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.

Application and New Developments in Fluorescence Spectroscopic Techniques in Studying Individual Molecules

“…Numerical modeling of the FCS observation volume combined with measurements has revealed that the standard assumption of a three-dimensional Gaussian FCS observation volume is not a valid approximation under many common measurement conditions (127). Combination of capillary electrophoresis and patterned FCS can perform electrophoretic analysis independently of the initial length of injected analyte plugs (128). FCS can measure diffusion on the cell surface with unparalleled sensitivity and can be the most sensitive and accurate method for measuring receptor interaction and oligomerization (129).…”

Recent developments in detection for microfluidic systems

“…Sonehara et al [14] used a laser to produce a stationary interference pattern across a capillary. The pattern was used as a multiplexed detection scheme to record fluorescence signals from passing samples.…”

On‐column detection of multiphoton‐excited fluorescence in CE using hyphenated cylindrical‐square capillaries

“…Due to its ultra-low detectability [4], LIF represents one of the most popular detection schemes in CE. Moreover, in combination with some other optical techniques such as polarization [5,6], total internal reflection [7], correlation spectroscopy [8] and multiphoton excitation [9][10][11][12][13], and applications of lowcost light-emitting diode [14][15][16] as well, LIF becomes more attractive, and shows various capabilities to meet the requirements of different samples for analysis.…”