Macroporous Thermosensitive Imprinted Hydrogel for Recognition of

Anal. Chem. 2009, 81, 7206–7216

Macroporous Thermosensitive Imprinted Hydrogel for Recognition of Protein by Metal Coordinate Interaction Lei Qin,† Xi-Wen He,† Wei Zhang,† Wen-You Li,*,† and Yu-Kui Zhang*,†,‡ Department of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China, and National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Acadamy of Sciences, Dalian 116011, People’s Republic of China A thermosensitive macroporous hydrogel showing selectivity for the lysozyme was developed by an imprinting procedure that is based on metal coordinate interaction. A metal chelate monomer [N-(4-vinyl)-benzyl iminodiacetic acid] forming coordination complex with the template protein in the presence of Cu ions co-polymerized with N-isopropylacrylamide and acrylamide, using N,N-methylenebisacrylamide as the cross-linker to prepare the thermosensitive protein-imprinted hydrogel. The synergetic combination of the smart property of the macroporous thermosensitive hydrogel with the merits of the coordinate interaction improved the selectivity and adsorption capacity, with respect to template lysozyme. The macropores were created by the frozen polymerization, and the influences of frozen polymerization and the chelate monomer content on the hydrogel affinity were investigated. The imprinted hydrogel can respond not only to external stimuli, but also to the template protein with a certain degree of shrinking. In recognition of the protein, the interaction of the imprinted thermosensitive hydrogel to the protein can be switched between the coordinate effect and the electrostatic effect by adding or not adding Cu ions. Finally, this imprinted hydrogel was used to purify the template lysozyme from the mixture of proteins and the real sample, which demonstrated its high selectivity. Proteomics research is a long-established part of biochemistry. The isolation of low-abundance proteins is often significant in diagnosis and therapy. The molecular imprinting technique is one of various promising and facile separation methods to create molecular recognition sites of predetermined selectivity in a synthetic polymer.1,2 Molecularly imprinted polymers (MIPs) have already been used successfully for mimicking natural receptors and have previously been shown to have several applications in various small molecules.3-5 Recently, considerable interest has * To whom correspondence should be addressed. Tel.: (86) 22-23494962. Fax: (86) 22-23502458. E-mail addresses: [email protected] (W.-Y. Li); ykzhang@ dicp.ac.cn (Y.-K. Zhang). † Department of Chemistry, Nankai University. ‡ National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Acadamy of Sciences. (1) Wulff, G. Angew. Chem., Int. Ed. Engl. 1995, 34, 1812–1832. (2) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9–14. (3) Sellergren, B. Angew. Chem., Int. Ed. 2000, 39, 1031–1037.

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been shown in the imprinting of biomacromolecules and particular proteins for its potential applications as biomaterials for separations, biosensors, and mimicking enzymes and antibodies.6-11 Compared to the affinity matrices prepared using antibodies or enzymes, the man-made protein-imprinted polymers possess unique properties, such as high stability, low cost, and facility of manipulation to existing techniques. However, when imprinting proteins, there are many inherent problems to address, relative to the molecular size, complexity, conformational flexibility, solubility, and sensitivity to environment of the proteins.12 Consequently, more and more researchers are engaged in exploring alternative methods to overcome these problems.13-18 The metal coordination procedure to imprint protein was first developed by Kempe.19 A similar approach was applied to protein with the surface imprinting method.20-22 Distributions of charge and polarity on protein surfaces are very complicated, whereas the surface histidines of certain proteins are known. Employment of the high selectivity of the metal coordination to recognize protein may be an easy way to imprint the target protein. (4) Hu, S.-G.; Li, L.; He, X.-W. J. Chromatogr. A 2005, 1062, 31–37. (5) Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O’Mahony, J.; Whitcombe, M. J. J. Mol. Recognit. 2006, 19, 106–180. (6) Mallik, S.; Plunkett, S. D.; Dhal, P. K.; Johnson, R. D.; Pack, D.; Shnek, D.; Arnold, F. H. New J. Chem. 1994, 18, 299–304. (7) Ye, L.; Mosbach, K. J. Am. Chem. Soc. 2001, 123, 2901–2902. (8) Shiomi, T.; Matsui, M.; Mizukami, F.; Sakaguchi, K. Biomaterials 2005, 26, 5564–5571. (9) Turner, N. W.; Jeans, C. W.; Brain, K. R.; Allender, C. J.; Hlady, V.; Britt, D. W. Biotechnol. Prog. 2006, 22, 1474–1489. (10) Bossi, A.; Bonini, F.; Turner, A. P. F.; Piletsky, S. A. Biosens. Bioelectron. 2007, 22, 1131–1137. (11) Bergmann, N. M.; Peppas, N. A. Prog. Polym. Sci. 2008, 33, 271–288. (12) Ge, Y.; Turner, A. P. F. Trends Biotechnol. 2008, 26, 218–224. (13) Nishino, H.; Huang, C.-S.; Shea, K. J. Angew. Chem., Int. Ed. 2006, 45, 2392–2396. (14) Li, Y.; Yang, H.-H.; You, Q.-H.; Zhuang, Z.-X.; Wang, X.-R. Anal. Chem. 2006, 78, 317–320. (15) Miyata, T.; Jige, M.; Nakaminami, T.; Uragami, T. Proc. Natl. Acad. Sci. 2006, 103, 1190–1193. (16) Lin, H.-Y.; Rick, J.; Chou, T.-C. Biosens. Bioelectron. 2007, 22, 3293–3301. (17) Hansen, D. E. Biomaterials 2007, 28, 4178–4191. (18) Takeuchi, T.; Hishiya, T. Org. Biomol. Chem. 2008, 6, 2459–2467. (19) Kempe, M.; Glad, M.; Mosbach, K. J. Mol. Recognit. 1995, 8, 35–39. (20) Vaidya, A. A.; Lele, B. S.; Kulkarni, M. G.; Mashelkar, R. A. J. Appl. Polym. Sci. 2001, 81, 1075–1083. (21) Odabas¸i, M.; Say, R.; Denizli, A. Mater. Sci. Eng., C 2007, 27, 90–99. (22) Bereli, N.; Andaç, M.; Baydemir, G.; Say, R.; Galaev, I. Y.; Denizli, A. J. Chromatogr. A 2008, 1190, 18–26. 10.1021/ac900676t CCC: $40.75  2009 American Chemical Society Published on Web 08/05/2009

