the sample and incubated for different durations at room temperature. After washing with 200 mM phosphate buffer containing 0.05% Tween 20 (pH 7.4) for 2−3 times, 200 μL of TMB solution was added and the absorbance at 650 nm was recorded precisely after 5 min of incubation. Binding Isotherm and Imprinting Efficiency Experiments. HRP samples of different concentrations, dissolved in 200 mM phosphate buffer (pH 7.4), were applied. Each HRPimprinted well was filled with 50 μL of the sample and incubated for 40 min at room temperature. After washing with 200 mM phosphate buffer containing 0.05% Tween 20 (pH 7.4) for 2−3 times, 200 μL of TMB solution was added and the absorbance at 650 nm was recorded precisely after 5 min of incubation. To roughly estimate the HRP amount captured by the phenylboronic acid-functionalized well, each well was incubated with 5 μg/mL HRP (200 mM phosphate buffer, pH 8.5) for 2 h and then washed with 200 mM phosphate buffer (pH 8.5) for 5 times; 200 μL of TMB solution was added, and after 5 min of incubation, absorbance at 650 nm was immediately recorded and averaged as Bused. ELISA of AFP. The procedure for the preparation of AFPimprinted MIP was the same as that for HRP-imprinted MIP at the optimized conditions, except that the template was different. For ELISA, each well was filled with 100 μL of the sample being tested or 200 mM phosphate buffer (pH 7.4) and incubated for 40 min. Then, each well was filled with 100 μL of BSA solution (1 mg/mL dissolved in 200 mM phosphate buffer, pH 7.4) and incubated for 5 min at room temperature. After that, each well was supplemented with 100 μL of HRPlabeled anti-AFP solution (250 ng/mL, diluted 2000-fold from stocking solution) and incubated for 5 min at room temperature. After washing with phosphate buffer (200 mM, pH 7.4, containing 0.05% Tween 20) for 2−3 times, 200 μL of TMB solution was added and the absorbance at 650 nm was recorded after incubation for 2 min.

room temperature for 24 h. Finally, the solutions in the wells were disposed, and the wells were washed with anhydrous methanol for 5−10 times. The modified microplate was dried by air and then kept at 4 °C for later experiments. Characterization of a Phenylboronic Acid-Modified 96-Well Microplate. The HRP capture experiment was carried out to confirm the modification of phenylboronic acid on the inner surface of the 96-well microplate; 150 μL/well of 10 ng/mL HRP dissolved in 200 mM phosphate buffer (pH 4.7 or 8.5) was, respectively, added to unmodified, amino-modified, and phenylboronic-acid modified microplate wells and slightly shaken at room temperature for 30 min. After washing with the same buffer for 5 times, each well was filled with 200 μL of TMB solution (100 μL of A solution and 100 μL of B solution added successively) and gently shaken at room temperature for 10 min. Then, absorbance at 650 nm for each well was measured, and the measurement for each condition was performed in parallel in 3 wells. After the measurement, the microplate was washed successively with 1% HAC and water (250 μL/well) to remove the reactants. Preparation of HRP-Imprinted MIP Layer on the 96Well Microplate. After the wells of a phenylboronic acidmodified 96-well microplate were filled with 100 μL/well of 5 μg/mL HRP dissolved in 200 mM phosphate buffer (pH 8.5), the microplate was sealed and slightly shaken at room temperature for 2 h. Then, each well was washed with 150 μL of 200 mM phosphate buffer (pH 8.5) for 3 times. Each well was filled with 50 μL of aniline of the desired concentration dissolved in 200 mM phosphate buffer at a certain pH and gently shaken at room temperature for 5 min. After each well was supplemented with 50 μL of APS of the desired concentration dissolved in 200 mM phosphate buffer (pH 8.5), the microplate was sealed and slightly shaken at room temperature for the desired time (5−80 min). After polymerization, the microplate was washed successively with water, 5% HAC containing 0.1% Tween 20, and water (250 μL/well each for 3−5 times) to remove reactants, template, and acid. The prepared HRP-imprinted microplate was dried by air and kept at 4 °C for later experiments. For the preparation of NIP (nonimprinted polymer) layer-modified microplate, 100 μL/ well of 200 mM phosphate buffer (pH 8.5) was added instead of HRP solution while the other steps were the same. Effects of Imprinting Conditions. With the above procedure, imprinting conditions, including polymerization pH, the ratio of aniline to APS, reactant concentration, and polymerization time, were investigated and optimized successively, using a set of starting conditions (polymerization pH, 7.4; molar ratio of aniline to APS, 3:1; aniline concentration, 600 mM; polymerization time, 60 min). Template solution of 100 ng/mL HRP containing 200 mM phosphate (pH 7.4) was applied. Each well was filled with 50 μL of template solution and incubated for 30 min at room temperature. After washing with phosphate buffer (200 mM, pH 7.4, containing 0.05% Tween 20) for 2−3 times, 200 μL of TMB solution was added and the absorbance at 650 nm was recorded precisely after 5 min of incubation. For all experiments, 3 wells of the same conditions were prepared and data were collected and averaged. For background subtraction (the same hereinafter) of the MIP or NIP layer itself, the absorbance at 650 nm was collected as background signal. Binding pH. To investigate the dependence of the target binding capability of the MIP at different pH, solutions of 100 ng/mL HRP dissolved in 0.1% HAC (pH 3.5) or 200 mM

