A Simple and Novel Colorimetric Optimized Assay for Determination of Hydrogen Peroxide Concentration Under Oxidative Stress Conditions

Background: Reactive oxygen species are formed through the electron transfer reactions in the mitochondria and chloroplasts and rapidly converted to H2O2.Therefore, H2O2 as a more stable ROS can be considered as an indicator of cellular stress and it can be used in a steady state to monitor intracellular stress level. In this regard, the increasing use of various nanoparticles, most of which are associated with biological systems, are essential to be studied for their possible adverse effects. We measured the concentration of hydrogen peroxide in samples collected before and after the treatment with silver nanoparticles by a novel method and optimized this method for the living tissue. Methods: In this study, we evaluated the endogenous H2O2 production from Pyricularia oryzae tissue under normal and stress conditions (such as after treatment with nanoparticles) by spectrophotometric assay. The method used is based on instant reaction of hydrogen peroxide with vanadium pentoxide in sulfuric acid solution, forming a peroxovanadate complex that has a maximum absorption at 454 nm. This method was also compared with other methods. Results: The results of this method compared with other methods in the same tissue showed that the method is simple, inexpensive and more efficient, and the complex is stable for several hours and can be used for a variety of H2O2 concentrations. Also, the detection range of the mentioned method equals with high-sensitivity methods such as available commercial kits. Furthermore, this method can also measure higher values of H2O2. Conclusion: The optimized methods for measuring the H2O2 concentration with vanadium pentoxide in sulfuric acid solution by the colorimetric method are simple, efficient, rapid, accurate, cost-effective and do not have problems of other methods. The measurements using this method in Pyricularia oryzae under oxidative stress showed that the created oxidative stress caused by the use of silver nanoparticles increased H2O2 in fungal tissue. H2O2 is the SOD reaction product that is later decomposed by CAT. This method is able to measure H2O2 in different ranges and under normal and stress conditions which are indicative of antioxidant defense. Therefore, we recommend it to the researchers in similar conditions.