Peroxidase inhibition assay for the detection of some thiols by carbon screen printed electrodes based on square wave voltammetry
Cassava peroxidase (CSP) from leaves of cultivar KU50 was used as an alternative peroxidase for the electrochemical detection of thiram and thiourea.Square-wave voltammetry (SWV) incorporated with screen-printed carbon electrode was applied for the investigation of peroxidase activity. It was based on the measurement of 3, 3¢-diaminobenzidine (DAB) electro active product catalyzed by CSP and horseradish peroxidase (HRP) in 0.04 M Britton-Robinson buffer, pH 6, containing urea hydrogen peroxide in 96-well microplate. The reaction mixture in the presence of either thiourea or thiram was performed in order to study their effects on DAB oxidation catalyzed by both peroxidases. From the result of SWV signals obtained from PalmSens potentiometric instrument, it was found that the reduction peak of DAB-product catalyzed by peroxidase occurred at -0.24±0.02 V versus Ag/AgCl electrode. In the presence of thiols, decrements of SWV current peak heights were clearly observed as inhibitory effects by both peroxidases. Thiram was a stronger inhibitor than thiourea. CSP inhibition assays elucidated well-correlated relationship between thiram log concentration and its relative activity as a linear function which could potentially be applied for thiram detection in the range of 10-100 mM under the SWV optimal condition. For HRP inhibition assays, a non-linear relationship with a short detectable range of 5-50 mM of thiram was also observed. The portable set of electrochemical device used in this study showed sufficient sensitivity for the detection of some thiol peroxidase inhibitors by electrochemical measurement with small sample needed. Its compact design could be advantageous for chemical analysis in the field use.
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