Tetramethylbenzidine
[CAS# 54827-17-7]

Identification

• Classification: Chemical reagent >> Organic reagent >> Aromatic hydrocarbon reagent
• Name: Tetramethylbenzidine
• Synonyms: 3,3',5,5'-Tetramethylbenzidine; TMB Quick
• Molecular Formula: C₁₆H₂₀N₂
• Molecular Weight: 240.35
• CAS Registry Number: 54827-17-7
• EC Number: 259-364-6
• SMILES: CC1=CC(=CC(=C1N)C)C2=CC(=C(C(=C2)C)N)C

Properties

• Melting point: 168-171 °C
• Water solubility: Slightly soluble. <0.1 g/100 mL at 20 °C

Safety Data

• Hazard Symbols: GHS07;GHS08 Warning
• Risk Statements: H302-H315-H319-H335-H341
• Safety Statements: P203-P261-P264-P264+P265-P270-P271-P280-P301+P317-P302+P352-P304+P340-P305+P351+P338-P318-P319-P321-P330-P332+P317-P337+P317-P362+P364-P403+P233-P405-P501

Hazard Classification

Hazard	Class	Category Code	Hazard Statement
Eye irritation	Eye Irrit.	2	H319
Skin irritation	Skin Irrit.	2	H315
Specific target organ toxicity - single exposure	STOT SE	3	H335
Acute toxicity	Acute Tox.	4	H302
Germ cell mutagenicity	Muta.	2	H341
Carcinogenicity	Carc.	2	H351
Specific target organ toxicity - single exposure	STOT SE	3	H336
Chronic hazardous to the aquatic environment	Aquatic Chronic	3	H412
Chronic hazardous to the aquatic environment	Aquatic Chronic	1	H410
Acute hazardous to the aquatic environment	Aquatic Acute	1	H400
Chronic hazardous to the aquatic environment	Aquatic Chronic	4	H413

Discovery and Applications

Tetramethylbenzidine (TMB) is a chromogenic substrate introduced in the 1970s as a safer alternative to benzidine, which was found to be carcinogenic. Researchers sought a compound that could effectively act as a peroxidase substrate, producing a measurable color change without the health risks associated with earlier compounds. The discovery of TMB marked a major advance in biochemical assays, especially immunoassays.

The chemical formula of tetramethylbenzidine is C16H20N2. It is commonly used in the form of 3,3',5,5'-tetramethylbenzidine. TMB is a white to pale yellow crystalline powder that is freely soluble in organic solvents and slightly soluble in water. It is light sensitive and should be stored in a cool place. Its structure features a benzidine backbone with four methyl groups that enhance its stability and reactivity as a chromogenic substrate.

The chromogenic reaction of TMB involves oxidation by HRP in the presence of hydrogen peroxide. The reaction produces a diimine compound that appears blue. When the reaction is stopped with an acid, the diimine compound converts to a yellow product. The intensity of the color is proportional to the amount of HRP and therefore the target analyte in the sample. This property makes TMB an important tool for quantitative assays.

MB is widely used in ELISA (enzyme-linked immunosorbent assay) kits as a substrate for horseradish peroxidase (HRP). TMB produces a blue color upon oxidation by HRP in the presence of hydrogen peroxide. The reaction is stopped by adding an acid, causing the color to change to yellow. This color change can be measured spectrophotometrically, allowing the analyte in the sample to be quantified.

TMB is used in a variety of diagnostic tests to detect antibodies or antigens in biological samples. Its ability to produce a distinct color change upon enzyme interaction is useful for visual confirmation of test results. For example, TMB is used in home pregnancy tests and rapid infectious disease tests.

In research settings, TMB is used to detect the presence of specific proteins or nucleic acids. Its high sensitivity and rapid color development are essential for accurate and timely performance of western blots, immunohistochemistry, and other assays.
TMB is used in environmental testing to detect the presence of pollutants, such as pesticides and heavy metals. Its ability to produce a color change in response to specific enzymes makes it a useful tool for monitoring environmental contamination.

TMB is safer than benzidine, but should still be handled with care. It can irritate the skin, eyes, and respiratory tract. Appropriate personal protective equipment, such as gloves and goggles, should be used when handling TMB. Additionally, TMB solutions should be prepared in a well-ventilated area to avoid inhaling dust or vapors.

References

2024. Highly Selective Ag-Ca@CuO Nanocomposite-Based Sensor for Trace-Level Cr (VI) Detection in Water. Water, Air, & Soil Pollution, 235(12).
DOI: 10.1007/s11270-024-07662-9

2010. Protection against multiple influenza A virus subtypes by intranasal administration of recombinant nucleoprotein. Archives of Virology, 155(10).
DOI: 10.1007/s00705-010-0756-3

2005. Inhibition of peroxidase-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine by aminophenols. Biochemistry. Biokhimiia, 70(2).
DOI: 10.1007/s10541-005-0118-z