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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