Development of a Microfluidic Based Portable Analyzer for Continuous Monitoring of Glutamate and other Amino Acid Neurotransmitters

Abstract

The amino acid glutamate (Glu) is one of the most ubiquitous neurotransmitters in the brain and the chief excitatory neurotransmitter. As a neurotransmitter, Glu is integral to the normal workings of the brain and is involved in many functions, such as memory formation and long-term potentiation, via action on multiple receptors. Two primary classes of Glu receptors, metabotropic and ionotropic respond to the concentration of Glu in the extracellular space of the brain in a dose dependent manner. Large excesses of Glu have been shown to produce an excitotoxic effect, which can lead to the long-term neuronal damage seen in many neurological disorders including stroke and traumatic brain injury (TBI). Following an event such as these, methods for continuous monitoring of Glu concentrations in the brain can be very useful to clinicians for determining the best timing for pharmacological intervention, provided the acquisition of that information can itself be performed in a timely manner. With that in mind, this thesis focuses on the development of analytical methods that will provide information on the extracellular concentration of glutamate and other amino acids in a timely manner and thereby providing actionable information for a clinician. Microdialysis (MD) is an in vivo sampling method that can be used to monitor multiple analytes simultaneously while also enabling the delivery of a pharmaceutical intervention directly to the site of the probe This technique can provide a powerful window into tissue function and health when combined with a separation-based analytical method. However, due to the need for very low flow rates, a trade off exists with regard to sample concentration and time. In order to maximize the concentration and minimize the time required, sensitive methods of detection must be used such as laser induced fluorescence (LIF) detection. To minimize the time required for sample analysis (and make point of care analysis possible), a portable fluorescence detection system for use with microchip electrophoresis was developed. With this system, six neuroactive amines commonly found in brain dialysate (arginine, citrulline, taurine, histamine, glutamate, and aspartate) were derivatized offline with naphthalene-2,3-dicarboxaldehyde/cyanide, separated electrophoretic ally, and detected by fluorescence. It was found that this system was able to detect these analytes of interest within a range of 250 nM – 1.3 µM, which was adequate for subsequent detection in a microdialysis sample collected from the brain of an anesthetized rat. Finally, the design and evaluation of a microfluidic device for coupling microdialysis to microchip electrophoresis with on-line derivatization (MD-ME) is discussed. By coupling sampling directly to the microchip, elements that would otherwise delay analysis such as the need to transport volumes to the analysis system or the wait for the generation of larger sample volumes can be avoided. The MD-ME device was modeled first using COMSOL Multiphysics™ in an effort to optimize the device geometry, allowing on-line sampling with minimal back pressure, but with complete sample derivatization prior to analysis. Following this, the device was evaluated experimentally to detect Glu samples collected via microdialysis over an extended time period. While the limits of detection for Glu were found to be slightly high for immediate use for in vivo brain sampling, it is hoped that modifications to materials used to construct the microchip may eliminate this problem.