Detection Of Methyltransferase And Mirna Based On Isothermal Cyclic Amplification Strategy

In this paper,combining surface enhanced Raman spectroscopy(SERS),fluorescence spectroscopy with isothermal cycle amplification reaction such as DNA strand displacement reaction(SDR)and Catalytic hairpin assembly(CHA),the methods for the detection of DNA methyltransferase(MTase)and miRNA were developed.This thesis mainly includes the following aspects.A novel and sensitive surface-enhanced Raman scattering(SERS)method is proposed for the assay of DNA methyltransferase(MTase)activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy.This method is based on DNA strand displacement reaction(SDR),and has many advantages,for instance,high efficiency,sensitivity,non-radioactivity.By using of a duplex substrate for MTase,two hairpin templates and a Raman probe,multiple signal amplification mode is achieved.Once recognized by Dam MTase,the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction.Consequently,a wide linear range and remarkably high sensitivity are obtained under isothermal conditions with a detection limit of 2.57×10-4 U mL-1.The assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase.In addition,this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase.The biomarkers of miRNA display great potential for cancer diagnosis and prognosis.The development of rapid and specific methods for miRNA detection has become a hotspot.Herein,hairpin DNA-templated silver nanoclusters(Ag NCs/HpDNA)were prepared and integrated into Catalytic hairpin assembly(CHA)for miRNA detection.The light-up platform was established based on guanine(G)-rich DNA sequences fluorescence enhancement.In the study,the fluorescence signal intensity of DNA-Ag NCs was linearly related to the concentration of miRNA in a certain range and a detection limit of 1 nM could be obtained.In addition,the assay exhibits an excellent selectivity.Simultaneously,stable DNA silver nanoclusters(DNA-Ag NCs)also has a good prospect in biosensing and biotechnology.