An automatic flow-injection analysis system for determining phosphate ion in river water using pyruvate oxidase G (from Aerococcus viridans)

Abstract

An automated flow-injection system combining a pyruvate oxidase reaction and chemiluminescence for the detection of phosphate ion in river water has been developed. In this research, we used pyruvate oxidase G (PyrOxG), from Aerococcus viridans, immobilizing it on N-hydroxysuccinicacidimido beads without a cross-linker. In this sensor system, which was constructed as a trial system of desktop type, the temperature was precisely controlled. After the sensor system was optimized, a calibration curve was obtained with a detection limit of 96 nM phosphate ion, a range between 96 nM and 32 μM phosphate ion, and a relative standard deviation of 2.3% ( n=5) at 25°C. The sensitivity of this sensor was sufficient to determine the maximal permissible phosphate-ion concentration in the environmental waters of Japan (0.32 μM). In addition, the sensor could determine the calibration curves between 0.16 and 32 μM phosphate ion (five points, n=3; averaged correlation, r=1.00) for at least 2 weeks, demonstrating enough stability for practical use. Furthermore, we investigated the influence on the sensor response of dissolved substances in river water such as metal ions, heavy metal ions, inorganic ions, and organic compounds. Treatment with activated carbon could improve the response of the sensor when inhibited by dissolved substances in river water, except for manganese ion and uric acid. The sensor system could determine the concentrations of phosphate ion in various samples of river water from the Tone River. The results obtained by this sensor system and the modified molybdenum blue method were compared, and good correlation ( r=0.94) was obtained.