This is a magical use of 3,3',5,5'-tetramethylbenzidine!

3,3',5,5'-tetramethylbenzidine is a new type of safe chromogen reagent, generally referred to as TMB, which is a white or light yellow crystalline powder. It is odorless, tasteless, insoluble in water, and soluble in organic solvent such as acetone, diethyl ether, dimethyl sulfoxide or dimethylformamide. The basic reaction principle of blood-staining test is that hemoglobin or heme in blood has peroxidase activity, which can decompose hydrogen peroxide and release new ecological oxygen. The oxidation of the latter makes the blood trace reagent discolor or visible, which is characterized by convenient and safe use, high sensitivity and good stability.

1. How does tetramethylbenzidine (CAS 54827-17-7) show occult blood?

The TMB development solution is usually prepared by using anhydrous ethanol as a solvent. The usual formulation is:

 Liquid B: 3% hydrogen peroxide (H2O2), 20 mL.

Before the appearance of the osmotic object, such as occult blood on various textiles, natural woods, light-colored papers, etc., the occult blood fingerprints are first fixed by absolute ethanol or anhydrous methanol, and then visualized with tetramethylbenzidine. In an acidic environment, the peroxidase of heme or hemoglobin in the blood can catalyze the decomposition of hydrogen peroxide to release free oxygen and water, and the free oxygen can oxidize the colorless tetramethylbenzidine to blue-green, which reveals potential blood marks.

2. Problems in the application of TMB

(1) Distinguish the blood of human and animal

The reaction principle of tetramethylbenzidine is to use the catalase and hematoporphyrin contained in the blood, and the animal blood and human blood both contain the components and react with tetramethylbenzidine to form blue color. Therefore, pay attention to the surrounding environment when testing to distinguish human blood and animal blood.

(2) Excluding interference from other substances

As long as there is substance that can release nascent oxygen, there is interference with the appearance of blood. For example, TMB can react with fruit juice.

Compared with traditional chromogen reagents, TMB has the advantages of high detection sensitivity, low toxicity, safety and reliability. It has also been widely used in clinical testing and environmental testing in addition to criminal detection. Clinical testing, as a new substrate for peroxidase in enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA), for the detection of diseases such as hepatitis, rapid and accurate; environmental aspects for water Chlorine detection.