3,3',5,5'-Tetramethylbenzidine

3,3′,5,5′-Tetramethylbenzidine
Names
	Preferred IUPAC name 3,3′,5,5′-Tetramethyl[1,1′-biphenyl]-4,4′-diamine
	Other names 3,3′,5,5′-Tetramethylbiphenyl-4,4'-diamine
Identifiers
CAS Number	54827-17-7 ;
3D model (JSmol)	Interactive image;
ChEMBL	ChEMBL1331128;
ChemSpider	37605 ;
ECHA InfoCard	100.053.949
EC Number	259-364-6;
PubChem CID	41206;
UNII	3B3T5CB8EO ;
CompTox Dashboard (EPA)	DTXSID5026120 ;
	InChI InChI=1S/C16H20N2/c1-9-5-13(6-10(2)15(9)17)14-7-11(3)16(18)12(4)8-14/h5-8H,17-18H2,1-4H3 Key: UAIUNKRWKOVEES-UHFFFAOYSA-N ; InChI=1/C16H20N2/c1-9-5-13(6-10(2)15(9)17)14-7-11(3)16(18)12(4)8-14/h5-8H,17-18H2,1-4H3 Key: UAIUNKRWKOVEES-UHFFFAOYAM;
	SMILES Cc1cc(cc(c1N)C)c2cc(c(c(c2)C)N)C;
Properties
Chemical formula	C16H20N2
Molar mass	240.3482 g/mol
Melting point	168 to 171 °C (334 to 340 °F; 441 to 444 K)
Hazards
	GHS labelling:
Pictograms
Signal word	Warning
Hazard statements	H302, H315, H319, H335
Precautionary statements	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). verify (what is ?) Infobox references

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.

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

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.
"TMB Substrate Solution".
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.

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[1] Sigma Aldrich Catalog Entry for 3,3′,5,5′-Tetramethylbenzidine

[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.

[3] "TMB Substrate Solution".

[Holland1974-4] 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] 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] 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] 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] 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.