Ultrasensitive electrochemical microRNA-21 biosensor coupling with carboxylate-reduced graphene oxide-based signal-enhancing and duplex-specific nuclease-assisted target recycling

An electrochemical biosensor for ultrasensitive detection of microRNA-21 (miRNA-21) coupling with carboxylate-reduced graphene oxide (COOH-rGO)-based signal-enhancing and duplex-specific nuclease (DSN)-assisted target recycling. Capture DNA (cDNA) was self-assembled on the surface of gold electrode (GE) and hybridized with the target miRNA-21 to form cDNA/miRNA-21 hybrids. COOH-rGO was further aggregated on cDNA/miRNA-21 hybrids through pi - pi stacking interaction, leading to a accumulation of electroactive methylene blue (MB) molecules and a significant differential pulse voltammetry (DPV) response of MB with peak potential of -0.28 V (vs. Ag/AgCl). At the present of DSN, DSN digested the cDNA/miRNA-21 hybrids and released miRNA-21 to hybridize with another cDNA, resulting in a significant decrease of the length of cDNA sequence. The amount of MB on the electrode surface decreased dramatically, following with the decrease of the DPV signal of MB. Under optimal experimental conditions, this approach displayed an ultrahigh sensitivity (with a 0.01 fM detection limit) and excellent selectivity among similar miRNAs sequences even with single-base difference. This biosensor further exhibited excellent applicability for target miRNA-21 in human serum samples with satisfactory results.