Langmuir 2008, 24, 5773-5780
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Thermosensitive and Salt-Sensitive Molecularly Imprinted Hydrogel for Bovine Serum Albumin ZhenDong Hua, ZhiYong Chen, YuanZong Li, and MeiPing Zhao* Beijing National Laboratory for Molecular Sciences (BNLMS), The Key Laboratory of Bioorganic Chemistry & Molecular Engineering, College of Chemistry and Molecular Engineering, Peking UniVersity, Beijing 100871, China ReceiVed December 18, 2007. ReVised Manuscript ReceiVed February 22, 2008 A novel stimuli-responsive protein imprinted polymer for selective recognition of bovine serum albumin is presented. N-[3-(Dimethylamino)propyl]-methacrylamide, which is positively charged in neutral solution and is able to selfassemble onto the template protein through electrostatic interaction, was chosen as the functional monomer. Polymerization was carried out in the presence of N-isopropylacrylamide as an assistant monomer, which resulted in a stimuliresponsive protein imprinted polymer. The template proteins were easily removed by treatment with 500 mmol L-1 NaCl solution. The influences of the external stimuli, such as temperature and ionic strength, on the polymer affinity were investigated, and a clear conformational memory was observed. The association constant (Ka) and binding capacity (Qmax) for the specific interaction between the protein imprinted polymer and the template protein were determined by Scatchard plots and found to be 9.6 × 104 L mol-1 and 4.7 µmol g-1, respectively. Several proteins different in molecular weight and isoelectric point were employed as reference, and it was shown that the charge effect and the shape memory effect were the major factors affecting the imprint formation and template recognition. Finally, this imprinted polymer was used to purify the bovine serum albumin from the protein mixture and real sample, which demonstrated its high selectivity.
Introduction Molecular imprinting is a technique which creates an affinity matrix toward template molecules by copolymerization of the functional monomers and cross-linkers around them. Upon removal of the template species, cavities are formed in the polymer matrix, which are chemically and sterically complementary to the template.1 The imprinting of small molecules has achieved great success since the noncovalent method and covalent method were formulated by Mosbach and Wulff, respectively.2–5 In recent years, much attention has been paid to the imprinting of biomacromolecules and in particular proteins for its potential applications as biomaterials for separations, biosensors, and mimicking enzymes and antibodies.6 Compared with the affinity matrices prepared using cell receptors or mono- or polyclonal antibodies, the man-made protein imprinted polymers offer a highly stable, labor-extensive, cost-effective, and time-saving alternative to the existing techniques. However, the flexible structure, water-solubility, and number of functional groups of the protein make it a challenging task to fabricate accessible binding sites with high specificity and affinity toward the target protein.7,8 Many artifices have been adopted to overcome these problems, such as surface imprinting8–11 and epitope mediated * To whom correspondence should be addressed. Fax: +86-10-62751708. E-mail:
[email protected]. (1) Haupt, K.; Mosbach, K. Chem. ReV. 2000, 100, 2495–2504. (2) Norrlow, O.; Glad, M.; Mosbach, K. J. Chromatogr. 1984, 299, 29–32. (3) Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645–647. (4) Wulff, G.; Sarhan, A. Angew. Chem., Int. Ed. Engl. 1972, 11, 341–344. (5) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812–1832. (6) Bossi, A.; Bonini, F.; Turner, A. P. F.; Piletsky, S. A. Biosens. Bioelectron. 2007, 22, 1131–1137. (7) Mallik, S.; Plunkett, S. D.; Pradeep, K. D.; Johnson, R. D.; Pack, D.; Shnek, D.; Arnold, F. H. New J. Chem. 1994, 18, 299–304. (8) Bossi, A.; Piletsky, S. A.; Piletska, E. V.; Righetti, P. G.; Turner, A. P. F. Anal. Chem. 2001, 73, 5281–5286. (9) Shiomia, T.; Matsuia, M.; Mizukamib, F.; Sakaguchia, K. Biomaterials 2005, 26, 5564–5571.