Smart hydrogels (sensitive hydrogels) have attracted much attention for their applications to drug delivery systems, tissue engineering, cell encapsulation, and so on.23,24 The smart hydrogels are sensitive, with regard to their dimension or structure, to a small stimulation from the environment, such as temperature, ionic strength, or specific chemicals. This type of stimuli-sensitive recognition is very similar to the recognition of proteins in natural systems.25-27 Thus, the biocompatibility and the smart response to external stimulation of the sensitive hydrogel could have a potential application in the molecularly imprinted polymer. The stimuli-responsive molecularly imprinted polymers using metal ions27 and small molecules28-30 as templates have been successfully prepared. To date, there have been several encouraging works focusing on the design of protein-imprinted materials based on stimuli-responsiveness.15,31-34 The thermosensitive proteinimprinting system consists of a major thermosensitive monomer component that allows for response to the environmental stimulus and a minor functional monomer component that offers interaction with the target molecule.31-33 In addition, temperature-sensitive hydrogels have many advantages, such as enhanced mass transfer and rebinding percentage.31,33 It can especially be used as a temperature gate to control the uptake and release of target molecules. It is a new way to imprint protein that would partly solve the difficulties in protein imprinting. In the object of this study, an alternative thermosensitive imprinted hydrogel was prepared for lysozyme through metal coordinate interaction. In preparing the imprinted hydrogel, the N-isopropylacrylamide was introduced as a temperature-sensitive element that allowed for swelling and shrinking in response to temperature changes to realize recognition and release of lysozyme, the acrylamide (AAm) was introduced to improve the mechanical strength of the hydrogel, and the N-(4-vinyl)-benzyl iminodiacetic acid (VBIDA) via Cu ion formed a coordinate complex with the surface-exposed histidine of the template. To increase the mass transfer of the protein, the ice crystal was chosen as a porogen. The optimized proportion of the chelate monomer was investigated. In this work, the prepared thermosensitive imprinted hydrogel showed sensitive responses to temperature and a clear conformational memory of the template protein. The notable binding capacity and selectivity of the imprinted hydrogels were (23) Qiu, Y.; Park, K. Adv. Drug. Delivery Rev. 2001, 53, 321–339. (24) De las Haras Alarcoń, C.; Pennadam, S.; Alexander, C. Chem. Soc. Rev. 2005, 34, 276–285. (25) Hiratani, H.; Alvarez-Lorenzo, C.; Chuang, J.; Guney, O.; Grosberg, A. Y.; Tanaka, T. Langmuir 2001, 17, 4431–4436. (26) Alvarez-Lorenzo, C.; Hiratani, H.; Tanaka, K.; Stancil, K.; Grosberg, A. Y.; Tanaka, T. Langmuir 2001, 17, 3616–3622. (27) 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. (28) 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. (29) Gong, C.; Lam, M. H.; Yu, H. Adv. Funct. Mater. 2006, 16, 1759–1767. (30) Kanekiyo, Y.; Naganawa, R.; Tao, H. Angew. Chem., Int. Ed. 2003, 42, 3014–3016. ¨ zçetin, G.; Turan, E.; C (31) Demirel, G.; O ¸ aykara, T. Macromol. Biosci. 2005, 5, 1032–1037. (32) Chen, Z.-Y.; Hua, Z.-D.; Xu, L.; Huang, Y.; Zhao, M.-P.; Li, Y.-Z. J. Mol. Recognit. 2008, 21, 71–77. (33) Hua, Z.-D.; Chen, Z.-Y.; Li, Y.-Z.; Zhao, M.-P. Langmuir 2008, 24, 5773– 5780. (34) Bergmann, N. M.; Peppas, N. A. Trans. Soc. Biomater. 2003, 29, 457– 458.

