Nanographite-based fluorescent biosensor for detecting microRNA using duplex-specific nuclease-assisted recycling

The development of a nanographite (NG)-based fluorescent biosensor for detecting microRNA (miRNA) is reported. Duplex-specific nuclease (DSN)-assisted signal amplification was key to its function. In the absence of a target, with the assistance of p-stacking interactions, the NG adsorbed the double carboxyfluorescein (FAM)-labelled probe (DFP) whose surface was perfectly complementary to miRNA, leading to quenching of FAM fluorescence. In the presence of a target, double-stranded DNA/RNA hybrids were repelled by the NG and fluorescence was restored. Meanwhile, the considerable increase in signal strength and sensitivity suggests DSN-mediated target recycling as an application. The detection limit of the proposed biosensor for miRNA was 10 pmol/L; there was a linear correlation when the miRNA concentration ranged from 50 pmol/L to 5 nmol/L. Additionally, the method could distinguish let-7b from most let-7 miRNA family members and was successfully used in a sample assay. This biosensor is a novel and highly sensitive tool for miRNA detection and has great potential for biochemical research, disease diagnosis, and therapy.