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Molecular imprinting of protein by coordinate interaction
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Chinese Chemical Letters 20 (2009) 747–750 www.elsevier.com/locate/cclet
Molecular imprinting of protein by coordinate interaction Jun Wang, Zhen Dong Hua, Zhi Yong Chen, Yuan Zong Li, Mei Ping Zhao * The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 12 October 2008
Abstract In this article, a novel strong interaction by forming complex between bovine serum albumin (BSA) and copper ion was utilized for the preparation of molecular imprinted hydrogel in aqueous solution. Results show that the inclusion of copper ion in preparation can bridge the template BSA and functional monomers together and improve the imprinting effect compared to the polymer made without copper ion added. High selectivity factor and large adsorption capacity are also observed for the obtained BSA-imprinted hydrogel. # 2008 Mei Ping Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Molecular imprinted hydrogel; Bovine serum albumin; Copper ion
Molecular imprinting technology is one of the most attractive methods to fulfill the aim to build the structure capable of recognizing and binding desired molecular targets. Although the recognition toward small molecules has been achieved by molecular imprinted polymers (MIPs) successfully [1–3], the attempts to imprint macromolecules, like proteins [4] or viruses [5] are still a significant challenge. Since a lot of properties of proteins influence their imprinting effect deeply, such as protein’s flexible conformation, large size, complex surface structure and so on, limited successful research papers have been published so far [6,7]. As an approach of protein imprinting, proteinmetal coordination interaction was first described by the group of Mosbach [6]. The selectivity for RNase A enzyme was enhanced by a surface imprinting procedure based on metal complex. According to our previous work [8,9], some small molecules could be imprinted effectively in the presence of metal ions, via the method of bridging template and functional monomer together. Based on the same consideration, it is possible that macromolecules, such as proteins, can be imprinted by the strong coordination bonds in aqueous phase too, so that the difficult problem of protein imprinting would be partly solved by this approach. Besides the aqueous environment, which is fit for protein molecules, the materials of imprinted polymer should also be bio-compatible. N-Isopropylacrylamide (NIPA) can meet such need. Acting as a kind of smart functional material usually, the polymer using NIPA would response to the external stimulation and shrink or swell under different conditions. In this work, we use bovine serum albumin (BSA, MW 66 kDa) as the template protein which can form a complex with copper ion at its N-terminal site [10]. By binding with functional monomer 4-vinylpyridine via copper
* Corresponding author. E-mail address: [email protected] (M.P. Zhao). 1001-8417/$ – see front matter # 2008 Mei Ping Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2008.12.035
748
J. Wang et al. / Chinese Chemical Letters 20 (2009) 747–750
ion, the whole protein-metal complex would be imprinted in polymers. Also, the polymer without adding copper ion is synthesized for comparison. 1. Experimental NIPA (200 mg, 1.77 mmol), acrylamide (AAm, 5.0 mg, 0.070 mmol), 4-vinylpyridine (4-VPy, 3.2 mL, 0.028 mmol), N,N-methylenebisacrylamide (BIS, 4.0 mg, 0.026 mmol), Cu(Ac)2H2O (2.0 mg, 0.010 mmol) and template protein BSA (130 mg, 2.0 mmol) were dissolved in 2.0 mL of Tris–HCl buffer solution (10 mmol/L, pH 7.0). A volume of 1 mL N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) and 10 mL 10% ammonium persulfate were added and the solution was deoxygenated by purging with nitrogen for 3 min. Immediately the solution was transferred into the space between two glass plates (Bio-rad, USA) with an interval of 0.75 mm. Polymerization was carried out at 25 8C for 12 h to prepare P(Cu-BSA). After the reaction finished, P(Cu-BSA) hydrogels were washed with 100 mL NaCl (300 mmol/L, pH 1.5) and deionized water three times, respectively. The complete removal of BSA from the imprinted hydrogel was confirmed by a Cary-1E UV–vis spectrophotometer and HPLC method. Then the resultant hydrogels were cut into disks (10 mm in diameter). They were immersed in deionized water at room temperature for 48 h, and dried under vacuum (60 8C). Correspondingly, P(BSA) polymer, without adding copper acetate, and non-imprinted control polymer (P(Blank)), in the absence of copper acetate and BSA, were generated in the same way. A piece of dry disk hydrogel (about 5 mg) was first put in 5.0 mL solution at specified pH and temperature to allow the hydrogel to swell for 24 h. Then the wet hydrogel was shaken for 24 h in 8.0 mL of protein–copper complex solution, or only protein solution without copper ion, at the same conditions as swelling. The protein concentration in the supernatant was measured by the same UV-spectrometer at 280 nm, and the amount of adsorbed protein can be determined by the difference before and after the adsorption. Recognition ability is evaluated by static distribution coefficient (D) and relative selectivity factor (ar) [11]. 2. Results and discussion The loosely crosslinked NIPA polymer is a type of smart hydrogel that is bio-compatible and also sensitive in its dimension or structure toward a small stimulation from the environment. The influence of temperature on the adsorption of BSA on the imprinted hydrogel was investigated and the results are shown in Fig. 1. As can be seen, the imprinting factor (IF) reaches the maximum at 25 8C, at which the polymer is prepared. This is mainly because that the internal structure of the hydrogel reserves the most similar pore size and shapes as it is formed at this temperature. So the following experiments were all conducted under the temperature of 25 8C. The effect of adding cupric ion in preparation solution on the binding capacity of the imprinted hydrogel can be observed from the comparison results shown in Fig. 2. As can be seen clearly, DBSA of P(Cu-BSA) in the presence of Cu2+ is much higher than those of P(BSA) and P(Blank). It ascertained that the protein-metal ion binding was an effective interaction for obtaining polymers with high binding capacity of the target protein. The results also show that
Fig. 1. IF values of BSA at different temperature in the presence of copper ion. The concentration of BSA is fixed at 10 mmol/L and the experiment is performed in pH 7.0 buffer solution.
