Detection Wavelength
λmax (chromogenic wavelength) = 666 nm, ε/H₂O₂ (molar absorption coefficient per H₂O₂ molecule) = 9×10⁴
Principle
Hydrogen peroxide is an important intermediate metabolite in the analysis of trace components in organisms, food quality monitoring, environmental substance detection and other applications.DA-64 and DA-67 are water-soluble oxidative chromogenic reagents that enable highly sensitive detection of hydrogen peroxide in the presence of peroxidase.
Traditional water-soluble hydrogen peroxide chromogenic reagents suffer from low molar absorption coefficients of the chromophores. Although some reagents offer high sensitivity, they are difficult to handle due to poor solubility.DA-64 and DA-67 achieve both water solubility and high sensitivity.
In the past, luminescent or fluorescent reagents had to be used to achieve high sensitivity.Now, by using DA-64 and DA-67, the target analytes detected by instruments can be measured with a simple colorimeter.
This product is less affected by coexisting substances and is especially suitable for the detection of biological samples.
Application Examples
Quantitative Determination of H₂O₂
1、Preparation of Chromogenic SolutionDissolve 4.08 mg DA-67 and 1 mL of 100 units/mL peroxidase (POD) in PIPES buffer (0.1 mol/L, pH 7, containing 0.5% Triton X-100), and adjust the volume to 100 mL (100 μmol/L DA-67 solution).
2、Mix 3 mL of the above solution with 10 μL of sample solution (H₂O₂: 0–5 mmol/L), and incubate at 37 °C for 5 minutes.
3、Measure the absorbance at 666 nm using a spectrophotometer.Use the absorbance difference relative to the blank (sample solution replaced with distilled water) as the detection value, and determine the H₂O₂ content in the sample using a separately prepared calibration curve.
Detection of POD Activity
1、Preparation of Chromogenic SolutionDissolve 4.08 mg DA-67 and 1 mL of 2% H₂O₂ in Mcllvaine buffer (0.05 mol/L citric acid, 0.1 mol/L disodium phosphate, pH 5.7), and adjust the volume to 100 mL (100 mmol/L DA-67 solution).
2、Mix 2 mL of the above solution with 2 mL of sample solution (1 fmol–1 pmol POD per tube), and incubate at 37 °C.
3、Measure the absorbance at 666 nm using a spectrophotometer.Use the absorbance difference after subtracting the blank (sample solution replaced with distilled water) as the detection value, and determine the POD content in the sample using a separately prepared calibration curve.
Usage
Colorimetric quantitative determination of hydrogen peroxide
Features
Combines high sensitivity and water solubility
