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Enhancing detection sensitivity for measuring amino neurotransmitters with flow-gated capillary electrophoresis
Maddukuri, Naveen
Maddukuri, Naveen
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dissertation
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2020-07
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Abstract
Capillary electrophoresis (CE) commences as a promising technique for analyzing a variety of
biological substances and has been used in bioanalytical and biopharmaceutical applications.
Flow-gated CE has numerous advantages over conventional and microchip CE. It has the
advantage of fresh background electrolyte, improved separation efficiency, and PDMS flow-gate
is easy for visual troubleshooting. However, it fails to have enough sensitivity in numerous
valuable applications. The goal of the present research is to enhance the detection sensitivity in
flow-gated CE for the measurement of amino neurotransmitters by using an integrated
microfluidic system to perform in vitro and in vivo analysis of biological samples. This
dissertation focuses on the development of novel methods to enhance the signal sensitivity for
the detection of important molecular components in biological samples using laser-induced
fluorescence detection. First, an efficient tagging strategy was developed to tag amino acids by
taking advantage of the fast kinetics of the derivatization of primary amines with ophthalaldehyde
(OPA) in the presence of a highly fluorescent thiol. Experimental results show
that this strategy was capable of performing rapid fluorescent derivatization, thus providing
detection sensitivity, reproducibility, accuracy, and robustness. Second, an on-line sample
preconcentration technique based on vacuum-assisted electrokinetic supercharging (EKS) was
developed for flow-gated CE to enhance the signal sensitivity of low abundant amino
neurotransmitters, such as gamma-aminobutyric acid (GABA), glutamate and aspartate. The
developed vacuum-assisted EKS technique was used to determine the GABA concentration in
cerebrospinal fluid (CSF) samples by using the one-point standard addition method. Both
techniques are anticipated to be valuable for in vivo measurements of neurotransmitters in
neuroscience research and drug development.
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Thesis (Ph.D.)-- Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry
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Wichita State University
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Copyright 2020 by Naveen Maddukuri
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