Quantitative determination of trace hydrogen peroxide in the presence of sulfide using the Amplex Red/horseradish peroxidase assay.
The Amplex Red/horseradish peroxidase (AR/HRP) assay for H2O2is one of the most sensitive and simple approaches for H2O2quantification, which is effected by measuring the highly-fluorescent resorufin formed from oxidation of AR by the oxidizing intermediates generated by reaction of HRP and H2O2. The direct reactions of S(-II) with both H2O2and resorufin are too slow to be of relevance on analytical timescales, however, the reaction between S(-II) and the HRP/H2O2oxidizing intermediates is rapid enough to compete with the desired reaction of these oxidizing intermediates with AR, suppressing formation of the resorufin analyte. As this mode of interference can be considered simply a competition between the AR reagent and S(-II) for the intermediate oxidizing species, a simple equation is derived in this work enabling one to correct for this interference and obtain a good estimate of the true H2O2concentration after measuring the apparent H2O2concentration and the S(-II) concentration. This mode of interference is general to any compound that can act as a HRP substrate even if not directly reactive with H2O2. As such, the approach described is widely applicable to many potential reducing interferents and opens up the use of the AR/HRP assay to a much wider range of conditions, as well as demonstrating the utility of explicitly considering the mechanism of the analytical process.
School of Mechanical Engineering, Beijing Institute of Petrochemical Technology, Beijing 102617, PR China; School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR China.
Nannan
N
Wang
School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW 2052, Australia.
Christopher J
CJ
Miller
School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, PR China; State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, PR China. Electronic address: pwang73@hit.edu.cn.
Peng
P
Wang
School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: d.waite@unsw.edu.au.
T David
TD
Waite
D015374:Biosensing Techniques / Q000379:methods*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000032:analysis*; D057230:Limit of Detection; D010078:Oxazines / Q000737:chemistry*; D010084:Oxidation-Reduction; D013440:Sulfides / Q000737:chemistry*
D010078:Oxazines; D013440:Sulfides; C470430:Amplex Red; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase
Amplex Red; Horseradish peroxidase; Hydrogen peroxide; Sulfide