Application principle and example of chromogenic substrate DA64 and DA67.

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1. The application principle of DA64-DA67.

Hydrogen peroxide is an intermediate for the analysis of trace components in organisms and the detection of environmental substances.

DA-64 and DA-67 can detect hydrogen peroxide with high sensitivity after the coordination of peroxidase. It is a water-soluble chromogenic reagent.

Compared with traditional chromogenic reagents, DA-64 and DA-67 have advantages in water solubility and high sensitivity, so it is not necessary to choose luminescent and fluorescent reagents to improve the sensitivity.

The result can be obtained by using a colorimeter, which is not only simple but also fast.

The advantage of DA-67 is that it is more resistant to the influence of coexisting substances than DA-64, and it is especially suitable for the detection of biological composition samples.

Instructions for the use of DA64-DA67.

One:Preparation of chromogenic solution.

1 dissolve the peroxidase (POD) of 4.08 mg DA67 and 1 mL100 units/mL in a configured PIPES buffer and fix the solution to 100 mL (DA67 100 μ mol/L solution).

2 3 mL of the prepared solution was mixed with 10 μ L sample solution (H2O2:0~5 mmol/L) and cultured at 37 °C for 5 minutes.

3 the absorbance at 666 nm was detected by spectrophotometer, the absorbance difference of blank (the mixed solution of sample solution was replaced by distilled water) was used as the detection value, and the calibration curve prepared separately was used to determine the content of the sample.

Two:Preparation of chromogenic solution for detection of POD activity.

1 add 4.08 mg DA67 and 1 mL H2O2 (2%) to the configured Mcllvaine buffer to dissolve (PH value 5.7), fixed volume to 100 mL.

2 the prepared solution 2 mL was incubated with 2 mL sample solution (1 fmol~1 pmol/tube POD) at 37 °C.

3 the absorbance at 666 nm was detected by spectrophotometer, the blank absorbance difference was subtracted as the detection value, and the content value in the sample could be determined by another calibration curve.