Studies On Electrochemical Biosensors Via The Hybridization Chain Reaction Signal Amplification

The hybridization chain reaction(HCR)is based on the chain reaction of recognition and hybridization events between two hairpin DNA probes,in which a long nicked dsDNA molecule is formed by a cascade of toehold mediated DNA strand displacement events triggered by an initiator DNA.Each copy of the initiators DNA can induce an HCR amplication event to form numerous DNA double helices which can realize signal amplication for target detection.As a promising amplification technique for ultrasensitive bioanalysis,HCR has attracted considerable attention owing to its enzyme-free and isothermal operation.Currently,much attention has been focused on developing HCR,which are used for signal amplification in various biosensors.Among these biosensors,electrochemical biosensors attracted substantial research interest owing to the advantages of high sensitivity,simple instrumentation,ease of miniaturization,low-cost manufacture,and fast response and so on.Especially,the integrated with signal amplification strategies has also attracted a lot of attention.In this context,the developments of HCR-based electrochemical biosenors are highly inspired to improve the sensitivity of target detection.Therefore,aiming at the development of high sensitivity method for target assay,this thesis mainly focuse on constructing electrochemical biosensor for the analysis of biological molecules and enzymes utilizing HCR-based signal enhancement.The main works are listed as follows:1.Triple-helix molecular switch-induced hybridization chain reaction amplification for developing a universal and sensitive electrochemical aptasensorIn this work,a universal and sensitive "signal-on" electrochemical aptasensor platform has been developed based on a triple-helix molecular switch(THMS)-induced hybridization chain reaction(HCR)amplification.This aptasensor platform system consists of a THMS-based molecular recognition process in a homogeneous solution and HCR amplification on a gold electrode.In the absence of a target,the aptamer sequence is flanked by two arm segments(APT)and the triplex-forming oligonucleotide(TFO),forming a rigid THMS.It is in the eT off state.However,upon the introduction of a target,the interaction between the target and the APT leads to the dissociation of the THMS and thereby liberates the TFO,allowing the TFO to hybridize with the capture probe(CP)DNA and trigger the formation of dsDNA polymers through in situ HCR amplification.The dsDNA polymers cause the electrostatic attraction of numerous electroactive indicators[Ru(NH3)6]3?,resulting in significantly amplified electrochemical signal output.It is in the eT on state.As proof-of-principle,we use this approach to detect adenosine and human a-thrombin(Tmb),achieving lowest limit of detection values of 0.6 nM and 70.9 pM,respectively.As an electrochemical aptasensor platform,its universality can be easily realized by altering only the sequence of the APT,which provides a promising alternative to the electrochemical detection of a variety of analytes and may have potential applications in biomedical research and clinical diagnosis.2.? exonuclease-induced hybridization chain reaction amplification for electrochemical detection of T4 polynucleotide kinase activityIn this work,we describe a new highly sensitive strategy for electrochemical detection of T4 polynucleotide kinase(T4 PNK)by means of a coupled ?,exonuclease(?,exo)cleavage reaction and hybridization chain reaction(HCR)signal amplification.Taking advantage of the efficient enzyme reactions,namely the phosphorylation of hairpin probe(HP)by T4 PNK and the ? exo cleavage reaction,the DNA trigger chain(UT)can be released from HP and then hybridize with the capture probe(CP)self-assembled on gold electrode surface.As a result,the presence of the two stable hairpin probe(H1 and H2)leads to the formation of extended dsDNA polymers through HCR on the electrode surface.The in situ,HCR-generated dsDNA polymers cause the intercalation of numerous electroactive indicators[Ru(NH3)6]3+ into the dsDNA grooves,resulting in significantly amplified electrochemical signal output.The developed methody shows high selectivity for the detection of T4 PNK,and the limit of detection value was 7.4 mU/ml.At the same time,it is proved that this method can realize the investigation of T4 PNK inhibitors.The method provides a simple,sensitive and efficient method for the determination of kinase activity and the screening of inhibitors.