Research On New Biosensor Methods Based On Nucleic Acid Catalytic Hairpin Assembly Reaction

As a new analysis and detection technology,biosensor integrates the principles and knowledge of biology,chemistry,physics and other disciplines.It realized the detection and analysis of various types of targets,and thus provides a powerful tool for the development of life science,medical research and other fields.Enzyme-free nucleic acid amplification technology is one of the commonly used methods in the field of biosensing.Based on the principle of specific coding Crick-Watson base complementary pairing,it achieves signal amplification through chain hybridization and chain substitution reaction.Catalytic hairpin assembly reaction is a common enzyme-free amplification method,it does not need enzyme-assisted.And because of its high sensitivity,good selectivity and stability,it has been widely used in the detection of nucleic acids,proteins,metal ions,small molecules and other substances by combining with other research methods.Part of our work has focused on the establishment of sensitive and selective biosensors for the detection of the mercury ions,glutathione in water samples and intracellular mRNAs based on the catalytic hairpin assembly reaction combining with the nucleic acid molecule chromogenic reaction and the nucleic acid fluorescence probe techniques.The details are as follows:In chapter 1,focusing on biosensing technology and nucleic acid amplification technology,enzyme-assisted nucleic acid amplification and enzyme-free nucleic acid amplification are discussed in detail.At the same time,the research content and significance of this topic are briefly introduced.In chapter 2,based on the catalytic hairpin assembly reaction and the coordination between mercury ion and thymine base pair,we designed a colorimetric method for mercury ions detection.In the presence of mercury ions,catalytic hairpin assembly will be initiated,and the conformational changes of nucleic acid molecules will be induced,resulting in the formation of G-quadruplex DNAzyme with peroxidase activity.The detection of heavy metal mercury ions can be achieved by color reaction and the change of ultraviolet-visible absorption peak.By means of gel electrophoresis analysis,ultraviolet spectrum analysis and the change of color reaction,it is concluded that the method can detect mercury ions with high sensitivity.The detection limit is 17 pM,and the selectivity performance is excellent.It is suitable for the quantitative detection of mercury ions in actual water samples.At the same time,based on thecoordination interaction between mercury ion and thiol,a biosensor was designed to detect glutathione in water samples with a detection limit of 16 pM.The results show that the method can realize the qualitative and quantitative analysis of mercury ions and glutathione,so it is a highly sensitive and simple biosensor.In Chapter 3,we designed a DNA nanostructure based on SA scaffold for efficient transporting of nucleic acid,and developed a sensitive method for detecting multicomponent mRNAs in living cells mediated by DNA loop based on cross-linking catalytic hairpin assembly.The results show that DNA nanostructures can effectively enter cells through temperature-related endocytosis and exhibit excellent lysosome escape ability.In vitro detection,when target RNA exists,cross-linking catalytic hairpin assembly reaction will be triggered to achieve fluorescence recovery,which has the potential of simultaneous quantitative detection of multiple target mRNA molecules;intracellular fluorescence imaging has high contrast and high resolution.Therefore,the method designed in this chapter develops an efficient nucleic acid delivery platform,as well as a sensitive detection and high contrast imaging method for intracellular multi-component low abundance biomarkers,which provides a new research tool for the diagnosis and precise treatment of diseases.