Development and Application of Enzymatic Biosensors in the Investigation of Organophosphate Toxicity and Neurotransmission

Abstract

Neurotransmitters are an extensive group of chemical messengers essential to the proper function of both physiological and psychological processes in humans. The comprehensive electrochemical detection of these compounds can provide insight into neuronal transmission mechanisms and metabolic dysregulation following toxicant exposure. This dissertation presents the development and application of multiple enzymatic biosensors to study a variety of neurotransmitters and their actions within biological systems. First, a glutamate biosensor was integrated into a microfluidic system for the real-time analysis of glutamate uptake. PC12 cells were shown to uptake glutamate for use as an energy source following glucose starvation. The dysregulation of glutamate uptake mechanisms in astrocytes with Tuberous Sclerosis Complex was also investigated—with diseased astrocytes demonstrated to uptake significantly less glutamate than non-diseased astrocytes. Additionally, an enzymatic acetylcholine sensor was characterized in the presence of the organophosphate pesticide chlorpyrifos (CPF) and used to analyze the dysregulation of acetylcholine metabolism in an organotypic blood-brain barrier—the neurovascular unit (NVU)—exposed to CPF. Electrochemical analysis of eluate collected from the NVU indicated significant metabolic disruption following CPF treatment. The integration of multiple neurotransmitter biosensors onto an electrode array can expand the amount of information gained through a single experiment. In addition to glutamate and acetylcholine biosensors, the enzymatic detection of adenosine and dopamine was demonstrated, and all four biosensors were adapted for use with a microfluidic electrode array with eight working electrodes. This 8-channel electrode is then used to investigate the effects of CPF on glutamate uptake in astrocytes. The addition of these biosensors downstream of organotypic devices like the NVU provides the opportunity to monitor real-time changes in neuronal transmission and metabolism. The final portion of this dissertation explores the challenges of performing electrochemical analysis downstream of these devices by investigating the effects of diffusion, crosstalk, and permeability on analyte quantification. The studies presented here demonstrate the effectiveness of electrochemical biosensors in the analysis of complex biological processes, while also laying the foundation for the further integration of electrochemical biosensors with microfluidic organotypic devices.