华中科技大学
硕士学位论文
基于生物素-亲和素系统引入的蛋白质芯片研究
姓名：崔浩巍
申请学位级别：硕士
专业：软件工程
指导教师：于军
2010-05-26
华中科技大学硕士学位论文
摘要
生物芯片是上世纪90年代发展起来的一项具有划时代意义的微量分析检测技术，它综合了分子生物学、免疫学、半导体微电子、激光科学等学科。作为生物芯片的一种，蛋白质芯片是随着DNA芯片不断成熟而发展起来的一种新的检测技术，它是按预先设计的方式将抗原或抗体固定在基片上，形成蛋白质的微阵列，然后带有特殊标记对应的抗体或抗原与之特异性结合，通过对标记物的检测来实现抗原抗体的检测。但是到目前为止蛋白质芯片的检测灵敏度不是很高，导致结果假阴性率升高，本文正是针对这一问题展开研究，通过较好的载体表面修饰及生物素-亲和素系统的引入，使得所研制的芯片的检测灵敏度得到了很大的提高。
鉴于光盘的表面性质很好，本文用镀Au膜的光盘片这种有机塑料作为蛋白质芯片的基片。为了后续实验的顺利进行，还需要对基片进行表面修饰，首先在Au表面自组装一层单分子膜，本文选用的自组装试剂是11-巯基十一烷酸(MUA)，分子式为HS-(CH2)10-COOH，其一端的巯基(-SH)可以和Au发生化学吸附，形成的S-Au键键能约177kJ/mol，这种强的键合作用使得巯基化合物在金表面吸附有明显的优越性，另一端的羧基(-COOH)则暴露在基片的表面。另外从分子式还可以看出它是一个具有11个碳原子的长链有机分子，它一端的巯基与Au结合，另一端则可以与待测蛋白质分子共价结合，这样一来待测蛋白质分子与基体表面就有11个碳原子的距离，很好的保持了待测蛋白质分子原来的空间构象，从而也就降低了检测的假阴性率，提高了检测的准确性。自组装之后，由于基片表面的羧基不能直接与人IgG上的氨基(-NH2)反应，需首先将羧基活化，使它变为活泼酯，进而才可以与氨基反应。
为了提高检测的灵敏度，本文尝试了在蛋白质芯片上引入生物素-亲和素系统，它是以生物素-亲和素之间具有很高的结合能力为基础，二者还可以偶联抗原、抗体、酶、荧光素等大分子生物活性物质，它们的结合迅速、专一、稳定。其中亲合素是由4个相同的亚单位构成的四聚体，每个亲合素亚单位通过其结构中的色氨酸残基与生物素中的咪唑酮环结合。这样1个亲合素分子具有4个与生物素分子结合的位点，使其具有放大效应。亲合素与生物素之间的亲和力极强，二者的亲和系数(Ka)为
华中科技大学硕士学位论文
1015mol-1，比抗原与抗体的亲和力(Ka＝105~11mol-1)至少高104倍，所以能高速结合，而且反应不受外界干扰，具有高度特异性和稳定性。
免疫学实验部分是本文的重要内容，实验中用到了酶标仪，荧光扫描仪等仪器，主要内容分为两大部分：一是以酶作为标记的在96孔板上做的微孔板ELISA实验；二是以荧光分子作为标记的在自制基片上做的微阵列实验，每种实验又分为常规实验和加了生物素-亲和素的实验。实验设计方案不仅证明微阵列实验的优越性，而且进一步验证了引入生物素-亲和素系统提高检测灵敏度的优点。
关键词：蛋白质芯片，自组装分子膜，生物素-亲和素系统，抗原-抗体，ELISA
华中科技大学硕士学位论文
ABSTRACT
Biochip is an emerging technology developed with Human Genome Project(HGP) and as first used in 1990s, which marks a new area of micro-analysis technology. It combines many subjects such as molecular biology, immunology, semiconductor microelectronics, laser optics, and so on. As a kind of biochip, protein chip developed a new detection technology along with the mature of the DNA chip. It works as follows: firstly, the pre-designed manner antigen or antibody fixed on the substrate to form a protein micro-array, and then react specifically with the marked corresponding antibody or antigen, through the detection of markers to infer the antigen or antibody. But until now the protein chip detection sensitivity is not very high, which results in false negative rate increasing, this article is precisely to deal with this problem through good surface modification and the introduction of biotin-avidin system, which greatly improve the detection sensitivity.
This paper use of this gold-plated plastic film as a protein chip substrates, in view of the surface of disc of good nature. In order to smooth the follow-up experiments, we also need to modify the surface of the substrate, firstly self-assembly monolayers are deposited on the surface of gold film, and the self-assembly kit is 11-mercapto-undecanoic acid (MUA), the formula HS-(CH2)10-COOH, whose the end of the thiol (-SH) can be a chemical adsorption with Au, the formation of S-Au chemical bond energy is about 177kJ/mol, this strong bond with the thiol compounds on gold film has obvious advantages, and then the other end, the carboxyl (-COOH) were exposed to the substrate surface. It also can be seen from the formula that the organic molecules has a 11 carbon atoms long chain, with a combination of Au film through thiol at one end, and a covalent combination of test protein at the other. so there is a distance of 11 carbon atoms between the test protein molecule and the substrate surface, which maintains original test protein conformation, reduces the false negative rate of detection and improves the detection accuracy. As the carboxyl on the substrate surface can not be directly react on the amino(-NH2) of human IgG, firstly it needs to be activated to active ester, and then can react on amino.
In order to enhance detection sensitivity, the article introduces the biotin-avidin system,