also demonstrated via application to the mixture of proteins and real sample. To the best of our knowledge, the adsorption behavior of this type of hydrogel system has not been reported to date and is of greater significance for component separation in proteomics. EXPERIMENTAL SECTION Reagents. All reagents used were of at least analytical grade. N-isopropylacrylamide (NIPAAm) and 4-vinylbenzyl chloride were supplied from Acros Organics USA (Morris Plains, NJ). The NIPAAm was recrystallized in benzene/n-hexane before being used. Glucose oxidase [Gox, pI (isoelectric point) ) 4.2, MW ) 90.0-100.0 kDa], bovine hemoglobin (BHb, pI ) 6.9, MW ) 64.0 kDa), bovine pancreas ribonuclease A (RNase A, pI ) 9.4, MW ) 13.7 kDa), cytochrome c (Cyt c, from bovine heart, pI ) 9.8, MW ) 12.3 kDa) and N,N,N′,N′-tetramethylenediamine (TEMED) were obtained from Sigma (St. Louis, MO). Lysozyme (pI ) 11.2, MW ) 13.4 kDa), bovine serum albumin (BSA, pI ) 4.9, MW ) 66.0 kDa), trypsin inhibitor (TI, pI ) 4.2, MW ) 20.1 kDa), and ovalbumin (pI ) 4.7, MW ) 43.0 kDa) were purchased from DingGuo Biotech (Beijing, PRC). Acrylamide (AAm), iminodiacetic acid (IDA), N,N-methylenebisacrylamide (MBAA) and ammonium persulfate (APS) were obtained from Institute of GuangFu Fine Chemicals (Tianjin, PRC). Tris-HCl buffer solution (10 mmol/ L, pH 7.0) was used as a working medium. Synthesis of N-(4-vinyl)-Benzyl Iminodiacetic Acid. As the chelate monomer, the N-(4-vinyl)-benzyl iminodiacetic acid (VBIDA) was synthesized according to a previously published procedure, with some modifications.35 One hundred milliliters of 50% methanol solution containing iminodiacetic acid (6.65 g, 0.05 mol) and sodium hydroxide (3.3 g, 0.083 mol) was stirred at 60 °C, to which vinylbenzyl chloride (7.65 g, 0.05 mol) was added dropwise. After one-half of the vinylbenzyl chloride had been added in 30 min, another 3.3 g of sodium hydroxide was introduced and the addition of vinylbenzyl chloride was continued. Heating was continued for another 30 min. After the addition was complete, the methanol solution was distilled under vacuum until two-thirds of the original volume remained. Extraction of the methanol solution with ether removed the thick oil. Acidification of the aqueous phase to pH 2.5 with hydrochloric acid then gave a white precipitate. After washing the white precipitate through filtration and drying under a vacuum, the VBIDA product was obtained (yield, 31%; mp > 200 °C).35 NMR analysis: CH2dCH-Ph-CH2-N-(-CH2COOH)2 (CD3DH). δ 3.4 (4H, s, N-CH2-); δ 3.8 (2H, s, -CH2-); δ 5.2 (1H, d, CH2d); δ 5.7-5.7 (1H, d, CH2d); δ 6.7 (1H, m, dCH-); δ 7.3-7.4 (4H, m, -C6H4-). Preparation of Thermosensitive Imprinted Hydrogels. For comparison, three types of hydrogels were prepared: imprinted without coordinate interaction [P(Lyz)], imprinted with coordinate interaction [P(Cu+Lyz)], and the correspondence nonimprinted hydrogel [P(Blank)]. The imprinted hydrogel was synthesized by free-radical cross-linking co-polymerization of NIPAAm, AAm, and VBIDA, using a small amount of MBAA as the cross-linker (see Scheme 1). N,N,N′,N′-tetramethylethylenediamine (TEMED) was used as the accelerator and ammonium persulfate (APS) was used as the initiator. The monomers and (35) Morris, L. R.; Mock, R. A.; Marshall, C. A.; Howe, J. H. J. Am. Chem. Soc. 1959, 81, 377–382.


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Scheme 1. Schematic Representation of Protein Imprinting Processes of the P(Cu+Lyz)a

a The bulk polymerization method was adopted. N-(4-vinyl)-benzyl iminodiacetic acid (VBIDA) chelated the metal Cu ion and then coordinated the imidazole groups (Im) of surface-exposed histidine on the protein. The protein was removed by treatment with NaCl (1.0 mol/L) and EDTA (0.2 mol/L). The metal chelate monomer was positioned on the imprinting cavity so as to selectively recognize and rebind, in the presence of a Cu ion, the template protein.

Table 1. Preparative Composition of Different Hydrogels hydrogel

NIPAAm (g)

AAm (g)

VBIDA+Cu2+ (mmol)

MBAA (g)

lysozyme (mmol)

APS(20%)/TEMED

P(Cu+Lyz1) P(Cu+Lyz2) P(Cu+Lyz3) P(Lyz1) P(Lyz2) P(Lyz3) P(Blank1) P(Blank2) P(Blank3)

0.2008 0.2008 0.2008 0.2008 0.2008 0.2008 0.2008 0.2008 0.2008

0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050 0.0050

0.01 0.02 0.03

0.0042 0.0042 0.0043 0.0042 0.0042 0.0043 0.0042 0.0042 0.0043

0.01 0.02 0.03 0.01 0.02 0.03

50 µL/10 µL 50 µL/10 µL 50 µL/10 µL 50 µL/10 µL 50 µL/10 µL 50 µL/10 µL 50 µL/5 µL 50 µL/5 µL 50 µL/5 µL

0.01 0.02 0.03

the concentrations used were shown in Table 1. Using preparation of the P(Cu+Lyz1) as an example, NIPAAm (0.2008 g, 1.77 mmol), AAm (0.0050 g, 0.070 mmol), VBIDA (0.0026 g, 0.010 mmol), CuSO4 (0.0024 g, 0.010 mmol), N,N-methylenebisacrylamide (MBAA, 0.0042 g, 0.0272 mmol), and template lysozyme (0.1440 g, 0.010 mmol) were dissolved in 2.0 mL of Tris-HCl buffer (10 mmol/L, pH 7.0). The prepolymerization mixture was deoxygenated by purging with nitrogen, and then certain volumes of 20% APS and TEMED were added to the solution. The mixture was immediately transferred into a poly(vinyl chloride) straw (70 mm × 6 mm i.d.) and sealed. The polymerization was conducted at 25 or -20 °C with different reaction times. The objective of frozen polymerization (-20 °C) was to reach the effect of ice crystal porogen. Hydrogels, obtained in long, cylindrical shapes, were cut into 2-mmthick pieces. The obtained hydrogels were extensively washed by the following procedure: deionized water, NaCl (1.0 mol/L), deionized water, EDTA (0.2 mol/L), and 7208


deionized water in sequence three times to elute the template.The wash liquids were taken to determine the amount of lysozyme extracted by the UV-vis spectrophotometry. As shown in Table S1 of the Supporting Information, the degree of extraction of the lysozyme from the P(Cu+Lyz2) was high and reached a value of 93.2% (w/w). The hydrogels were immersed in Tris-HCl buffer (10 mmol/L, pH 7.0) at 4 °C before use. Correspondingly, the P(Lyz) hydrogel without adding chelate monomer and the nonimprinted control hydrogel [P(Blank)], with the same monomers as the P(Cu+Lyz) with the exception of lysozyme, were generated in the same way. Characterization. Fourier transform infrared (FT-IR) spectra (4000-500 cm-1) in KBr were recorded with an Avatar 360 instrument (Nicolet, Waltham, MA). Scanning electron microscopy (SEM) of the imprinted hydrogels with different polymerization temperatures and times were studied with a Quanta 200 (FEI, Eindhoven, The Netherlands).

