A Self-Luminous Bifunctional Bacteria Directed Fluorescent Immunosensor for the Simultaneous Detection and Quantification of Three Pathogens in Milk

Abstract Herein, we report a novel multiplex fluorescent competitive immunosensor using self-luminous bacteria as a bifunctional probe for the simultaneous detection and quantification of multiple pathogens. The self-luminous bacteria are prepared through the facile one-step co-culture of bacteria and organic fluorescent dyes. Cronobacter muytjensii, Listeria monocytogenes, and Escherichia coli O157:H7 are fluorescently labelled with 7-hydroxycoumarin, fluorescein isothiocyanate, and rhodamine B, respectively, due to the dyes small spectral overlap in excitation/emission. The obtained fluorescent bacteria retain specific immunorecognition activity, but exhibit excellent fluorescent sensing ability. By using these fluorescent bacteria as bifunctional probes for competitive recognition and optical sensing, we achieved the sensitive and simultaneous quantitative determination of three pathogens with sensitivities of below 8.6 × 103 CFU/mL. The sensitivities can be significantly enhanced to 1 CFU/mL in real milk by integrating with a pre-incubation step, showing great potential for the ultrasensitive determination of trace bacteria and even single colony with the overall detection time of less than 10 h. In summary, this work provides an alternative multiplexing analytical platform as a powerful and rapid diagnostic tool for monitoring foodborne pathogen contamination.