Analysis of amino acids in human vascular endothelial (ECV-304) cells by microchip electrophoresis with fluorescence detection.
A rapid and sensitive method was developed for the analysis of amino acids by microchip electrophoresis with Hg-lamp excitation fluorescence detection. Fluorescein-isothiocyanate (FITC) was chosen to estimate the sensitivity of this system, and the detection limit (S/N = 3) with FITC was 1.7 nM, which showed that the system was sensitive as well as simple. Two derivatizing agents, FITC and ortho-phthalaldehyde (OPA) were employed to label amino acids and were compared in the same fluorescence detection system with an Hg lamp as the excitation source. The separation parameters were optimized in detail. Optimum separation of OPA-labeled amino acids was obtained in less than 200 s with 20 mM borate buffer (pH 9.0) containing 20% acetonitrile and 10 mM beta-cyclodextrin. Detection limits for amino acids (alanine (Ala), taurine (Tau), glycine (Gly), glutamic acid (Glu), and aspartic acid (Asp)) of 0.38-1.0 muM were achieved. The method was successfully applied to analysis of amino acids in human vascular endothelial cells (ECV-304). The average amount of amino acids in single ECV-304 cells is estimated to be 5.84 fmol for Ala, 2.78 fmol for Tau, 1.15 fmol for Gly, 3.10 fmol for Glu, and 1.30 fmol for Asp.
Shi
Baoxian
B
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
Huang
Weihua
W
Cheng
Jieke
J
D000596:Amino Acids / Q000032:analysis*; D002021:Buffers; D002460:Cell Line; D046209:Electrophoresis, Microchip / Q000379:methods*; D042783:Endothelial Cells / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008628:Mercury; D017785:Photons; D012680:Sensitivity and Specificity; D047392:beta-Cyclodextrins
D000596:Amino Acids; D002021:Buffers; D047392:beta-Cyclodextrins; D008628:Mercury; C031215:betadex