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

生物物理化学 上一篇    下一篇

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. 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-Wen1,2, LIAN Jie1,2, HU Ke-Jia1,2, GAO Yun-Hua1, XU Bai1   

  1. 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:

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

MSC2000: 

  • O647