The increasing incidence of foodborne illnesses highlights the need for rapid, sensitive, and portable methods to detect pathogenic bacteria in food. In this work, we develop a portable method that utilizes a ratiometric fluorescence nanoprobe for adenosine triphosphate (ATP) quantification. The nanoprobe is constructed by encapsulating Ru(bpy) within a zirconium-based metal-organic framework, followed by functionalization of double-stranded DNA (dsDNA). This design permits SYBR Green I (SGI) to intercalate into dsDNA, conferring the nanoprobe with dual-emission property. The presence of ATP disrupts dsDNA structure, quenching SGI fluorescence while maintaining Ru(bpy) fluorescence, providing a stable reference signal. This differential response enables the nanoprobe to achieve ratiometric ATP detection with high precision and sensitivity, with a detection limit of 0.63 μM. Since ATP is a reliable biomarker for viable bacterial cells, a portable hydrogel kit was further developed by integrating the ratiometric fluorescence nanoprobe into an agarose hydrogel matrix. The validation of the kit was conducted using a smartphone application for color recognition, enabling the rapid and on-site detection of pathogens in milk samples. The kit exhibits exceptional sensitivity with a detection limit of 10 CFU/mL, making it a promising tool for real-time bacteria detection in food safety monitoring.