Transition temperatures of the hydrogels were measured by thermal analysis using differential scanning calorimetry (DSC) (Netzsch, Selb, Germany). In the DSC analysis, ∼10 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. Measurements of Swelling Ratio of Hydrogels. For the temperature-response studies, the swelling weight of the hydrogel was measured as a function of temperature. The hydrogel was initially immersed in water at 23 °C, and the excess water on the hydrogel surface was removed with wet filter paper. The equilibrium weight of the hydrogel was then measured with a microbalance at various predetermined temperatures, ranging from 23 °C to 53 °C. The average values of three measurements were taken for each hydrogel, and the equilibrium swelling ratio was calculated as follows:

Swelling ratio )

Ms - Md Md

(1)

where Md and Ms are the masses of the dry and swollen hydrogels, respectively. Protein Adsorption Experiments. A mass of 5.0 mg of the dry hydrogels was first allowed to swell in the Tris-HCl buffer (10 mmol/L, pH 7.0) to equilibrium. The wet state hydrogels then were immersed for 12 h in either 1.0 mL of protein-copper complex solution or an only-protein solution (without Cu ions) at the desired temperature. In the solution with the protein-copper complex, the molar ratio between the exposed histidines of protein and the Cu ion was 1:1. The concentration of protein in the supernatant was measured by an UV-2450 UV-vis spectrophotometer (Shimadzu, Kyoto, Japan) at a wavelength of 280 nm. The amount of adsorbed protein can be determined by the difference in concentration before and after the adsorption. The adsorption capacity (Q, expressed in units of mg/g) of the protein or analogue bound to the hydrogel is calculated by

Q)

(C0 - Ct)V W

(2)

where C0 and Ct are the initial concentration and the residual concentration of the protein or analogue, respectively (each given in units of mg/mL), V is the volume of the initial solution (in milliliters), and W is the weight of the hydrogel (in milligrams). The specific recognition property of the imprinted hydrogel is evaluated by the imprinting factor (IF), which is defined as

IF )

QMIH QNIH

(3)

Here, QMIH and QNIH are the adsorption capacities of the protein or analogue on imprinted hydrogel and the corresponding nonimprinted hydrogel, respectively. The selectivity factor (R) is defined as

R)

IFtem IFana

(4)

where IFtem is the imprinting factor toward the template protein and IFana is the imprinting factor toward the analogous protein. To investigate the effect of temperature on the adsorption of lysozyme, the hydrogel was first swollen to equilibrium and then immersed in 1.0 mL of a 2.0 mg/mL lysozyme-copper complex in Tris-HCl buffer (10 mmol/L, pH 7.0) and the temperature was controlled by a thermostat water bath ranging in temperature from 23 °C to 53 °C. The amount of adsorbed lysozyme was measured. After the isotherm adsorption experiments, the data obtained were linearized with a variation of the Langmuir plot, using the following equation: Ce Ce 1 ) + Q QmaxK Qmax

(5)

where Q and Qmax are the experimental adsorption capacity to the template protein and the theoretical maximum adsorption capacity of the hydrogel, respectively (both expressed in units of mg/g), Ce is the concentration of protein in equilibrium solution (given in terms of mg/mL), and K is the dissociation constant for the template protein to the hydrogel (expressed in units of mL/mg). Selectivity Experiments. In the competitive adsorption experiment, three types of hydrogels were used to investigate the selectivity to the template protein from the mixture of proteins (GOx, BSA, ovalbumin, TI, RNase A, and lysozyme, each with a concentration of 2.0 mg/mL) and certain concentrations of Cu ion. Ten microliters of the residual mixture after adsorption was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, using 15.0% polyacrylamide gel (Miniprotean, Bio-Rad, Hercules, CA). In the study of the real sample, chicken egg white was separated from a fresh egg and diluted to 50% (v/v) with TrisHCl buffer (50 mmol/L, pH 7.0). After the diluted solution was immersed in an ice bath and centrifuged at 10 000 rpm for 30 min,36 the supernatant solution was used as a lysozyme source. The imprinted hydrogel was applied to purify the lysozyme from a 20-fold diluted real sample of egg white at 28 °C. The hydrogels were then treated with washing procedure to elute the specifically adsorbed protein. The eluate was desalted and concentrated 10fold, using an ultrafiltration membrane (molecular weight cutoff ) 3000), and 10 µL of each sample was used for SDS-PAGE analysis, using 15.0% polyacrylamide gel (Mini-protean, Bio-Rad, Hercules, CA). RESULTS AND DISCUSSION Characterization Studies. The FT-IR characteristic peaks of the monomers NIPAAm, AAm, and VBIDA were compared to the imprinted hydrogel (see Figure 1). Although the percentages of AAm and VBIDA in the P(Cu+Lyz2) were very low, the presence of AAm and VBIDA in the hydrogel could be shown in the FT-IR spectra. Figure 1a shows that the characteristic single peak of NIPAAm (-NH, 3299.87 cm-1) overlapped with the characteristic double peak of AAm (-NH2, 3355.94 cm-1 and 3191.03 cm-1), which appeared as a shoulder peak at a wavenumber of ∼3300 cm-1 in the FT-IR spectra of the P(Cu+Lyz2). Also, (36) Arica, M. Y.; Yilmaz, M.; Yalcin, E.; Bayramoglu, G. J. Chromatogr. B 2004, 805, 315–323.


