Sol-Gel Optical Sensors for Glutamate
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Sol-Gel Optical Sensors for Glutamate Jenna L. Rickus1, Esther Lan2, Allan J. Tobin3, Jeffery I. Zink4, Bruce Dunn2 1 Neuroengineering 2 Department of Materials Science and Engineering 3 Brain Research Institute 4 Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, CA 90095, U.S.A ABSTRACT The amino acid glutamate is the major excitatory neurotransmitter used in the nervous system for interneuronal communication. It is used throughout the brain by various neuronal pathways including those involved in learning and memory, locomotion, and sensory perception. Because glutamate is released from neurons on a millisecond time scale into sub-micrometer spaces, the development of a glutamate biosensor with high temporal and spatial resolution is of great interest for the study of neurological function and disease. Here, we demonstrate the feasibility of an optical glutamate sensor based on the sol-gel encapsulation of the enzyme glutamate dehydrogenase (GDH). GDH catalyses the oxidative deamination of glutamate and the reduction of NAD+ to NADH. NADH fluorescence is the basis of the sensor detection. Thermodynamic and kinetic studies show that GDH remains active in the sol-gel matrix and that the reaction rate is correlated to the glutamate concentration. INTRODUCTION Neurons are the fundamental building blocks of the brain. They form signaling networks that process sensory information, provide motor commands, and store memories. One neuron communicates with another by releasing neurotransmitters into the synaptic space between the two cells. The level and pattern of neurotransmitter release partially determines the activity of the second cell. [1] No analytical methods are currently available for the detection of glutamate on a physiologically relevant timescale of milliseconds. Microdialysis is a widely accepted method of time resolved in vivo glutamate monitoring, but it is limited by poor temporal resolution and a lack of real-time data output [2]. Enzyme modified electrochemical sensors have been developed for both in vivo and in vitro use. They provide a significant improvement in temporal resolution over traditional methods, but are often disadvantaged by an oxygen dependence, toxicity in tissue, and electrochemical interference from other molecules, such as ascorbate. [3]. The results presented here demonstrate the feasibility of using enzyme doped sol-gel materials as the basis for developing an optical glutamate sensor. The enzyme glutamate dehydrogenase catalyses the oxidative deamination of glutamate to α-ketoglutarate and the simultaneous reduction of NAD+ to NADH [4]. NADH fluoresces (~ 460nm) when excited with UV light (~ 340nm) while its oxidized form, NAD+, exhibits no fluorescence [5]. Monitoring reaction rates by NADH fluorescence has been a frequently exploited strategy for the development of biosensors such as those for glucose and lactate [5]. Adapting this approach to sol-gel encapsulation methods offers promise of creating a novel fiber optic glutamate sensor.
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