Research Of Colorimetric Biosensing Technology Based On Terminal Protection

Protein is a kind of important biological macromolecules. It is the basic substance of life event, and also is the most basic structure constitute substance and functional material in organisms. The acknowledgement of structure and function of proteins could facilitate to explain the chemical nature of life phenomenon at the molecular level, but also boost the developments of other fields, such as biology, medicine. As a new biological analysis method, biosensor has good selectivity, high sensitivity, strong stability, low cost and can also be employed in online monitoring complex system, etc. So biosensor is widely used in fields of biology, medicine, environmental science, food, etc. This thesis reports several new biosensing technology analysis methods, which are used for the detection of some important enzymatic activity and the research of interactions between small biomolecule and binding protein based on terminal protection.1. A simple, rapid and highly sensitive biosensing technology based on terminal protection has been proposed and applied to detect catalytic activity of DNA (cytosine-5)-methyltransferase 1(Dnmt 1) and 8-oxoguanine DNA glycosylase (hOGG 1) via gold nanoparticle’s color changes. The specific binding of Dnmt 1 or hOGG 1 and gold nanoparticle labeled DNA substrates forms covalent complexes, which can effectively protect DNA from being hydrolyzed by Exo I and Exo III. The catalytic activity of Dnmt 1 and hOGG 1 can be gained through detecting the plasma resonance absorption of protected gold nanoparticle. This method might provide a convenient, quick, highly sensitive and specific detection platform for catalytic activity detection of Dnmt 1 and hOGG 1.2. Two biosensor technologies based on terminal protection have been proposed and applied to study interactions between folate and folate receptor via colorimetric analysis and real-time quantitatie polymerase chain reaction (RT-QPCR). The specific binding of folate labeled DNA and folate receptor forms covalent complexes, which can effectively protect DNA from being hydrolyzed by Exo I and Exo III. Folate receptor can be quantitated through colorimetric analysis of protected G-quadruplex-hemin complexes catalyting 2,2’-Azinobis-3-ethylbenzthiazoline-6- sulphonate (ABTS) and real-time quantitative polymerase chain reaction (RT-QPCR) of protected folate labeled DNA. The colorimetric analysis has high specificity and can be detected by visualization without need of special equipment; the real-time quantitatie polymerase chain reaction (RT-QPCR) method is not only convenient but also has low detection limit.