Two-beam fluorescence cross-correlation spectroscopy for simultaneous analysis of positive and negative ions in continuous-flow capillary electrophoresis

Abstract

Two-beam fluorescence cross-correlation spectroscopy was used for multicomponent electrophoretic analysis of positive and negative ions flowing continuously, and in opposite directions, through a polymer-coated electrophoresis capillary. Cross-correlation analysis of the fluorescence monitored from two spatially offset microscopic detection volumes revealed the magnitude and direction of the electrophoretic flow velocity of each analyte. This enabled resolution of a three-component mixture containing nanomolar concentrations of cationic rhodamine 6G and anionic 5-carboxytetramethylrhodamine (TAMRA) and TAMRA-labeled single-stranded DNA in aqueous buffer. The relative concentrations of each analyte, determined from the cross-correlation analysis, agreed with known values within experimental error. This technique is sensitive to molecular processes occurring on a millisecond time scale and will prove useful for monitoring rapid fluctuations in the magnitude and direction of the electrophoretic flow velocity caused by spontaneous chemical reactions or conformational transformations at thermodynamic equilibrium.