Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system.
Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system.
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DOI：
10.1016/j.talanta.2004.06.021
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发表时间：
2004-12
期刊：
影响因子：
6.1
通讯作者：
Y. Iida;M. Ikeda;M. Aoto;I. Satoh
中科院分区：
文献类型：
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作者：
Y. Iida;M. Ikeda;M. Aoto;I. Satoh
将酸性脲酶柱应用于荧光流动注射分析（FIA）系统，作为测定黄酒中尿素的识别元件。与来自刀豆的脲酶相比，酸性脲酶具有在低pH范围内表现出催化活性和对乙醇的耐受性的特殊性质。将酶共价固定在孔径受控的多孔玻璃珠上，然后装入小聚合物柱中。流动型生物传感系统由样品注射阀、固定化酶柱和连接到荧光分光光度计的流通石英池组装而成。柠檬酸盐缓冲液（50mM，pH 5.0）作为载体溶液连续泵入系统。样品溶液通过旋转进样阀引入系统。通过监测荧光异吲哚衍生物的荧光强度变化来测量标准尿素溶液，所述荧光异吲哚衍生物是通过与尿素和邻苯二甲醛试剂的酶水解中释放的氨分子偶联而形成的。在λex=415nm和λem=485nm的条件下测量荧光强度。尿素浓度 (1.0–100μM) 和荧光强度变化之间存在广泛的线性关系。监测并未受到黄酒中所含乙醇及各种氨基酸的影响。将经过离子交换树脂预处理以去除内源氨的真实样品引入 FIA 系统，并测定样品中的尿素。将这些结果与使用 F-kit 方法获得的结果进行比较。拟议的 FIA 系统应该能够对酒精饮料中的尿素进行灵敏、选择性和方便的分析。
An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines. The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of λex= 415nm and λem= 485nm. A wide, linear relationship was obtained between the concentration of urea (1.0–100μM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.