J. Wang et al. / Chinese Chemical Letters 20 (2009) 747–750
749
Fig. 2. Static distribution coefficients on P(Cu-BSA), P(BSA) and P(blank) in the presence and absence of copper ion. The error bars are based on three parallel experiments.
Table 1 Relative selectivity of different proteins on different polymers. Polymers
DBSA (mL/g dry gel)
DOVA (mL/g dry gel)
a(DBSA/DOVA)
P(Cu-BSA) P(BSA) P(Blank)
107 7 67 3 31 9
49 2 72 10 105 10
2.2 0.94 0.30
Relative selectivity (ar = ai/an)
7.4
imprinting with BSA in the absence of cupric ion is not effective and the adsorption capacity is very low under present experimental conditions, although BSA was imprinted previously [12]. The effect of adding cupric ion in preparation solution on the selectivity of the imprinted hydrogel was also investigated. As shown in Table 1, the distribution coefficient (D) of ovalbumin (OVA, MW 44 kDa) on P(Cu-BSA) is much lower than that of BSA, but on P(Blank), DOVA shows much higher value than that of BSA. The relative selectivity (ar) is as high as 7.4. These results indicate that P(Cu-BSA) possesses good template recognition capability compared to the control polymer P(Blank). The high adsorption of OVA on P(Blank) is presumably due to the small size of OVA which allows its easy access to the cavities where coordination bond may form. The maximum adsorption capacity of BSA on the imprinted polymer P(Cu-BSA) was found to be 1.42 mg/mg dry gel in batch experiment. Comparing with the maximum adsorption capacity of our previously prepared BSAimprinted hydrogel without using protein-metal ion coordinate interaction [7], which is 0.825 mg/mg dry gel, the presented approach may offer more and stronger interaction sites to adsorb protein molecules. In summary, the inclusion of cupric ion in polymer preparation remarkably enhanced the imprinting effect of BSA by strengthening the interaction between functional monomers and the template proteins. Acknowledgment The work was supported by the National Natural Science Foundation of China (Nos. 20575007 and 30872109). References [1] [2] [3] [4] [5] [6] [7] [8]
L.I. Andersson, Anal. Chem. 68 (1996) 111. T.L. Zhang, F. Liu, W. Chen, J. Wang, Anal. Chim. Acta 450 (2001) 53. J.L. Urraca, A.J. Hall, M.C. Moreno-Bondi, B. Sellergren, Angew. Chem. Int. Ed. 45 (2006) 5158. K. Haupt, Anal. Chem. 75 (2003) 377A. O. Hayden, P.A. Lieberzeit, D. Blaas, F.L. Dickert, Adv. Funct. Mater. 16 (2006) 1269. M. Kempe, M. Glad, K. Mosbach, J. Mol. Recognit. 8 (1995) 35. Z.D. Hua, Z.Y. Chen, Y.Z. Li, M.P. Zhao, Langmuir 24 (2008) 5773. M.L. Yang, Y.Z. Li, J. Mol. Recognit. 18 (2005) 103.
750 [9] [10] [11] [12]
J. Wang et al. / Chinese Chemical Letters 20 (2009) 747–750 L.Q. Wu, Y.Z. Li, Anal. Chim. Acta 482 (2003) 175. P.J. Sadler, A. Tucker, J.H. Viles, Eur. J. Biochem. 220 (1994) 193. Y.W. Liu, X.J. Chang, D. Yang, Y. Guo, S.M. Meng, Anal. Chim. Acta 538 (2005) 85. X.S. Pang, G.X. Cheng, S.L. Lu, E.J. Tang, Anal. Bioanal. Chem. 384 (2006) 225.
