Graphene oxide-based fluorometric determination of methicillin-resistant Staphylococcus aureus by using target-triggered chain reaction and deoxyribonuclease-assisted recycling

Abstract

The authors describe a method for the fluorometric determination of methicillin-resistant Staphylococcus aureus (MRSA) by exploiting target-triggered chain reactions and deoxyribonuclease I (DNase I)-aided target recycling. It is making use of a carboxy-fluorescein (FAM)-labeled single-stranded probe containing two sections. One is complementary to the 5' terminus of the target, while the 3' terminus of the other target is adsorbed on the surface of graphene oxide (GO) via π-stacking interactions without the target (16S rRNA). This adsorption results in quenching of the fluorescence of the label and protects it from being cleaved by DNase I. However, upon addition of the target, DNA/RNA hybrids are repelled by GO. This leads to fluorescence recovery as measured at excitation/emission wavelengths of 480/514 nm due to a chain reaction that is triggered by the target. The signal is strongly amplified by using DNase I-mediated target recycling. The 16S rRNA of MRSA can be detected by this method in the 1 to 30 nM concentration range, and the detection limit is 0.02 nM. The method was applied to analyze bacterial samples, and the detection limit is as low as 30 CFU . mL^-1. The assay is highly sensitive and selective and in our percpetion has a large potential in diagnosis of drug-resistant bacteria. Graphical abstract Schematic of the graphene oxide-based fluorescent bioassay for Methicillin-resistant Staphylococcus aureus detection by using target-triggered chain reaction and deoxyribonuclease I-aided signal amplification.

Keywords: 16S rRNA; Carboxyfluorescein; DNA probe; DNA/RNA hybrids; Drug-resistant bacteria; Enzymatic reaction; Fluorescence resonance energy transfer; Fluorometry; Quenching.