Characterization of biological chromophores using fast electrophoretic analyses and multiphoton-exited fluorescence
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A fast and highly sensitive technique has been developed for characterizing mixtures of biological molecules that should be useful for various applications, including investigations of enzymatic reactions with fast chemical kinetics, analysis of transient photochemical reactions, and probing neurotransmitter secretion. In this dissertation work, multiphoton excitation (MPE), a high sensitivity detection approach, and capillary electrophoresis (CE), a fast separation strategy, have been coupled to rapidly characterize primary amine neurotransmitters, to perform fast electrophoretic analyses of photochemical reactions, and to probe changing biochemical microenvironments. Nonfluorescent neurotransmitter molecules have been separated and detected by virtue of fluorogenic-labeling strategies with mass detection limits (< 50,000 molecules) that should prove useful for various single-cell studies. Also, this technique has been extended to rapidly probe spectroscopically indistinct components in solution. Extremely short capillary lengths and large applied electric fields are used to extend the speed of CE to sub-second time regimes. Previously, others have demonstrated that fast CE can be accomplished using optical gating, a technique in which sample injection is controlled by modulation of photobleaching at the separation channel inlet. In these studies, this strategy has been adapted to an "inverse" optical photogating mode with CE, where fluorescent packets of photogenerated molecules are created and detected by MPE. Two instrumental configurations, defined by the alignment of the laser light with the capillary ("end on" and "side on" geometries) and the separation distance, are constructed to achieve fast separations. Using these approaches, this work demonstrates the capacity to analyze dynamic biochemical microenvironments and track transient reaction products on a several-hundredmicrosecond to several-hundred-millisecond time scale. These fast analysis strategies have provided additional information on transient species on millisecond and faster time scales, and may prove valuable in analyzing cellular processes in real-time.