硅基芯片表面化学性质对蛋白质固定化的影响

doi:10.3866/PKU.WHXB20101016

物理化学学报 >> 2010, Vol. 26 >> Issue (10): 2821-2827.doi: 10.3866/PKU.WHXB20101016

硅基芯片表面化学性质对蛋白质固定化的影响

周稳稳^1,2, 廉洁^1,2, 胡科家^1,2, 高云华¹, 徐百¹

1. 中国科学院理化技术研究所光化学转换和功能材料重点实验室, 北京 100190;
2. 中国科学院研究生院, 北京 100049

收稿日期:2010-04-22 修回日期:2010-07-09 发布日期:2010-09-27
通讯作者: 高云华, 徐百 E-mail:yhgao@mail.ipc.ac.cn, nanobioic@gmail.com
基金资助:
国家自然科学基金(20975106), 中国科学院知识创新工程重要方向项目(KJCX2-YW-M15) 及国家重大专项(2008ZX08012-001) 资助

Effect of Surface Chemical Properties of a Silicon Chip on Antibody Immobilization

ZHOU Wen-Wen^1,2, LIAN Jie^1,2, HU Ke-Jia^1,2, GAO Yun-Hua¹, XU Bai¹

1. Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry,Chinese Academy of Sciences, Beijing 100190, P. R. China;
2. Graduate University of Chinese Academy of Sciences,Beijing 100049, P. R. China

Received:2010-04-22 Revised:2010-07-09 Published:2010-09-27
Contact: GAO Yun-Hua, XU Bai E-mail:yhgao@mail.ipc.ac.cn, nanobioic@gmail.com
Supported by:
The project was supported by the National Natural Science Foundation of China (20975106), Funds of the Chinese Academy of Sciences for Key Topics in Innovation Engineering (KJCX2-YW-M15) and Important National Science & Technology Specific Projects, China (2008ZX08012-001).

摘要/Abstract

摘要：

制备蛋白质芯片的关键在于将蛋白质固定到芯片表面并保持其生物学活性. 本实验中, 我们分别采用物理吸附、直接化学固定、加入间隔臂化学固定和生物亲和作用固定的方法将癌胚抗原(CEA)抗体固定到硅基芯片的二氧化硅表面. 基于抗原-抗体的特异性相互作用, 利用双抗体夹心酶联免疫法(ELISA)评价各种方法固定抗体的效果. 实验结果表明, 在修饰有氨基的表面采用戊二醛作为偶联试剂固定CEA 抗体具有最高的偶联效率, 引入多聚赖氨酸(poly-L-lysine)作为间隔臂可以显著增强固定效果, 并可进一步降低非特异性吸附. 而利用生物亲和作用固定CEA抗体也可获得较好的固定效果,但是非特异性吸附较严重.

关键词: 蛋白质芯片, 抗体固定, 夹心反应, 酶联免疫吸附分析, 氨基表面, 戊二醛, 多聚赖氨酸

Abstract:

The key to constructing a protein microarray is the stable immobilization of proteins and the retention of their biological activities. In this study, immobilization of the carcinoembryonic antigen (CEA) antibody onto a silicon dioxide surface was investigated by physical adsorption, direct chemical covalent conjugation, spacer-added chemical covalent conjugation, and biological affinity interactions. Based on the specific antibody-antigen interactions, the sandwich reaction, enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate various immobilization strategies. The most efficient immobilization strategy was with glutaraldehyde as a coupling reagent between the CEA antibody and the amino surface. The attachment of a spacer-armcomprising poly-L-lysine significantly improved the immobilization efficiency and simultaneously decreased nonspecific adsorption. High immobilization efficiency and stronger nonspecific adsorption were also observed when the CEA antibody was immobilized by bioaffinity interactions.

Key words: Protein microarray, Antibody immobilization, Sandwich reaction, Enzyme-linked immunosorbent assay, Amino surface, Glutaraldehyde, Poly-L-lysine

周稳稳, 廉洁, 胡科家, 高云华, 徐百. 硅基芯片表面化学性质对蛋白质固定化的影响[J]. 物理化学学报, 2010, 26(10), 2821-2827. doi: 10.3866/PKU.WHXB20101016

ZHOU Wen-Wen, LIAN Jie, HU Ke-Jia, GAO Yun-Hua, XU Bai. Effect of Surface Chemical Properties of a Silicon Chip on Antibody Immobilization[J]. Acta Phys. -Chim. Sin. 2010, 26(10), 2821-2827. doi: 10.3866/PKU.WHXB20101016

