New diode laser-excitable green fluorescent label and its application to detection of bovine serum albumin via microchip electrophoresis

A novel amino-reactive fluorescent label is presented that is based on a yellow daylight chromophore and fluorophore. Its absorption band is wide and peaks at 431 nm in water solution, thus well matching the lines of either the 375-nm and the 431-nm diode lasers and of many frequency-variable dye lasers. When conjugated to bovine serum albumin (BSA), the fluorescence peaks at 501 nm with a quantum yield of 0.21. Its large Stokes' shift of 70 nm facilitates the discrimination of undesired excitation light which is particularly important for sensitive detection in miniaturized separation techniques such as microchip capillary electrophoresis (MCE). Unlike several other fluorophores, the fluorescence intensity of the new label is independent of pH over a broad range (3 to 9). The applicability of the label is demonstrated by labeling the amino acid lysine and the 66 kD protein BSA, and by separating BSA from the free label via MCE within 90 s. The limit of detection is in the order of 12 nM at an optically active path length of 20 A mu m.