Hydrogen Peroxide (H2O2) is a significant reactive oxygen species (ROS) playing essential roles in redox signaling and oxidative stress. It's generated during various cellular processes, including respiration, protein folding, peroxisome activity, and oxidase catalysis. High doses of hydrogen peroxide can be toxic to eukaryotic cells, leading to the oxidation of DNA, lipids, and proteins, potentially causing mutagenesis and cell death. Peroxide-induced cellular damage is associated with the development of numerous pathological conditions, such as aging, asthma, arthritis, diabetes, cardiovascular disease, atherosclerosis, Down's Syndrome, and neurodegenerative diseases.

The RayBio® Hydrogen Peroxide (H2O2) Colorimetric Assay Kit offers a straightforward, reproducible, and sensitive method for measuring hydrogen peroxide concentrations in plasma, serum, cell lysates, urine, and other biological fluid samples, without the need for prior treatment. This colorimetric probe reacts with H2O2 and the horseradish peroxidase enzyme (HRP), producing a pink-colored product with less background interference and enhanced stability compared to the commonly used Xylenol Orange (FOX) colorimetric assay for H2O2. The intensity of the pink color, measured at 520nm, corresponds to the amount of hydrogen peroxide present in the sample.

• Microplate reader capable of measuring absorbance at 520 nm
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Multi-channel pipettes to deliver 20 μl to 200 μl volumes
• Tubes to prepare sample dilutions
• 50 ml conical tubes

Each hydrogen peroxide standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 50 μL diluted Hydrogen Peroxide standards or unknown samples to the 96-well microtiter plate.
2. Add 150 μL Chromogenic Working Solution (See “Reagent Preparation”) to each well. Incubate the plate at room temperature for 15 minutes gently mixing.
3. Measure the absorbance at 520nm using a plate reader.