Studies On The Fluorescent And Electrochemical Biosensors Based On The Nucleic Acid-mediated Transition And Amplification Of Molecule

The nucleic acid-mediated biosensing analysis use nucleic acid molecules as identification components,or signal transduction and amplification components.It has been widely used for the detection of various analytes,including nucleic acids,proteins,small molecules,and even cells.These nucleic acid-mediated biosensing analysis make use of the design of nucleic acid sequences,the modification of functional groups,and base-pair hybridization,realise the goal of recognition of target molecules,or signal transduction and amplification of recognition signals.Generally,they have the advantages of selectivity and high sensitivity,arousing the great interest of researchers.In order to construct three kinds of biological sensing systems,respectively for the determination of biotin,DNA polymerase and miRNA,we use fluorescent and electrochemical detection methods,combining with nucleic acid-mediated molecular recognition technology,or signal transduction and amplification technology.The specific research contents are as follows:1.On condition that specific binding between terminal small molecules and their corresponding receptor proteins can protect nucleic acids from degradation by exonuclease,we establish a homogeneous fluorescence system that utilizing 2-amino purine molecule as a fluorescent reporter group to detect biotin.When the target molecule concentration is in the range of 1×10^-7 mol/L～2×10^-7 mol/L,the fluorescence intensity signal exhibits a good linear relationship with biotin concentration.And this method can be used for biotin detection in complex sample systems such as human serum samples.This method shows the advantages of short analysis time,good selectivity,and convenience.Therefore,this method has certain application prospect in biomedical related research.2.Based on the ability of fluorescent indicator ATMND molecule inserting into the nucleoside deletion site in the double-strands,we utilize Klenow Fragment exo-polymerase（KF）as an label-free fluorescent assay model to detect DNA polymerase activity.The proposed method has good sensitivity to the detection of KF activity,showing good linearity in the range of 0.05 U/mL～5U/mL.The limit of detection is approximately 0.0349 U/mL.The method is simple and convenient,and the design of the nucleic acid substrate is simple and has no use of the additional label of signal molecules.In conclusion,it has a good application prospect in DNA polymerase-related disease diagnosis,drug screening and biomedical research.3.Bsed on the catalyze hairpin assembly mediated target recycle and signal amplification assisted by hairpin self-assembling nucleic acid nanostructures,and taking advantage of methylene blue as an electroactive molecule,a label-free electrochemical method has been developed for the sensitivity determination of miRNA-21.The method is with a good linear relationship between peak current and miRNA-21 concentration in the range of 5×10^-13 to 2.5×10^-8 mol/L miRNA-21.The limit of detection of this proposed method is 4.8284×10^-13 mol/L,and this method has good selectivity.In addition,we utilize this established miRNA-21 assay to perform successfully in hepatoma cells HepG2,and compare the differences of miRNA-21 expression in hepatoma cells and normal hepatocytes.Therefore,the developed electrochemical methods is expected to be used in the diagnosis of related diseases.