Single-molecule electrophoresis: applications to biomolecular detection

Abstract

A new method for the detection and identification of biological molecules at the single molecule level of sensitivity has been developed. The technique involves measuring the electrophoretic velocity of each molecule present in a sample. Since different chemical species generally exhibit different electrophoretic velocities, identification is accomplished by classification according to electrophoretic velocity. The solution is contained in a capillary to which an external voltage is applied, and the velocity is determined by measuring the time taken by an individual molecule to travel the distance between two tightly focused laser beams due to the electrophoretic effect. The detection of the migration of individual molecules through each laser beam was accomplished by a modified version of our recently developed technique of single fluorescent molecule detection. Monte Carlo computer simulations of the process were performed beforehand in order to estimate the experimental feasibility of the method, and to determine the optimum values for the various experimental parameters. The method has been applied to the analysis of single-fluorophore molecules such as rhodamine 6G, and to biological macromolecules, such as mixtures of nucleic acids and of proteins. Applications of the technique to the detection of specific DNA sequences are discussed.