New Electrochemical Tools to Study Exocytosis
Sammanfattning: The work described in this thesis has the focus on the development of new analytical tools to study processes related to cellular secretion (exocytosis) in cell models. Four novel techniques were developed, allowing new ways to study processes related to exocytosis, and gain previously unattainable knowledge. The methods were applied to single cells as well as to populations of cells in culture. In the first work A novel enzyme based biosensor, capable of detection of rapid fluctuations in acetylcholine concentration was developed. The work was motivated by limitations found in current electrochemical methods, for monitoring of single vesicle neurotransmitter release, which to date has been unable to detect electroinactive substances. Selective detection of the analyte was performed, based on sequential digestion by acetylcholine-esterase (AChE) and choline oxidase (CHO) enzymes together producing hydrogen peroxide in the presence of acetylcholine. The enzymes were immobilized on a nanostructured, high curvature, electrode surface, promoting retention of enzymatic activity, and the transduction of hydrogen peroxide concentration into amperometric current relied on electrochemical reduction, by the negatively polarized electrode. The sensor structure, catalytic function, and sensor temporal performance were characterized. In the second work, a new method was developed, with the aim of being able to answer the question of the prevalence, of two fundamentally different modes of exocytosis: ‘full fusion’, where the whole content of neurotransmitter containing vesicles is ejected from cells, and ‘kiss and run’, which may result in a partial release. The method was based on amperometric quantification of the total neurotransmitter content of single isolated vesicles releasing their neurotransmitter content when, collapsing at a microelectrode surface, resulting in a current spike for each collapse. Comparison showed that only a smaller fraction of the total neurotransmitter content is released from each vesicle during stimulated secretion of chromaffin cells. The goal of the third work was to develop an amperometric method which show the location of single vesicle release with both high temporal and spatial resolution, a combination which has previously been difficult to obtain. A novel microelectrode array (MEA) probe, lithographically microfabricated on the tip of a transparent glass substrate was developed. The MEA was capable of approaching single isolated chromaffin cells, there detecting exocytotic release in 16 different areas across the cell surface with high temporal resolution. Data from the probe was first used to estimate the thickness of the glycocalyx, separating the cell and the MEA probe. Secondly, exocytotic release from single vesicle release was ‘imaged’, in the areas in between the electrodes. The technique used was based on comparing the amperometric currents, passing through multiple detecting electrodes, with random-walk computer simulations. A resolution was obtained, sufficiently high for resolving hot-spots in activity, with distributions smaller than 120 nm. Finally, the question if quartz crystal microbalance (QCM-D) can detect structural changes, related to exocytosis was addressed. A new method where the QCM-D response and amperometric measurement were applied in parallel, monitoring neurotransmitter release and structural changes in a population of cultured PC12 cells stimulated by high K+. Perturbation of the secreted amounts using the drugs L-dopa and reserpine suggested that measurement by QCM-D reflects processes related to exocytosis, validating previous claims.
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