COMPARISON OF METHODS FOR FOLLOWING ALKALINE-PHOSPHATASE CATALYSIS - SPECTROPHOTOMETRIC VERSUS AMPEROMETRIC DETECTION

被引:89
作者
THOMPSON, RQ
BARONE, GC
HALSALL, HB
HEINEMAN, WR
机构
[1] UNIV CINCINNATI,BIOMED CHEM RES CTR,CINCINNATI,OH 45221
[2] UNIV CINCINNATI,DEPT CHEM,CINCINNATI,OH 45221
基金
美国国家科学基金会;
关键词
D O I
10.1016/0003-2697(91)90190-5
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2·5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 m, pH 9.0. Tris buffer is 56 μm, while it is 82 μm for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nm for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 μg/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay. © 1991.
引用
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页码:90 / 95
页数:6
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