imprinting.12,13 Also, polyacrylamide gels14–16 are often used for protein imprinting in aqueous solution with a low degree of cross-linking to ensure the mobility of large molecules, but difficulty in removing the templates and stability of the gel still seem to be the limitations. The sensitive hydrogels first proposed by Tanaka and coworkers are regarded as excellent materials for controlled uptake and release of molecules.17–19 Their stimuli-response to external stimuli makes it possible to alter their volume as well as their affinity to the target molecules by changing the environmental conditions, such as temperature,17–19 solvent composition,20 pH,21,22 ionic strength,23,24 light,25,26 and specific chemicals.27 This property can be introduced into the molecularly imprinted (10) Li, Y.; Yang, H. H.; You, Q. H.; Zhuang, Z. X.; Wang, X. R. Anal. Chem. 2006, 78, 317–320. (11) Shi, H. Q.; Tsai, W. B.; Garrison, M. D.; Ferrari, S.; Ratner, B. D. Nature 1999, 398, 593–597. (12) Nishino, H.; Huang, C. S.; Shea, K. J. Angew. Chem., Int. Ed. 2006, 45, 2392–2396. (13) Tai, D. F.; Lin, C. Y.; Wu, T. Z.; Chen, L. K. Anal. Chem. 2005, 77, 5140–5143. (14) Hjerten, S.; Liao, J. L.; Nakazato, K.; Wang, Y.; Zamaratskaia, G.; Zhang, H. X. Chromatographia 1997, 44, 227–234. (15) Ou, S. H.; Wu, M. C.; Chou, T. C.; Liu, C. C. Anal. Chim. Acta 2004, 504, 163–166. (16) Kimhi, O.; Bianco-Peled, H. Langmuir 2007, 23, 6329–6335. (17) Alvarez-Lorenzo, C.; Guney, O.; Oya, T.; Sakai, Y.; Kobayashi, M.; Enoki, T.; Takeoka, Y.; Ishibashi, T.; Kuroda, K.; Tanaka, K.; Wang, G. Q.; Grosberg, A. Y.; Masamune, S.; Tanaka, T. J. Chem. Phys. 2001, 114, 2812– 2816. (18) Alvarez-Lorenzo, C.; Hiratani, H.; Tanaka, K.; Stancil, K.; Grosberg, A. Y.; Tanaka, T. Langmuir 2001, 17, 3616–3622. (19) Hiratani, H.; Alvarez-Lorenzo, C.; Chuang, J.; Guney, O.; Grosberg, A. Y.; Tanaka, T. Langmuir 2001, 17, 4431–4436. (20) Katayama, S.; Ohata, A. Macromolecules 1985, 18, 2781–2782. (21) Philippova, O. E.; Hourdet, D.; Audebert, R.; Khokhlov, A. R. Macromolecules 1997, 30, 8278–8285. (22) Zhang, J.; Yuan, K.; Wang, Y. P. J. Bioact. Compat. Polym. 2007, 22, 207–218. (23) Kumar, A.; Galaev, I. Y.; Mattiasson, B. Biotechnol. Bioeng. 1998, 59, 695–704. (24) Yang, J.; Fang, L.; Wang, F.; Tan, T. W. J. Appl. Polym. Sci. 2007, 105, 539–546. (25) Suzuki, A.; Tanaka, T. Nature 1990, 346, 345–347.
10.1021/la703963f CCC: $40.75 2008 American Chemical Society Published on Web 05/07/2008
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polymer (MIP) by polymerization in the presence of a thermosensitive monomer. Namely, the MIP consists of a major thermosensitive monomer component that allows for swelling and shrinking of the polymers and a minor functional monomer component that captures target molecules via multiple-point electrostatic interaction. By self-assembly of the functional monomers and the template molecules, the functional groups will be placed with a low-energy spatial arrangement in the flexible polymer chains and the resulting MIP has the global energy minimum in the original conformation. In contrast to the traditional MIP with a rigid matrix, the relative position of the functional groups can be controlled via the stimuli-response of the sensitive hydrogel matrix. The recognition of the template will be specific when the MIP maintains a 3D structure similar to the imprinting state, whereas the memory of the template will be lost when external stimuli break the imprinting state and lead to shrinkage or swelling of the MIP. By this approach, the metal ions28,29 and small molecules30,31 are successfully imprinted. Also, a sensitive imprinted hydrogel for bovine serum albumin (BSA) was reported by Caykara and co-workers.32 The imprinted hydrogel showed higher adsorption capacity for the template compared with a hydrogel prepared by the usual procedure. However, when studying the effect of temperature, the conformational memory against temperature was not observed. The reason may be that the functional monomer (maleic acid) and template (BSA, pI 4.8) they used are both negatively charged in neutral solution and the hydrogen bonds existing between them is very weak. In this work, a sensitive protein-imprinted hydrogel was synthesized in aqueous solution using BSA as the template. In order to form a stable complex with the template molecules via multiple-point electrostatic interaction, N-[3-(dimethylamino) propyl]methacrylamide (DMAPMA) with a tertiary amine group which is positively charged in neutral solution was chosen as the functional monomer. The assistant monomer N-isopropylacrylamide (NIPA) was introduced as a thermosensitive element. NIPA is widely used to fabricate sensitive hydrogels, and the choice is motivated by its inert nature, biocompatibility, and