Single-microsphere biosensors based on UiO-66 confinement-enhanced fluorescence emission for sensitive detection of antibiotics and pathogenic bacteria.

To meet the urgent need for sensitive detection of foodborne pathogens and antibiotic residues-key to preventing outbreaks and curbing antimicrobial resistance-we developed a stable fluorescent probe, H4TCPE@UiO-66 (H@U). The probe retains strong fluorescence under harsh conditions (18.70% intensity change at pH 3, 11.36% at 80 °C) and exhibits a 6.90-fold signal amplification over H4TCPE. In addition, H@U is able to be a sensitive probe integrated with millimetre-scale polystyrene microspheres (mPS) and realize rapid detection of chloramphenicol within 20 min with a detection limit of 33.52 pg/mL, representing a 26.61-fold improvement in sensitivity over the enzyme-linked immunosorbent assay. Building on this, we harnessed the trans-cleavage activity of CRISPR/Cas12a to develop a CRISPR/Cas12a-enhanced H@U mPS (H@U-C mPS) biosensor for the quantitative detection of Staphylococcus aureus, achieving a linear range of 10-105 CFU/mL (two orders of magnitude lower than qPCR) and a detection limit of 6.97 CFU/mL. Furthermore, the practical applicability of H@U-C mPS biosensors was successfully demonstrated through validation using 30 real-world samples. Together, these platforms offer a robust, rapid, and highly sensitive strategy for monitoring chemical hazards and microbial in food safety and clinical diagnostics.