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RESULTS AND DISCUSSION Characterization of the Modification of Phenylboronic Acid. The boronic acid functionalization step is a key for the oriented surface imprinting method. Therefore, the phenylboronic acid-modified microplate should be evaluated using an appropriate method. Due to the low density of modified functional groups as well as a high background signal

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thick or too thin, the target binding properties of the imprinted cavities would be unsatisfied. Using HRP as a representative target, the imprinting conditions, including polymerization pH, molar ratio of aniline to APS, concentration of aniline, and polymerization time, were investigated and evaluated in terms of imprinting factor (IF), which was calculated according to the ratio of the absorbance value at 650 nm of HRP captured by MIP over that by NIP. The concentration of the template protein used for immobilization was fixed at 5 μg/mL and was not optimized because concentration ranging within 1−10 μg/ mL is widely applied to coat capture antibody or antigen on the microplate by nonspecific adsorption. Figure 3 shows the absorbance signals for MIP and NIP as well as the IF values at different imprinting conditions. Polymerization at pH 7.4 gave a better imprinting factor and more reproducible signal as compared with pH 8.5. This can be explained because the polymerization reaction at a more basic pH was faster so the controllability was worse. As the ratio of aniline to APS increased, the NIP layer showed gradually reduced HRP adsorption while the MIP layer first exhibited increased HRP adsorption from 2:1 to 3:1 and then the HRP adsorption gradually decreased after the ratio exceeded 3:1. This can be explained by the thickness of the imprinting layer increasing with an increase in the aniline/APS ratio. A thicker imprinting layer can more efficiently prevent nonspecific adsorption. However, if the imprinting layer is too thick, the total number of accessible imprinted cavities becomes less. The best ratio of aniline to APS was found to be 4:1, which provided the best IF value (24.5). The effect of the aniline concentration was more complicated. Too high or too low concentration generated much worse target binding properties. The optimal aniline concentration was found to be 800 mM, giving the best imprinting factor (24.5). After the above imprinting conditions were optimized, the effect of polymerization time seemed simpler. When increasing the polymerization time from 5 to 20 min, the HRP amount captured by the NIP decreased while that by the MIP increased. However, when the polymerization time exceeded 20 min, the HRP amounts captured by both the MIP and NIP were kept nearly constant. The imprinting factor increased as the polymerization time increased from 5 to 20 min but dropped slightly after the polymerization time was over 20 min. At the beginning, we expected that too long of a polymerization time might result in a significantly reduced imprinting factor. This unexpected experimental result suggests that the catalyst APS can last for only a certain period, and within this period, the thickness of the imprinting layer can be controlled by adjusting the polymerization time. The optimal conditions for HRP imprinting were found to be (1) template immobilization: 100 μL/well of 5 μg/mL HRP solution (dissolved in 200 mM phosphate buffer, pH 8.5) for 2 h; (2) oriented imprinting by self-polymerization of aniline: 50 μL/ well of aniline solution (800 mM, dissolved in 200 mM phosphate, pH 7.4) and 50 μL/well of APS solution (200 mM, dissolved in 200 mM phosphate buffer, pH 7.4) for 20 min. Under these conditions, the best IF was found to be 25.8, which was much higher than that provided by the photolithographic imprinting method (9.2).39 The apparent improvement in imprinting factor can be attributed to the fact that there were no boronic acid moieties outside of the imprinted cavities in the current method but there were in the previous method. For the imprinting of AFP, we directly applied the optimized conditions without further optimization and found that these conditions worked very well. However, for other glycoprotein

