Use of capillary electrophoresis and laser-induced fluorescence for attomole detection of amino acids.
A capillary electrophoresis and laser-induced fluorescence (CE-LIF) method was developed to identify and quantitate at amol (10(-18)) concentration. Amino acids were derivatized with 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde prior to CE-LIF analysis. The assay was developed by varying the sodium borate concentration, buffer pH, operating voltage, and operating temperature. A run buffer system containing 6.25 mM borate, 150 mM sodium dodecyl sulfate, and 10 mM tetrahydrofuran (pH 9.66) at 25 degrees C, and 24 kV provided analysis conditions for a high-resolution, sensitive, and repeatable assay of amino acids. The rate of derivatization, stability of the labeled amino acids, and amino acid quantitation varied for each amino acid. Amino acids were detected with greater efficiency by this method than automated HPLC amino acid analysis. The repeatability of the assay ranged from 0.3 to 0.9% within a day and 0.7 to 1.5% between analysis days. Bacterial amino acid utilization in a chemically defined medium was successfully monitored using this method. This work defines a sensitive and repeatable method for the detection of amino acids during bacterial metabolism.
Ummadi
M
M
Western Dairy Center, Center for Microbe Detection and Physiology, Department of Nutrition and Food Sciences, Utah State University, Logan, UT 84322-8700, USA.
Weimer
B C
BC
D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D001951:Brevibacterium / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D000596:Amino Acids