New Electroanalytical Tools for Exocytosis: From Single Cells Toward Single Vesicles

Abstract

This dissertation focuses on the development and application of new electroanalytical tools for the measurement of neurotransmitter release (e.g. dopamine, norepinephrine, epinephrine) from vesicles. Advancements in biosensor design for more sensitive, higher spatial resolution measurements of exocytosis at neuroendocrine cells and stem-cell derived neurons are detailed. New techniques for the characterization of redox-filled liposomes (here used as an analog forbiologically-derived vesicles) are described and new insights into their electrochemical detection discussed. The application of these techniques to the measurement of catecholamine-containing large dense-core vesicles are also presented. Chapter 1 introduces the relevant background for understanding the present state-of-the art in exocytosis measurements, focusing on the information obtained from amperometric measurements. Chapter 2 details the development of carbon nanoprobes and their application to exocytosis from single rat pheochromocytoma (PC12) cells and stem-cell derived human dopaminergic neurons. Chapter 3 discusses the development of a facile gold electrodeposition method for improving the sensitivity of carbon-fiber microelectrodes to catecholamine exocytosis. Beginning in Chapter 4, we transition toward liposome/vesicle studies. Chapter 4 presents the initial validation of our new nanopore/microelectrode technique for simultaneous measurement of relative size and redox contents of single, redox-filled liposomes. Due to challenges in quantitation with the technique presented in Chapter 4, Chapter 5 details the use of a simpler technique, collision amperometry, to understand how these liposomes accumulate and releasemolecules.