STUDY OF REAGENTLESS PHOSPHATE SENSING SYSTEM

本研究的目的是建立一种利用磷酸结合蛋白（PBP）的无试剂磷酸盐传感器。首先从土样中提取PBP，并被净化将PBP固定在硝酸纤维素膜上，测定了PBP的活性，结果与文献报道的结果基本一致。使用Ag/AgCl电极观察到该膜向磷酸盐的电位变化。磷酸根离子浓度超过1 mM时，传感器的电位变化依赖于磷酸根离子浓度的变化。通过对膜载体材料、固定化方法的考察以及FT-IR对蛋白质固定化的评价，采用聚乙烯醇缩丁醛树脂包埋法将PBP固定化，并在亚mM级范围内观察到PBP固定化ISFET对磷酸钾的响应。然而，传感器对钾离子也有响应。据推测，应通过减去对钾的响应来校正响应，以获得对磷酸盐的净响应。最后采用包埋法将PBP固定在PVA-SbQ中，并将固定化的PBP膜固定在铂电极上。对Ag/AgCl反电极测量膜电位。在该体系中，磷酸根的电位变化比其它阴离子的电位变化大。在10- 100 mM范围内，该体系对磷酸根的电位变化与磷酸根浓度呈线性关系，表明无试剂磷酸根传感体系是可行的。

The purrpose of this study is to establish the pliciple of reagentless phosphate sensor utilizing phosphate binding protein (PBP). First of all PBP was extracted from Eschericia Coli., and purified. Activity of obtained PBP was assayd and it was almost same as the previously reported data.PBP was immobilized on a sheat of nitrocellulose membrane. Potential change of this membrane was observed to phosphate, with use of Ag/AgCl Electrode. The potential change depended on the concentration of phosphate more than 1mM.An atttempt of miniaturization of phosphate sensor was done by immobilizing PBP on an ion sensitive field effect transistor (ISFET). As the result of examination of membrane support materials, immobilization method, and the estimation of immobilization of the protein by FT-IR,PBP was immobilized in a layr of polyvinylbutyral resin entrapment method.Response of the PBP immobilized ISFET to potasium phosphate was observed at the range of sub mM order. However, the sensor responded to potasium ion either. it was supposed that the response should be corrected by subtraction of the response to potasium, to obtain net response to phosphate. Moreover thestability of immobilized PBP was not satisfactory to ascertain its response to various ions.At last PBP was immobilized in PVA-SbQ by entrappment method, and the immoibilized PBP membrane was fixed on a platinum electrode. The membrane potential was measured against Ag/AgCl counter electrode. The higher potential changer to phsophate was observed than to other anions with this system. The potential change of this system to phosphate showed linear relationship to the concatration of phosphate at the range of 10-100mM.From these results, the reagentless phosphate sensing is shown to be possible system.