A mediated polyphenol oxidase biosensor immobilized by electropolymerization of 1,2-diamino benzene

A biosensor based on a partially purified polyphenol oxidase (PPO) enzyme was developed by using electropolymerization of [(2,2′-bipyridine)(chloro)(p-cymene)rutenium(II)]chloride] mediator complex and 1,2-diamino benzene (DAB) on a screen printing Pt electrode (1 mm diameter). The electropolymerization was carried out at   +0.7 V for 45 min in phosphate buffer (50 mM, pH 7.0) which contained 14.0 U/10 mL polyphenole oxidase, 200 mM DAB and 2.5 mM Ru-mediator complex solutions. Measurement is based on the detection of the oxidation current of the Ru-mediator complex that related to the enzymatic reaction catalyzed by PPO at +0.65 V. The phosphate buffer (50 mM, pH 7.0 containing 0.1 M KCl) and 30 °C were established as being the optimum working conditions. Under the optimum experimental conditions a linear calibration curve was obtained between 5 and 100 μM catechol concentration. The detection limit of the biosensor is 2.385 μM. In the characterization studies of the biosensor some parameters such as effect of Ru-mediator types on the biosensor response, substrate specificity, reproducibility and storage stability were studied. From the experiments, the average value (x), standard deviation (SD) and coefficient of variation (CV%) were found to be 48.75 μM, ± 1.56 μM, and 3.2% respectively for 50 μM catechol standard.