Do copper ions activate tyrosinase enzyme? A biosensor model for the solution

Abstract

Some metal ions play a cofactor role for the activity of tyrosinase enzyme and one of them is copper ion. In this study an amperometric biosensor was developed in order to investigate the effect of the copper ions on the activity of tyrosinase enzyme. In the construction of the biosensor tyrosinase enzyme was immobilized on a Clark-type dissolved oxygen probe which was covered with a oxygen sensitive teflon membrane, by using a chemical covalent immobilization method based on gelatine and bifunctional reagent, glutaraldehyde. The principle of the measurement was based on the determination of the differentiation of dissolved oxygen level in the enzymatic reaction catalyzed by tyrosinase in the absence and the presence of copper ions. Differences between the dissolved oxygen concentrations were related to copper ion concentration which was added in to the reaction medium. The biosensor response depends linearly on copper ion concentration between 2.5-20.0microM with a response time 1min. The detection limit of the biosensor is 0.95microM. In the optimization studies of the biosensor, the most suitable amounts of tyrosinase, gelatin and glutaraldehyde ratio were determined to be 69.0U/cm(-2), 4.21mg/cm(-2), and 2.5%, respectively. In the optimization studies of the biosensor, phosphate buffer (pH 7.0 ,50mM) and 30 degrees C were detected to be working conditions. For the characterization of the biosensor some parameters such as reproducibility, thermal and pH stability were carried out.