Bermingham, Kobi P. (2022) The Development of a Gamma-(γ)-Aminobutyric Acid (GABA) Biosensor and Characterisation of an L-Glutamate Biosensor for Neurochemical Analysis. Masters thesis, National University of Ireland Maynooth.

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Abstract

The research presented in this thesis started off as a PhD project with the aim to develop and characterise an in vitro biosensor appropriate for in vivo detection and monitoring of gamma- (γ)-aminobutyric acid (GABA). The ambition was simultaneous monitoring of GABA and Lglutamate. It was also hoped that simultaneous D-serine monitoring would be performed using a newly developed and validated sensor for this co-agonist of the glutamatergic N-methyl Daspartate (NMDA) receptor. The development of a GABase-based biosensor was found to be too large an undertaking and consequently, the research plan converted to refinement of an Lglutamate biosensor. Some development work (pH and temperature studies) was also performed on the D-serine biosensor. Gamma-(γ)-aminobutyric acid is the major inhibitory neurotransmitter but has yet to receive wide examination in the scientific community. In contrast, L-glutamate, the major excitatory, neurotransmitter has not only experienced vast amounts of research but is also present in the public eye unlike GABA. As neurotransmitters are chemicals producing electrical stimulation in the brain, electrochemical techniques offer a unique insight into their operation and reactions. The development of a first generation GABA biosensor used an underlying L-glutamate biosensor as GABA is the precursor to L-glutamate. An appropriate enzyme unit activity for the GABase solution was the first barrier to be overcome. After this, the position of the GABase in the composite design was investigated. This experimentation didn’t garner any results that suggested a response would be produced. The active surface was examined to ensure that the production of hydrogen peroxide would be detected. An alternate reaction scheme was also investigated which didn’t produce any response either. This suggested the enzyme solution was, at least in part, at fault. Further exploration and refinement of the enzyme solution could potentially alleviate the issues encountered during this development work. The characterisation of the L-glutamate biosensor then became the priority. The optimal composite design was found to be: Pt/IrC – PoPD – (Sty – GluOx(100 U/mL) – BSA:GA(1.0:0.1 %) – PEI(1%))15 After the optimal design was found and had appropriate sensitivity (90.4 ± 2.0 nA∙cm−2∙μM−1) comparable to previously reported sensors, the in vitro characterisation was performed. This consisted of ensuring the biosensor would remain operational after implantation in the extracellular fluid i.e. under the chemical and physical parameters present in the brain. The shelf-life was found to be several weeks (28 days) and there was no recorded loss in sensitivity after repeated calibrations, or exposure to ex vivo rodent brain tissue. The sensor performed as desired under all physiologically relevant pH and temperature ranges. The interference was mitigated with the use of poly-ortho-phenylenediamine (PoPD) (interferent species were typically < 5% of the basal glutamate (10 μM) glutamate response) and reliable detection of L-glutamate was still observed. Preliminary in vivo characterisation performed in freely moving animals suggested the suitability of this sensor design for in vivo use. Expected signal changes were observed and a stable baseline over 16 days. Future work will include further in vivo characterisation and validation of this biosensor. Tentatively, the dual monitoring of L-glutamate and D-serine would be examined because of their co-agonist role at the NMDA receptor.

Item Type:	Thesis (Masters)
Keywords:	Gamma-(γ)-Aminobutyric Acid; GABA; Biosensor; L-Glutamate Biosensor; Neurochemical Analysis;
Academic Unit:	Faculty of Science and Engineering > Chemistry
Item ID:	17082
Depositing User:	IR eTheses
Date Deposited:	03 Apr 2023 11:02
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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