3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in
immunohistochemistry
Immunohistochemistry (IHC) is the most common application of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to an ...
as well as being a visualising reagent used in enzyme-linked immunosorbent assays (
ELISA
The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presen ...
). TMB is a white solid that forms a pale blue-green liquid in solution with
ethyl acetate
Ethyl acetate ( systematically ethyl ethanoate, commonly abbreviated EtOAc, ETAC or EA) is the organic compound with the formula , simplified to . This colorless liquid has a characteristic sweet smell (similar to pear drops) and is used in glues ...
. TMB is degraded by sunlight and by
fluorescent lights
A fluorescent lamp, or fluorescent tube, is a low-pressure mercury-vapor gas-discharge lamp that uses fluorescence to produce visible light. An electric current in the gas excites mercury vapor, which produces short-wave ultraviolet lig ...
.
Enzymatic assay
TMB can act as a hydrogen donor for the reduction of
hydrogen peroxide
Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscous than water. It is used as an oxidizer, bleaching agent, and antiseptic, usually as a dilute solution (3%†...
to water by
peroxidase
Peroxidases or peroxide reductases ( EC numberbr>1.11.1.x are a large group of enzymes which play a role in various biological processes. They are named after the fact that they commonly break up peroxides.
Functionality
Peroxidases typically ca ...
enzymes such as
horseradish peroxidase.
The resulting one-electron oxidation product is a
diimine
Diimines are organic compounds containing two imine (RCH=NR') groups. Common derivatives are 1,2-diketones and 1,3-diimines. These compounds are used as ligands and as precursors to heterocycles. Diimines are prepared by condensation reactions ...
-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
Sulfuric acid (American spelling and the preferred IUPAC name) or sulphuric acid ( Commonwealth spelling), known in antiquity as oil of vitriol, is a mineral acid composed of the elements sulfur, oxygen and hydrogen, with the molecular formu ...
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.
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.
References
{{DEFAULTSORT:Tetramethylbenzidine, 3,3,5,5-
Biphenyls
Anilines