Photochemical Enzyme Co-Factor Regeneration: Towards Continuous Glutamate Monitoring with a Sol-Gel Optical Biosensor
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Photochemical Enzyme Co-Factor Regeneration: Towards Continuous Glutamate Monitoring with a Sol-Gel Optical Biosensor Jenna L. Rickus1, Allan J. Tobin2, Jeffrey I. Zink3, Bruce Dunn4 1
Neuroscience IDP, Neuroengineering Program Brain Research Institute 3 Department of Chemistry and Biochemistry 4 Department of Materials Science and Engineering 2
ABSTRACT Sol-gel encapsulation has recently surfaced as a successful approach to biomolecule immobilization. Proteins, including enzymes, are trapped in the pores of the sol-gel derived glass while retaining their spectroscopic properties and biological activity. Our current work extends the unique capabilities of biomolecule-doped sol-gel materials to the detection of glutamate, the major excitatory neurotransmitter in the central nervous system. We are developing an in vivo fiber optic biosensor for glutamate along with methods to achieve continuous monitoring. In our research to date we have encapsulated GDH in a silica sol-gel film on the tip of an optical fiber. GDH catalyzes the oxidative deamination of glutamate to αketoglutarate and the simultaneous reduction of NAD+ to NADH. To quantify the glutamate concentration, we observe the rate of change of NADH fluorescence as a function of time. An important consideration for continuous in vivo monitoring is the incorporation of a selfsustaining NAD+ source. We have adopted a photochemical means of regenerating NAD+ from NADH, by irradiating thionine (3,7-diaminophenothiazin-5-ium) which we incorporate into the sol-gel sensor material. When excited with visible light (λabs ~ 596 nm), thionine undergoes a reaction with NADH resulting in a non-fluorescent form of thionine and NAD+. We have characterized the kinetics of this reaction in the sol-gel matrix, and have shown that the reaction results in regenerated co-factor that is usable by GDH for the oxidation of glutamate. INTRODUCTION Glutamate is the most prominent excitatory neurotransmitter in the central nervous system. It is present throughout the entire brain, but acts locally to produce different types of signals that vary in their spatial and temporal characteristics. Glutamate signaling is an important component of networks involved in attention, learning and memory, and motor control. Current detection techniques are limited in their ability to achieve the required resolution to observe these signals. New types of sensors are needed; optical sol-gel sensors could help to fill the gap. Enzymes are commonly employed in biosensors because they provide both biochemical recognition and transduction of the recognition event into a reaction. Redox enzymes in particular are convenient because the electron transfer provides a measurable current or a measurable change in the spectroscopic properties of the substrate or co-factor. Dehydrogenases are the largest class of redox enzymes, but their requirement of an enzyme co-factor has hindered their use in continuous sensors [1,4,10]. Because the co-factor serves as an electron acceptor or donor, a replenishing source of co-f
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