neutrality. Nonspecific interactions with proteins are expected to be minimized with this matrix. Such a polymer was easily synthesized by chemical oxidation of a mixture of NIPA, DMAPMA, N,N-methylenebisacrylamide (BIS), and also the template protein in Tris-HCl buffer (pH 7.0). The resultant polymer showed sensitive responses to both temperature and ionic strength and a clear conformational memory of the template protein. The notable selectivity and binding capacity of the stimulisensitive MIP were also demonstrated via application to synthetic protein mixtures and real serum samples. (26) Mamada, A.; Tanaka, T.; Kungwatchakun, D.; Irie, M. Macromolecules 1990, 23, 1517–1519. (27) Hoffman, A. S.; Stayton, P. S.; Bulmus, V.; Chen, G. H.; Chen, J. P.; Cheung, C.; Chilkoti, A.; Ding, Z. L.; Dong, L. C.; Fong, R.; Lackey, C. A.; Long, C. J.; Miura, M.; Morris, J. E.; Murthy, N.; Nabeshima, Y.; Park, T. G.; Press, O. W.; Shimoboji, T.; Shoemaker, S.; Yang, H. J.; Monji, N.; Nowinski, R. C.; Cole, C. A.; Priest, J. H.; Harris, J. M.; Nakamae, K.; Nishino, T.; Miyata, T. J. Biomed. Mater. Res. 2000, 52, 577–586. (28) Hendri, J.; Hiroki, A.; Maekawa, Y.; Yoshida, M.; Katakai, R. Radiat. Phys. Chem. 2001, 60, 617–624. (29) Morris, G. E.; Vincent, B.; Snowden, M. J. J. Colloid Interface Sci. 1997, 190, 198–205. (30) Liu, X. Y.; Guan, Y.; Ding, X. B.; Peng, Y. X.; Long, X. P.; Wang, X. C.; Chang, K. Macromol. Biosci. 2004, 4, 680–684. (31) Liu, X. Y.; Ding, X. B.; Guan, Y.; Peng, Y. X.; Long, X. P.; Wang, X. C.; Chang, K.; Zhang, Y. Macromol. Biosci. 2004, 4, 412–415. (32) Demirel, G.; Ozcetin, G.; Turan, E.; Caykara, T. Macromol. Biosci. 2005, 5, 1032–1037.
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Experimental Section Materials. N-Isopropylacrylamide (NIPA) and N-[3-(dimethylamino)propyl]-methacrylamide (DMAPMA) were purchased form Aldrich. Acrylamide (AAm; ultrapure bioreagent) and Tris (base) (ultrapure bioreagent) were purchased from J.T. Baker (Phillipsburg, NJ). N,N-Methylenebisacrylamide (BIS), ammonium persulfate (APS), and N,N,N′,N′-tetramethylethylenediamine (TEMED) were obtained from Sigma. Bovine serum albumin (BSA; MW 66 kDa, pI 4.8), ovalbumin (OVA; MW 44 kDa, pI 4.7), hemoglobin (Hb; MW 64.5 kDa, pI 6.8-7.0), lysozyme (Lys; chicken egg white, MW 14.4 kDa, pI 10.7), myoglobin (Mb; MW 17.5 kDa, pI 6.8-7.2), and human serum albumin (HSA; MW 69 kDa, pI 4.8) were obtained from Sigma. Deionized water was produced by a Millipore water system composed of Milli-RO 60 and Milli-Q SP. All other chemicals used in this study were of analytical reagent grade or better. Preparation of Imprinted and Nonimprinted Hydrogels. NIPA (400 mg, 3.5 mmol), AAm (5.0 mg, 0.070 mmol), DMAPMA (16 µL, 0.085 mmol), BIS (18.0 mg, 0.117 mmol), 10% APS (50 µL), and template protein BSA (100 mg, 0.0015 mmol) were dissolved in 2 mL of Tris-HCl buffer solution (10 mmol L-1, pH 7.0). The solution was deoxygenated by purging with nitrogen for 5 min, and a volume of 5 µL of TEMED was added. The solutions were then immediately placed into the space between two glass plates (Biorad, Miniprotein-3) with an interval of 0.75 mm. The polymerization was carried out at 35 °C for 3 h. After the reaction, the resultant hydrogels were cut into disks with a diameter of 10 mm. The disks were washed first with deionized water twice to remove the unreacted monomers and then with 100 mL of NaCl (500 mmol L-1) and deionized water three times each to elute the template protein. The removal of template protein from the imprinted hydrogels was confirmed by using a UV/vis spectrophotometer at 280 nm (Varian, Cary-1E). The unreacted monomers, which have little adsorption at 280 nm, were proven to be entirely washed away by the deionized water in the first two washing cycles and could not be detected in the template protein eluate by HPLC. After the washing process, the hydrogel disks were equilibrated in deionized water at room temperature for at least 48 h then and dried under vacuum (60 °C). The corresponding nonimprinted control polymer (NIP) was generated in the same way but in the absence of the template protein. The influences of the functional monomer-to-template ratio, crosslinking degree, and polymerization temperature on the property of the resultant MIP were all investigated and optimized. Characterization of the Hydrogels. The hydrogels were observed on an environmental scanning electron microscope (ESEM; FEI Quanta 200FEG). The hydrogels containing water without drying were mounted on metal stubs and at low vacuum degree (∼10-3 atm) and relatively low temperature (near 4 °C). Transition temperatures of the MIP and NIP were measured by thermal analysis with a differential scanning calorimeter (DSC; TA Instruments, Q100). In the DSC