Electrochemical Biosensor For Ultrasensitive Exosomal MiRNA Analysis By Cascade Primer Exchange Reaction And MOF@Pt@MOF Nanozyme

Exosomal miRNAs have been discovered as important and reliable biomarkers for early diagnosis of tumors.Currently,conventional quantitative reverse transcription polymerase chain reaction（qRT-PCR）is the dominating method for highly sensitive quantification of exosomal miRNAs.Nevertheless,the availability of qRT-PCR is substantially limited due to the complicated operation,long detection time and difficulty in point-of-care testing.Therefore,it is still urgent to develop a novel,sensitive,rapid and simple biosensing strategy for exosomal miRNAs detection.Herein,we report an electrochemical strategy based on the cascade primer exchange reaction（PER）with MOF@Pt@MOF nanozyme for ultrasensitive detection of exosomal miRNA.Target-triggered PER that only includes a gated hairpin,a primer,and DNA polymerase can produce a long single strand in an autonomous and isothermal manner.Then,the nascent strand releases the protector B used to blockade capture probes,resulting in the binding of the nanozyme to sensing interface.Under the catalysis of the nanozyme,hydrogen peroxide（H₂O₂）is decomposed into H₂O and O₂,thus producing an amplified electrochemical signal.Benefiting from the cascade PER and the noticeable catalytic activity of the multiple-layered nanozyme,the established biosensor shows high sensitivity with limit of detection down to 0.29 f M and high specificity that can distinguish homologous miRNAs with single base mismatch.The developed strategy allows discrimination of tumor cells and breast cancer patients by detecting exosomal miRNA-21,and the results of this biosensor are consistent with qRT-PCR.Therefore,the electrochemical strategy has wide potential in the early screening of tumors.