of polystyrene of blank microplate, IR spectrometry failed to confirm the modification of phenylboronic acid on the microplate (data not shown). We turned to detection of HRP captured by the phenylboronic acid-modifed microplate through TMB colorimetric reaction, which is highly sensitive and capable of detecting trace HRP. Amino-modified or unmodified microplate is capable of noncovalently adsorbing capture antibodies or antigens in alkaline aqueous solution, which is widely used in ELISA to coat capture antibodies or antigen. Under the same conditions, covalent binding should be able to capture more target molecules than noncovalent adsorption. The HRP-captured amount is proportional to the absorbance value of TMB colorimetric reaction in the same time period. As shown in Figure 2, under an alkaline condition

Figure 2. Comparison of HRP capture capability of different modified microplates.

(pH 8.5), the phenylboronic-acid modified microplate captured the highest amount of HRP as compared with the aminomodified and unmodified microplates, while under an acid condition (pH 4.7) the HRP amount captured by the three types of microplates were all very limited. These results are in good agreement with the pH-dependent binding property of boronic acid and suggest that phenylboronic acid groups have been successfully immobilized onto the microplate. Template Removal. Template removal is an important aspect in molecular imprinting. If the template molecules cannot be effectively removed, the number of imprinted cavities formed will be inadequate. Meanwhile, the residual template molecules in the MIP obtained will give rise to strong blank signal. Therefore, the template used for the imprinting should be removed as much as possible. As compared with another imprinting approach, boronate affinity-based imprinting allows for more efficient template removal, because the boronate affinity interaction between template glycoproteins and boronic acid ligands can be broken down easily by using acidic solution. To further facilitate template removal, Tween 20 was added to the acidic template removing solution. Once the template protein was covered by the surfactant, the template molecules became larger in size and thereby became difficult to rebind with the cavities. Using 5% HAC containing 0.1% Tween 20 as the template removing solution, washing the wells for only 3 times (1 min each time) was found sufficient to remove most of the template and the residual template was only about 3% (Figure S1 in the Supporting Information). Effects of Imprinting Conditions. The thickness of the imprinting layer is critical for the properties of the MIPs prepared by the proposed method. If the imprinting layer is too 962

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Figure 3. Effects of imprinting conditions of the target amount captured by the HRP-imprinted MIP and the imprinting factor. (A) Polymerization pH; (B) ratio of aniline to APS; (C) concentration of aniline; (D) polymerization time.

templates, a simple but effective way may be to first employ the optimized conditions reported here and tune the polymerization time. If they do not work well, a de novo optimization should be carried out. Characterization of MIP-Coated Microplate. After the 96-well microplate was processed by the imprinting procedure, a uniform thin layer in light brown was formed on the bottom and wall of each well (Figure S2 in the Supporting Information). Although the MIP layer was not completely transparent, it exhibited weak absorbance at 650 nm, which allowed for UV absorbance detection (Figure S3 in the Supporting Information). Scanning electron microscopic (SEM) images (Figure S4 in the Supporting Information) show that the bare microplate was smooth at the surface while the surface of NIP and MIP-coated microplates, particularly the latter, was rough. On the other hand, the contact angle test (Figure S5 in the Supporting Information) indicates that both MIP and NIP layers were hydrophilic, which further suggests that MIPs prepared using polyaniline as the imprinting layer could provide good specificity. Binding pH. The affinity of boronic acids toward cis-diol containing compounds is pH dependent. Usually, for substantial binding, the surrounding pH should be ≥ the pKa value of the boronic acid used. For commonly used boronic acids, such as phenylboronic acid (pKa 8.8), boronic acidfunctionalized materials can work only within alkaline pH range. This is an apparent disadvantage of conventional boronate affinity materials, because biomolecules may degrade at alkaline pH, while adjusting the pH of real samples

