3,3′-Diaminobenzidine (DAB) is an organic compound with the formula (C<sub>6</sub>H<sub>3</sub>(NH<sub>2</sub>)<sub>2</sub>)<sub>2</sub>. This derivative of benzidine is a precursor to polybenzimidazole, which forms fibers that are renowned for their chemical and thermal stability. As its water-soluble tetrahydrochloride, DAB has been used in immunohistochemical staining of nucleic acids and proteins.

Structure

DAB is symmetric about the central carbon bond between both ring structures. In the crystal, the rings of each molecule are co-planar and the amine units connect molecules to form an intermolecular 3-dimensional hydrogen bond network.

Preparation

Diaminobenzidine, which is commercially available, is prepared by treating 3,3′-dichlorobenzidine with ammonia with a copper catalyst at high temperature and pressure, followed by acidic workup.

An alternate synthesis route involves the diacylation of benzidine with acetic anhydride under basic conditions: The solubility of DAB in water allows for adaptability compared to other detection solutions which use toxic solvents. Improperly prepared tissue samples may give false positives.

In research, this reaction is used to stain cells that were prepared with hydrogen peroxidase enzyme, following common immunocytochemistry protocols. Relevant to Alzheimer's disease, Aβ protein amyloid plaques are targeted by a primary antibody, and subsequently by a secondary antibody, which is conjugated with a peroxidase enzyme. This will bind DAB as a substrate and oxidize it, producing an easily observable brown color. Plaques can then be quantified for further evaluation. One other method uses complexes of injected biocytin with avidin or streptavidin, biotin, and then peroxidase.

References

  • Fishersci.com
  • Polysciences.com