title: Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase.
abstract: 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.
authors.affiliation: Department of Chemical Pathology, Guy's, King's, and St. Thomas's School of Medicine, Guy's Hospital, London SE1 9RT, UK.
authors.forename: Faeiza
authors.initials: F
authors.lastname: Hussain
authors.forename: David J S
authors.initials: DJ
authors.lastname: Birch
authors.forename: John C
authors.initials: JC
authors.lastname: Pickup
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004800:Enzymes, Immobilized; D017022:Flow Injection Analysis; D005453:Fluorescence; D005782:Gels; D005947:Glucose / Q000032:analysis* / Q000737:chemistry; D006593:Hexokinase / Q000097:blood / Q000737:chemistry*; D006801:Humans; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012822:Silicon Dioxide / Q000737:chemistry*
chemical_list: D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D005782:Gels; D012822:Silicon Dioxide; D006593:Hexokinase; D005947:Glucose