Improvement of a CL-FIA System Using Maltose Phosphorylase for the Determination of Phosphate-Ion in Freshwater†

In a previous study, a phosphate ion (Pi) biosensor consisting of a system of chemiluminescence flow-injection analysis (CL-FIA) combined with a reaction system of maltose phosphorylase, mutarotase, and glucose-oxidase (MP-MUT-GOD) was developed without a detailed investigation of the measurements of real samples. Therefore, in this study, after characterization of the measuring conditions in the CL-FIA system for freshwater samples, we investigated the influences of dissolved substances, such as metal ions, heavy metal ions, inorganic ions, and organic substances, in freshwater on the sensor response. The results of the study indicated that there were no substances giving irreversible effects to the enzyme activities of MP, MUT, or GOD used in this sensor; however, some cations or organic substances influenced the sensor response. We then compared the effects of the elimination of the dissolved substances by a chelate reagent, ion exchange resin, or combination of ion exchange resin and UV irradiation on the sensor response. Consequently, a cation-exchange resin (sulfonated), which improved most effectively the sensor response, was chosen as a pre-treatment method. By utilizing this pre-treatment method, the sensor system could determine the Pi concentrations in 31 samples of river or pond waters in Japan. The results obtained by this sensor system and the molybdenum-blue method were compared, and a regression curve (y = 2.78x) with good correlation (r = 0.958) was obtained between them. The reason of the slope at the regression curve was discussed. Finally, we introduce a trial type of an automatic CL-FIA biosensor system for future Pi determination.