ABSTRACT
The immobilization of tyrosinase onto glutaraldehyde activated streptavidine magnetic particles and subsequent retention onto a magnetized carbon paste electrode for the amperometric assay of tyrosinase inhibitors is described. Tyrosine was used as substrate as it is the first substrate in the melanogenesis process. The sensing mode is based on monitoring the decrease of the amperometric signal corresponding to the electrochemical reduction of dopaquinone enzymatically generated. This current decrease is due to the presence of inhibitors acting directly on the enzyme or inhibitors acting on the product of the enzymatic reaction, i.e. dopaquinone. The methodology is designed for the evaluation of the inhibitory potency of the most frequently used active substances in cosmetic marketed products against hyperpigmentation such as kojic acid, azelaic acid and benzoic acid. These compounds bind to the tyrosinase active center. Ascorbic acid is also investigated as it interrupts the synthesis pathway of melanin by reducing the melanin intermediate dopaquinone back to l-dopa. By comparing the obtained IC(50), under the same experimental conditions, the order of their inhibitory potency was: kojic acid (IC(50)=3.7 × 10(-6)M, K(i)=8.6 × 10(-7)M), ascorbic acid (IC(50)=1.2 × 10(-5)M), benzoic acid (IC(50)=7.2 × 10(-5)M, K(i)=2.0 × 10(-5)M) and azelaic acid (IC(50)=1.3 × 10(-4)M, K(i)=4.2 × 10(-5)M) in close agreement with literature spectrophotometric inhibition data using the soluble tyrosinase.
Subject(s)
Biosensing Techniques/methods , Bleaching Agents/analysis , Enzyme Inhibitors/analysis , Magnetics , Monophenol Monooxygenase/chemistry , Nanotechnology/methods , Skin/drug effects , Agaricales/enzymology , Bleaching Agents/pharmacology , Electrochemistry , Enzyme Inhibitors/pharmacology , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Hydrogen-Ion Concentration , Kinetics , Melanins/biosynthesis , Monophenol Monooxygenase/metabolism , Reproducibility of Results , Skin/metabolism , Streptavidin/metabolismABSTRACT
The development of composite electrodes for biosensors construction based on HRP and zirconium alcoxide film for acetaminophen detection and finally, acetaminophen determination in pharmaceutical products is described. The enzyme immobilization is performed by retention in a polyetilenimine and zirconium alcoxide porous gel film, technique that offers a good entrapping and in the mean times a "protective" environment for the biocomponent. The operation principle of the biosensor is based on monitoring the amperometric signal generated by reduction at the electrode surface of the enzymatically generated quinoneimine from acetaminophen. The resulting biosensor shows a linear response towards acetaminophen with a linear range of 1.96 x 10(-5)M and 2.55 x 10(-4)M and a limit of detection of 1.17 x 10(-7)M. The proposed biosensor shows long term stability and good reproducibility.
