Single molecule detection: Analytical applications and fundamental studies

This thesis covers preliminary research done in the area of saliva diagnostics and protein detection as well as extensive research in the area of single molecule detection. In each case, fiber optic arrays were used to detect target molecules of interest. Preliminary screening of target analytes in saliva used to monitor end-stage renal disease patients is discussed in Chapter 2. The work in Chapter 3 is an extension of the saliva project, and demonstrates the first implementation of a sandwich-based immunoassay on our fiber array platform. This immunoassay technique will be useful for monitoring the levels of various proteins in patient saliva samples.; The bulk of the work presented in this thesis deals with single molecule detection. Traditionally, our fiber arrays are used to house microspheres. These microspheres are optically encoded and functionalized with sensing chemistry, permitting multiplexed detection of analytes using a single fiber array. The array platform was transitioned to an array of isolated reaction vessels by sealing across the face of an etched fiber array. Using a silicone gasket material and a precision mechanical platform, the individual microwells were converted into femtoliter sized reaction chambers. Each of these chambers was isolated from neighboring chambers and could be used to trap and interrogate single enzyme molecules.; In Chapter 5, the initial platform was setup and single enzyme molecule trapping experiments were conducted at various concentrations, which were calculated based on the Poisson distribution. Using the Poisson distribution, concentrations could be calculated that would result in a binary response when monitoring one reaction chamber at a time. By sealing a solution of enzyme and substrate in the array of reaction chambers, the presence of a single enzyme in a reaction chamber could be monitored. This binary 'on' or 'off' response of single reaction chambers enabled low enzyme concentrations to be quantified using a digital readout.; In Chapter 6, the digital readout format was maintained; however, single enzyme molecules were not trapped in the reaction chambers using low concentration solutions of enzyme. Instead, the surface of the reaction chambers was modified with the capture probe biotin. By incubating the modified fiber array with a solution of streptavidin-beta-galactosidase, single enzyme molecules were bound to the surface of the reaction chambers. After washing away any unbound enzyme, reaction chambers that had captured an enzyme molecule could be monitored by sealing the chambers with a substrate solution and monitoring fluorescence generation. This capture and readout approach could be useful for detecting low concentrations of DNA or protein targets.; Finally, Chapter 8 focuses on monitoring the kinetics of single enzyme molecules. A population of single enzyme molecules, each trapped in an isolated reaction chamber, was monitored over time. We have found that each enzyme molecule possesses a distinct activity state, suggesting each molecule has a slightly different structure that is stable on long time scales. This information is masked when studying enzymes with traditional bulk measurements. Our results explain the kinetic variability within enzyme molecule populations and offer a deeper insight into the unique properties of single enzyme molecules. Gaining a more fundamental understanding of how individual enzyme molecules work within a population should provide insight into how they affect downstream biochemical processes. If the results reported here can be generalized to other enzymes, then the stochastic nature of individual enzyme molecule kinetics should have a substantial impact on the overall metabolic activity within a cell.