Channel Electrophoresis for Kinetic Assays

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Abstract
A rectangular channel electrophoresis system and a cylindrical sampling capillary combination allows chemical changes in nanoliter-volume samples to be monitored as a function of time. The electrophoretic microseparation is carried out in a rectangular channel with a 7 -cm-long, 40-microm x 2.5-cm geometry and is coupled to a 50-microm-i.d. cylindrical sample introduction capillary. The channel width dimension is used as a time axis by moving the outlet of the sampling capillary across the entrance of the separation channel. Detection of the separated analyte bands is achieved with laser-induced fluorescence and spatially resolved detection based on a charge-coupled device. The system is characterized with a series of fluorescein thiocarbamyl amino acid derivatives; limits of detection are < 10(-8) M for amino acids and 10(-9)M (425 zmol) for fluorescein. The ability to achieve a time-based dynamic microseparation is demonstrated by monitoring fluorescent product formation during the enzyme-catalyzed hydrolysis of fluorescein di-beta-D-galactopyranoside (FDG), a commonly used fluorescent substrate for enzymological studies.