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Figure 1. FT-IR spectra of NIPAAm, AAm, VBIDA, and the imprinted hydrogel [P(Cu+Lyz2)] (a) from 3600 cm-1 to 2500 cm-1 and (b) from 1800 cm-1 to 1000 cm-1.

the isopropyl group of NIPAAm was prominently observed in the P(Cu+Lyz2) at a wavenumber of 2970.43 cm-1. In the P(Cu+Lyz2), the presence of a band at ∼1387.91 cm-1 was consistent with that of the VBIDA (see Figure 1b). The amide bond in the hydrogel was observed at a wavenumber of 1659.08 cm-1. These results indicated that all monomers were successfully incorporated into the backbone of the imprinted hydrogel. Temperature-sensitive hydrogels are well-known for swelling and shrinking reversibly in response to various environmental temperatures. Such properties allow the thermosensitive characters of the P(Cu+Lyz2), P(Lyz2), and P(Blank2) to be determined by recording the enthalpy of transition using differential scanning calorimetry (DSC). The P(Cu+Lyz2) presented a volume phasetransition temperature (Tvpt) of 49.9 °C, while the P(Blank2) showed a Tvpt value of 47.5 °C. The Tvpt value of P(Lyz2) was 38.1 °C. The lower critical solution temperature (LCST) of pure poly(N-isopropylacrylamide) is ∼32 °C.37 The increased Tvpt 7210


value of the P(Cu+Lyz2) resulted from the involvement of the hydrophilic monomer VBIDA, which contained a carboxyl group. The P(Cu+Lyz2) presented a Tvpt value that was slightly higher than that of the P(Blank2), even though the VBIDA content was the same for both of them. The difference between the Tvpt value of P(Cu+Lyz2) and that of P(Blank2) proved that the fabrication of microstructures was led by imprinting in the P(Cu+Lyz2). Study of Conditions of the Polymerization. Effect of Ice Crystal Porogen. The surface morphologies of the hydrogels prepared under different temperatures and times were observed by SEM after they were freeze-dried (Figure 2). It could be inferred that, at the temperature of -20 °C, the water formed an ice crystal that functioned as a porogen. The hydrogels prepared (37) Stile, R. A.; Burghardt, W. R.; Healy, K. E. Macromolecules 1999, 32, 7370– 7379.

Figure 2. SEM photomicrographs of the P(Cu+Lyz2) prepared at (a) 25 °C for 36 h, (b) -20 °C for 12 h, (c) -20 °C for 24 h, and (d) -20 °C for 36 h. (Scale bar ) 2.0 mm.)

Figure 3. SEM photomicrographs of the surface of the P(Cu+Lyz2) prepared at (a) 25 °C for 36 h and (b) -20 °C for 36 h. (Scale bar ) 500.0 µm.)

at -20 °C (see Figures 2b-d) had numerous pores several hundreds of micrometers in size, compared to the hydrogel prepared at 25 °C (see Figure 2a). In addition, it can be seen that the number of these macropores increased as the polymerization time increased at -20 °C. The impact of the frozen polymerization at -20 °C on the morphology of the hydrogel was obvious under a magnification of 150× (see Figure 3). The pore structure is also an important parameter in the adsorption process. These micrometer-sized interconnected pores could act as a channel for the protein, which benefitted the mass transfer of the protein. Therefore, the effects of pore structure on the lysozyme adsorption to the P(Cu+Lyz2) and P(Blank2) prepared with different frozen times were investigated. The adsorption capacity and variation of the imprinting factor (IF) for lysozyme are shown in Figure 4. It should be mentioned that the IF value increased with the prolonging of (38) Wong, J. W.; Albright, R. L.; Wang, N. H. L. Sep. Purif. Methods 1991, 20, 49–106.

the frozen time at -20 °C. In addition, the IF value almost doubled, from 4.67 when the hydrogel was prepared at 25 °C for 36 h to 7.87 at -20 °C for 36 h. The high adsorption capacity of template by the P(Cu+Lyz2) prepared at -20 °C for 36 h was attributed to the more macroporous structure, which facilitated the protein removal in the wash step and rebinding in the recognition step. Hence, -20 °C for 36 h was adopted as the polymerization condition in the subsequent experiments. Effect of the Amount of VBIDA. Lysozyme contains only one histidine residue (His-15) on its surface.38 One chelate monomer (VBIDA) with one Cu ion can self-assemble onto the histidine residue of one lysozyme. The molar ratio of chelate monomer to lysozyme should be 1:1, which ensured the formation of a stable proteinmonomer complex via coordinate interaction. Too many functional monomers may lead to excessive nonspecific recognition sites. The effect of using coordinate interaction in the adsorption process can be observed from the comparative data in Figure 5. It can be seen that, in the presence of Cu ions, the P(Cu+Lyz) Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

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Figure 4. Effect of polymerization time and temperature on the adsorption capacity of the P(Cu+Lyz2) and its corresponding nonimprinted hydrogel P(Blank2). Experimental conditions: 1.0 mL of 2.0 mg/mL lysozyme-copper complex with 5.0 mg of the hydrogel at 28 °C for 12 h.

Figure 5. Adsorption capacities of the P(Cu+Lyz), P(Lyz), and P(Blank) with different amounts of the chelate monomer in the presence and absence of the Cu ion. Experimental conditions: 1.0 mL of 2.0 mg/mL lysozyme with or without Cu ions, incubated by 5.0 mg of the hydrogel at 28 °C for 12 h.

had a higher adsorption capacity to the lysozyme through coordinate interaction than when there were no Cu ions, in which case the VBIDA performed as an acid monomer to interact with the lysozyme through electrostatic interaction. Results showed that the inclusion of Cu ions in adsorption can bridge the template lysozyme and chelate monomer together and reach the imprinting effect, compared to the situation without added Cu ions. In all cases, the P(Cu+Lyz) with coordinate interaction has a higher adsorption capacity than both the P(Lyz) without the coordinate interaction and the nonimprinted hydrogel P(Blank). For comparison, the control hydrogels for the P(Lyz) [P(Blank′)] were also prepared (see Table S2 in the Supporting Information). From the adsorption capacity data of the P(Lyz) and the P(Blank′) (see 7212


