V. Mekler, S. Bystryak
Jul 15, 1992
Citations
0
Influential Citations
44
Citations
Journal
Analytica Chimica Acta
Abstract
Abstract o -Phenylenediamine ( o -PD) is one of the most effective substrate for spectrophotometric horseradish peroxidase (HRP)-mediated enzyme-linked immunosorbent assay (ELISA), giving 2,3-diaminophenazine (DAP) as a product of o -PD oxidation. In this study it was found that the fluorescence of DAP in aqueous buffer solutions is considerably increased on adding a detergent (Triton X-100). DAP embedded in the micelles of Triton X-100 has absorption and fluorescence wavelength maxima at 428 and 554 nm, respectively, with a fluorescence quantum yield of 0.09. These properties make it possible to use high-purity o -PD as a fluorogenic substrate for HRP-mediated ELISA. The limit of detection of HRP by the spectrofluororimetric method in buffer solutions is 0.56 μU ml −1 . The applicability of the proposed technique is demonstrated by the determination of carcinoembryonic antigen.