analysis, about 5-7 mg of a sample was used, and pure water was adopted as the reference. The heating rate was 2 °C/min. All samples were analyzed under a 20 mL/min continuous flow of dry nitrogen gas. Determination of Equilibrium Swelling Ratio. For the ionresponsive studies, the hydrogels were incubated in Tris-HCl buffer solution (10 mmol L-1, pH 7.0) with different NaCl concentrations ranging from 1 to 10 mmol L-1 for 24 h at 25 °C. The gravimetric method was employed to study the hydrogel swelling ratio. After equilibrium, the hydrogels were removed from the buffer solution and blotted with filter paper for the removal of excess water on the surface. They were then weighed, and the swelling ratio was calculated from the following formula:32
swelling ratio ) (Ws - Wd) ⁄ Wd where Ws and Wd are the weights of the swollen and dry hydrogels, respectively. Rebinding and Measurement of the Dissociation Constant. Protein solutions were prepared in Tris-HCl buffer (10 mmol L-1, pH 7.0) with desired NaCl concentrations. About 5 mg of the dry hydrogels was first incubated in Tris-HCl buffer solution (10 mmol
MIP Imprinted Hydrogel for BSA Recognition L-1, pH 7.0) and then added into 8 mL of protein solutions and allowed to equilibrate for 24 h at the desired temperature. The supernatant solutions were then removed, and their concentrations were measured by using the UV/vis spectrophotometer. The amount of adsorbed protein was calculated from the following formula:
Q)
(c0 - c)V M
where Q is the amount of protein adsorbed onto a unit mass of dry gel (mg g-1), c0 and c are the concentrations of protein in the initial and equilibrium solutions, respectively (mg mL-1), V is the volume of the solutions treated (mL), and M is the mass of dry gel (g). The hydrogels were then incubated with 8 mL of 500 mmol L-1 NaCl solutions for 24 h, and the protein concentrations of supernatant solutions were also measured by using the UV/vis spectrophotometer to confirm the complete removal of the proteins adsorbed. All of the measurements were made in triplicate. To investigate the effect of temperature, the concentration of BSA was fixed at 0.5 mg mL-1, and no NaCl was added. The hydrogels were incubated in solutions at temperatures ranging from 20 to 50 °C controlled by a thermostat water bath. To investigate the influence of ionic strength, different concentrations of NaCl varying from 1 to 10 mmol L-1 were tested with the concentration of BSA and the temperature fixed at 0.5 mg mL-1 and 25 °C, respectively. To investigate the selectivity of the hydrogels, 0.5 mg mL-1 protein solutions of ovalbumin, hemoglobin, lysozyme, myoglobin, and human serum albumin were prepared with 1 mmol L-1 NaCl, and the equilibrium temperature was also controlled at 25 °C. The competitive adsorption was performed with a protein mixture containing 0.5 mg mL-1 BSA and 0.5 mg mL-1 OVA in Tris-HCl buffer (10 mmol L-1, pH 7.0) containing 1 mmol L-1 NaCl at 25 °C. The hydrogel was applied to purify BSA from real samples of bovine calf serum. The serum was 10-fold diluted with Tris-HCl buffer (10 mmol L-1, pH 7.0) containing 1 mmol L-1 NaCl and equilibrated at 25 °C. The hydrogels were then treated with TrisHCl buffer (10 mmol L-1, pH 7.0) containing 3 mmol L-1 NaCl to wash out the nonspecifically adsorbed proteins and then 500 mmol L-1 NaCl to elute the specifically adsorbed proteins. The eluates were desalted and 10-fold concentrated by using an ultra filtration membrane (molecular weight cutoff 3000), and then 10 µL of each sample was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis using 12.5% polyacrylamide MiniGels (Biorad, Miniprotein-3). To measure the dissociation constant for the template protein to the hydrogels, BSA solutions at concentrations of 0.15, 0.30, 0.50, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 mg mL-1 were prepared in TrisHCl buffer (10 mmol L-1, pH 7.0) containing 1 mmol L-1 NaCl. After the rebinding experiment, the data obtained were linearized with a variation of the Scatchard-Rosenthal plot, using the following equation:
Q Qmax Q ) c Kd Kd where Q and Qmax are the amount of protein adsorbed onto a unit mass of dry gel and the maximum adsorption capacity of a unit mass of dry gel, respectively (µmol g-1), c is the concentration of protein in equilibrium solutions (µmol L-1), and Kd is the dissociation constant for the template protein to the hydrogels.
Results and Discussion Synthesis of the Protein Imprinted Hydrogels. At pH 7.0, there are about 18 net negative charges for each BSA molecule,33,34 and thus, functional monomers with the same amount of positive charges may self-assemble onto the template protein (33) Peters, T. AdV. Protein Chem. 1985, 37, 161–245. (34) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203.
