Research Of Protein Chip Based On The Introduction Of Biotin-avidin System

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, which is based on high binding ability between biotin and avidin, both them can be coupled with antigen, antibody, enzyme molecules, fluorescent molecules rapidly, specificly, and stably. The avidin consists of four identical subunits, each of which combines with imidazole ring ketone of biotin through its tryptophan residues. Therefore, an avidin molecule has four conjunct sites of biotin molecules, making it has a multi-stage amplification effect. There is a strong affinity between biotin and avidin, and the affinity constant (Ka) is 1015mol^-1, with a magnitude at least 1 million times higher than the antigen-antibody affinity (Ka= 105¹1mol^-1), therefore they can combine at high-speed, and the reaction is free from outside interference, and with a high degree of specificity and stability.Immunologic experiment section is the important content of this article, the experiment used a ELISA Reader, fluorescence scanner and other instruments, the content can be mainly divided into two parts:one is the enzyme molecules marked detection experiment on microplates of 96-well; the other is the fluorescent molecules marked detection experiment on protein chip. The object is to prove the superiority of microarray experiments, in which each part is divided into conventional experiments and biotin-avidin participative experiments to indicate the high sensitivity advantages of biotin-avidin system.