| Description |
Aptamer-based sensors rely on the ability of nucleic acids to bind to target molecules with high affinity and specificity via molecular recognition. Due to the ease of production and high thermal stability of nucleic acids, these sensors are emerging as attractive candidates for detecting a wide range of chemical and biological targets. The purpose of this dissertation is to develop new methods that expand the repertoire of potential applications for aptamer-based sensors. While the utility of aptamer-based sensors has been demonstrated in a variety of bioanalytical applications, the ability to achieve temporal control over their sensing activity remained relatively unexplored. We envisioned that a covalent self-caging strategy can be employed in the design of aptamer-based sensors. As a proof of concept, we used the structure-switching (SS) biosensor for L-tyrosinamide (L-Tym) and installed an equilibrium-shifting photocleavable linker that renders the sensor functionally inert to the target molecule. We demonstrated the precise temporal control over sensing activity by cleaving the linker with UV light restoring the sensor into its native and functional state. Next, we demonstrated the utility of enantiomeric biosensors in microfluidic droplets and developed a method for enantiopurity analysis by flow cytometry. We fabricated microfluidic devices that allow us to generate droplets encapsulating the enantiomeric biosensors and their respective target molecules, iv L- and D-Tym. Taking advantage of the exceptionally high throughput provided by FACS, we were able to perform rapid enantiopurity analysis with high accuracy and precision. We envisioned our approach to be utilized as a screening method in evolution experiments for enantioselective enzymes, providing a significant increase in the throughput of library screening process. Finally, we demonstrated the utility of DiO as a fluorogenic probe for the measurement of surfactant CMC. We developed a fluorescence-based method using DiO, a fluorescence dye that undergoes a change in emission intensity based on the polarity of its environment, for CMC measurement of a variety of surfactants. For comparison purposes, we carried out parallel experiments using DiO and NR, with the latter being a common probe for CMC measurement, and demonstrated that DiO was more reproducible and user-friendly. |
