Single-Molecule Electrophoresis

The single-molecule electrophoresis technique consists of measuring the electrophoretic velocity of individual molecules-the velocity at which molecules move in solution under the influence of an electric field-and identifies them by comparing their measured velocity with the velocity characteristic of a particular molecular species. The electrophoretic velocity of a molecule is determined by its size, shape, and ionic charge and by the chemical environment of the solution in which it is contained. The electrophoretic velocity therefore provides a unique identification signature of each molecular species.

The apparatus for single-molecule electrophoresis consists of a laser source split into two beams, a sample compartment, light-collection optics, two single photon detectors, and detection electronics under computer control. The sample compartment contains two reservoirs, one of which contains a cathode and the other, an anode. The reservoirs hold the solution that is being analyzed and are connected by tubing to a the capillary cell. The two laser beams, which are focused at the capillary cell, produce two 5-micron spots separated by a distance of 250 microns.

When a voltage is applied to the electrodes, the molecules in the solution migrate toward the cathode or anode, depending on their charge. As the individual molecules in the solution pass through the two laser-illuminated spots, they emit bursts of fluorescence. The photons from each burst are then collected by a microscope objective and detected by a single-photon avalanche photodiode. The detection electronics reject Raman and Rayleigh scattering by the use of a time-gated window set to detect only delayed fluorescence photons. The instrument measures the time it takes for each molecule to travel the distance between the two laser beams and then uses this information to calculate the electrophoretic velocity of the molecule. The computer then produces a histogram of electrophoretic velocities which show a peak for every chemical species present in the sanple.

Although the single-molecule electrophoresis technique relies on measuring molecular fluorescence, non-fluorescent molecules may be detected by attaching a fluorescent tagging molecule to them. In addition, some of the experimental conditions such as buffer composition, pH, viscosity, inner-surface capillary coating, excitation and emission wavelengths, among others, can be optimized to achieve the best separation of the particular sample components being analyzed. In fact, many of the analytical protocols specially developed for capillary electrophoresis separations are directly applicable to the present technique. For many years, researchers have optimized various capillary electrophoresis methods for the separation of a large variety of chemical species ranging from small organic and inorganic ions, to various kinds of pharmaceutical drugs and natural products.

The new method described here promises to combine the advantages of free-solution capillary electrophoresis (system automation, speed, reproducibility) with the unsurpassed sensitivity of single-molecule detection. The sensitivity and versatility of the method may open the way to develop fluorescence immunoassay, hybridization, and DNA fingerprinting techniques without the need for extensive DNA amplification using the polymerase chain reaction (PCR) or other methods. Although PCR is a highly effective amplification mechanism, the use of many PCR cycles may introduce ambiguities arising from contamination and by mechanisms not yet fully understood. Besides the demonstrated ability for the analysis of single-fluorophores, mixtures of nucleic acids and of proteins, the technique may find applications in many other fields that require the ultrasensitive analysis of sample components.