A hybridization-gated ZIF-8 ratiometric nanoprobe for hydrogel-based portable detection of Staphylococcus aureus via ATP-triggered DNA duplex signaling

Hybridization gating reduces fluorophore ratio-control constraints. • Post-activation dye recruitment limits background from pre-integrated signal dyes. • Immunomagnetic enrichment provides target selectivity for ATP-based sensing. • Chitosan-modified hydrogel limits acid-induced background in portable readout. Rapid bacterial detection in dairy food safety monitoring requires portable platforms that combine analytical sensitivity with target selectivity. Here, we report a hybridization-gated zeolitic imidazolate framework-8 (ZIF-8) ratiometric nanoprobe, designated RSZ, for portable detection of Staphylococcus aureus ( S. aureus ) through adenosine triphosphate (ATP)-triggered DNA duplex signaling. The RSZ probe adopts a dual-zone architecture with Ru(bpy) 3 2+ and ssDNA1 embedded within the ZIF-8 particle interior and ssDNA2 adsorbed on the exterior surface. This architecture separates the stable reference channel from the formation of the responsive fluorescence channel, thereby reducing dependence on precise dual-fluorophore loading and limiting background associated with a pre-integrated responsive fluorophore. Upon ATP-induced framework disruption, ssDNA1 becomes accessible for hybridization with ssDNA2, and the resulting duplex recruits SYBR Green I from solution to generate the responsive fluorescence channel relative to the stable Ru(bpy) 3 2+ reference. To enable field-deployable detection, the nanoprobe is embedded in a chitosan-modified agarose hydrogel in which chitosan maintains a near-neutral microenvironment and limits acid-induced nonspecific activation. Fe 3 O 4 nanoparticles conjugated with porcine immunoglobulin G (Fe 3 O 4 -pIgG) are integrated upstream to selectively capture S. aureus via protein A-pIgG recognition, followed by smartphone red-green-blue analysis without specialized instrumentation. The integrated platform achieves a detection limit of 6 CFU/mL and recoveries of 98.8–101.7% in spiked milk. Therefore, this study demonstrates a hybridization-gated strategy for translating stimulus-induced ZIF-8 disruption into controlled ratiometric signal generation for portable bacterial sensing in food analysis. Download: Download high-res image (106KB) Download: Download full-size image