An electrochemical biosensor for microRNA-196a detection based on cyclic enzymatic signal amplification and template-free DNA extension reaction with the adsorption of methylene blue.

A simple and sensitive electrochemical biosensor was developed for microRNA-196a detection, which is of important diagnostic significance for pancreatic cancer. It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction. In the presence of microRNA-196a, duplex-specific nuclease (DSN) catalyzed the digestion of the 3'-PO4 terminated capture probe (CP), resulting in the target recycling amplification. Meanwhile, the 3'-OH terminal of CP was exposed. Then, template-free DNA extension reaction was triggered by terminal deoxynucleotidyl transferase (TdT), producing amounts of single-stranded DNA (ssDNA). After ssDNA absorbed numerous methylene blue (MB), 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 0.05 fM 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. In conclusion, with the excellent analytical performance, this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer.