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Highly sensitive chemiluminescence FIA of the minor components in relation to diabetes involving a bioreactor

涉及生物反应器的与糖尿病相关的微量成分的高灵敏化学发光 FIA

基本信息

批准号：
08672642
负责人：
TABATA Masayoshi
金额：
$ 1.34万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Increasing the sensitivity for chemiluminescence, and a chemiluminescence-FIA of serum 3-hydroxybutyrate (3-HBA) and 1,5-anhydroglucitol (1,5-AG) using a bioreactor were tried. The detection sensitivity for H_2O_2 was increased up to about 0.1 mu mol/mu 1 of sample by the introduction of a non-pulse flow pumps, an improvement of a flow cell, and intercepting the light which goes into the detecktor along the tubes from the outside For the determination of serum 3-HBA,the coimmobilized 3-hydroxybutyrate dehydrogenase (3-HBADH)/NADH oxidase (NOX) column with a weight ratio 5 : 1 was the most efficient for determining serum 3-HBA.The column hydrolyzed all the 1.5 mM 3-HBA,and showed linearity of the data up to 1.5 mM 3-HBA.Forever, the chemiluminescence intensity obtained by the column was about twice as much as those by the sequential and the mixed-bed 3-HBADH/NOX columns, because 3-HBADH and NOX in the coimmobilized 3-HBADH/NOX column were in such close proximity that both progresses proceeded quickly.Pyranose oxidase (PROD) oxidized not only glucose but also 1,5-AG to produce H_2O_2. The oxidation rate for 1,5-AG was far slower than that for glucose, but because the column contained a large quantity of PROD,the oxidation rate for 1,5-AG increased to 70% that for glucose. When the coimmobilized glucokinase (GK)/pyruvate kinase (PK) column was used for the removal of serum glucose, GK phosphorylated not only glucose but also 1,5-AG.But, the phosphorylation rate for 1,5-AG was far lower compared with that for glucose. The most efficient column for 1,5-AG was the coimmobilized GK/PK column with a weight ratio GK 1000U : PK 0.28ml. The column removed 99.99% glucose in a sample of 1 mu 1 injected. The results from serum 1,5-AG correlated satisfactorily with those by other method.

提高化学发光的敏感性，并采用生物反应器对血清3-羟基丁酸酯（3-HBA）和1,5-无氢葡萄糖醇（1,5- ag）进行化学发光- fia检测。通过引入无脉冲流泵、改进流动池、截流从管外进入检测器的光，将H_2O_2的检测灵敏度提高到0.1 μ mol/ μ 1左右。在血清3-HBA测定中，重量比为5:1的3-羟基丁酸脱氢酶(3-HBADH)/NADH氧化酶（NOX）共固定化柱测定血清3-HBA的效率最高。该柱可水解所有1.5 mM - 3-HBA，且数据在1.5 mM - 3-HBA以内呈线性。由于共固定的3-HBADH/NOX柱中3-HBADH与NOX的距离非常近，两者的反应都进行得很快，因此该柱获得的化学发光强度大约是顺序柱和混合床3-HBADH/NOX柱的两倍。吡喃糖氧化酶（PROD）不仅能氧化葡萄糖，还能氧化1,5- ag生成H_2O_2。1,5- ag的氧化速率远低于葡萄糖，但由于柱中含有大量的PROD， 1,5- ag的氧化速率提高到葡萄糖的70%。用共固定化葡萄糖激酶(GK)/丙酮酸激酶（PK）柱去除血清葡萄糖时，GK不仅磷酸化葡萄糖，还磷酸化1,5- ag。但是，1,5- ag的磷酸化速率远低于葡萄糖的磷酸化速率。以重量比为GK 1000U: PK 0.28ml的GK/PK共固定柱为测定1,5- ag的最有效柱。该柱在1 μ 1注射的样品中去除99.99%的葡萄糖。血清1,5- ag测定结果与其他方法吻合较好。