Navigable DNA walker coupled with exonucleases-assisted signal amplification for sensitive fluorescent detection of Staphylococcus aureus

A fluorescent aptasensor for Staphylococcus aureus (S. aureus) was constructed by integrating the mechanisms of fluorescence signal regulation and exonucleases-assisted two-step signal amplification into a navigable DNA walker. After the recognition of S. aureus, the first signal amplification step converts the aptamer-cDNA1 duplex to the hydrolysate of cDNA1 by exonuclease I (Exo I). Then the second signal amplification step converts the hydrolysate to the fuel chain of the navigable DNA walker with the help of exonuclease III (Exo III) reaction system containing hairpin probe and cDNA2. Driven by the fuel chain, the movement of the DNA walker triggers the fluorescence resonance energy transfer (FRET) to obtain an amplified fluorescent signal that correlates to the target concentration. The movement track is ingeniously designed and constructed by DNA self-assembly technology, which controls the motion direction and stride of the DNA walker. In addition, the movement triggers FRET, which effectively enhances the detection signal. The biosensor has a wide detection concentration range of 1 x 102 -2 x 107 CFU/mL, and the detection limit is 9 CFU/mL. This biosensor provides a reference method for customization of signal amplification strategy in DNA circuit machinery. And has potential applications in the fields of environmental and food safety monitoring.