Study Of Fluorescent Sensors Based On DNA Hybridization Chain Reaction

DNA hybridization chain reaction is a new isothermal amplification technology of nucleic acid in vitro. The principle of this technology is that the autonomous cross-opening of two stable hairpin probes could be triggered to the formation of DNA nanowires only in the present of the target DNA. Herein, DNA hybridization chain reaction consisting of numerous G-quadruplex was designed to develop enzyme-free and label-free fluorescent sensors by taking advantage of the fact that NMM can selectively bind to the G-quadruplex and enhance the fluorescence signal. Three chapters were involved in this thesis.In the first chapter, the fundamental and application of nucleic acid amplification techniques in vitro were briefly introduced. Especially, the application of DNA hybridization chain reaction was emphasized. The purpose and main content of this thesis were proposed at the end of this chapter.In the second chapter, based on hybridization chain reaction to amplify the signal and NMM as a fluorescent probe to indicate the formation of G-quadruplex, a sensitive, enzyme-free and label-free fluorescent sensor for HIV-1 DNA was proposed. Two DNA hairpins were designed according to the long terminal repeat sequence of HIV-1 U5. The target DNA could trigger the autonomous cross-opening of the two probes based on the strand displacement principle. This hybridization chain reaction led to the formation of DNA nanowires consisting of numerous G-quadruplexes, which could quantitatively analyze HIV-1 DNA after the binding of NMM. The enhancement of fluorescence intensity was proportional to the concentration of the target DNA. The linear range for HTV-1 DNA was ranged from 3 nM to 30 nM with a detection limit of 0.5 nM.In the third chapter, based on the specific binding of the aptamer for ATP and the hybridization chain reaction to amplify the signal, an enzyme-free and label-free fluorescent sensor for ATP was proposed by using NMM as the fluorescent probe to indicate the formation of G-quadruplex. In the present of ATP, the blocking probe which was originally hybridized with the aptamer was released after the specific binding of the aptamer for ATP. Then hybridization chain reaction triggered by the released blocking probe led to the formation of DNA nanowires consisting of numerous G-quadruplexes, which could quantitatively analyze ATP after the binding of NMM. The enhancement of fluorescence was proportional to the concentration of ATP. The linear range for ATP was ranged from 30 μM to 800 μM with a detection limit of 15 μM.