| Understanding of the central nervous system has advanced tremendously by the ability to link chemical fluctuations with behavioral, cognitive, and emotional states of animals. Electrochemical techniques are well-suited for these measurements, especially when utilizing background-subtracted fast-scan cyclic voltammetry (FSCV) coupled with carbon-fiber microelectrodes. This union combines the high spatial resolution of the microelectrode with the selectivity and the temporal resolution of fast-scan voltammetry, and has led to exciting progress in many fields of neuroscience. However, there are also many possible avenues to improve FSCV. Electrodes can be engineered to produce more sensitive and selective sensors, data analysis can be improved to extract more information from in vivo data, and applied waveforms can be altered to elicit more sensitivity and chemical selectivity.;This research aims to advance FSCV to applications monitoring non-electroactive molecules, as well as those that are traditionally difficult to detect. Hydrogen peroxide (H2O2) is an endogenously produced reactive oxygen species that has recently gained appeal as a neuromodulator and is widely as a reporter molecule in the biosensing community. The first voltammetric measurement of H2O2 on carbon electrodes in living tissue is described, and the chemical mechanisms underlying this detection are investigated. Electron paramagnetic spectroscopy identified that hydroxyl radicals were electrochemically generated at the electrode surface and anodic current was dependent on the presence of this radical. Carbon and platinum electrode substrates were investigated to determine the most appropriate sensor for monitoring H2O2 in biological samples. Carbon electrodes coated with Nafion, a cation-exchange polymer, showed enhanced sensitivity to H2O2 detection.;Additional research sought to advance electrode sensitivity, by evaluating electrode substrates, electrode coatings, and surface chemistry. Utilizing Raman Spectroscopy, it was found that higher anodic wavelimits increased the population of oxygen-containing functional groups, resulting in enhanced sensitivity to dopamine detection. Furthermore, FSCV was adapted to monitor methionine-enkephaline (mENK), a naturally-occurring opioid peptide and neuromodulatory that is present in very low concentrations in vivo. Additionally, peptides are inherently difficult to monitor electrochemically because of electrode fouling. An optimized waveform was developed to enable the detection of tyrosine-containing peptides, such as mENK, in brain tissue.;In all electrochemical experiments, current must be converted to analyte concentration by way of a calibration that usually takes place at the end of the experiment. However, studies in neuroscience also require verification of electrode placement in brain tissue. This is accomplished at the end of the experiment by passing a large current through the electrode to lesion the area, destroying the electrode. Advanced statistical methods were successfully developed to meet a critical need to eliminate traditional calibration methods. This calibration method will allow researchers to use non-faradaic background currents, collected during the experiment, to accurately predict calibration factors without removing the electrode from the brain, and will thus enable the electrode to be sacrificed for tissue lesion.;Finally, this dissertation describes the application of FSCV to the characterization of a bacterially-produced metal chelator, protochelin, which is associated with the sequestration and mobility of iron. This voltammetric approach provides a comprehensive functional analysis relating the redox chemistry of the chelator to structural changes in real-time. It was found that the ligand was easier to oxidize than the bound metal, suggesting an alternative metal release mechanism.;Overall, the fundamental experiments and advances described herein can be exploited to enable lower limits of detection, target new molecules, obtain higher spatial resolution, and simultaneously measure multiple analytes in a single location. These results will enable FSCV to fill critical needs across scientific disciplines ranging from neuroscience to environmental monitoring. |