Figure S1 in the Supporting Information), it can be seen that the P(Lyz) also had the imprinting effect, compared to the P(Blank′). However, the imprinting factor (IF) of the P(Lyz2) was only 2.61 when the initial concentration of lysozyme was 2.0 mg/mL in the presence of Cu ions. Obviously, it was not better than that of the P(Cu+Lyz2) (see Figure 4). Thus, it was further demonstrated that the imprinted hydrogel through the coordinate interaction exhibited a high imprinting effect and selectivity toward the template. The effect of different amounts of chelate monomer was investigated by the preparation of three types of imprinted hydrogelssP(Cu+Lyz1), P(Cu+Lyz2), and P(Cu+Lyz3)s with the same cross-linking degree fixed at 2.0%, but different amounts

Figure 6. Effect of the amount of chelate monomer on the imprinting factor (IF) of the imprinted hydrogel at different temperatures. The error bars were from three parallel experiments using different gels. Experimental conditions: 1.0 mL of 2.0 mg/mL lysozyme-copper complex, incubated by 5.0 mg of the hydrogel at different temperatures for 12 h.

of VBIDA (see Figure 6). The IF value to the target lysozyme molecule and the thermosensitive response were used to evaluate the suitable amount of chelate monomer. The imprinted hydrogels with different amounts of VBIDA alternate between a swollen water-rich phase and a collapsed water-poor phase, as a response to temperature. Figure 6 shows that the P(Cu+Lyz1), P(Cu+Lyz2), and P(Cu+Lyz3) all had the highest IF value at 28 °C, but the fluctuations of the IF value with temperature change were different. For the P(Cu+Lyz3), a high ratio of the chelate monomer led to more recognition sites and a high IF value. In contrast, the thermosensitive ability did not follow this trend, suggesting that a certain amount of chelate monomer was favorable for high imprinting effect and thermosensitive response. Thus, the compositions of P(Cu+Lyz2) was adopted as the optimum condition for preparing imprinted hydrogels, and 28 °C was adopted as the optimum temperature for specific recognition. Properties of the Imprinted Hydrogel. Intelligent Response to the Template. To confirm the intelligent mechanism, the swelling ratios of the P(Cu+Lyz2) and P(Blank2) were measured in the Tris-HCl buffer and the lysozyme-copper complex solution, respectively (see Figure 7). Figure 7 shows that the P(Blank2) had similar swelling ratios in both the Tris-HCl buffer and the lysozyme-copper complex solution. However, the P(Cu+Lyz2) had a special swelling ratio in the presence of the template lysozyme. This phenomenon confirmed that the spatial integrity of the recognition sites and the spatial orientation of the functional groups were formed in the P(Cu+Lyz2) during the imprinting process. The imprinted hydrogel with conformational memory could respond to the template protein with a volume transition, but the P(Blank2) could not. Binding Kinetics. Figure 8 shows the kinetics adsorption processes of template to the P(Cu+Lyz2), P(Lyz2), and P(Blank2). As shown in the kinetics curves, the P(Cu+Lyz2) reached the experimental maximum adsorption capacity after ∼10 h and a satisfactory imprinting effect of P(Cu+Lyz2) could be observed.

Figure 7. Effect of temperature on the swelling ratio of the P(Cu+Lyz2) (square symbols) and the P(Blank2) (circular symbols) in the Tris-HCl buffer (0.01 M, pH 7.0) (open symbols) and in the solution of 2.0 mg/mL lysozyme-copper complex (solid symbols).

Figure 8. Adsorption kinetics curves of the P(Cu+Lyz2), P(Lyz2), and P(Blank2). Experimental conditions: 1.0 mL of 2.0 mg/mL lysozyme-copper complex, incubated by 5.0 mg of the hydrogel at 28 °C for certain hours.

The P(Cu+Lyz2) was rebinding more template protein than the P(Lyz2). Because of the lack of the imprinting process, the functional groups were randomly distributed in the P(Blank2), which led to the low adsorption ability of the P(Blank2) to the lysozyme. The imprinting factor (IF) of the P(Cu+Lyz2) was 7.87 after adsorption equilibrium. Binding Isotherms. In all of the concentrations studied, the P(Cu+Lyz2) exhibited higher adsorption capacity to lysozyme than the P(Blank2) (see Figure 9). The surface histidine of the lysozyme is believed to have interacted, via the Cu ion, with the chelate monomer during the imprinting process. The arrangement of the chelate monomer units in the P(Cu+Lyz2) were sufficiently stable to construct recognition sites for lysozyme. The binding constants between the lysozyme and the P(Cu+Lyz2) were fitted by Langmuir analysis (R ) 0.9986). According to the linearized plot of Ce/Q vs Ce (see Figure S2 in the Supporting Information), the dissociation constant (K) and the theoretical maximum adsorption capacity (Qmax) for the specific interaction Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

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Figure 9. Adsorption isotherms of lysozyme on the P(Cu+Lyz2) and P(Blank2). Experimental conditions: 1.0 mL of 0.1-4.0 mg/mL lysozyme-copper complex, incubated by 5.0 mg of the hydrogel at 28 °C for 12 h. The error bars were from three parallel experiments using different gels.