链接本文: https://www.whxb.pku.edu.cn/CN/10.3866/PKU.WHXB20101016

https://www.whxb.pku.edu.cn/CN/Y2010/V26/I10/2821

参考文献

1. Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol., 2001, 5: 40
2. MacBeath, G. Nat Genet, 2002, 32(suppl.): 526
3. Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Võhringer, C. F.; Joos, T. O. Trends Biotechnol., 2002, 20: 160
4. Ng, J. H.; Ilag, L. L. Journal of Cellular and Molecular Medicine, 2002, 6: 329
5. Wilson, D. S.; Nock, S. Angew. Chem. Int. Edit., 2003, 42: 494
6. Angenendt, P. Drug Discovery Today, 2005, 10: 503
7. Kusnezow, W.; Hoheisel, J. D. Biotechniques, 2002, 33(suppl.): 14
8. Liotta, L. A.; Espina, V.; Mehta, A. I.; Calvert, V.; Rosenblatt, K.; Geho, D.; Munson, P. J.; Young, L.; Wulfkuhle, J.; Petricoin III, E. F. Cancer Cell, 2003, 3: 317
9. Lutanie, E.; Voegel, J. C.; Schaaf, P.; Freund, M.; Cazenave, J. P.; Schmitt, A. Proc. Natl. Acad. Sci. U. S. A., 1992, 89: 9890
10. Vijayendran, R. A.; Leckband, D. E. Anal. Chem., 2000, 73: 471
11. Rossier, J. S.; Gokulrangan, G.; Girault, H. H.; Svojanovsky, S.; Wilson, G. S. Langmuir, 2000, 16: 8489
12. Angenendt, P.; Glõkler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem., 2002, 309: 253
13. Kusnezow, W.; Hoheisel, J. D. J. Mol. Recognit., 2003, 16: 165
14. Seong, S. Y. Clin Diagn Lab Immunol, 2002, 9: 927
15. Kusnezow, W.; Jacob, A.; Walijew, A.; Diehl, F.; Hoheisel, J. D. Proteomics, 2003, 3: 254
16. Niemeyer, C. M.;Wacker, R.; Adler, M. Nucl. Acids Res., 2003, 31: e90
17. Peluso, P.;Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.;Wagner, P.; Nock, S. Anal. Biochem., 2003, 312: 113
18. Ramachandran, N.; Hainsworth, E.; Bhullar, B.; Eisenstein, S.; Rosen, B.; Lau, A. Y.;Walter, J. C.; LaBaer, J. Science, 2004, 305: 86
19. Oh, S. J.; Hong, B. J.; Choi, K. Y.; Park, J. W. OMICS, 2006, 10: 327
20. Iwata, R.; Satoh, R.; Iwasaki, Y.; Akiyoshi, K. Colloids Surf. B, 2008, 62: 288
21. Hermanson, G. T. Bioconjugate techniques. 2nd ed. Rockford, Illinois: Academic Press, 2008
22. Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol., 2001, 2: 0004.1
23. Stillman, B. A.; Tonkinson, J. L. Biotechniques, 2000, 29: 630
24. Qian,W.; Yao, D.; Yu, F.; Xu, B.; Zhou, R.; Bao, X.; Lu, Z. Clin. Chem., 2000, 46: 1456
25. MacBeath, G.; Schreiber, S. L. Science, 2000, 289: 1760
26. Piehler, J.; Brecht, A.; Geckeler, K. E.; Gauglitz, G. Biosens. Bioelectron., 1996, 11: 579
27. Penzol, G.; Armisén, P.; Fernández-Lafuente, R.; Rodés, L.; Guisán, J. M. Biotechnol. Bioeng., 1998, 60: 518
28. Afanassiev, V.; Hanemann, V.;Wolfl, S. Nucl. Acids Res., 2000, 28: e66
29. Piletsky, S. A.; Matuschewski, H.; Schedler, U.; Wilpert, A.; Piletska, E. V.; Thiele, T. A.; Ulbricht, M. Macromolecules, 2000, 33: 3092
30. Wilson, D. S.; Nock, S. Curr. Opin. Chem. Biol., 2002, 6: 81
31. Neubert, H.; Jacoby, E. S.; Bansal, S. S.; Iles, R. K.; Cowan, D. A.; Kicman, A. T. Anal. Chem., 2002, 74: 3677
32. Gupta, B.; Plummer, C.; Bisson, I.; Frey, P.; Hilborn, J. Biomaterials, 2002, 23: 863
33. Stine, R.; Cole, C. L.; Ainslie, K. M.; Mulvaney, S. P.;Whitman, L. J. Langmuir, 2007, 23: 4400
34. Gibson, T. D.;Woodward, J. R. Protein stabilization in biosensor systems//Biosensors and chemical sensors.Washington, DC: American Chemical Society, 1992: 40
35. Mateo, C.; Torres, R.; Fernández-Lorente, G.; Ortiz, C.; Fuentes, M.; Hidalgo, A.; López-Gallego, F.; Abian, O.; Palomo, J. M.; Betancor, L.; Pessela, B. C. C.; Guisan, J. M.; Fernández-Lafuente, R. Biomacromolecules, 2003, 4: 772
36. Lee, S. D.; Hsiue, G. H.; Chang, P. C. T.; Kao, C. Y. Biomaterials, 1996, 17: 1599
37. Kato, K.; Ikada, Y. Biotechnol. Bioeng., 1995, 47: 557
38. Lee,W.; Oh, B. K.; Bae, Y. M.; Paek, S. H.; Lee, W. H.; Choi, J. W. Biosensors and Bioelectronics, 2003, 19: 185
39. Turková, J. J. Chromatogr. B, 1999, 722: 11
40. Jung, Y.; Lee, J. M.; Jung, H.; Chung, B. H. Anal. Chem., 2007, 79: 6534
41. Võlimaa, L. Streptavidin———a versatile binding protein for solid- phase immunoassays [D]. Turku: University of Turku, 2008
42. Lue, R. Y. P.; Chen, G. Y. J.; Zhu, Q.; Lesaicherre, M. -L.; Yao, S. Q. Site-specific immobilization of biotinylated proteins for protein microarray analysis//Protein arrays. Totowa: Humana Press Inc., 2004: 85

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硅基芯片表面化学性质对蛋白质固定化的影响

Effect of Surface Chemical Properties of a Silicon Chip on Antibody Immobilization

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