In-situ optical sensing for the detection and quantification of pathogen indicator organisms

In-situ sensing of pathogens of concern in waters is a need for water monitoring agencies. The unpredictable, rapid fluctuations in indicator organism (Escherichia coli—EC) concentrations renders current standards and methods both untimely and unrepresentative of the water body as a whole. The use of glucuronides with a fluorescent label allows EC detection by identifying β-glucuronidase (GUS) expressed through EC's natural metabolic processes. This activity can be quantified in a stop-flow sensor and correlated via a quantification curve back to initial EC concentrations. A fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs is also necessary. The current lack of understanding of the factors that influence GUS synthesis and expression in natural waters is addressed in this dissertation. The information presented here increases the accuracy of an in-situ optical sensor.;This dissertation reports for the first time the Michaelis-Menten kinetic parameters of GUS using the substrate Carboxyumbelliferyl-β-D-glucuronide (CUG) as Km=0.910, Vmax = 0.041, Vmax/K m 0.045 mM min-1; optimal pH is 6.5±1.0; optimal temperature is 38°C; CUG hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps; GUS preferentially binds to MUG in comparison to CUG. Additionally, it is found fluorescent reagents facilitate the induction of GUS in EC; increases in GUS activity per culturable EC cell in marine water is likely due to breakdown of cell wall and release of intracellular enzymes; EC GUS expression in natural waters does not significantly correlate to initial physiochemical water parameters or EC concentrations. Benefits of this research include a better understanding of GUS production in natural waters; a better understanding of GUS expression in typical sensor growing conditions; the characterization of a soluble fluorescent reagent ideal for in-situ optical sensing; the use of an optical sensor for the detection and partial quantification of pathogen indicator organisms.