Time-Resolved Fluorescence Techniques for the Development and Characterization of Genetically-Encoded Biosensors.

Graduate Thesis Or Dissertation

Citations

Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/ws859f98m

Abstract

Fluorescent biosensors are important measurement tools for in vivo quantification of pH, concentrations of metal ions and other analytes, and physical parameters such as membrane potential. Both the development of these sensors and their implementation in examining cellular heterogeneity requires technology for measuring and sorting cells based on the fluorescence levels before and after chemical or physical perturbations. We developed a droplet microfluidic platform for the screening and separation of cell populations on the basis of the in vivo response of expressed fluorescence-based biosensors after addition of an exogenous analyte. Screening with this instrument reveals increased heterogeneity in an array of targeted Zn²⁺ biosensorsin HeLa cells that helps shed light on the complexities of these sensors in different chemical environments. Subsequently, the instrument is used to screen and assess diversity in a number of HeLa-cell based genetic linker libraries for a family of genetically-encoded Zn²⁺ sensors. Progress on sensor characterization is made using time-resolved fluorescence techniques to advance a deeper molecular understanding of these sensors to guide further development.

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