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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| Names | |
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| Preferred IUPAC name
3,3′,5,5′-Tetramethyl[1,1′-biphenyl]-4,4′-diamine | |
| Other names
3,3′,5,5′-Tetramethylbiphenyl-4,4'-diamine
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| Identifiers | |
3D model (JSmol)
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| ChEMBL | |
| ChemSpider | |
| ECHA InfoCard | 100.053.949 |
| EC Number |
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PubChem CID
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| UNII | |
CompTox Dashboard (EPA)
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| Properties | |
| C16H20N2 | |
| Molar mass | 240.3482 g/mol |
| Melting point | 168 to 171 °C (334 to 340 °F; 441 to 444 K) |
| Hazards | |
| GHS labelling: | |
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| Warning | |
| H302, H315, H319, H335 | |
| P261, P264, P270, P271, P280, P301+P312, P302+P352, P304+P340, P305+P351+P338, P312, P321, P330, P332+P313, P337+P313, P362, P403+P233, P405, P501 | |
| Safety data sheet (SDS) | External MSDS |
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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Enzymatic assay
editTMB 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,[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
editTMB 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
edit- ^ Sigma Aldrich Catalog Entry for 3,3′,5,5′-Tetramethylbenzidine
- ^ 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.
- ^ "TMB Substrate Solution".
- ^ a b 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.
- ^ 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.
- ^ 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.
- ^ 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.
- ^ 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.