A robotics-based automated assay for inorganic and organic phosphates
A robotics-based automated assay for inorganic and organic phosphates
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DOI：
10.1006/abio.1999.4100
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发表时间：
1999-06-15
影响因子：
2.9
通讯作者：
Griffith, OH
中科院分区：
文献类型：
--
作者：
Cogan, EB;Birrell, GB;Griffith, OH
磷酸盐分析是涉及无机磷酸盐和有机磷酸酯（例如磷脂、磷酸化蛋白质和核酸）的广泛生化应用的基础。本报告描述了一种利用机器人技术的实用自动化方法。测试了基于磷钼酸盐复合物形成且与自动测定兼容的五种磷酸盐分析比色方法，并讨论了基本化学。相对灵敏度为孔雀石绿 > 结晶紫 > 喹哪啶红 > 抗坏血酸还原 > 锑改性抗坏血酸还原，尽管从改进的抗坏血酸程序到孔雀石绿仅观察到四倍的改进。选择孔雀石绿来优化测定，因为这种染料提供了最高的灵敏度。然而，当颜色稳定性和低空白比灵敏度更重要时，抗坏血酸还原和喹哪啶红方法被发现是比孔雀石绿更好的选择。使用机器人液体处理系统的自动化大大减少了处理大量样品所需的劳动力。其结果是对无机磷酸盐进行高通量的灵敏、非放射性测定。在耐酸 96 孔板中开发了消化步骤，以将测定扩展到磷酸酯。使用无机磷酸盐和常见的磷脂（磷脂酰胆碱）演示了基于机器人的测定。 (C) 1999 年学术出版社。
Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine. (C) 1999 Academic Press.