A Bienzyme Electrode (Alkaline Phosphatase−Polyphenol Oxidase) for the Amperometric Determination of Phosphate

Abstract

The electropolymerization of an alkaline phosphatase (AP)−amphiphilic pyrrole ammonium mixture previously adsorbed on the electrode surface provides efficient entrapment of AP in a polypyrrole film. The resulting biosensor was applied to the detection of phenyl phosphate via amperometric oxidation at 0.6 V vs SCE of the enzymically generated phenol. The sensitivity and detection limit of the biosensor were 862 μA M-1 cm-2 and 20 μM, respectively. The co-immobilization of polyphenol oxidase and AP leads to a bienzyme electrode for the determination of phenyl phosphate on the basis of amperometric detection of the enzymically generated o-quinone at −0.2 V. An amplification factor of 9.7 was calculated as the ratio of the bienzyme to monoenzyme electrode sensitivities. In the presence of 5 mM MgCl2, the sensitivity and detection limit of the biosensor for phenyl phosphate were 36.82 mA M-1 cm-2 and 0.2 μM, respectively. Phosphate competitively inhibited the hydrolysis of phenyl phosphate. Consequently, its determination is based on the decrease of the biosensor response to phenyl phosphate. The sensitivity and detection limit of the biosensor for phosphate were 1.27 mA M-1 cm-2 and 2 μM, respectively.