Fluorescence reaction of bilirubin with zinc ion in dimethyl sulfoxide and its application to assay of total bilirubin in serum

A new fluorimetric method is described for the determination of total bilirubin in serum. The fluorescence with maximum excitation and emission at 470 and 545 nm, respectively, is based on the reaction of bilirubin with zinc acetate in dimethyl sulfoxide (DMSO). Serum (10 μl) is added to 2.5 ml of DMSO solution containing zinc acetate (0.01 M) and Tris (0.2 M). After 60 min, at 37°C, the fluorescence is measured. The blank is obtained by mixing serum (10 μl) and Tris-DMSO solution (2.5 ml) without zinc acetate. The method gave a linear calibration for 0.1–5 mg dl−1 bilirubin, a 3.3σ detection limit of 5 μg dl−1 and relative standard deviations for bilirubin in control sera of 1–2% (n=10). It is simple, specific and sensitive, and showed good correlation with the usual diazo-coupling method.