Enzymatic methods for determining biogenic compounds are a key technology in quantification of various analytes of clinical interest in the biomatrix and have found wide application in clinical diagnosis due to their high selectivity and rapidity. The enzymes employed for this purpose are primarily oxidases and dehydrogenases. Upon reacting with target compounds, these enzymes produce hydrogen peroxide or NAD(P)H, amounts of which are then determined by using oxidative or reductive chromogenic reagents, respectively. In chromogenic reagents, the following properties are desirable: 1) a long absorption wavelength, 2) strong resistance to interferences caused by various compounds (such as bilirubin) in the biomatrix, 3) stability of the stock solutions for long-term storage, 4) stability of the resulting dye, and 5) high water solubility. There have been no conventional reagents so far that meet all of these criteria. In the effort to develop such a compound, we recently synthesized the following new reagents for colorimetric enzymatic determination. A series of oxidative chromogenic reagents, Bis-MAPS, do not require any coupling reagents in the peroxidase-mediated oxidation reaction producing dyes that absorb at ∼650 nm with a large molar absorptivity. New coupling reagents, NCP, give a green color upon reaction with the aniline derivatives known as Trinder's reagents. Hydrogen donors, DADB, were found not to suffer from interference by bilirubin. As a reductive chromogen for use in clinical diagnosis like LDH analysis, we have developed a novel tetrazolium salt, WST-1, that produces a highly water soluble formazan dye. WST-1 proved to be also valuable as a chromogenic indicator in cell proliferation assays where MTT is used most widely.
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