title: Evaluation of seven cosubstrates in the quantification of horseradish peroxidase enzyme by square wave voltammetry.
abstract: The electrochemical detection for horseradish peroxidase-cosubstrate-H(2)O(2) systems was optimized. o-Phenilendiamine, phenol, hydroquinone, pyrocatechol, p-chlorophenol, p-aminophenol and 3,3'-5,5'-tetramethylbenzidine were evaluated as cosubstrates of horseradish peroxidase (HRP) enzyme. Therefore, the reaction time, the addition sequence of the substrates, the cosubstrate:H(2)O(2) ratio and the electrochemical techniques were elected by one-factor optimization assays while the buffer pH, the enzymatic activity and cosubstrate and H(2)O(2) concentrations for each system were selected simultaneously by response surface methodology. Then, the calibration curves for seven horseradish peroxidase-cosubstrate-H(2)O(2) systems were built and the analytic parameters were analyzed. o-Phenilendiamine was selected as the best cosubstrate for the HRP enzyme. For this system the reaction time of 60s, the phosphate buffer pH 6.0, and the concentrations of 2.5×10(-4)molL(-1) o-phenilendiamine and of 1.25×10(-4)molL(-1) H(2)O(2) were chosen as the optimal conditions. In these conditions, the calibration curve of horseradish peroxidase by square wave voltammetry showed a linearity range from 9.5×10(-11) to 1.9×10(-8)molL(-1) and the limit of detection of 3.8×10(-11)molL(-1) with RSD% of 0.03% (n=3).
authors.affiliation: Laboratorio de Sensores y Biosensores, Cátedra de Química Analítica I, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina.
authors.forename: Silvina V
authors.initials: SV
authors.lastname: Kergaravat
authors.forename: Maria Isabel
authors.initials: MI
authors.lastname: Pividori
authors.forename: Silvia R
authors.initials: SR
authors.lastname: Hernandez
mesh_terms: D002138:Calibration; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized; D006735:Horseradish Peroxidase / Q000032:analysis*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D057230:Limit of Detection; D010636:Phenols / Q000737:chemistry*; D010655:Phenylenediamines / Q000737:chemistry*; D025341:Principal Component Analysis; D013379:Substrate Specificity
chemical_list: D004800:Enzymes, Immobilized; D010636:Phenols; D010655:Phenylenediamines; C034193:1,2-diaminobenzene; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase