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 (1mm diameter). The electropolymerization was carried out at +0.7V for 45min in phosphate buffer (50mM, pH 7.0) which contained 14.0U/10mL polyphenole oxidase, 200mM DAB and 2.5mM 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 +0.65V. The phosphate buffer (50mM, pH 7.0 containing 0.1M KCl) and 30 degrees C were established as being the optimum working conditions. Under the optimum experimental conditions a linear calibration curve was obtained between 5 and 100microM catechol concentration. The detection limit of the biosensor is 2.385microM. 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 48.75microM,+/-1.56microM, and 3.2% respectively for 50microM catechol standard.