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

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

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis

Optimization of Virus-induced Gene Silencing to Facilitate Evo-devo Studies in the Emerging Model Species Mimulus guttatus (Phrymaceae)

Mimulus guttatus DC. (yellow monkey-flower; Phrymaceae) is an important model species for ecological and evolutionary studies, being locally adapted to a wide range of elevation, moisture and temperature gradients, soil types, and pollinator availabilities. In order to advance this species as a model for evolutionary genetic studies, we have developed virus-induced gene silencing (VIGS) using the tobacco rattle virus (TRV) to assay gene function. We demonstrate the effectiveness of Agrobacterium-mediated VIGS in two divergent populations of M. guttatus, Iron Mountain 767 (IM767) and Point Reyes (PR). Plants infected with a fragment of the carotenoid biosynthesis pathway gene PHYTOENE DESATURASE (PDS) cloned into the TRV2 vector exhibited endogenous PDS silencing and photobleached phenotypes. We further assayed for VIGS-induced floral phenotypes by silencing paralogous genes putatively affecting floral symmetry, CYCLOIDEA1 (CYC1) and CYCLOIDEA2 (CYC2). Simultaneous silencing of CYC1 and CYC2 resulted in organ number defects in the petal and stamen whorls; silencing of CYC1 affected petal margin growth; and silencing of CYC2 had no effect on flower development. Infection with TRV2 and TRV1 is significantly higher and more pervasive in the IM767 versus the PR population and is more efficient after vacuum infiltration. These results demonstrate the efficacy of VIGS for determining the function of developmental genes, including those involved in ecologically important reproductive traits

Evaluation of a Portable Microchip Electrophoresis Fluorescence Detection System for the Analysis of Amino Acid Neurotransmitters in Brain Dialysis Samples

A portable fluorescence detection system for use with microchip electrophoresis was developed and compared to a benchtop system. Using 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 electrophoretically, and detected by fluorescence. The limits of detection for the analytes of interest were 50 – 250 nM for the benchtop system and 250 nM – 1.3 μM for the portable system, both of which were adequate for most analyte detection in brain microdialysis samples. The portable system was then demonstrated for the detection of the same six amines in a rat brain microdialysis sample