particularly blood samples is inconvenient. In order to overcome this issue, many efforts have been made to reduce the binding pH of boronate affinity materials by using boronic acids with special structures28−30 or boronic acid teams.33−35 We have reported previously that the binding pH of boronate affinity-based MIPs can be as low as pH 5.0 even using a common boronic acid as a functional monomer.39 This is a highly desired feature. Since such a phenomenon was unusual, it is very necessary to further investigate whether it is just a special case or a universal rule. Figure 4 shows the target

Figure 4. Target binding capability of HRP-imprinted MIP and NIP at different pH. Sample: 100 ng/mL HRP dissolved in 0.1% HAC (pH 3.5) or 200 mM phosphate buffer (pH 5.0, 6.0, 7.4, or 8.5). 963

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MIP-coated microplate showed a faster equilibrium, taking only 40 min to reach equilibrium. This result indicates that the mass transfer speed in the MIPs prepared by the proposed method was fast and the prepared MIP-coated microplates are applicable to ELISA. Binding Isotherm and Imprinting Efficiency. The binding isotherms for HRP-imprinted MIP and NIP were investigated. As shown in Figure 7A, the binding isotherm

binding capability of HRP-imprinted MIP and NIP at different pH values. It can be seen that under all pH conditions the NIP exhibited very limited HRP binding capability while the MIP exhibited significant target binding capability within the pH range of 5.0−8.5. Thus, it was confirmed that boronate affinitybased MIPs can provide a widely applicable binding pH. Such a unique feature makes boronate affinity-based MIPs very promising for real sample applications since the pH of most frequently used biological samples such as blood, saliva, and tears are in such a pH range. Tolerance for Interference. We have demonstrated previously that boronate affinity-based MIPs were tolerant of severe interference. We further examined such a highly attractive feature using HRP-imprinted microplate. Figure 5

Figure 5. Target binding capability of HRP-imprinted MIP and NIP in the presence of different interfering agents. Sample: 100 ng/mL HRP containing 200 mM phosphate (pH 7.4) without or with 1 mg/mL of the specified interfering agent. The absorbance in the absence of interfering agent (control) was taken as 100%, and the percentage of the signals for other situations over the control were calculated accordingly and shown in the figure.

shows that, even under the presence of interfering agents such as OVA, BSA, glucose, and mannose at 10 000-fold higher concentration, the MIP still remained at least 60% of its original target binding capability. This result suggests that the asprepared MIP could be applied to complex biological samples. Binding Equilibrium. Binding dynamics of MIPs is an important factor for their feasibility of practical applications. In real antibody-based ELISA, the incubation within a 96-well microplate usually takes 1 h at 37 °C. As shown in Figure 6, the

Figure 7. (A) Binding isotherms for HRP-imprinted MIP and NIP. (B) Binding constant fitting according to the Hill equation.

showed a good linearity within 0−100 ng/mL (R2 = 0.99), which provided a solid basis for quantitative analysis. To determine the binding strength, the Hill equation41 as shown below was applied to fit the data for MIP. y = Bmax x n/(x n + Kd n)

