Abstract
An amperometric biosensor based on tyrosinase, immobilized onto a carbon black paste electrode using glutaraldehyde and BSA was constructed to detect competitive inhibitors. Three inhibitors were used in this study: benzoic acid, sodium azide, and kojic acid, and the obtained values for fifty percent of inhibition (IC50) were 119 µM, 1480 µM, and 30 µM, respectively. The type of inhibition can also be determined from the curve of the degree of inhibition by considering the shift of the inhibition curves. Amperometric experiments were performed with a biosensor polarized at the potential −0.15 V vs. Ag/AgCl and using 0.1 M phosphate buffer (pH 6.8) as an electrolyte. Under optimized conditions, the proposed biosensor showed a linear amperometric response toward catechol detection from 0.5 µM to 38 µM with a detection limit of 0.35 µM (S/N = 3), and its sensitivity was 66.5 mA M−1 cm−2. Moreover, the biosensor exhibited a good storage stability. Conversely, a novel graphical plot for the determination of reversible competitive inhibition was represented for free tyrosinase. The graph consisted of plotting the half-time reaction (t1/2) as a function of the inhibitor concentration at various substrate concentrations. This innovative method relevance was demonstrated in the case of kojic acid using a colorimetric bioassay relying on tyrosinase inhibition. The results showed that the t1/2 provides an extended linear range of tyrosinase inhibitors.
Highlights
Over the last decade, electrochemicalsensing systems have attracted much attention as a simple and effective alternative to traditional approaches foranalytical studies [1,2,3,4]
The results showed that the t1/2 provides an extended linear range of tyrosinase inhibitors
50% of a inhibitor determination allowsallows for a plot the time required to achieve of a substrate substrate conversion versus inhibitor concentrations
Summary
Electrochemical (bio)sensing systems have attracted much attention as a simple and effective alternative to traditional approaches for (bio)analytical studies [1,2,3,4]. The enzymatic biosensors were widely used because of their ability to detect a target substance/analyte with a high specificity through enzyme-catalyzed reactions [5,6,7]. Tyrosinase-based amperometric biosensors have been extensively reported in the literature for the detection of various compounds ( mono- and di-phenols) due to their rapid response, low cost and low energy consumption [12,13,14]. The immobilization and stability of tyrosinase is one of the most important aspects in the potential success of enzyme-based biosensors [16,17]. The carbon black paste electrode has been positioned as an attractive candidate for enzyme immobilization due to the high surface area of carbon black (CB) and long-term stability of enzyme electrodes in various applications of an electrochemical biosensor [18]
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