Langmuir, Vol. 24, No. 11, 2008 5775 Table 1. Efficiency of Template Removal of MIPs of Different Degrees of Cross-Linking percentage of template proteins washed out, %
degree of cross-linkinga
cycle 1
cycle 2
cycle 3
total
30% 10% 4.5% 2%
42.9 66.4 93.4 96.5
0.8 1.7 1.2 0.7
N/A N/A N/A N/A
43.7 68.1 94.6 97.2
a The degree of cross-linking is defined as the mass fraction of the crosslinking agent.
through electrostatic interaction. Different functional monomerto-template ratios were tested, and a molar ratio between 20:1 and 80:1 was proven to be appropriate to ensure the formation of stable protein-monomer complexes via multiple-point electrostatic interactions. Too many excessive functional monomers may lead to extensive nonspecific recognition sites. In the following experiments, the functional monomer-to-template molar ratio was fixed at 64:1. After polymerization, the MIP was washed with Tris-HCl buffer (pH 7.0) containing 0.5 mol L-1 NaCl to remove the templates. The template removal efficiency of MIPs with different cross-linking degrees is compared in Table 1. For the highly cross-linked MIPs (30% and 10%), a large amount of template proteins remained in the matrix and this quantity could hardly be decreased with more washing cycles. By contrast, most of the template proteins were extracted from the low cross-linked MIPs (4.5% and 2%), suggesting that relatively large pore sizes of the polymer networks are favorable for diffusion and transfer of proteins. It should also be mentioned that the extraction of template proteins was almost accomplished in the first washing cycle and few could be detected in the next two cycles. This high efficiency of template removal was attributed to the weakening of the electrostatic force between the protein and the functional monomer and the shrinkage of the soft 3D cross-linked polymer matrix, both of which were caused by the high ionic strength of the washing solution. Fortunately, this neutral solution was helpful to keep the activity of the protein, which made the recovery and reuse of the template protein possible. On the basis of this experiment, 4.5% was chosen as the cross-linking degree for the polymer preparation in order to obtain an MIP with fast mass transfer and easy removal of the template proteins. These weakly cross-linked polymer gels were of enough mechanical strength for the experiments, and also a satisfactory imprinting effect could be observed in the rebinding of the template proteins. The influence of the polymerization temperature was investigated by preparation of four imprinted polymers with the same amount of reagents but at different temperatures varying from 20-35 °C. Nonimprinted polymers were synthesized without template protein to provide a reference. After the washing and desiccation procedures, rebinding of BSA from aqueous solutions was performed at their synthetic temperatures to maintain the state of imprinting. The results shown in Figure 1 indicate that the ability of the MIP to rebind BSA increased with increasing polymerization temperature, while that of the NIP remained almost the same. We infer that, in a free radical initiated polymerization, a higher polymerization temperature will result in shorter polymer chains and thus a more rigid polymer network, which is helpful to maintain the spatial integrity after template removal. The adsorption of the NIP is nonspecific, so it is not affected by this factor. The MIP prepared at a temperature higher than 35 °C was also tried, but the polymer became very crispy when dried and was not suitable for practical applications. Hence, 35 °C was adopted as the polymerization temperature in the subsequent experiments.
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Figure 1. Influence of polymerization temperature on the rebinding ability of the MIP and NIP to BSA. Rebinding measurements were performed in Tris-HCl buffer (10 mmol L-1, pH 7.0) using 0.5 mg mL-1 BSA at the temperature they were synthesized.
Figure 2. DSC heating thermograms for MIP and NIP. Heating rates were 2 °C/min.
Characterization of the Protein Imprinted Hydrogels. The thermosensitive characters of the MIP and NIP were determined by recording the enthalpy of transition using differential scanning calorimetry (DSC), and the curves are shown in Figure 2. As the temperature increased from 25 to 90 °C, the MIP presented an endothermal peak with a lower critical solution temperature (LCST) of 59.4 °C, while the NIP showed an endothermal peak with a LCST of 55.6 °C. The broad endothermal peak showed that the transition became broader with introduced DMAPMA, which indicated a continuous shrinking with the increase of temperature. The LCST of pure poly(N-isopropylacrylamide) is about 32 °C.35 The increase of the LCST resulted from the involvement of the hydrophilic monomer DMAPMA, which contains a tertiary amine group. The hydrophilic monomer DMAPMA strongly influenced changes in the hydrophilic/hydrophobic nature of the polymer, which hindered the dehydration of the polymer chains and acted to expand the collapsed structure.35,36 However, the (35) Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32, 7370–7379. (36) Stile, R. A.; Healy, K. E. Biomacromolecules 2001, 2, 185–194.
Figure 3. ESEM images of MIP (A) and NIP (B) in Tris-HCl buffer (10 mmol L-1, pH 7.0).
