New fluorescence tools for investigating enzyme activity

Novel fluorescence-based enzyme-substrate probes have been fabricated which incorporate a unique utilization of chemically modified micron-sized particles in conjunction with a single-excitation dual-emission wavelength ratio technique. By chemically modifying micron-sized particles with both an enzyme-specific substrate and a reference fluorophore the effects of source intensity fluctuations, fluorophore diffusion, and variances in substrate loading inherent in in situ biological fluorescence assays can be reduced. Thus these probes have the potential to provide more sensitive and less invasive fluorescence detection of enzyme activity in solution, in microorganisms and in single cells. In addition, proper selection of particle size facilitates selective targeting of microorganisms through natural ingestion processes. Examples of source fluctuation and substrate loading corrections are provided for in in vitro experiments with a common esterase species. The in situ application of these probes in individual microorganisms which are used as biosensors is also discussed.