Hussain Faeiza, Birch David J S, Pickup John C
Department of Chemical Pathology, Guy's, King's, and St. Thomas's School of Medicine, Guy's Hospital, London SE1 9RT, UK.
Anal Biochem. 2005 Apr 1;339(1):137-43. doi: 10.1016/j.ab.2005.01.016.
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.
在本研究中，我们研究了酵母己糖激酶的固有荧光测量，以此作为葡萄糖检测方法，并将该酶固定在硅溶胶 - 凝胶基质中，作为用于糖尿病患者的潜在体内葡萄糖传感器。溶液中己糖激酶的固有荧光（激发波长 = 295 nm，发射波长 = 330 nm）在1 mM饱和葡萄糖浓度（解离常数Kd = 0.3 mM）下降低了23%，但血清消除了与葡萄糖相关的荧光响应。当包埋在原硅酸四甲酯衍生的溶胶 - 凝胶中时，己糖激酶保留了活性，固有荧光最大降低25%与葡萄糖相关，且饱和点增加到50 mM葡萄糖（Kd = 12.5 mM）。通过聚甲基丙烯酸2 - 羟乙酯覆盖膜，葡萄糖响应范围进一步增加（至120 mM，Kd = 57 mM）。与游离酶不同，对于溶胶 - 凝胶固定的己糖激酶，无论有无覆盖膜，缓冲液和血清中对葡萄糖的荧光响应相似。我们得出结论，溶胶 - 凝胶包埋的酵母己糖激酶的荧光监测是一种适合开发为体内葡萄糖生物传感器的系统。
