A graphene oxide (GO)-based fluorescent bioassay was developed to quantify agrA gene transcription (its mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method is based on the use of Klenow fragment (KF)-assisted target recycling amplification and hybridization chain reaction (HCR). A triple complex was designed that contained a capture probe (CP), a trigger probe (TP), and a help probe (HP), which were partially complementary to one another. In the absence of the target, all the oligonucleotides labeled with carboxyfluorescein (FAM) are adsorbed onto the surface of GO by π-stacking interactions. This adsorption quenches the FAM signal. On the contrary, the target RNA causes the triple complex to disintegrate and initiates strand-displacement polymerization reaction (SDPR) and HCR in the presence of the appropriate raw materials, including the primer, KF, dNTPs, hairpin 1 (H1), and hairpin 2 (H2), generating double-stranded DNA (dsDNA) products. These dsDNA products are repelled by GO and produce strong fluorescence, measured at excitation/emission wavelengths of 480/514 nm. The fluorescent signal is greatly amplified by SYBR Green I (SGI) due to the synergistic effect of dsDNA-SGI. The target was assayed with this method at concentrations in the range 10 fM to 100 pM, and the detection limit (LOD) was 10 fM. This method also displayed good applicability in the analysis of real samples. It provides a new way of monitoring biofilm formation and studying the mechanisms of drug actions. Graphical abstract Schematic representation of the graphene oxide-based fluorescent bioassay for agrA gene transcription in methicillin-resistant Staphylococcus aureus by using strand-displacement polymerization recycling and hybridization chain reaction.
Keywords: Biofilms; Drug-resistant bacteria; Dual fluorescence; Enzymatic reaction; Fluorescent assay; Quenching.