Target-triggered Strand Displacement Reaction Coupled With HCR-assisted Synthesis Of CuNPs For Electrochemical Detection Of MiRNA

MicroRNA(miRNA)is an endogenous non-coding RNA which plays an important role in almost all biological processes.A mass of studies has proved that the imbalance expression of miRNA is closely related to a variety of human diseases,including cancer,which was widely used as a tumor marker.Therefore,developing the methods of sensitive and selective miRNA tumor marker detection is of great significance.Traditional methods of miRNA detection generally showed disadvantages such as high cost,cumbersome operation,or complicated design.Electrochemical biosensing technology displayed unique advantages in the detection of miRNA tumor markers due to its simple operation,low cost,good sensitivity,strong specificity,ease of miniaturization and commercialization.In addition,the specificity and sensitivity of miRNA tumor marker detection can be further improved by introducing nucleic acid signal amplification and the functional nanomaterials in electrochemical detection.Based on thorough literature research,the following work is planned to focus on the electrochemical detection of miRNAs:1.DNA-templated CuNPs as an electrochemical indicator for the detection of miRNA based on target triggered strand displacement reaction and hybridization chain reactionIn this work,an electrochemical biosensing strategy was proposed for the detection of miRNA-155,which synthesising copper nanoparticles as electrochemical indicator based on target triggered DNAzyme strand displacement reaction coupled with the assistance of hybridization chain reaction(HCR).In this system,the Locker DNA,a complementary strand of miRNA-155,was hybridizes with the DNAzyme to block its active site.In the presence of target miRNA,the Locker DNA competitively hybridizes with miRNA-155,which led to the liberation of the DNAzyme.The liberated DNAzyme can then hybridized with the capture probe(SH-CP)onto the electrode surface and activate the DNAzyme cleavage reaction with the assistance of Mn²⁺.The cleavage SH-CP residue then exposed trigger sequence and catalyze the HCR of H1 and H2 on the electrode surfa ce.In the same time,the electroactive CuNPs will in situ synthesized utilized the HCR long dsDNA template.A large amount of Cu²⁺released from acid dissolution can oxidize o-phenylenediamine(OPD)to diaminophenazine(DAP),which produces a significant electrochemical signal.Benifiting from amplification of nucleic aicd reaction and the excellent electrochemical indicating ability of CuNPs,this sensing strategy can realize the detection of miRNA-155 down to 24.3 pM.Cell lysates were taken as complex samples to further investigate the application of this strategy.2.DNA-templated CuNPs as an electrochemical indicator for the detection of dual-miRNA based on DNA Logic Gate triggered strand displacement reaction and hybridization chain reactionA single target cannot meet the need of accurate diagnosis of cancer due to the dynamic changes of multi markers in its generation.Compared with the single target sensing,the detection of dual-miRNA can effectively avoid false positive signals and improve the accuracy of diagnosis.To carry out this,an electrochemical strategy was proposed for dual-miRNA detection based on target-regulated"AND"DNA logic gate triggered strand displacement reaction and hybridization chain reaction with CuNPs as the electrochemical indicator.In this assay,the trigger DNA FT and Linker DNA were hybridized together to form a complex with an exposed toehold.This complex could only be disaggregated in the presence of both two target.The FT could be released through strand displacement reaction(SDR)in the presence of dual-target,then open the capture probe(SH-CP)and trigger the HCR on the electrode surface,which formate the dsDNA long chain for in situ synthesizingof CuNPs with obviously electrochemical response.Benefiting from the target-triggered SDR and HCR with the excellent indicating ability of CuNPs,this sensing strategy can simultaneously detect miRNA-21 and miRNA-122 with detection limits of 14.5 pM and 16.8 pM,respectively.Further,we will demonstrate the practical applications of our sensing strategy in complex samples.