Development of Ultra-fast Biosensors for Detection of Non-electroactive Neurotransmitters

During neuronal communication neurotransmitters are released through the process exocytosis that occurs on the time scale of sub-milliseconds to milliseconds. Neuronal activity by neurotransmitters that are non-electroactive cannot easily be monitored due to limitations in the temporal resolutions of the sensor probes for these analytes. In order to achieve a fast detection of the rapid transients derived from these nonelectroactive neurotransmitters e.g. acetylcholine and glutamate, enzyme nanoparticle conjugates was carefully characterized and optimal enzymatic conditions from these studies were used to design and construct a nanostructured enzyme-based electrochemical biosensor for acetylcholine. This sensor was constructed from a micro-sized carbon fiber (5 μm to 30 μm in diameter) sealed into a glass capillary and functionalized with gold nanoparticle (AuNP) hemispheres at the electrode surface. The nanoparticle structure at the electrode surface increases the electrode surface area and allows immobilization of larger amount of enzyme. In addition the nanoparticle provides a surface with high curvature that may prevent enzyme denaturation compared to the flat surface. The enzymes subsequently immobilized onto the AuNPs-structured surface will catalyze the non-electroactive molecules of interests and produce H2O2 that is electroactive and can be detected using amperometry. We have found that the key for providing high temporal resolution by these sensors is to limit the enzyme coverage at the electrode surface to a monolayer. In Paper I, an acetylcholine sensor is constructed with a two-enzyme system consisting of acetylcholinesterase and choline oxidase. This sensor provided a temporal resolution that was fast enough to detect single vesicle release of acetylcholine in the millisecond time scale by an artificial cell model for exocytosis. For the design and construction of a microelectrode glucose sensor, the characterization of the conjugation of the enzyme glucose oxidase (GOx) to the surface of AuNP was studied in Paper II to achieve the optimal conditions for the GOx. The work shows that the structure of GOx adsorbed on AuNP changes to much less extent than if GOx adsorbs on to a flat surface and that the enzymatic activity is maintained to the same extent as for GOx in solution. Keywords: Biosensor, amperometry, acetylcholine, acetylcholine esterase, choline oxidase, artificial cell, exocytosis, glucose oxidase, gold nanoparticles, enzyme monolayer, adsorption, quantification, deformation, enzymatic activity, stability, nanoparticle tracking analysis, dynamic light scattering