FLUORESCENCE CORRELATION SPECTROSCOPY FOR RAPID MULTICOMPONENT ANALYSIS IN A CAPILLARY-ELECTROPHORESIS SYSTEM

We describe a new technique for performing multicomponent analysis usi ng a combination of capillary electrophoresis (CE) and fluorescence co rrelation spectroscopy (FCS), which we refer to as CE/FCS, FCS is a hi ghly sensitive and rapid optical technique that is often used to perfo rm multicomponent analysis in static solutions based on the different diffusion times of the analyte species through the detection region of a tightly focused laser beam. In CE/FCS, transit times are measured f or a mixture of analytes continuously flowing through a microcapillary in the presence of an electric field. Application of an electric fiel d between the inlet and outlet of the capillary alters the transit tim es, depending on the magnitude and polarity of the applied held and th e electrophoretic mobilities of the analytes, Multicomponent analysis is accomplished without the need to perform a chemical separation, due to the different electrophoretic mobilities of the analytes, This tec hnique is particularly applicable to ultradilute solutions of analyte. We have used CE/FCS to analyze subnanomolar aqueous solutions contain ing mixtures of Rhodamine 6G (R6G) and R6G-labeled deoxycytosine triph osphate nucleotides, Under these conditions, fewer than two molecules were typically present in the detection region at a time. The relative concentrations of the analytes were determined with uncertainties of similar to 10%, Like diffusional FCS, this technique is highly sensiti ve and rapid. Concentration detection limits are below 10(-11) M, and analysis times are tens of seconds or less. However, CE/FCS does not r equire the diffusion coefficients of the analytes to be significantly different and can, therefore, be applied to multicomponent analysis of systems that would be difficult or impossible to study by diffusional FCS.