The goal of the proposed research is to develop chemical sensors of micrometer dimensions which are capable of selectively monitoring small changes in the concentrations of a variety of neurotransmitters in picoliter samples with millisecond time resolution. The initial target molecules will be the amino acid neurotransmitters. These transmitters are intimately involved in the etiology of. seizure, stroke and ischemic brain damage. Therefore,-there is great need to better understand the processes which control their release and deactivation. Most amino acids have very few physical properties which can be utilized for chemical analysis (i.e. UV or visible absorption, fluorescence, electrochemical activity). in vivo measurements of very small quantities of these species (attomoles) in very small sample volumes (subnanoliter) limit the utility of these methods, especially when the analysis must be performed on a subsecond time scale. Therefore, detection of these species in real time will be accomplished after reaction with enzymes immobilized to the surface of carbon-fiber ultramicroelectrodes. The enzyme-catalyzed reactions which oxidize amino acids and simultaneously reduce NAD+ to NADH are very rapid and can be used to provide the selectivity necessary to make physiologically relevant measurements. The NADH generated in this manner acts as an electron transfer mediator and can be monitored with fast-scan cyclic voltammetry at the ultramicroelectrode surface. Considerable effort will be directed toward the characterization of the electrode surface and the modifications induced by chemical derivatization. Correlation of the spatial distribution of chemical functional groups on the electrode surface to the electrochemical activity observed at the electrode will be attempted. This information is critical to the rational design of novel sensors based on surface modification of ultramicroelectrodes. Ultimately, an array of well-characterized chemical sensors will be developed, which could be used interchangeably to monitor a wide variety of neurotransmitters in vivo in the mammalian brain. Synchronization of the measurement process to well defined, externally-applied physiological stimuli will allow characterization of the complex interactions that occur between different neuronal systems.
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