Enhancing detection sensitivity for measuring amino neurotransmitters with flow-gated capillary
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.