Experimental Procedure for a Hydrogen Peroxide Assay Based on the Peroxidase‐Oxidase Reaction

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Abstract
A low‐cost assay method that is able to measure H2O2 concentrations as low as the nano‐molar range is described. The assay solution contains NADH, horseradish peroxidase, and superoxide dismutase at pH 7.5. After the addition of the sample, the decrease in NADH concentration measured by spectrophotometry is proportional to the H2O2 concentration. Because of superoxide dismutation, a high amplification factor defined as moles NADH oxidised per mole H2O2 added is obtained, which allows the sensitivity limit of the method to be greatly improved. We have established the conditions under which the amplification factor can be stabilised at a high level: the best compromise is to increase both the horseradish peroxidase and superoxide dismutase concentrations. Finally, we have also shown that coupled to specific oxidases, our assay method is suitable for measuring very low concentrations of biochemicals that can be oxidized by oxygen with H2O2 production.

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