Nozaki Osamu, Munesue Motonori, Kawamoto Hiroko
Department of Clinical Laboratory Medicine, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan.
Luminescence. 2007 Sep-Oct;22(5):401-6. doi: 10.1002/bio.976.
The reactivity of flow-injection (FI)-horseradish peroxidase (HRP)-catalysed imidazole chemiluminescence (CL) was studied for continuous determination of hydrogen peroxide (H(2)O(2)) and serum glucose with immobilized glucose oxidase. Light emission by the HRP-catalysed imidazole CL was obtained when immobilized HRP, alkaline imidazole (in Tricine solution, pH 9.3) and H(2)O(2) were reacted at room temperature. The optimal pH for the CL reaction was 9.3 and the optimal concentration of imidazole was 100 micromol/L. When no imidazole was added, the light intensity of the same H(2)O(2) specimen decreased to a level that could not be quantitatively determined. The spectrum of the light emitted by imidazole CL was in the range 400-600 nm with a peak at 500 nm. The calibration equation for determination of H(2)O(2) was y = 9860x(2) + 3830x + 11,700, where y = light intensity (RLU) and x = concentration of H(2)O(2) (micromol/L). The detection limit of H(2)O(2) was 5 pmol, and the reproducibility of the H(2)O(2) assay was 2.3% of the coefficient of variation (H(2)O(2) 48 micromol/L, n = 13). The CL method was successfully applied to assay glucose after on-line generation of H(2)O(2) with the immobilized glucose oxidase column, resulting in good reproducibility (CV = 3.3% and 1.0% for the standard glucose and the control serum, respectively).
研究了流动注射（FI）-辣根过氧化物酶（HRP）催化的咪唑化学发光（CL）反应，用于通过固定化葡萄糖氧化酶连续测定过氧化氢（H₂O₂）和血清葡萄糖。当固定化HRP、碱性咪唑（在pH 9.3的三羟甲基甘氨酸溶液中）和H₂O₂在室温下反应时，可获得HRP催化的咪唑CL发光。CL反应的最佳pH为9.3，咪唑的最佳浓度为100 μmol/L。不添加咪唑时，相同H₂O₂样品的光强度降低到无法定量测定的水平。咪唑CL发射光的光谱范围为400 - 600 nm，峰值在500 nm。测定H₂O₂的校准方程为y = 9860x² + 3830x + 11700，其中y = 光强度（相对光单位），x = H₂O₂浓度（μmol/L）。H₂O₂的检测限为5 pmol，H₂O₂测定的重现性为变异系数的2.3%（H₂O₂ 48 μmol/L，n = 13）。该CL方法成功应用于通过固定化葡萄糖氧化酶柱在线生成H₂O₂后测定葡萄糖，标准葡萄糖和对照血清的重现性良好（CV分别为3.3%和1.0%）。
