Development of an Automated Dual Type of Micro Stopped-Flow/Flow-Injection Apparatus Applicable for Detection of Minute Alteration of Inorganic Phosphate or Phosphoric Group in Protein Level

通过两套停止流动/流动注射装置（单台SF-FIA装置）的组合，构建了一台自动双SF-FIA装置，用于连续测量与正常/癌细胞硬组织钙化或生长控制系统磷酸化/去磷酸化周期相关的蛋白质水平中无机磷酸盐（Pi）或磷酸基的微小变化。在双SF-FIA装置的基础上，安装智能自动进样器，对样品进行一些复杂的前处理。通常情况下，分别通过磷酸酶或激酶磷酸化/去磷酸化在蛋白水平上的定量变化，可以通过用孔雀石绿/钼酸盐试剂直接释放Pi的方法或在连续酶分析系统中间接测量NADH化学计量学反映ATP/ADP变化的方法来分光光度法检测。此外，双SF-FIA装置可用于微孔膜过滤的^<32> p标记方法所需的快速（毫秒级）停止工具。每个样品和分析溶液每一个周期测量29.4 ul，使用双SF-FIA仪器进行50次连续测量，每个样品和分析溶液的总容积为3.0 ml。双SF-FIA装置串行测量的时间间隔可以设置在超过4秒的任意范围内。

By combination of two sets of stopped-flow/flow-injection apparatus (single SF-FIA apparatus), an automated dual SF-FIA apparatus was constructed for serial measurements of minute alteration of inorganic phosphate (Pi) or phosphoric groups in protein level related to calcification of hard tissues or phosphorylation/dephosphorylation cycle of growth control system in normal/cancer cells. The dual SF-FIA apparatus was further installed by an intelligent auto sampler in order to carry out some complex pre-treatments of sample. Normally, the quatitative change of phosphorylation/dephosphorylation in protein level by phosphatase or kinase, respectively, can be detected spectrophotometrically through a direct method to free Pi using Malachite Green/molybdate reagent or an indirect method measuring NADH stoichiometrically reflecting ATP/ADP change in consecutive enzyme assay system. Furthermore, the dual SF-FIA apparatus could be applied as a rapid (msec order) stopping tool required for ^<32>P-labeling method by using filtration with milipore membrane. With 29.4 ul for each of sample and assay solution per one cycle measuring, half-hundred of serial measurements by using the dual SF-FIA apparatus are possible for the total volume of 3.0 ml for each of sample and assay solution. The time interval among the serial measurements with the dual SF-FIA apparatus can be set up within an arbitrary range longer than 4 sec.