3,3',5,5'-Tetramethylbenzidine
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Through Acid and Sulfuric acid
Enzymatic assay
TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase.
The resulting one-electron oxidation product is a diimine-diamine complex, which causes the solution to take on a blue colour, and this colour change can be read on a spectrophotometer at the wavelengths of 370 and 650 nm.
The reaction can be halted by addition of acid or another stop reagent. Using sulfuric acid turns TMB yellow, with a peak absorbance of 450 nm. The amount of converted TMB may be indexed by the amount of 450 nm light it absorbs.
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Material safety
TMB should be kept out of direct sunlight as it is photosensitive. It is not known if TMB is carcinogenic and the evidence is contradictory: TMB is not mutagenic by the Ames test, and did not induce formation of tumors in a single-arm study of 24 rats. On that evidence, it has been used as a replacement for carcinogenic compounds such as benzidine and o-phenylenediamine.