Dual-signal assay integrating colorimetric and pH biosensing pathways for simultaneous detection of multiple pathogenic bacteria

The increasing prevalence of multi-pathogen contamination highlights the critical need for advanced diagnostic technologies capable of accurately detecting multiple pathogens simultaneously. In this study, we developed a dual-signal assay that synergistically integrates colorimetric and pH biosensing pathways to detect multiple pathogenic bacteria simultaneously. This assay employs the unique properties of rhodamine B (RhB) and urease to create two distinct and independent signaling transduction pathways: RhB provides a characteristic colorimetric absorbance, while urease catalyzes the hydrolysis of urea, generating a pH-modulating signal. The spatiotemporal separation between RhB absorbance and the pH signal can effectively eliminate crosstalk interference, ensuring exceptional accuracy and reliability. Implemented through a dual-recognition strategy, the assay uses Fe 3 O 4 -functionalized phenylboronic acid nanoparticles to capture a wide range of bacteria. Pathogen-specific probes, featuring immunoglobulin G- and aptamer-conjugated ZIF-90 composites encapsulating either RhB or urease, enable precise bacterial identification. The assay exhibits remarkable sensitivity, with detection limits as low as 6 CFU/mL for Staphylococcus aureus and 8 CFU/mL for Escherichia coli . Moreover, it demonstrates excellent accuracy in actual samples, with recovery rates exceeding 97.75 % and a relative standard deviation below 4.99 %. We believe that this study can offer a new insight into the development of multiplex assays for simultaneous multi-target detection.