between the P(Cu+Lyz2) and the template lysozyme were determined to be 1.23 mL/mg and 233.64 mg/g dry gel, respectively. A comparison of the K, Qmax, and the experimental maximum adsorption capacity of the imprinted hydrogel with some reported works21,22,32,39-41 are listed in Tables S3 and S4 in the Supporting Information. It was demonstrated that the thermosensitive lysozyme imprinted hydrogel exhibited relatively high selectivity and adsorption capacity. Temperature Swing Adsorption. The thermosensitive hydrogel can undergo a reversible volume transition between the swollen and collapsed phases, which can be triggered by external stimuli such temperature, pH, and specific chemicals. The influence of temperature on the adsorption of lysozyme onto the P(Cu+Lyz2) was investigated and the results are shown in Figure 10. The adsorption capacity of the P(Cu+Lyz2) was first increased and then decreased as the temperature changed over the range of 28-43 °C. The experiments show that both the swollen and collapsed states happened in this process, and the imprinting cavity also underwent a volume change. It can be seen that the adsorption capacity reached the maximum at 28 °C. Through the conformational memory, the shape of the imprinting cavity and the distribution of the chelate group corresponded to the protein at 28 °C, so that the highest affinity was obtained. Also, because the imprinting cavity was in a swollen state at 28 °C, the chelate monomer chelated with the Cu ion, which was easily accessible for the protein. When the temperature increased to 43 °C, the cavity was in a collapsed state and the Cu ion was blocked; thus, the adsorption of the protein was lower.42 Selectivity of the Imprinted Hydrogel. Adsorption Capacity to Single Protein. Seven types of proteins (GOx, BSA, BHb, ovalbumin, RNase A, lysozyme, and Cyt c) with different molecular (39) Kimhi, O.; Bianco-Peled, H. Langmuir 2007, 23, 6329–6335. (40) Tan, C. J.; Wangrangsimakul, S.; Bai, R.; Tong, Y. W. Chem. Mater. 2008, 20, 118–127. (41) Lu, S. L.; Cheng, G. X.; Pang, X. S. J. Appl. Polym. Sci. 2006, 99, 2401– 2407. (42) Tokuyama, H.; Yanagawa, K.; Sakohara, S. Sep. Purif. Technol. 2006, 50, 8–14.

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Figure 10. Changes of the adsorption capacity of P(Cu+Lyz2) for the template protein by a temperature swing over a range of 28-43 °C. Experimental conditions: 1.0 mL of 2.0 mg/mL lysozyme-copper complex, incubated by 5.0 mg of the hydrogel for 12 h. The error bars were from three parallel experiments using different gels.

Figure 11. Adsorption capacities of the imprinted hydrogel [P(Cu+Lyz2) and P(Lyz2)] and the nonimprinted hydrogel [P(Blank2)] for the template protein and other proteins. Experiment conditions: 1.0 mL of 2.0 mg/mL protein-copper complex, incubated by 5.0 mg of the hydrogel at 28 °C for 12 h.

weights and isoelectric points were involved to reveal the selectivity of the imprinted hydrogel (see Figure 11). In the case of the P(Cu+Lyz2), the GOx, BSA, BHb, and ovalbumin were all larger than the template lysozyme, so their access to the imprinting sites might be limited by the steric hindrance of large proteins by hydrogel chains. Although the RNase A and Cyt c have the similar molecular weight with the lysozyme and also have exposed histidines, the spatial arrangement of the chelate functional monomer in the cavity was not suitable for them. The P(Cu+Lyz2) only had a high adsorption capacity for the template lysozyme, which showed that the coordinate interaction and the shape memory effect were the major factors affecting the imprinting formation and template recognition. For the P(Lyz2), the selectiv-

Table 2. Selectivity of the Thermosensitive Imprinted Hydrogel P(Cu+Lyz2) Using Lysozyme as a Template Protein Value parameter imprinting factor, IF selectivity factor, Rb

a

lysozyme

Cyt c

RNase A

ovalbumin

BHb

BSA

GOx

7.87

0.39 20.15

0.76 10.41

0.86 9.21

0.60 13.01

0.26 30.11

0.86 9.11

a The imprinting factor was calculated using the following formula: IF ) QMIH/QNIH. b The selectivity factor was calculated using the following formula: R ) IFtem/IFana.

Figure 12. SDS-PAGE analysis of the results for the isolation of lysozyme from a mixture of six proteins using the thermosensitive hydrogel of P(Cu+Lyz2), P(Lyz2), and P(Blank2). Lane 1, 10 µL of a mixture of proteins containing GOx, BSA, ovalbumin, TI, RNase A, and lysozyme; lane 2, the mixture after adsorption by the P(Cu+Lyz2) with the Cu ion; lane 3, the mixture after adsorption by the P(Lyz2) with the Cu ion; lane 4, the mixture after adsorption by the P(Blank2) with the Cu ion; lane 5, the mixture after adsorption by the P(Cu+Lyz2) without the Cu ion; lane 6, the mixture after adsorption by the P(Lyz2) without the Cu ion; and lane 7, the mixture after adsorption by the P(Blank2) without the Cu ion.

ity was poor and it had approximately the same adsorption capacities for the lysozyme, Cyt c, and RNase A. The adsorption capacities for the seven proteins on the P(Blank2) were all small, because of a short imprinting process and the random arrangement of the chelate monomer. Table 2 showed the imprinting factor (IF) and the selectivity factor (R) of the P(Cu+Lyz2) for different proteins in the presence of a Cu ion. It was affirmed that adequate cavities with properly placed functional groups for lysozyme have been fabricated in the matrix of P(Cu+Lyz2) during polymerization in the presence of lysozyme as a template. Adsorption Capacity to the Mixture of Proteins. For comparison, the selectivity of the imprinted hydrogel to the mixture of proteins was also measured (see Figure 12). In this system, the monomer VBIDA offered different interactions with the protein. In the presence of a Cu ion, the VBIDA chelated with the Cu ion, which acted as a coordinate monomer to the protein. In the absence of a Cu ion, the VBIDA that contained a free carboxyl group acted as an electrostatic monomer to the protein. When there was a Cu ion, the P(Cu+Lyz2) with VBIDA could chelate with the Cu ion and form a coordinate interaction with the lysozyme. As a result, the imprinting effect was triggered. In this situation (lane 2), the P(Cu+Lyz2) had high selectivity to the template lysozyme in the presence of the other five protein types. The P(Lyz2) with an imprinting cavity for lysozyme could nonspeciphically adsorb some Cu ion. Thus, the P(Lyz2) not only adsorbed some lysozyme, but also randomly adsorbed some RNase A, which has surface exposed histidines and is of a similar size to the lysozyme (lane 3). With or without a Cu ion, the P(Blank2) did not have a high adsorption ability for the six proteins studied (lanes 4 and 7), which was consistent with the single adsorption data.