Chinese Chemical Letters 20 (2009) 747–750 www.elsevier.com/locate/cclet
Molecular imprinting of protein by coordinate interaction Jun Wang, Zhen Dong Hua, Zhi Yong Chen, Yuan Zong Li, Mei Ping Zhao * The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China Received 12 October 2008
Abstract In this article, a novel strong interaction by forming complex between bovine serum albumin (BSA) and copper ion was utilized for the preparation of molecular imprinted hydrogel in aqueous solution. Results show that the inclusion of copper ion in preparation can bridge the template BSA and functional monomers together and improve the imprinting effect compared to the polymer made without copper ion added. High selectivity factor and large adsorption capacity are also observed for the obtained BSA-imprinted hydrogel. # 2008 Mei Ping Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. Keywords: Molecular imprinted hydrogel; Bovine serum albumin; Copper ion
Molecular imprinting technology is one of the most attractive methods to fulfill the aim to build the structure capable of recognizing and binding desired molecular targets. Although the recognition toward small molecules has been achieved by molecular imprinted polymers (MIPs) successfully [1–3], the attempts to imprint macromolecules, like proteins [4] or viruses [5] are still a significant challenge. Since a lot of properties of proteins influence their imprinting effect deeply, such as protein’s flexible conformation, large size, complex surface structure and so on, limited successful research papers have been published so far [6,7]. As an approach of protein imprinting, proteinmetal coordination interaction was first described by the group of Mosbach [6]. The selectivity for RNase A enzyme was enhanced by a surface imprinting procedure based on metal complex. According to our previous work [8,9], some small molecules could be imprinted effectively in the presence of metal ions, via the method of bridging template and functional monomer together. Based on the same consideration, it is possible that macromolecules, such as proteins, can be imprinted by the strong coordination bonds in aqueous phase too, so that the difficult problem of protein imprinting would be partly solved by this approach. Besides the aqueous environment, which is fit for protein molecules, the materials of imprinted polymer should also be bio-compatible. N-Isopropylacrylamide (NIPA) can meet such need. Acting as a kind of smart functional material usually, the polymer using NIPA would response to the external stimulation and shrink or swell under different conditions. In this work, we use bovine serum albumin (BSA, MW 66 kDa) as the template protein which can form a complex with copper ion at its N-terminal site [10]. By binding with functional monomer 4-vinylpyridine via copper
* Corresponding author. E-mail address: [email protected] (M.P. Zhao). 1001-8417/$ – see front matter # 2008 Mei Ping Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. doi:10.1016/j.cclet.2008.12.035
748
J. Wang et al. / Chinese Chemical Letters 20 (2009) 747–750
ion, the whole protein-metal complex would be imprinted in polymers. Also, the polymer without adding copper ion is synthesized for comparison. 1. Experimental NIPA (200 mg, 1.77 mmol), acrylamide (AAm, 5.0 mg, 0.070 mmol), 4-vinylpyridine (4-VPy, 3.2 mL, 0.028 mmol), N,N-methylenebisacrylamide (BIS, 4.0 mg, 0.026 mmol), Cu(Ac)2H2O (2.0 mg, 0.010 mmol) and template protein BSA (130 mg, 2.0 mmol) were dissolved in 2.0 mL of Tris–HCl buffer solution (10 mmol/L, pH 7.0). A volume of 1 mL N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) and 10 mL 10% ammonium persulfate were added and the solution was deoxygenated by purging with nitrogen for 3 min. Immediately the solution was transferred into the space between two glass plates (Bio-rad, USA) with an interval of 0.75 mm. Polymerization was carried out at 25 8C for 12 h to prepare P(Cu-BSA). After the reaction finished, P(Cu-BSA) hydrogels were washed with 100 mL NaCl (300 mmol/L, pH 1.5) and deionized water three times, respectively. The complete removal of BSA from the imprinted hydrogel was confirmed by a Cary-1E UV–vis spectrophotometer and HPLC method. Then the resultant hydrogels were cut into disks (10 mm in diameter). They were immersed in deionized water at room temperature for 48 h, and dried under vacuum (60 8C). Correspondingly, P(BSA) polymer, without adding copper acetate, and non-imprinted control polymer (P(Blank)), in the absence of copper acetate and BSA, were generated in the same way. A piece of dry disk hydrogel (about 5 mg) was first put in 5.0 mL solution at specified pH and temperature to allow the hydrogel to swell for 24 h. Then the wet hydrogel was shaken for 24 h in 8.0 mL of protein–copper complex solution, or only protein solution without copper ion, at the same conditions as swelling. The protein concentration in the supernatant was measured by the same UV-spectrometer at 280 nm, and the amount of adsorbed protein can be determined by the difference before and after the adsorption. Recognition ability is evaluated by static distribution coefficient (D) and relative selectivity factor (ar) [11]. 2. Results and discussion The loosely crosslinked NIPA polymer is a type of smart hydrogel that is bio-compatible and also sensitive in its dimension or structure toward a small stimulation from the environment. The influence of temperature on the adsorption of BSA on the imprinted hydrogel was investigated and the results are shown in Fig. 1. As can be seen, the imprinting factor (IF) reaches the maximum at 25 8C, at which the polymer is prepared. This is mainly because that the internal structure of the hydrogel reserves the most similar pore size and shapes as it is formed at this temperature. So the following experiments were all conducted under the temperature of 25 8C. The effect of adding cupric ion in preparation solution on the binding capacity of the imprinted hydrogel can be observed from the comparison results shown in Fig. 2. As can be seen clearly, DBSA of P(Cu-BSA) in the presence of Cu2+ is much higher than those of P(BSA) and P(Blank). It ascertained that the protein-metal ion binding was an effective interaction for obtaining polymers with high binding capacity of the target protein. The results also show that
Fig. 1. IF values of BSA at different temperature in the presence of copper ion. The concentration of BSA is fixed at 10 mmol/L and the experiment is performed in pH 7.0 buffer solution.
