Biosensing of Aromatic Amines in Reversed Micelles with Self‐Generation of Hydrogen Peroxide at Glucose Oxidase‐Peroxidase Bienzyme Electrodes

Abstract

Abstract The amperometric biosensing of aromatic amines using a composite glucose oxidase (GOD)‐peroxidase (HRP) biosensor in reversed micelles is reported. Rigid composite pellets of graphite and Teflon, in which GOD and HRP were coimmobilized by simple physical inclusion, were employed for the biosensor design. This design allows the in situ generation of the H 2 O 2 needed for the enzyme reaction with the aromatic amines, thus preventing the negative effect that the presence of a high H 2 O 2 concentration in solution has on HRP activity. The H 2 O 2 in situ generation is performed by oxidation of glucose catalyzed by GOD. The effect of the composition of the reversed micelles, i.e., the nature of the organic solvent used as the continuous phase, the nature and concentration of the surfactant used as emulsifying agent, the aqueous 0.05 mol L −1 phosphate buffer percentage used as the dispersed phase, and the glucose concentration in the aqueous phase, on the biosensor response was evaluated. Reversed micelles formed with ethyl acetate, a 5% of phosphate buffer (pH 7.0) containing 3.0×10 −3 mol L −1 glucose, and 0.1 mol L −1 AOT (sodium dioctylsulfosuccinate), were selected as working medium. Well‐defined and reproducible amperometric signals at 0.00 V were obtained for p ‐phenylenediamine, 2‐aminophenol, o ‐phenylenediamine, m ‐phenylenediamine, 1‐naphthylamine, o ‐toluidine and aniline. The useful lifetime of one single biosensor was of 60 days. The trend in sensitivity observed for the aromatic amines is discussed considering the effect of their structure on the stabilization of the radicals formed in the enzyme reaction which are electrochemically reduced. The behavior of the composite bienzyme electrode was also evaluated in a FI (flow injection) system using reversed micelles as the carrier. The suitability of the composite bienzyme electrode for the analysis of real samples was demonstrated by determining aniline in spiked carrots.