Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluorescence detection for the determination of a few beta-galactosidase molecules.
beta-Galactosidase can be assayed by monitoring the generation of the fluorescent products, fluorescein-mono-beta-D-galactopyranoside and fluorescein, when the fluorogenic substrate fluorescein-di-beta-D-galactopyranoside is used. We have used capillary electrophoresis with ultrasensitive laser-induced fluorescence detection to monitor the formation of the fluorescent products of off-column enzymatic reactions. By analyzing as little as 40 pl of the enzymatic mixture we obtain limits of detection of 6.5 x 10(-14) M beta-galactosidase or 1.6 molecules based on the detection of fluorescein-mono-beta-D-galactopyranoside.
Craig
D
D
Department of Chemistry, University of Alberta, Edmonton, Canada.
Arriaga
E A
EA
Banks
P
P
Zhang
Y
Y
Renborg
A
A
Palcic
M M
MM
Dovichi
N J
NJ
D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D007834:Lasers; D012815:Signal Processing, Computer-Assisted; D001616:beta-Galactosidase / Q000032:analysis*
D005452:Fluoresceins; D001616:beta-Galactosidase