Figure 13. SDS-PAGE analysis of the results for isolation of lysozyme from egg white. Lane 1: 10 µL of 20-fold dilution of egg white before adsorption; lane 2: 10 µL of 20-fold dilution of egg white after adsorption; and lane 3: 10 µL of the eluate protein from the P(Cu+Lyz2).

When there was no Cu ion, the VBIDA with a carboxyl group was negatively charged at a pH of 7.0 while the template protein lysozyme (pI 11.2) and RNase A (pI 9.4) were positively charged. The increase in adsorption capacity for the RNase A can be attributed to the electrostatic interaction between the negatively charged P(Cu+Lyz2) and positively charged protein. Therefore, in lane 5, the P(Cu+Lyz2) has a certain adsorption ability for both the lysozyme and RNase A. The adsorption ability of P(Lyz2) for lysozyme just through hydrophobic interaction and hydrogen bond was not high, as shown in lane 6. Separation of Lysozyme from Egg White. To further ascertain the ability of P(Cu+Lyz2) to discriminate between lysozyme and other co-existence proteins, egg white as a real sample was used to purify the lysozyme. The concentration of lysozyme in egg white is ∼3% (from all proteins).21 The SDS-PAGE analysis (see Figure 13) shows that the eluate of P(Cu+Lyz2) exhibited a single band with a molecular mass of ∼14.4 kDa, in excellent agreement with that of the template lysozyme (lane 3). All other proteins in the egg white, such as ovalbumin and ovotransferrin, displayed no cross-adsorption by the P(Cu+Lyz2) and did not interfere with the binding of lysozyme, which suggested the potential practical application of the P(Cu+Lyz2). This high selectivity of the P(Cu+Lyz2) to the template was attributed to the synergistic combination of the imprinting effect with the smart response of the thermosensitive hydrogel to the template. A remarkable merit of using this type of molecularly imprinted hydrogel for the separation of complex biological samples is that expensive antibody and tedious sample treatment procedures can be avoided. Cross-Reactivity of the Thermosensitive Imprinted Hydrogels. The generality of the proposed molecular imprinting method was further measured through imprinting the RNase A. The RNase A has two exposed histidines, and its molecular weight and isoelectric point are similar to those of the lysozyme. The P(Cu+RNase A) was synthesized in the same way with the Analytical Chemistry, Vol. 81, No. 17, September 1, 2009

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P(Cu+Lyz2), except for the addition of template RNase A (see Table S2 in the Supporting Information). To investigate the imprinting effect of the P(Cu+Lyz2) and P(Cu+RNase A), the aforementioned two types of imprinted hydrogels were dedicated to the adsorption of the single solution of the lysozyme or RNase A, respectively. Each type of imprinted hydrogel exhibited specific selectivity to the template protein over structurally similar proteins (see Figure S3 in the Supporting Information). In addition, these thermosensitive imprinted hydrogels were also observed to have a higher template protein binding capacity than the control hydrogel. The P(Cu+Lyz2) and P(Cu+RNase A) were further investigated to adsorb the mixture solution of lysozyme and RNase A, respectively (see Figure S4 in the Supporting Information). From Figure S4 in the Supporting Information, it was apparent that, when the mixture solution was loaded upon the P(Cu+Lyz2), most of the nontemplate protein (RNase A) remained in the solution (lane 2). The same phenomenon was observed on the P(Cu+RNase A) (lane 3). This clearly showed that the imprinted hydrogel displayed specificity to the original template protein and had a relatively low level of cross-reactivity toward structurally similar species. It also demonstrated that the imprinting cavities were discriminating proteins, on the basis of molecular shape rather than size. CONCLUSIONS A novel thermosensitive protein-imprinted hydrogel for the selective recognition of lysozyme by self-assembling with the chelate monomer and Cu ions was prepared. Frozen polymerization was adopted to synthesize the thermosensitive imprinted hydrogel. The obtained macroporous thermosensitive imprinted hydrogel was endowed with a higher adsorption capacity for the template protein, compared to the hydrogel prepared without the chelate monomer. Meanwhile, the recognition ability of the imprinted hydrogel for template could be controlled by temperature stimuli. Both the imprinting effect and the smart response

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to the template are reasons for the high selectivity of the imprinted hydrogel to the lysozyme from the mixture of proteins and real samples. Although the research on the applications of the proteinimprinted hydrogel is still in its infancy, it will have wide analytical applications in the near future, because of its advantages. ACKNOWLEDGMENT We would like to thank the National Nature Science Foundation of China (No. 20875049), the National Basic Research Program of China (973 Program, Nos. 2007CB914100 and 2006CB705703), and Tianjin Natural Science Foundation (No. 06YFJMJC02800) for financial support. We also appreciate the contribution of Ms. Anatola S. He (Harvard College, 2012), who provided help with the language of this paper. SUPPORTING INFORMATION AVAILABLE Information regarding the adsorption capacity of P(Lyz) and P(Blank′), relative to the presence/absence of Cu ions (Figure S1). Langmuir linearized plot of lysozyme on P(Cu+Lyz2) (Figure S2). Information regarding the adsorption capacity of the imprinted hydrogels P(Cu+Lyz2) and P(Cu+RNase A) and the nonimprinted hydrogel P(Blank′), relative to the the corresponding proteins (Figure S3). SDS-PAGE analysis of the results from the isolation of protein from a mixture of lysozyme and RNase A, using the hydrogels P(Cu+Lyz2), P(Cu+RNase A), and P(Blank′) (Figure S4). Degree of extraction of the template protein from the macroporous thermosenstitive protein-imprinted hydrogels (Table S1). Composition of various hydrogels (Table S2). Comparison of K and Qmax variables with those from other reported works for imprinting lysozymes (Table S3). Comparison of maxiumum adsorption capacities of different imprinting methods (Table S4). (PDF) This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 1, 2009. Accepted July 22, 2009. AC900676T

Macroporous Thermosensitive Imprinted Hydrogel for Recognition of

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