A bifunctional ZIF-8 nanoprobe with spatially decoupled ratiometric channels for hydrogel-based portable detection of Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a major foodborne pathogen in the dairy industry that produces heat-stable enterotoxins capable of surviving pasteurization. However, rapid, pathogen-specific, and instrument-free field detection remains a significant challenge. Here, we report a bifunctional zeolitic imidazolate framework-8 (ZIF-8) nanoprobe (RZF) for portable ratiometric detection of S. aureus. The probe exploits the spatially distinct interior and exterior of a single ZIF-8 nanoparticle: Ru(bpy)32+ is encapsulated within the pore interior as a photostable reference fluorophore, while 6-carboxyfluorescein (FAM)-labeled single-stranded DNA (ssDNA-FAM) is adsorbed onto the positively charged outer surface, where its fluorescence is quenched via Förster resonance energy transfer (FRET). This spatially decoupled architecture overcomes the ratio-control limitation of conventional co-encapsulation strategies, enabling independent optimization of the reference and responsive channels within a single nanoparticle. Upon bacterial lysis, released adenosine triphosphate (ATP) serves both as a biochemical indicator of viable bacteria and as a competitive Zn2+ ligand, triggering ZIF-8 disintegration and selectively restoring FAM fluorescence at 514 nm while leaving the Ru(bpy)32+ emission at 594 nm unchanged, thereby generating a self-calibrating ratiometric signal. The nanoprobe is immobilized within an agarose hydrogel kit, enabling smartphone-based RGB readout without cold-chain storage. Upstream immunomagnetic separation using Fe3O4-pIgG probes confers pathogen specificity through protein A recognition. The integrated platform achieves a limit of detection of 6 CFU/mL, with recoveries of 99.72-100.63% in spiked milk. This spatially decoupled single-particle probe strategy establishes a generalizable and reproducible design principle for ratiometric sensing in resource-limited food safety monitoring.