3,3',5,5'-Tetramethylbenzidine

3,3',5,5'-Tetramethylbenzidine

3,3',5,5'-Tetramethylbenzidine

Chemical compound

3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (ELISA).[1] TMB is a white solid that forms a pale blue-green liquid in solution with ethyl acetate. TMB is degraded by sunlight and by fluorescent lights. Used to detect hematuria as it turns blue in contact with hemoglobin.

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Enzymatic assay

TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase.

Shows the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to 3,3',5,5'-tetramethylbenzidine diimine

The resulting one-electron oxidation product is a diimine-diamine complex, which causes the solution to take on a blue colour,[2] 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.[3]

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,[4][5][6][7] and did not induce formation of tumors in a single-arm study of 24 rats.[4] On that evidence, it has been used as a replacement for carcinogenic compounds such as benzidine[8] and o-phenylenediamine.

References

  1. [1]
    Sigma Aldrich Catalog Entry for 3,3′,5,5′-Tetramethylbenzidine
  2. [2]
    Zhang X; Yang Q; Lang Y; Jiang X; Wu P (2020). "Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis". Anal. Chem. 92 (18): 12400–12406. doi:10.1021/acs.analchem.0c02149. PMID 32794705. S2CID 221132691.
  3. [3]
    "TMB Substrate Solution".
  4. [4]
    Holland VR; Saunders BC; Rose FL; Walpole AL (1974). "A safer substitute for benzidine in the detection of blood". Tetrahedron. 30 (18): 3299. doi:10.1016/S0040-4020(01)97504-0.
  5. [5]
    Ashby J; Paton D; Lefevre PA; Styles JA; Rose FL (1982). "Evaluation of two suggested methods of deactivating organic carcinogens by molecular modification". Carcinogenesis. 3 (11): 1277–1282. doi:10.1093/carcin/3.11.1277. PMID 6758975.
  6. [6]
    Chung K-T; Chen S-C; Wong TY; Li YS; Wei CI; Chou MW (2000). "Mutagenicity studies of benzidine and its analogs: Structure-activity relationships". Toxicol Sci. 56 (2): 351–356. doi:10.1093/toxsci/56.2.351. PMID 10910993.
  7. [7]
    Chung K-T; Chen S-C; Claxton LD (2006). "Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes". Mutation Research/Reviews in Mutation Research. 612 (1): 58–76. doi:10.1016/j.mrrev.2005.08.001. PMID 16198141.
  8. [8]
    Yang J; Wang H; Zhang H (2008). "One-pot synthesis of silver nanoplates and charge-transfer complex nanofibers". J Phys Chem C. 112 (34): 13065–13069. doi:10.1021/jp802604d.
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