Peptide electrophoresis by two-beam fluorescence cross-correlation spectroscopy

This dissertation presents the concept, development, and characterization of a new methodology for both qualitative and quantitative analysis of protein digests in solution. Two beam fluorescence cross correlation spectroscopy is used to characterize the migration rates of fluorescently labeled peptides present in a poly(methyl methacrylate) (PMMA) microfluidic system. To achieve ultimate sensitivity, a two beam confocal microscope is employed to allow low background, single molecule detection. Two spatially separated laser beams are focused to near-diffraction limited spots and then positioned a few microns apart within a narrow region of a PMMA microdevice. Mobility measurements of the protein fragments are determined by the transit time for a single peptide to traverse through both detection volumes. Cross correlation of the fluorescence intensity signals from each confocal volume is used characterize the distribution of transit times. Electrophoresis conditions are employed and each peptide in a mixture will migrate at a characteristic velocity that depends on its size and charge. The cross correlation analysis yields a distribution of velocities reminiscent of an electropherogram in that each peak is evidence of an individual peptide. For a specific peptide digest, one can generate a fingerprint spectrum from the cross correlation data. The fingerprint could then be matched to a library of individual protein spectra allowing the rapid identification of the protein from whence the peptide mixture was derived. Our proposed method eliminates some of the shortcomings associated with current microfluidic technology. For example, analytes are monitored in free solution without actually separating the mixture this eliminates the need for generating an analyte plug or migration over long distances. Also, since single molecule fluorescence is utilized it is possible to analyze multiple complex species at sub-nanomolar concentrations, in turn minimizing sample consumption. The two-beam fluorescence cross correlation method has the potential to be a high speed, highly sensitive alternative approach for protein and peptide analysis.