This application describes a systematic program of research aimed at the development of micron-sized sensors for the measurement of neurotransmitter release and uptake in """"""""real-time"""""""" using biotin/avidin technology. The objective of this work is to perform submicrometer-sized modification of the surface through generation of oxidized regions of carbon for use as covalent bonding sites for further modification. Direct attachment of functional molecules is also proposed. Experiments with oxidative modification are aimed towards generating microscopic regions with a specific type and density of surface oxide to provide a constructed surface that is optimized for use as a sensor device. Spatially-localized modification of carbon fiber electrodes is proposed on the sub-micron scale to allow spatial segregation of enzyme-binding sites from electron transfer sites. Specifically, photocleavable reagents can be used to locally protect/deprotect derivatization sites on carbon electrode surfaces to allow spatial segregation of enzyme attachment sites from redox sites. The use of photolithographic techniques will produce sub- micron segregation of redox and enzyme sites. Latex nanoparticles (30 - 200 nm dia.) will serve as sites of attachment for redox enzymes to increase the surface loading of the enzyme without interfering with electron-transfer sites on the electrode. Fluorescence microscopy with high resolution will be employed to characterize the spatial localization of modified sites on electrodes as well as electrodes prepared using global modification methods, such as electrochemical pretreatment. The distribution and electron-transfer activity of the attached enzyme will be mapped with fluorescence and chemiluminescence microscopy and compared to electrode activity prior to enzyme attachment. An objective is to construct redox-enzyme based sensors with improved sensitivity and time response. Ideally, this will allow the construction of microscopic arrays of active enzyme sites on a carbon-fiber substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators. Thus, enzyme activity can be maximized while detection of the enzyme mediator can be optimized on the unmodified carbon. The GDH- modified electrode will be used to monitor glutamate efflux following depolarization of glutamate-containing neurons of the larval fly (Musca domestica) neuromuscular junction. These axons can be stimulated independently and recording electrodes will be placed in close proximity to the neuromuscular junction, where the efflux of glutamate from adjacent nerve terminals should be observed. Therefore, this should be an ideal preparation to determine the basic characteristics of the presynaptic moduation of glutamate release without the use of vertebrate animals.
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