MIP presented a higher LCST than the NIP, although the content of the monomer DMAPMA was the same for them. We inferred that, through the imprinting process, many tertiary amine groups were assembled around the cavity created by the BSA. This distribution was then fixed through the polymerization. After removal of the template protein, these tertiary amine groups were exposed to the solvent and such an ordered distribution led to a higher LCST. On the other hand, in the NIP, the tertiary amine groups were distributed randomly. So, the LCST was relatively low. The difference of the LCST between the MIP and NIP proves the fabrication of microstructures led by imprinting, which is the foundation of the specific recognition. In order to study the detailed morphology of the MIP and NIP in the wet state, environmental scanning electron microscopy was employed and the images are shown in Figure 3. Both of them show a rough surface and thus a high specific surface area, which is beneficial for the adsorption of the proteins. Also, some micrometer sized interstitial pores could be seen on the surfaces, which could act as the passages of the proteins and accelerate the adsorption kinetics.
MIP Imprinted Hydrogel for BSA Recognition
Figure 4. Effect of temperature on the swelling ratio (A) and BSA adsorption (B) of the MIP and NIP. The swelling ratio was determined by incubating the hydrogels in Tris-HCl buffer solution (10 mmol L-1, pH 7.0) without BSA. Readsorption was performed in Tris-HCl buffer (10 mmol L-1, pH7.0) using 0.5 mg mL-1 BSA.
Influences of Temperature and NaCl Concentration on the Specific Rebinding of the Template Protein. Smart hydrogels can undergo a reversible volume transition between the swollen and collapsed phases, which can be triggered by external stimuli such as temperature, solvent composition, pH, ionic strength, light, and specific chemicals. The MIP synthesized with NIPA monomer may also respond to these factors. The effects of temperature and ionic strength on the adsorption of BSA were investigated for both MIP and NIP prepared under 35 °C. Figure 4 shows the effect of temperature on the swelling ratio and BSA adsorption of MIP and NIP, respectively. As shown in Figure 4B, the adsorption capacity of the MIP first increased and then decreased as the temperature increased from 20 to 50 °C. From both Figure 4A and the DSC experiment, it could be seen that a continuous shrinking happened in this process, and thus, the cavity formed by imprinting also underwent a volume change. The maximum adsorption was around 40 °C with a swelling ratio of 6.7, and we inferred that at this temperature the cavity came to the state of imprinting. By the conformational memory, the shape of the cavity and the distribution of the charged groups should accord with the protein at this state accurately,
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Figure 5. Effect of NaCl concentration on the swelling ratio (A) and BSA adsorption (B) of the MIP and NIP. The swelling ratio was determined by incubating the hydrogels in Tris-HCl buffer solution (10 mmol L-1, pH 7.0) without BSA at 25 °C. Readsorption was performed in Tris-HCl buffer (10 mmol L-1, pH 7.0) using 0.5 mg mL-1 BSA at 25 °C.
and thus, a highest affinity was obtained.37 This temperature was slightly higher than the temperature at which the polymerization was carried out, which could be explained by the swelling of the hydrogel after the removal of the template protein and also the unreacted monomers. When the temperature reached 50 °C, the collapse of the polymer networks led to destruction of the spatial integrity of the recognition site and the specific binding of the template could hardly be observed compared with the case of the NIP. The NIP showed a slight increase of adsorption capacity with increasing temperature, which was attributed to the enhancement of the hydrophobic interactions between the proteins and the polymer chains. Temperatures above 50 °C were tried, but the deposition of BSA was observed, which indicated the thermal denaturation of the protein. Salt ions also play an important role in the recognition process in aqueous solutions, which can screen the electrostatic interactions between the charged polymer chains and protein molecules. The swelling ratio of the MIP and NIP and their ability to rebind the template protein were measured in Tris-HCl buffer with different NaCl concentrations varying from 0 to 10 mmol L-1 (Figure 5). As the pH of the buffer was set at 7.0, the polymer chains with tertiary amine groups were positively charged while the template protein BSA (pI 4.8) was negatively charged, which (37) Aburto, J.; Borgne, S. L. Macromolecules 2004, 37, 2938–2943.
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Table 2. Ka and Qmax Calculated from the Curves of the Scatchard Plot polymer a
MIP-H MIP-Lb NIP
Ka (mol
-1
9.6 × 104 2.1 × 104 2.3 × 104
)
Qmax (µmol g-1)
R2
4.7 7.8 6.0
0.981 0.989 0.934
a MIP-H: Left slope of the biphasic curve of MIP with high association constant. b MIP-L: Right slope of the biphasic curve of MIP with low association constant.
