Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis.

Abstract

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.