Development of Analytical Methodology for Neurochemical Investigations

Abstract

David J. Fischer Department of Pharmaceutical Chemistry University of Kansas Neurochemical Applications of Microchip Electrophoresis The development of sensitive and selective analytical tools has facilitated the investigation of complex neurological pathways and enhanced our understanding of neurodegenerative diseases. The development of sensitive analytical methodology for the determination of neurotransmitters and proteins related to neurodegenerative disease is described. The goal of the work performed in the first part of this dissertation was to develop analytical methodology for the analysis of catecholamine neurotransmitters (NTs) by microchip electrophoresis with electrochemical (EC) detection. Much of this work focused on the fabrication and characterization of the novel carbon-based electrode material, pyrolyzed photoresist. The fabrication of pyrolyzed photoresist film (PPF) electrodes was optimized for use in microchip electrophoresis and analytical performance was characterized using catecholamine NTs. In addition, an extensive comparison of the analytical performance of several commonly used electrode materials and electrode alignment schemes and the PPF electrode material was performed. Aspects such as sensitivity, limit of detection (LOD), resolution, reproducibility, and ease of fabrication were examined. In addition to the development of EC detection methods for catecholamine NTs, analytical methods for the determination of myc-tagged proteins were developed. The development of an electrophoretic immunoaffinity assay for the detection of a myc-tagged protein expressed in cell culture is described. While this is a general assay that can be applied to a variety of myc-tagged proteins, mutant huntingtin protein (mHtt) was used as a specific example. The development and optimization of capillary and microchip electrophoresis assays were performed for this purpose. In addition, the results obtained using these methods were directly compared to traditional analysis by Western blotting. The long term goal of this project is integrate both of these assays into a single lab-on-a-chip device capable of detecting NT release and mHtt protein in single cells.