Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase

In this study, we investigated measurements of the intrinsic fluorescence of yeast hexokinase as an assay for glucose and immobilization of the enzyme in a silica sol-gel matrix as a potential in vivo glucose sensor for use in patients with diabetes. The intrinsic fluorescence of hexokinase in solution (excitation = 295 nm, emission = 330 nm) decreased by 23% at a saturating glucose concentration of 1 mM (Kd = 0.3 mM), but serum abolished the glucose-related fluorescence response. When entrapped in tetramethylorthosilicate-derived sol gel, hexokinase retained activity, with a 25% maximal glucose-related decrease in intrinsic fluorescence, and the saturation point was increased to 50 mM glucose (Kd = 12.5 mM). The glucose response range was increased further (to 120 mM, Kd = 57 mM) by a covering membrane of poly(2-hydroxyethyl) methacrylate. Unlike free enzyme, the fluorescence responses to glucose with sol-gel immobilized hexokinase, with or without covering membrane, were similar for buffer and serum. We conclude that fluorescence monitoring of sol-gel entrapped yeast hexokinase is a suitable system for development as an in vivo glucose biosensor.