Semiconductor laser-induced fluorescence detection in capillary electrophoresis using a cyanine dye.
Cy5, an activated carboxyl cyanine fluorophore, was characterized by capillary 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 dicarboxylic acid. A Cy5-labeled oligonucleotide M13 primer for DNA sequencing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using UV-visible detection, and by CE, monitored by laser-induced fluorescence (LIF) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the purity 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.
Chen
F T
FT
Beckman Instruments Inc., Fullerton, CA 92634.
Tusak
A
A
Pentoney
S
S
Konrad
K
K
Lew
C
C
Koh
E
E
Sternberg
J
J
D001483:Base Sequence; D002232:Carbocyanines / Q000737:chemistry*; D004396:Coloring Agents; D017931:DNA Primers; D004586:Electrophoresis / Q000379:methods*; D007834:Lasers; D008969:Molecular Sequence Data; D012666:Semiconductors; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet
D002232:Carbocyanines; D004396:Coloring Agents; D017931:DNA Primers; C085321:cyanine dye 5