A biosensor based on the release of the enzyme substrate from its structure was developed for the inhibitive detection of benzoic acid. A polyurethane support comprising two perforated microcapsules (800 μm in diameter) filled with methylene blue as a model compound and covered with a conductive deposit of multiwalled carbon nanotubes, continuously released this stored dye for 24 h. An increase in methylene blue concentration of 0.5-0.75 μmol L-1 h-1 and 1.5-2 μmol L-1 h-1, in the presence and absence of the multiwalled carbon nanotube coating, respectively, was demonstrated by UV-vis spectroscopy in a 2 mL UV cuvette. The same configuration with microcapsules filled with catechol was modified by a laponite clay coating containing tyrosinase enzyme. The resulting biosensor exhibits a constant cathodic current at -0.155 V vs AgCl/Ag, due to the reduction of the ortho-quinone produced enzymatically from the released catechol. The detection of benzoic acid was recorded from the decrease in cathodic current due to its inhibiting action on the tyrosinase activity. Reagentless biosensors based on different deposited quantity of tyrosinase (100, 200, 400 and 600 μg) were investigated for the detection of catechol and applied to the detection of benzoic acid as inhibitor. The best performance was obtained with the 400 μg-based configuration, namely a detection limit of 0.4 μmol L-1 and a sensitivity of 228 mA L mol-1. After the inhibition process, the biosensors recover 97-100% of their activity towards catechol, confirming a reversible inhibition by benzoic acid.
Keywords: Benzoic acid; Catechol; Enzyme electrode; Microcapsule; Tyrosinase.
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