Indirect fluorescence detection of amino acids on electrophoretic microchips.

Microfabricated devices enable rapid separations of a variety of clinically significant analytes, including DNA, proteins, and amino acids. However, absorbance detection has been difficult to achieve on these devices, prohibiting analysis of nonfluorophore-bearing or nonfluorescently tagged analytes. An alternative detection technique exploiting indirect fluorescence has been adapted to the electrophoretic microchip to provide fast analysis of amino acids, bypassing the need for absorbance detection or fluorescence derivitization procedures. Nineteen of the standard amino acids could be detected with an average detection limit of 32.9 microM (approximately 1.6 amol). Despite the fact that the detection sensitivity was lower than that achievable by labeling the amino acids with fluorescein isothiocyanate (approximately 1 nM), circumventing sample preparation and the difficulties inherent with tagging complex samples make this technique attractive for a variety of assays where sensitivity is not critical. To demonstrate the applicability to real sample matrixes, the analysis of urine with elevated amino acid levels is used as a model system where the elevated levels are indicative of a variety of pathologies including amino acid metabolism disorders and kidney malfunction. The minimal sample handling and rapid separations achievable by employing indirect detection on microchips provides the potential for high-throughput applications for certain amino acid analyses.