SEMICONDUCTOR LASER-INDUCED FLUORESCENCE DETECTION IN CAPILLARY ELECTROPHORESIS USING A CYANINE DYE

Cy5, an activated carboxyl cyanine fluorophore, was characterized by c apillary electrophoresis (CE) using a semiconductor laser at 652 nm to induce fluorescence. Hydrolysis of the activated Cy5 in the presence of ammonia results in the formation of a mono- and diamide and a dicar boxylic acid. A Cy5-labeled oligonucleotide M(13) primer for DNA seque ncing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using U V-visible detection, and by CE, monitored by laser-induced fluorescenc e (LIE) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the pu rity of the major Cy5-oligonucleotide primer was greater than 90%. The detection sensitivity for Cy5-based CE-LIF detection system with a 2. 5-mW red semiconductor laser is about 10(-10) M.