A Graphene Oxide-based Assay for Sensitive Osteonecrosis of the Femoral Head (ONFH) related microRNA Detection via Exonuclease-III Assisted Dual Signal Cycle.

Abstract

Accurate detection of circulating microRNAs (miRNAs) plays a vital role in the diagnosis of various diseases. The current miRNA detection methods, however, are widely criticized for their low sensitivity and excessive background signal. Herein, we propose a graphene oxide (GO) based fluorescent biosensor for sensitive and reliable miRNA analysis with a low background signal by utilizing exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction (HCR). To initiate Exo-III-assisted dual signal cycles, a hairpin DNA probe (H probe) was developed for selective miRNA binding. Dye quenching occurred when carboxyfluorescein (FAM)-labeled hairpins (HP1 and HP1) were unable to bind to their intended target and instead adsorb onto the surface of GO via p-stacking interactions. Exo III sequentially cleaved the 3'-strand of the H probe and the S probe upon attachment of the target miRNA, resulting in the release of the miRNA and the autonomous production of a "g" sequence. The released target miRNA then hybridized with a second H probe and progressed to the subsequent reaction phase. With the help of the HP1 and HP2 probes, a lengthy dsDNA product was produced when the "g" sequence triggered HCR. The dsDNA product was not absorbed by GO, and the material instead fluoresced brightly. As a result, the amount of miRNA of interest was measured. With a LOD of only 5.6 fM, this bioassay demonstrated excellent selectivity and great sensitivity.