Electrochemical Strategy For Ultrasensitive Detection Of MicroRNA Based On Mnazyme-mediated Rolling Circle Amplification On A Gold Electrode

MiRNAs,the short RNA sequences regulating gene expression,liberate from long RNA polymerase II transcribed precursors by a series of sequential endonuclease-mediated cleavage events.Recent researches show that the expression profiles of miRNAs are associated with several diseases,including cancer,cardiovascular disease,and neurological disorders.Analytical methods that are capable of identifying sequence-specific miRNAs and measuring their concentrations at biologically relevant levels in specimens may become beneficial for studying and diagnosing disease.However,it has been proven difficult to sensitively and specifically analyze miRNA due to its high sequence homology,small size,and low natural abundance.The currently most used techniques,such as polymerase chain reaction(PCR),microarray,and northern blotting,do not sensitively and specifically detect miRNA.The authors describe an electrochemical strategy for ultrasensitive and specific detection of micro RNA(miRNA).It is based on both multicomponent nucleic acid enzyme(MNAzyme)amplification and rolling circle amplification(RCA).In the presence of target miRNAs,partial enzyme A(partzyme A)and partial enzyme B(partzyme B)are assembled to form active MNAzymes.Once formed,the MNAzymes catalyze the cleavage of the hairpin substrates to liberate biotinylated fragments which hybridized with the capture probes immobilized on a gold electrode.The RCA is then initiated to form a product that binds many detection probes.Finally,the amperometric signal(best acquired at a working voltage of 0.22 V vs.Ag/Ag Cl)is obtained by employing the streptavidinylated alkaline phosphatase as the enzyme.This biosensor has a 1.66 f M detection limit,and a dynamic range that extends from 10 f M to 1 n M.It displays specificity down to single mismatch discrimination of target miRNAs and good reproducibility.It was successfully applied to the determination of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells.