An Electrochemical Biosensor For MicroRNA-196a Detection Based On Cyclic Enzymatic Signal Amplification And Template-free DNA Extension Reaction

Microrna-196a is a biomarker of pancreatic cancer and has important diagnostic significance for pancreatic cancer.Electrochemical biosensors have the advantages of sensitivity,low cost and low sample consumption.In this work,a simple and sensitive electrochemical biosensor was developed for microRNA-196a detection.It was based on cyclic enzymatic signal amplification and template-free DNA extension reaction.In the presence of microRNA-196a,duplex-specific nuclease catalyzed the digestion of the 3'-PO₄ terminated capture probe,resulting in the target recycling amplification.Meanwhile,the 3'-OH terminal of capture probe was exposed.Then,the DNA extension reaction was triggered by terminal deoxynucleotidyl transferase,producing amounts of single-stranded DNA.After single-stranded DNA absorbed methylene blue,an ultrasensitive electrochemical readout was obtained.Based on this dual amplification mechanism,the proposed biosensor exhibited a high sensitivity for detection of microRNA-196a down to 15 aM with a linear range from 0.05fM to 50 pM.This biosensor displayed high specificity,which could discriminate target microRNAs from one base mismatched microRNAs.It also showed good reproducibility and stability.Furthermore,it was successfully applied to the determination of microRNA-196a in plasma samples.Therefore,this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer.