Method for Analyte Identification Using Isotachophoresis and a Fluorescent Carrier Ampholyte Assay

We present a novel method for identification of unlabeled analytes using fluorescent carrier ampholytes and isotachophoresis (ITP). The method is based on previous work where we showed that the ITP displacement of carrier ampholytes can be used for detection of unlabeled (non-fluorescent) analytes. We here propose a signal analysis method based on integration of the associated fluorescent signal. We define a normalized signal integral which is equivalent to an accurate measure of the amount of carrier ampholytes which are focused between the leading electrolyte and the analyte. We show that this parameter can be related directly to analyte effective mobility. Using several well characterized analytes, we construct calibration curves relating effective mobility and carrier ampholyte displacement at two different leading electrolyte (LE) buffers. On the basis of these calibration curves, we demonstrate the extraction of fully ionized mobility and dissociation constant of 2-nitrophenol and 2,4,6-trichlorophenol from ITP experiments with fluorescent carrier ampholytes. This extraction is based on no a priori assumptions or knowledge of these two toxic chemicals. This technique allows simultaneous identification of multiple analytes by their physiochemical properties in a few minutes and with no sample preparation.