led to the electrostatic attraction between the polymer and the protein and also the electrostatic repulsion between the polymer chains. When the salt concentration increased, repulsive electrostatic interactions between the polymer chains were gradually screened and a similar degree of shrinkage could be observed for both the MIP and NIP. However, the dependence of their rebinding of BSA on the salt concentration showed a quite different trend. As we know, specific recognition sites were formed in the imprinting process, but in NIP the functional groups were distributed randomly. As shown in the previous experiment, the MIP showed the maximum adsorption of the template protein when the swelling ratio was 6.7. On the one hand, an increase of the NaCl concentration led to shrinkage of the MIP which helped to maintain the imprinting state; on the other hand, the electrostatic interaction was weakened. At NaCl concentrations below 2 mmol L-1, the MIP showed almost constant affinity to the template proteins via multiple interactions. Contrarily, the NIP exhibited a faster decrease of affinity than the MIP because of less effective binding sites. However, when ionic strength increases to a higher level, a gradual loss of specificity could be observed for the MIP. The imprinting factor (IF, QMIP/QNIP) reached its maximum at 2.5 when 1 mmol L-1 NaCl was added to the buffer, and the swelling ratio of the MIP was about 8.4 in this state. It should be mentioned that, in the experiments of the temperature effect, the MIP with the same swelling ratio gave an IF of about 1.3, indicating that an optimum ionic strength is beneficial to inhibit the nonspecific interaction and enhance the selectivity of the MIP. The results obtained from above study on the influences of temperature and NaCl concentration confirmed that the spatial integrity of the recognition site and the spatial orientation of the functional groups are the foundation of molecule recognition. Also, this type of MIP with conformational memory could respond to external stimuli with a volume transition, and thus, its affinity toward the template protein could be controlled by them. Binding Parameters. Further studies were carried out to determine the binding constants between BSA and the MIP by Scatchard analysis (Figure 6A). Considering the stability of the protein, Tris-HCl buffer containing 1 mmol L-1 NaCl at 25 °C was adopted as the optimum condition for specific recognition other than Tris-HCl buffer at 40 °C. For comparison, the binding parameters of BSA with NIP were also measured (Figure 6B). When BSA was bound to the MIP, a biphasic curve was obtained, indicating two distinct populations of binding sites in the MIP. The calculated association constants (Ka) and the binding capacity (Qmax) corresponding to each part of the curves in Figure 6 are summarized in Table 2. As can be seen, the association constant (Ka) of the left slope (MIP-H) in Figure 6A is 9.6 × 104 L mol-1, which is about 5 times higher than that of the right slope (MIP-L). And it was noticeable that the right slope gave a similar association constant as that of the NIP, although the binding capacity of the former was higher than the latter. These results indicated that the left and right slopes of the binding curves in Figure 6A corresponded to specific and nonspecific recognition sites in the MIP. Also,
Figure 6. Scatchard plot of the readsorption assay of the MIP (A) and NIP (B). Readsorption was performed in Tris-HCl buffer (10 mmol L-1, pH 7.0) with 1 mmol L-1 NaCl at 25 °C. Table 3. Specificity of the BSA-MIP in the Binding of Proteins amount of the proteins adsorbed, mg g-1 dry gel MIP NIP Ra a
Lys
Mb
Hb
OVA
HSA
BSA
6(1 6(1 1.0
13 ( 3 12 ( 2 1.1
26 ( 2 24 ( 1 1.1
61 ( 4 65 ( 2 0.9
70 ( 3 49 ( 2 1.4
126 ( 4 52 ( 4 2.4
R ) MIP/NIP.
it was affirmed that adequate cavities with properly placed groups for BSA could be fabricated in the polymer matrix during polymerization in the presence of BSA as the template. The difference of the nonspecific binding capacity between the MIP and NIP might also be attributed to the imprinting effect, which enlarged the specific surface area of the MIP and thus increased the nonspecific binding sites. Selectivity of the BSA-MIP. As electrostatic forces, hydrophobic forces, and other interactions may also exist between nontemplate proteins and the BSA-MIP, the selectivity of the MIP was tested using five proteins with different molecular weights and isoelectric points. Table 3 shows the adsorption capacities of the MIP and NIP for different proteins in pH 7.0 buffered solutions with protein concentration of 0.5 mg mL-1. For all the proteins except BSA and HSA, the differences of the
MIP Imprinted Hydrogel for BSA Recognition
Langmuir, Vol. 24, No. 11, 2008 5779
Figure 7. SDS-PAGE analysis of the results for the isolation of BSA from a mixture of BSA and OVA (A) and from bovine calf serum (B). For A: line 1, protein molecular weight marker; line 2, 10 µL of BSA solution (0.5 mg mL-1); line 3, 10 µL of OVA solution (0.5 mg mL-1); line 4, 10 µL of MIP eluate; and line 5, 10 µL of NIP eluate. For B: line 1, protein molecular weight marker; line 2; 10 µL of BSA solution (0.5 mg mL-1); line 3, 10 µL of MIP eluate; line 4, 10 µL of 10-fold dilution of bovine calf serum before adsorption; and line 5, 10 µL of 10-fold dilution of bovine calf serum after adsorption.
