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

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 d...