Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers

We present a method to achieve separation and indirect detection of nonfluorescent species using fluorescent mobility markers. This technique leverages isotachophoresis (ITP) for both preconcentration and separation. We employ a leading electrolyte (LE), trailing electrolyte (TE), and a set of fluorescent markers of mobilities designed to bound those of nonfluorescent analytes of interest. Fluorescent markers and nonfluorescent analytes are initially mixed homogenously and ITP is initiated. The dynamics of isotachophoresis cause the analyte and fluorescent marker mixture to segregate into respective zones between the LE and TE in the order of reducing mobility. Unlabeled analytes are detected as gaps (regions with local minimums in intensity) in the fluorescent signals of mobility markers. We have successfully demonstrated preconcentration, separation, and detection of unlabeled amino acids serine, glycine, and phenylalanine; and of acetic acid, aspartic acid, and 3-phenylpropionic acid. We show detection of 12 microM concentration of analytes with signal-to-noise ratio of 4.0 and with a high degree of repeatability. We discuss methods for encoding mobility marker identity using marker fluorescence intensity level and alternating fluorescence emission wavelengths. We present example experimental results of fluorescence intensity level encoding.