An ultrasensitive electrochemical miRNAs sensor based on miRNAs-initiated cleavage of DNA by duplex-specific nuclease and signal amplification of enzyme plus redox cycling reaction

• We report a simple and ultrasensitive electrochemical miRNAs sensing platform.
• Biotin group in DNA allows streptavidin-conjugated alkaline phosphatase to be attached.
• MiRNAs trigger the cleavage of DNA by duplex-specific nuclease on electrode surface.
• The method is based on signal amplification of phosphatase plus recycling reaction.
• The detection limit of 0.2 fM is lower than that of other enzyme-amplified methods.

In this work, we reported a simple and ultrasensitive electrochemical sensing platform for microRNAs (miRNAs) detection by combination of duplex-specific nuclease (DSN) with signal amplification of alkaline phosphatase (ALP) plus redox cycling reaction. Specifically, biotinylated single-stranded DNA capture probes (CPs) immobilized on gold electrodes allowed streptavidin-conjugated alkaline phosphatase (SA-ALP) to be attached, facilitating the production of electrochemically active p-aminophenol (p-AP) from p-aminophenyl phosphate (p-APP) substrate. The resulting p-AP could be cycled by tris(carboxyethyl)phosphine (TCEP) after its electro-oxidization, enabling an increase in the anodic current of p-AP (known as p-AP redox cycling). If the CPs formed duplexes with miRNAs by hybridization, DNA in the DNA-miRNAs duplexes would be cleaved by DSN, making the biotin groups stripping from the electrode surface and the SA-ALP molecules incapable of attachment onto the electrode. In this process, miRNAs strands remained intact and could be released back to the sample solution for recycling. The oxidation current of p-AP decreased significantly with the increase of miRNAs concentration. As a result, a detection limit of 0.2 fM for miRNA-21 detection was achieved.