Construction Of Electrochemiluminescence MicroRNA Sensor Based On Multiple Nucleic Acid Signal Amplification Strategies

In recent years,the incidence of cancer has increased year by year,and most of the current clinical detection methods are for the diagnosis of cancer in the middle and late stages.The cure rate of advanced cancer is very low and the treatment cost is very high,so the development of early cancer diagnosis technology becomes particularly critical.MicroRNAs(miRNAs)are a kind of endogenous non-coding small molecules.Studies have shown that miRNAs,as biomarkers,have early diagnostic,prognostic and predictive roles in cancer biology.However,the fragments of miRNAs in the human body are short and the content is very low,which causes great difficulties in detection.Electrochemiluminescence(ECL)biosensors have the advantages of high sensitivity,good stability,low background signal,simple operation,etc.,which have attracted extensive attention and research over the years.In order to improve the detection limit and sensitivity of cancer marker miRNAs,we mainly combined nucleic acid amplification strategies such as catalytic hairpin self-assembly reaction,strand displacement cycle amplification and DNA walker to construct ECL biosensors.The work of this thesis is as follows:1.Combining bipedal DNA walking machine and catalytic hairpin self-assembly amplification technology applied to the electrochemiluminescence detection of miRNA-141.First,ZAIS/ZnS NCs with good optical properties and low biological toxicity are connected to the electrode surface through Hairpin DNA 3(H3).Then miRNA-141 acted as the trigger chain to initiate the self-assembly reaction of the catalyzed hairpin.Hairpin DNA 1(H1)and Hairpin DNA 2(H2)were turned on to form a bipedal DNA walking machine.The feet are paired with the "head" of the H3 connected to the electrode through complementary base pairs,and the H3 is cut under the action of the restriction endonuclease.At this time,the H3 with ZAIS/ZnS NCs fell off the electrode,and the electrochemiluminescence intensity was weakened.Under the combined action of the catalytic hairpin self-assembly and the bipedal DNA walker,the miRNA-141 signal is amplified,thereby preparing an "on-off" biosensor for ultra-sensitive detection of miRNA-141.It provides a new design idea for the biosensor.2.Combining toehold-mediated strand displacement reaction(TMSDR)and catalytic hairpin self-assembly reaction for the ultra-sensitive electrochemiluminescence detection of miRNA-21.In this process,only when the sensor specifically recognizes the base sequence of miRNA-21,the primer DNA(P1)can be released by strand displacement.P1 further cycles to connect ferrocene to the electrode,which causes the quenching of the ZAIS/ZnS NCs ECL signal.In this experiment,the quenching of luminescence is achieved by using ferrocene as the quencher and interface regulation of the ZAIS/ZnS NCs luminescence system,so the sensitivity of the sensor can be improved.The experiment combines the sticky end-mediated strand displacement reaction and the catalytic hairpin self-assembly reaction to double-amplify the nucleic acid signal,which shows excellent performance in terms of sensitivity and selectivity.It provides a potential and practical miRNA detection strategy for the early diagnosis and treatment of cancer.3.Combining MZAIS/ZnS NCs and catalytic hairpin self-assembly amplification technology to achieve electrochemiluminescence detection of miRNA-19 a.First,miRNA-19 a acts as a trigger chain to develop DNA 1(H1).The single-stranded part of H1 replaces the single-stranded DNA 2(S2)and single-stranded DNA 3(S3)on the magnetic beads,S2 and S3 enter the catalyzed hairpin self-assembly cycle reaction.The hairpin DNA 2(H2)connected with MZAIS/ZnS NCs is connected to the electrode,and S2 and S3 enter the next cycle.On the one hand,by referring to the quantum dot synthesis method of Zou’s research group,synthesizing MZAIS/ZnS five-element quantum dots with good cycle stability to improve the cycle stability of the sensor.On the other hand,strand replacement and catalytic hairpin self-assembly realize the double-cycle amplification of the tumor marker miRNA-19 a signal,thereby improving the sensitivity of the sensor.This sensor establishes an enzyme-free cyclic amplification strategy,which overcomes the shortcomings of traditional enzyme amplification technology that the protease is easily interfered by the external environment(ionic strength,temperature,pH).And it has shown good performance in terms of sensitivity and cycle stability.The ultra-sensitive detection of miRNA-19 a is realized,which opens up new ideas for enzyme-free ECL biosensors.