Enzyme-free hybridization chain reaction-based signal amplification strategy for the sensitive detection of...

In this study, based on hybridization chain reaction (HCR) amplification and graphene oxide (GO), we developed a facile enzyme-free signal amplification strategy for sensitive detection of Staphylococcus aureus (S. aureus). Two hairpin probes (HP₁ and HP₂) labeled by fluorophore 6-carboxyfluorescein (FAM) are designed. The HP₁ and HP₂ can not only trigger to the HCR but also form a long nicked double strand DNA (dsDNA) with the target (16S rRNA). In the absence of target (16 s RNA), the free FAM-labeled HP1 and HP2 are adsorbed by the GO via π-π stacking, the fluorescence signal is quenched. In the presence of target (16 s RNA), the HCR is triggered and dsDNA complexes are generated. As a result, the fluorescence signal can be strongly amplified by the synergistic effect of FAM and the dsDNA dye SYBR Green I. Based on this mechanism, a fluorescence method is designed for the detection of 16S rRNA of S. aureus. Under the optimal conditions, it has low detection limit (50 pM) and a linear response in a concentration range of 50 pM to 100 nM for 16S rRNA. Furthermore, this method has also been successfully applied to the detection of S. aureus in milk sample with the detection limit of 4 × 10² CFU·mL^-1.