Immobilized capillary tyrosinase microreactor for inhibitor screening in natural extracts by capillary electrophoresis.

ABSTRACT

A method for creating an immobilized capillary tyrosinase (TRS) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. Tyrosinase was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte hexadimethrine bromide (HDB). Then, HDB solution with the same plug length as the TRS was injected again into the capillary to cover the immobilized enzyme by forming HDB-TRS-HDB sandwich-like structure. Then, the substrate of l-tyrosine was introduced into the capillary and on-line enzyme inhibition study was performed by capillary electrophoresis (CE). The enzyme activity was determined by the quantification of peak area of the product of l-DOPA. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The immobilized enzyme could withstand 25 consecutive assays by only losing 12% activity. A known TRS inhibitor, kojic acid was employed as a model compound for the validation of the inhibitor screening method. Finally, screening 19 natural extracts of traditional Chinese drugs was demonstrated. The results indicated that inhibition activity could be straightforwardly identified with the system.