Graphene-based fluorometric determination of agrD gene transcription in methicillin-resistant Staphylococcus aureus using exonuclease III-aided target recycling and DNA walker cascade amplification.

A graphene-based bioassay is described for the fluorometric determination of agrD gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method includes exonuclease III (Exo III)-assisted target recycling and DNA walker cascade amplification. Hairpin1 (HP1) consists of a capture probe (CP) and DNA walker sequence. In the absence of the target, 5'-amino modified hairpin2 (HP2) labeled with carboxyfluorescein (FAM) at its 3' terminus is covalently linked to graphene via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS) catalysis, resulting in the quenching of the FAM signal. The stem-loop structure of HP1 opens when the target is added to form partially complementary DNA/RNA hybrids. Exo III then initiates the target recycling process by cleaving the CP and DNA walker cascade reaction by automatic walking. This iterative reaction causes the FAM to dissociate from the graphene, and the fluorescence can be measured at excitation/emission wavelengths of 480/514 nm. Therefore, the target can be assayed by fluorescence. This method has a linear relationship with the concentration of target within the range 1 fM to 100 pM with a detection limit of 1 fM. The developed bioassay was used to monitor biofilm formation and investigate the mechanism of drug action with satisfactory results. Schematic representation of the graphene-based fluorescent bioassay for agrD gene transcription in methicillin-resistant Staphylococcus aureus by using exonuclease III-aided target recycling and DNA walker cascade amplification.