In enzymatic colorimetric detection, there are many substrates for peroxidase, such as commonly used new Trinder's reagents, ABTS, DAB, OPD, and TMB commonly used in ELISA. In addition, a substrate with higher sensitivity and higher color wavelength, DA64, has gradually been developed and used. Physical and chemical properties of DA64 The chromogen substrate DA64 has more advantages in the detection system of peroxidase, with molecular formula C19H23N4NaO3, molecular weight 378.39, full name N - (carboxymethyl amino carbonyl) -4,4 '- bis (dimethylamino) diphenylamine sodium, appearance white to green brown or gray green, crystalline powder to powder. Every 100g of water can dissolve 40mg of DA64, dissolved in water at a concentration of 1 mM or more. The advantages of DA64 From a molecular structure perspective, DA64 is similar to Trinder's reagent or TMB, both containing aniline structures. After POD catalytic oxidation, it can generate color products with specific absorption wavelengths. The maximum absorption wavelength of DA64 λ Max (color rendering wavelength)=727 nm, ε/ H2O2 (extinction coefficient of one H2O2 molecule)=9 × 104. Most chromogenic substrates have wavelengths in the range of 400 to 550, while DA64 is as high as 727 nanometers, much higher than MAOS known for its long chromogenic wavelength (630nm). It has stronger anti-interference ability for complex samples and higher sensitivity for trace detection.