In situ Tracking of Exoenzyme Activity Using Droplet Luminescence Concentrators for Ratiometric Detection of Bacteria

[Image: see text] We demonstrate a novel, rapid, and cost-effective biosensing paradigm that is based on an in situ visualization of bacterial exoenzyme activity using biphasic Janus emulsion droplets. Sensitization of the droplets toward dominant extracellular enzymes of bacterial pathogens is realized via selective functionalization of one hemisphere of Janus droplets with enzyme-cleavable surfactants. Surfactant cleavage results in an interfacial tension increase at the respective droplet interface, which readily transduces into a microscopically detectable change of the internal droplet morphologies. A macroscopic fluorescence read-out of such morphological transitions is obtained via ratiometrically recording the angle-dependent anisotropic emission signatures of perylene-containing droplets from two different angles. The optical read-out method facilitates detection of marginal morphological responses of polydisperse droplet samples that can be easily produced in any environment. The performance of Janus droplets as powerful optical transducers and signal amplifiers is highlighted by rapid (<4 h) and cost-effective antibody and DNA-free identification of three major foodborne pathogens, with detection thresholds of below 10 CFU mL(–1) for Salmonella and <10(2) to 10(3) CFU mL(–1) for Listeria and Escherichia coli.