adsorption capacities between the MIP and NIP were negligible. Lysozyme, which has an isoelectric point of 10.7, is positively charged at pH 7.0. Thus, the electrostatic repulsion occurred between the lysozyme and the tertiary amine group of the polymer chains, and little could be adsorbed by the MIP as well as by the NIP. Also, the proteins with a neutral isoelectric point showed low adsorption capacities, such as hemoglobin and myoglobin. OVA and HSA, both of which have approximately the same isoelectric point as BSA, could be adsorbed efficiently. However, the MIP and NIP showed no significant difference between their affinities toward OVA. HSA is homologous to BSA,38 and the amino acid sequence homology may be the reason that the MIP showed certain cross-reactivity toward HSA. However, the adsorption capacity of HSA on the MIP was smaller than those of BSA and OVA, which could be explained by the idea that the cavity in the MIP was created by the BSA and therefore access to imprinted sites might be limited by the steric hindrance of polymer chains for larger proteins, such as HSA. From above, it can be inferred that the recognition of the MIP depended on both the charge effect and the shape memory effect. In order to determine the ability of MIP to discriminate the template from other proteins, OVA was used as the competitor because it showed relatively high adsorption values on both MIP and NIP. From the SDS-PAGE analysis (Figure 7A), it could be seen that most of the proteins adsorbed by the MIP were BSA. By contrast, both BSA and OVA were adsorbed by the NIP; however, the amount of BSA bound to the MIP was much larger than that of NIP. To demonstrate the applicability of the MIP, bovine calf serum was employed to perform direct purification of the target protein from real samples (Figure 7B). The eluate of MIP exhibited a single band with a molecular mass of 66 kDa, in excellent agreement with that of the BSA standard. All other proteins in the complex serum sample, such as immunoglobulin G and plasma proteins, displayed no cross-adsorption to the MIP and did not interfere with the binding of BSA. As revealed in the competition experiments, the MIP validated high selectivity toward the template protein, which suggested its potential in practical applications. Storage and Reusability of the BSA-MIP. After each adsorption process, the MIP and NIP were washed by Tris-HCl buffer (pH 7.0) containing 0.5 mol L-1 NaCl to release the bound proteins. Tris-HCl buffer (pH 7.0) without NaCl was then used to remove the remaining salt. After the washing process, the MIP could be used for adsorption again or be dried for storage. (38) Hirayama, K.; Akashi, S.; Furuya, M.; Fukuhara, K. Biochem. Biophys. Res. Commun. 1990, 173, 639–646.
Figure 8. Influence of the regeneration cycles on BSA adsorption of the MIP and NIP. Readsorption was performed in Tris-HCl buffer (10 mmol L-1, pH 7.0) with 1 mmol L-1 NaCl at 25 °C.
Figure 8 shows the change of the amount of adsorbed BSA after several regeneration cycles. It could be seen that over six cycles the MIP lost about 10% of its affinity on average, while the affinity of NIP remained unchanged. The decrease in affinity resulted mainly from the partial destruction of the recognition sites, since it underwent a shrinking-swelling process during the washing. Some cavities could not recover their shape after washing, and thus, they were not fit for the template protein anymore. On the other hand, the affinity of the NIP was nonspecific and the effect of washing is negligible. Also, the MIP was stored in both the wet state (Tris-HCl buffer, pH 7.0) and dry state for a month. Over this period of time, the MIP in the wet state lost 20% of its affinity but the MIP in the dry state kept its affinity unchanged, which proved the dry state was suitable for long-term storage.
Conclusions In summary, a new type of stimuli-responsive protein imprinted polymer was synthesized by self-assembly of a basic functional monomer (DMAPMA) with an acidic template protein (BSA) and then polymerized in the presence of NIPA. The MIP provided high efficiency of template removal by using a mild NaCl solution. In contrast to the traditional MIP with a rigid matrix, the volume of the MIP as well as the ability of MIP to recognize the target
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protein could be controlled by external stimuli such as temperature and ionic strength. Under the optimal binding conditions, which are very close to the original imprinting state, the obtained MIP showed maximal affinity toward the template, which proved that the origin of the specificity relied on the spatial integrity of the recognition site formed in the imprinting process. In this work, basic groups were introduced to create strong interactions with the acidic template protein and thus led to a high binding capacity but also to a certain degree of nonspecific adsorption. Fortunately, when NaCl was added to adjust the volume of the MIP, nonspecific adsorption was also inhibited. The selectivity of the MIP was verified by direct adsorption of the template protein from the
Hua et al.
protein mixture and real samples, and quite pure BSA was recovered. The easy preparation, stimuli-responsiveness, high selectivity, and binding capacity of the MIP suggest the presented approach is an attractive and broadly applicable way to develop solid-phase extraction devices, sensors, and especially protein delivery agents in the controlled-release system. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Grant No. 20575007) and National 863 Program (No. 2006AA10Z435). LA703963F
