| Molecular beacon as a kind of nucleic acid hybridization probe has a unique stem ring structure, at the same time showed sensitively detection, good stability and specificity in a complex environment, so attract the attention and favor by the scientific research personnel. Molecular beacon technology combined with other chemical analysis, such as fluorescence, electrochemistry, etc., had realized the rapid detection of a variety of target molecules with high sensitive. It has become a hot topic in current research. With the continuous development and progress, molecular beacon technology plays a crucial role in disease diagnosis, biochemistry, clinical treatment and other fields.According to the different classification of signal detection, Molecular beacon is mainly divided into electrochemical beacon and fluorescence beacoa The fluorescent molecular beacon as initial widespread use of molecular beacon contains a lot of advantages:simple operation, high sensitivity, time, etc. It has been achieved real-time online monitoring detection of DNA/RNA, protein and other biological small molecules in vivo. But the instability of fluorescent molecular beacon in the complex system and the characteristics of easy bleaching their fluorescence, limits its further development. Compared with the traditional optical methods, electrochemical molecular beacon does not have problem such as fluorescent beacons, and has the advantages of simple, rapid, high sensitivity, good selectivity, no turbidity of the sample. But the traditional electrochemical molecular beacon generally need by means of chemical bonds in order to fix to the electrode surface as modified. At the same time the production process involves a number of relatively complex fixed step.The distances between the electrode surfaces and the tag electrochemical active material will affect the electrochemical signals, and finally cause the poor reproducibility. In addition, due to the electrochemical molecular beacon need to modify on the electrode surface, it is difficult to achieve real-time online detection of target molecules within living cell. These curb the further development of electrochemical beacon.In this thesis we use hemin double marking electrochemical molecular beacon (Hs-MB) implementation to detection the oligonucleotide chain and protein material in homogeneous solution. Studies have shown that SNP single-base mutations have close relations with most cancers, so the study of the SNP single-base mutation becomes a hot spot. This thesis using electrochemical molecular beacon has made the further research of SNP single-base mutation, conducted for the SNP single-base mutation analysis and testing research. On this basis, in order to further apply the molecular beacon to analys the protein material, we developed a method detection of insulin based on nucleic acid-aptamer specificity binding, endonuclease and DNAzyme double signal amplification. Electrochemical method is simple and easy to operate, without limiting by large instruments and equipment, so it has the hope to realize the application of living cells.The first chapter introductionIn this chapter, we mainly introduced some basic knowledge about molecular beacon including the structure of molecular beacon and its principle, the advance of molecular beacon and its application.Second, we focus on the DNAzyme techniques, DNAzyme molecular beacon. And we also focus on the principle, structure and catalytic activity influence factors of the 8-17 DNAzyme. Finally we elaborated the design idea, theory support, purpose and innovation of electrochemical molecular beacon.The second chapter Detected of MP53 by the high sensitive electrochemical activity switch MB (Hs-MB) system based on EXO Ⅲ and DNAzyme double signal amplificationIn this chapter, we build high sensitive electrochemical activity switch MB (Hs-MB) based on EXO Ⅲ and DNAzyme double signal amplification system for the detection of Mp53. The synthesized Hs-MB by our group is both tag hemins in stem end. We detected the MP53 by the difference electrochemical activity between hemin dimers and hemin monomer. In the absence of Mp53, Hs-MB exists in hairpin structure, hemin exists in the form of dimers, the switch activity of Hs-MB is closed, and electrochemical activity is extremely low, shows low electrochemical signals. When there is a MP53; Mp53 hybrize with the probe, under the shearing action of Exonuclease Ⅲ enzymes,8-17 DNAzyme release. And Hs-MB specificity hybridization with it, in the presence of Zn2+, Hs-MB fracture, hemin exists in the form of monomer, the Hs-MB activity switch open, produce a great electrochemical signal. The experimental results show that the method cans high sensitive detection of Mp53, the lowest detection limit as low as 13 fM. The test of SNP of MP53 specificity showed good specificity, at the same time it also has a good stability in a complex environment, is expected to be applied to the actual biological sample detection.The third chapter Detected of insulin by the high sensitive electrochemical activity switch MB (Hs-MB) system based on EXO III and DNAzyme double signal amplificationBuild in this chapter we design a kind of based on aptamer combined with insulin, the high sensitive electrochemical activity switch (Hs-MB) system based on EXO III and DNAzyme double signal amplification to detect the insulin. The synthesized Hs-MB by our group is both tag hemins in stem end. We mainly detected the insulin by the difference electrochemical activity between hemin dimers and hemin monomer. In the absence of insulin, Hs-MB with hairpin structure exists, hemin exists in the form of dimers, Hs-MB activity of the switch is closed, electrochemical activity is extremely low. When there is insulin, insulin hybridization with probel, the releases DNA I will hybridization with probe2, in under the action of shear Exonuckase III enzymes,8-17 DNAzyme release. And Hs-MB hybridization specificity recognition, in the presence of Zn2+, Hs-MB fracture, hemin exists in the form of monomer, the Hs-MB activity switch open, produce a great electrochemical signaL The experimental results show that the method cans high sensitive detection of insulin, the lowest detection limit as low as 0.5 pM. This method can specificity detects insulin, even at low concentrations of insulin environment still showed good anti-jamming. We test Continuous Monitoring of Time-Dependent Insulin Secretion from Cultured Mouse Pancreatic p-Cells, the results are consistent with ELISA method, and so our method is of high reliability and is expected to be applied to real biological sample detection. |
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