Two -photon excited fluorescence in microbore capillaries
Abstract
Taking advantage of the high selectivity and reduced path-length dependence of two-photon excited fluorescence, a capillary electrophoresis system employing TPE fluorescence detection has been constructed. Characterization of the instrument, including the effects of beam astigmatism, is performed. Additionally, a comparison between 90° and epi-excitation geometries is conducted in order to optimize the instrument's performance. Major findings include the presence of a two-photon fluorescent impurity in the capillary walls, the nonideal behavior of the two-photon excitation volume in cylindrical capillaries, and application of the detection system to capillary microchip devices. The fluorescent impurity originating from the capillary walls is reduced by spatial filtering and time-filtered detection. The loss in sensitivity of TPE fluorescence, when switching from a 1-cm cell format to capillaries, is determined to result from a shortened path length and astigmatism. Despite the ability to use higher numeric aperture objectives, a comparison between 90° and epi-excitation geometries reveals that due to the increased background, both orientations offer similar performance. Application of TPE to microchips is shown to offer detection limits in the submicromolar domain. Additionally, electrophoretically mediated microanalysis is performed for the first time on a capillary microchip device. The enzyme, leucine aminopeptidase, is assayed on a microchip using TPE fluorescence detection.
Degree
Ph.D.
Advisors
Lytle, Purdue University.
Subject Area
Analytical chemistry
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