Photochemical enzyme co-factor regeneration: Towards continuous glutamate monitoring with a sol-gel optical biosensor

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 α-ketoglutarate 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 self-sustaining 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 (λ_abs ∼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.