Application of a tyrosinase microreactor - detector in a flow injection configuration for the determination of affinity and dynamics of inhibitor binding

- Original tyrosinase microreactor with amperometric detection optimized with an experimental design.
- Determination of inhibitor strength potency of several tyrosinase inhibitors.
- Investigation of the mechanism of inhibition and the recovery rate of the enzyme activity after inhibition.

An original self-contained enzymatic microreactor − detector in a thin layer flow-through configuration has been developed for the screening of tyrosinase inhibitors. The microreactor- detector consisted of a gold disk for tyrosinase immobilization adjacent to a glassy carbon disk as working electrode (GCE). Using l-tyrosine as substrate, the enzymatic product formed (dopaquinone) was detected amperometrically at the GCE. Experimental parameters most affecting the l-tyrosine response were optimized with a central composite design. Measurements were performed in phosphate buffer pH 6.0 with an applied potential of +50 mV vs Ag/AgCl and a flow rate of 100 μL/min. The determined Kmapp was 1.9 ± 0.1 mM. The setup permitted the determination of the efficiency and the duration of inhibition of twelve compounds known as tyrosinase inhibitors namely kojic acid, gallic acid, azelaic acid, benzoic acid, l-ascorbic acid, hydroquinone, glabridine, resveratrol, quercetin dihydrate, captopril, l-cysteine and l-glutathione reduced. l-Ascorbic acid and kojic acid were found to be the most potent inhibitors for the two studied criteria with IC50 of 32 μM and 156 μM, respectively.

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