(1)

where Bmax is the maximum specific binding, Kd is the dissociation constant, and n is Hill slope; when n > 1, it means positively cooperative binding. The measured Kd value was 1.2 nM or 54.3 ng/mL (R2 = 0.98, Bmax = 0.59, n = 1.33). The experimental binding strength was better than that by the previous method for the same template (85 nM).39 This was likely due to the fact that the formation of imprinted cavities was better controlled by the current method. The nM level binding constant of the prepared MIP was comparable to that of real antibodies (usually 10−7−10−9 M). The imprinting efficiency was estimated by comparing the maximum HRP amount captured by the MIP over the HRP amount used for the imprinting. The absorbance at 650 nm (Bused) after the phenylboronic acid-functionalized well was saturated with HRP was measured to be 3.13 while the maximum signal from the above Hill equation fitting (Bmax) was 0.59. Thus, the imprinting efficiency was calculated to be 18.7%. Such a value was lower than that provided by the

Figure 6. Binding equilibrium of HRP-imprinted MIP. Sample: 100 ng/mL HRP containing 200 mM phosphate, pH 7.4. 964

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determined to be 12 ± 2.0 ng/mL (Figure S7 in the Supporting Information), which is in good agreement with the value by radioimmunoassay by the supplier (10 ng/mL).

photolithographic imprinting.39 This can be attributed to the lower polymerization pH (7.4) in this study. During polymerization under such a condition, some immobilized target may have dissociated from the microplate surface because pH 7.4 was not the best binding pH for phenylboronic acid. Therefore, in future studies, a more favorable boronic acid that can bind glycoproteins around neutral pH, such as benzoboroxole,29 will be a better choice. Reusability. Usually, the microplates for ELISA are designed for disposable use. The reusability of the MIP-coated microplate prepared by the proposed method was evaluated. As shown in Figure S6 in the Supporting Information, the MIPcoated microplate could be reused at least 3 times. MIP-Based ELISA. Because of the excellent binding properties demonstrated above, the MIPs prepared by the proposed method can be promising substitutes for real antibodies for practical applications. To demonstrate such a feasibility, AFP-imprinted microplate was prepared using the above optimized conditions and the prepared microplate was used for sandwich ELISA of AFP. AFP has been routinely used as a biomarker in clinical screening for liver cancer.42 The procedure of the sandwich ELISA is shown in Figure 8A. It

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CONCLUSIONS In this work, a new version of the boronate affinity molecular imprinting method, called boronate affinity-based oriented surface imprinting, has been developed to prepare highperformance MIPs for ELISA of glycoproteins. The new method inherited all merits of the previous version39 and meanwhile overcame its limitation and thereby was applicable to wider substrates. Moreover, because the imprinting layer contained no boronic acid moieties, the current method provides better specificity. The prepared MIPs exhibited fast incubation equilibrium and reusability. The usefulness of the MIP-coated 96-well microplate in ELISA analysis has been well demonstrated by the analysis of AFP in human serum. The method can be expanded to other glycoprotein disease biomarkers. It is worth pointing out that, to improve the linear detection range of the MIP-based ELISA, the total binding capacity of each well should be increased, which can be realized by roughening the well wall.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 25 8368 5639. Fax: +86 25 8368 5639. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the Ministry of Science and Technology of China (Grant No. 2013CB911202), the National Natural Science Foundation of China (Grant Nos. 21275073, 21075063, and 21121091), and the Natural Science Foundation of Jiangsu Province, China (Grant No. KB2011054). We greatly appreciate the kind assistance of Ms. Zhiping Zhou for SEM characterizaiton.

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Figure 8. Sandwich ELISA based on AFP-imprinted MIP. (A) Procedure of the sandwich ELISA. (B) Response curve. Inset: linear fit within the range of 0−50 ng/mL, R2 = 0.93.

includes the following major steps: (1) incubation of the sample in the MIP-coated well; (2) blocking the remaining area of the MIP layer with BSA; (3) incubation with HRP-labeled anti-AFP IgG; (4) staining with TMB solution; and (5) measurement of the absorbance at 650 nm. The purpose of the BSA blocking step was to prevent nonspecific interaction between HRP-labeled IgG and unoccupied cavities. The response curve for AFP in phosphate buffer is shown in Figure 8B. Within the range of 0−50 ng/mL, it showed a good linearity (R2 = 0.93). By using the standard addition method, the AFP concentration in a human serum sample was 965

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