Fluorescence-based enzymatic assay by capillary electrophoresis laser-induced fluorescence detection for the determination of a few β-galactosidase molecules

β-Galactosidase can be assayed by monitoring the generation of the fluorescent products, fluorescein-mono-β-D-galactopyranoside and fluorescein, when the fluorogenic substrate fluorescein-di-β-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 × 10^-14 M β-galactosidase or 1.6 molecules based on the detection of fluorescein-mono-β-D-galactopyranoside.