Graphene-oxide-based bioassay for the fluorometric determination of agrC gene transcription in methicillin-resistant Staphylococcus aureus that uses nicking-enzyme-assisted target recycling and a hybridization chain reaction.

Herein, we report the development of a graphene-oxide-based (GO-based) fluorescent bioassay for determining agrC gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). The design is based on nicking-enzyme-assisted (Nb.BbvcI-assisted) target recycling amplification (NATR) and a hybridization chain reaction (HCR). The system consists of a helper probe (HP), a molecular beacon (MB) probe, four hairpins, and endonuclease Nb.BbvcI, which plays a role in target recycling and signal amplification. In the absence of the target, all of the carboxyfluorescein-labeled (FAM-labeled) hairpins are adsorbed through π-stacking interactions onto the surface of GO, resulting in FAM signal quenching. When the target is added, three nucleic acid chains hybridize together to form a triple complex that is recognized by Nb.BbvCI. The MB probe is then cleaved by Nb.BbvCI to generate an HP/target complex and two new DNA fragments; the former is hybridized to another MB probe and enters the next round of reaction. The two newly reproduced DNA fragments induce a HCR with the assistance of hairpins 1-4 to create double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and generate strong fluorescence at excitation/emission wavelengths of 480/514 nm. Importantly, synergy between FAM and the dsDNA-SYBR Green I duplex structure led to significantly amplified fluorescence and enhanced sensitivity. The bioassay showed a detection limit of 7.5 fM toward the target and a good linearity in the 10 fM to 100 pM range. The developed method was applied to monitor biofilm formation and study the mechanism of drug action, with satisfactory results obtained.