Electrocheemical Detectiong Of Single Enzyme Molecules Based On Nanochannel

The technology of single molecule detection is widely applied in the field of analysis, this paper mainly researchs the activity of enzyme by the technology of single molecule detection. The research of this paper is based on the single molecular electrochemical detection of hemolysin nanopores to discuss the activity of restriction endonuclease and thrombin. Using the principle of endonuclease’s restriction of a specific site and the affinity function of thrombin and thrombin aptamer, the different specific DNA chains are designed, when restriction endonuclease or thrombin acts on the dsDNA, the microelectric current when DNA throughing nanopores will bedifferent, so as to achieve the purpose of researching the activity of restriction endonuclease and thrombin. This thesis is divided into five chapters:The first chapter, this paper summarizes the introduction and function of nanochannel, endonuclease and nanoparticles of and simply introduces the analytical method of single molecule detection, microgel electrophoresiss and the patch clamp amplifier detection. This paper especially illustrates the single molecule detection and nanochannel, at the same time the prospects of the single molecule detection is presented.The second chapter, the single molecule detection is used to preliminary study the DNA sequencing and mainly study the activity of restriction enzymes. Using the characteristics of hemolysin aperture to control the different current when the single-strand DNA and double-stranded DNA through the pore, this paper preliminary understands the DNA sequence by using the biological nanopores of hemolysin and lecithin free assembed. Under the action of restriction enzymes, the double-stranded DNA can smoothly through the nanopore. By comparing the current change before and after restriction enzymes function, the restriction enzyme’s activity is studied.The third chapter, by microgel electrophoresiss technology and the the TEM images of DNA tagged by AuNPs, the restrictive endonuclease’s activity is studied, as well as the mismatch base T and T can stably exist under the condition of Hg2+. Firstly studing the activity of the restriction enzyme by observing the separation, which is the DNA under the microgel electrop ho res iss before and afte the restriction enzyme’s action. Secondly after the DNA is tagged by AuNPs, studing the activity of the restriction enzyme by observing nanoparticles’s different characters in TEM. At the same time in the above experiments, which have mismatch base T and T the restriction enzymes can not act on the DNA. Only when under the condition of Hg2+, formed the stable structure of T-Hg-T, the restriction enzymes can work.The fourth chapter, through the hemolysin nanopores, the activity of thrombin molecule is studied. Using the characteristics of hemolysin aperture to control the different current when the single-strand DNA and double-stranded DNA through the pore, this paper preliminary understands the DNA sequence by using the biological nanopores of hemolysin and lecithin free assembed. This paper researchs the affinity of thrombin and thrombin aptamer. When under the action of thrombin, the ssDNA can smoothly through the hemolysin nanopores. At the same time, the time of melting the chains of neckring structure is relevant to the voltage.The fifth chapter, it was summarized for the whole thesis.