Study On Construction And Performance Of Highly Sensitive Electrochemical Nucleic Acid Sensor Based On Proximity-dependent Surface Hybridization

In this thesis,we have constructed three electrochemical DNA biosensor based on universal dynamic DNA assembly-programmed surface hybridization effect strategy,realized the highly sensitive and selective detection of target DNA associated with disease.The main contents are as follows:1.Herein,we present the proof-of-principle based on DNA-fueled target recycling machine-programmed surface hybridization,which confers the single-step,and enzyme-free amplified electrochemical nucleic acid analysis.Wherein the DNA fuel target cycling mechanism is driven by Toe-hold mediated isothermal strand displacement reaction(TSDR).In dynamic DNA self-assembly processes,DNA-fueled target recycling is flexibly harnessed to convey the homogenous target recognition and amplification events into various DNA scaffolds-double stand complxes(TS+FS)for the autonomous proximity-based surface hybridization with SH-DNA captured by electrode.Then the ferrocene labels of the tail sequence are drawn close to the electrode surface,producing a readily detectable redox current.A low detection limit of 0.29 fM towardtarget DNA could be achieved.2.Herein,HCR-programmed surface hybridization strategy is investigated for the dynamic,sensitive,non-enzyme electrochemical detection of DNA.Two hairpin-like DNAs(HP1 and HP2)are designed and the HP2 contains the methylene blue(MB)-labled tail sequence at 5'-terminus,which is complementary to the surface-tethered DNA strand.In the presence of target DNA,the alternate assembly between HP1 and HP2 by the sequential TSDRs leads to the formation of linear DNA concatamer,which accordingly brings the dangling tail sequence of HP2 into close proximity to allow the pair of tail sequences to hybridize together with the surface-tethered DNA strands with their local concentration increased substantially.The electrochemical response of the methylene blue toward target DNA was achieved,owing tothe methylene blue labels of the tail sequenceare drawn close to the electrode surface,producing a readily detectable redox current.A low detection limit of 50 a M towardtarget DNA could be achieved.3.Herein,target induced strand displacement amplification-programmed surface hybridization effect to regulate DNA dynamic assembly and achieve ultra-sensitive detection of target DNA analysis.For one thing,the enzyme-assisted isothermal strand displacement reaction(SDA)can be used to amplify the target cyclic signal,the target triggered SDA which could produce amounts of free ssDNA with accurate and comprehensive nucleotide sequence for another.Then,the free ssDNA could hybridize with SP labled the methylene blue,forming ds DNA-Proximity probe,which accordingly brings the dangling tail sequence of SP into close proximity to allow the tail sequences to hybridize together with the surface-tethered DNA strands.The electrochemical response of the methylene blue toward target DNA was achieved.