Date of Award

Summer 8-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Micro and Nanoscale Systems

Abstract

Continuous simultaneous measurement of glutamate (GLU), an excitatory neurochemical, and γ-aminobutyric acid (GABA), an inhibitory neurochemical, constitutes one of the major challenges in neuroscientific research. Maintaining appropriate levels of GLU and GABA is important for normal brain functions. Abnormal levels of GLU and GABA are responsible for various brain dysfunctions, like epilepsy and traumatic brain injury. GLU and GABA being non-electroactive are challenging to detect in real-time. To date, GABA is detected mainly via microdialysis with a high performance liquid chromatography (HPLC) system that employs electrochemical (EC) and spectroscopic methodologies. However, these systems are bulky and unsuitable for real-time continuous monitoring. As opposed to microdialysis, biosensors are easy to miniaturize and are highly suitable for in-vivo studies. Unfortunately, this method requires a rather cumbersome process that relies on externally applied pre-reactors and reagents. Here, we report the design and implementation of a GABA microarray probe that operates on a newly conceived principle. It consists of two microbiosensors, one for GLU and one for GABA detection, modified with glutamate oxidase and GABASE enzymes, respectively. The detection of GABA by this probe is based upon the in-situ generation of α-ketoglutarate from the GLU oxidation that takes place at both microbiosensor sites. By simultaneously measuring and subtracting the H2O2 oxidation currents of GLU microbiosensor from GABA microbiosensor, GABA and GLU can be detected continuously in real-time in vitro and ex vivo. This mechanism happens without the addition of any externally applied reagents. We optimized our novel approach in commercially available ceramic-based probes. The GABA probe was successfully tested in an adult rat brain slice preparation. However, those electrodes are geometrically limited (we cannot have a sentinel site at the same spatial level as GLU and GABA sites). Keeping theseissues in mind, we have developed a microwire array sensor that is not only capable of simultaneous measurement of GLU and GABA, but is also able to track signal resulting from interferents (e.g. Ascorbic Acid, AA). The unique geometry enables these microwire probes to measure GLU, GABA and interferents in the same spatial level. A Simple fabrication procedure and easy integration with the existing amperometric systems allow us to use them in cell culture, brain tissue, and in vivo recordings as an inexpensive alternative to our planar electrodes. We demonstrated the effectiveness of the probes in rat brain tissue. We were able to get. Additionally, we determined the excitation/inhibition (E/I) ratios for different stimulations which have clinical relevance. Our results about this E/I balance can help refine electrical stimulation parameter for different clinical purposes (e.g. deep brain stimulation). Finally, we successfully tested our probe in awake-free behaving rats. In summary, our results suggest that microwire probes have the potential to become a powerful tool for measuring GLU and GABA in various ex-vivo and in-vivo disease models, such as epilepsy.

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