J. Wang et al. / Chinese Chemical Letters 20 (2009) 747–750
749
Fig. 2. Static distribution coefficients on P(Cu-BSA), P(BSA) and P(blank) in the presence and absence of copper ion. The error bars are based on three parallel experiments.
Table 1 Relative selectivity of different proteins on different polymers. Polymers
DBSA (mL/g dry gel)
DOVA (mL/g dry gel)
a(DBSA/DOVA)
P(Cu-BSA) P(BSA) P(Blank)
107 7 67 3 31 9
49 2 72 10 105 10
2.2 0.94 0.30
Relative selectivity (ar = ai/an)
7.4
imprinting with BSA in the absence of cupric ion is not effective and the adsorption capacity is very low under present experimental conditions, although BSA was imprinted previously [12]. The effect of adding cupric ion in preparation solution on the selectivity of the imprinted hydrogel was also investigated. As shown in Table 1, the distribution coefficient (D) of ovalbumin (OVA, MW 44 kDa) on P(Cu-BSA) is much lower than that of BSA, but on P(Blank), DOVA shows much higher value than that of BSA. The relative selectivity (ar) is as high as 7.4. These results indicate that P(Cu-BSA) possesses good template recognition capability compared to the control polymer P(Blank). The high adsorption of OVA on P(Blank) is presumably due to the small size of OVA which allows its easy access to the cavities where coordination bond may form. The maximum adsorption capacity of BSA on the imprinted polymer P(Cu-BSA) was found to be 1.42 mg/mg dry gel in batch experiment. Comparing with the maximum adsorption capacity of our previously prepared BSAimprinted hydrogel without using protein-metal ion coordinate interaction [7], which is 0.825 mg/mg dry gel, the presented approach may offer more and stronger interaction sites to adsorb protein molecules. In summary, the inclusion of cupric ion in polymer preparation remarkably enhanced the imprinting effect of BSA by strengthening the interaction between functional monomers and the template proteins. Acknowledgment The work was supported by the National Natural Science Foundation of China (Nos. 20575007 and 30872109). References [1] [2] [3] [4] [5] [6] [7] [8]
L.I. Andersson, Anal. Chem. 68 (1996) 111. T.L. Zhang, F. Liu, W. Chen, J. Wang, Anal. Chim. Acta 450 (2001) 53. J.L. Urraca, A.J. Hall, M.C. Moreno-Bondi, B. Sellergren, Angew. Chem. Int. Ed. 45 (2006) 5158. K. Haupt, Anal. Chem. 75 (2003) 377A. O. Hayden, P.A. Lieberzeit, D. Blaas, F.L. Dickert, Adv. Funct. Mater. 16 (2006) 1269. M. Kempe, M. Glad, K. Mosbach, J. Mol. Recognit. 8 (1995) 35. Z.D. Hua, Z.Y. Chen, Y.Z. Li, M.P. Zhao, Langmuir 24 (2008) 5773. M.L. Yang, Y.Z. Li, J. Mol. Recognit. 18 (2005) 103.
750 [9] [10] [11] [12]
J. Wang et al. / Chinese Chemical Letters 20 (2009) 747–750 L.Q. Wu, Y.Z. Li, Anal. Chim. Acta 482 (2003) 175. P.J. Sadler, A. Tucker, J.H. Viles, Eur. J. Biochem. 220 (1994) 193. Y.W. Liu, X.J. Chang, D. Yang, Y. Guo, S.M. Meng, Anal. Chim. Acta 538 (2005) 85. X.S. Pang, G.X. Cheng, S.L. Lu, E.J. Tang, Anal. Bioanal. Chem. 384 (2006) 225.