The Development of Microelectrochemical Choline and Acetylcholine Biosensors for Real-Time Neurochemical Monitoring

The aim of this thesis was the development of a choline biosensor for the electrochemical detection of choline in the brain, which could subsequently be modified for acetylcholine detection. The choline biosensor was characterised in-vitro to optimise sensitivity towards choline which resulted in two biosensor designs (Chapter 4); Sty-(ChOx)(BSA)(GA)(PEI) and MMA-(ChOx)(BSA)(GA)(PEI) which suitably detected choline with sensitivities of 0.03 ± 0.0003 nA/μM, n=3 and 0.03 ± 0.001 nA/μM, n=4 respectively, and were subsequently progressed for further characterisation. Sensitivity to O2 was also investigated as the biosensor was developed using an oxidase enzyme which utilises O2 as a co-substrate (Chapter 5). In the in-vivo environment O2 fluctuations can cause interference in the sensors response to substrate. The styrene design demonstrated high levels of O2 interference which could not be improved upon. The MMA design was modified with cellulose acetate to (MMA)(CelAce)(MMA)-(ChOx)(BSA)(GA)(PEI) with a sensitivity of 0.58 nA/μM and experienced O2 interference of 3 % at 20 μM choline. Further in-vitro characterisation was performed (Chapter 6) which demonstrated that the shelf life of the sensor for 14 days was subject to a decrease in sensitivity of 10 %. Upon exposure to brain tissue for 14 days the sensor experienced a decrease in sensitivity of 26 %. The effect of a variety of interferent species on the selectivity of the sensor was examined. The total average response attributed to 12 interferents was 0.182 ± 0.019 nA, (n = 4). The limit of detection of the sensor was determined to be 0.11 ± 0.02 μM, (n = 8) with subsecond recording.Chapter 7 utilises the choline biosensor in the in-vivo environment. Using microdialysis, the ability of the sensor to detect exogenous choline is examined with the perfusion of concentrations of choline between 20 and 1000 μM. As this was successful, pharmacological manipulations were utilised to determine the O2 dependence of the sensor using chloral hydrate, Diamox and L-Name. Also, the sensors ability to detect changes in choline, as a result of manipulations of aspects of the cholinergic system were undertaken using HC-3, neostigmine and atropine. The choline biosensor was characterised in both the in-vitro and the in-vivo environment and demonstrated detection of choline in the striatum of a freely moving rat. This sensor was then further modified for the detection of Acetylcholine. A preliminary investigation into the modification of the choline biosensor with Acetylcholinesterase to detect acetylcholine is presented in chapter 8. It was demonstrated that the sensor can successfully detect acetylcholine in the in-vitro environment with a sensitivity of 0.26 ± 0.01 nA/μM.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

National University of Ireland Maynooth

Year dc:date.issued

2013

Author dc:creator

• Baker, Keeley

Language dc:language

en