An improved enzymatic assay for glucose determination in blood serum using a l,1′-dimethylferricinium dye
An improved enzymatic assay for glucose determination in blood serum using a l,1′-dimethylferricinium dye
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DOI：
10.1007/bf02787800
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发表时间：
1997-12
影响因子：
3
通讯作者：
K. Male;J. Luong
中科院分区：
文献类型：
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作者：
K. Male;J. Luong
l,l'-二甲基铁鎓 (DMFe+) 是一种稳定且 pH 不敏感的蓝色染料，通过酶氧化 1,1'-二甲基二茂铁 (DMFe):2-羟丙基-β-环糊精 (HPCD) 水溶性包合物，使用通过戊二醛活化固定在多孔氨丙基玻璃珠上的胆红素氧化酶来制备。在葡萄糖存在下，DMFe+通过与还原型葡萄糖氧化酶（FADH2）反应被还原为DMFe，并且在650 nm处吸光度降低。在醋酸盐 pH 5.2 缓冲液中，由于 DMFe+染料和氧气之间对还原葡萄糖氧化酶的竞争，对血清中葡萄糖的响应是非线性的，尤其是在低浓度范围内。在此 pH 值下，还观察到内源性铜蓝蛋白氧化染料制剂中残留的 DMFe (16%)，导致 650 nm 处的吸光度增加。然后使用马来酸盐缓冲液（pH 6.5）开发了测定方案，以克服这些干扰以及 α-D-葡萄糖的变旋。血清样品中葡萄糖的结果与参考己糖激酶/6-磷酸葡萄糖脱氢酶方法的结果非常吻合。
l,l′-dimethylferricinium (DMFe+),a stable and pH-insensitive blue dye, was prepared via enzymatic oxidation of a 1,1′-dimethylferrocene (DMFe):2-hydroxypropyl-β-cyclodextrin (HPCD) watersoluble inclusion complex, using bilirubin oxidase immobilized onto porous aminopropyl glass beads via glutaraldehyde activation. In the presence of glucose, DMFe+was reduced to DMFe by reacting with the reduced glucose oxidase (FADH2), and the absorbance decrease was followed at 650 nm. In acetate pH 5.2 buffer, the response to glucose in blood serum was nonlinear, especially in the low concentration range, because of a competition for the reduced glucose oxidase between the DMFe+dye and oxygen. At this pH, endogenous ceruloplasmin was also observed to oxidize residual DMFe (16%) in the dye preparation, causing an increase in absorbance at 650 nm. An assay protocol was then developed using maleate buffer, pH 6.5, to overcome these interferences as well as mutarotation of α-D-glucose. The results obtained for glucose in the blood serum samples agreed well with those of the reference hexokinase/glucose-6-phosphate dehydrogenase method.