Baykov A A, Evtushenko O A, Avaeva S M
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow, State University, USSR.
Anal Biochem. 1988 Jun;171(2):266-70. doi: 10.1016/0003-2697(88)90484-8.
An improved procedure for phosphate determination based on a highly colored complex of phosphomolybdate and malachite green is described. All necessary reagents are combined in one concentrated solution, making the assay sensitive and convenient. The procedure is based on the finding that the dye is easily soluble and stable in the presence of 6 N acid. The addition of Tween 20 is required to stabilize the dye-phosphomolybdate complex at phosphate concentrations above 10 microM. The time of color development at 25 degrees C is about 3 min. The procedure was adopted to measure alkaline phosphate activity in heterogeneous enzyme immunoassay with rho-nitrophenyl phosphate and pyrophosphate as substrates. In both cases, a 4-fold increase in sensitivity in terms of absorbance readings was obtained compared to the standard method based on rho-nitrophenol measurement. In visual analysis, the gain in sensitivity was as high as 20-fold, due to contrast color change (yellow to greenish blue).
本文描述了一种基于磷钼酸盐与孔雀石绿形成的高显色络合物的改进的磷酸盐测定方法。所有必要试剂都混合在一种浓缩溶液中，使该测定方法灵敏且方便。该方法基于这样一个发现：在6N酸存在下，该染料易溶解且稳定。当磷酸盐浓度高于10 microM时，需要添加吐温20来稳定染料 - 磷钼酸盐络合物。在25℃下显色时间约为3分钟。该方法被用于以对硝基苯磷酸酯和焦磷酸为底物的异相酶免疫测定中碱性磷酸酶活性的测定。在这两种情况下，与基于对硝基苯酚测量的标准方法相比，吸光度读数的灵敏度提高了4倍。在目视分析中，由于对比色变化（黄色变为绿蓝色），灵敏度提高高达20倍。
