Rapid determination of superoxide free radical in hepatocellular carcinoma cells by MCE with LIF.

A method for determination of superoxide free radical (O(2) (-*)) based on MCE with LIF was developed. Fluorescent reagent 2-chloro-1, 3-dibenzothiazolinecyclohexene, which was synthesized in our laboratory, was employed as the labeling reagent, the highest derivatization efficiency was obtained in 20 mM HEPES buffer (pH 7.4) for 10 min at 37 degrees C. Optimal determination of O(2) (-*) was achieved on a glass microchip, using 50 mM HEPES buffer (pH 7.4). Under the optimized conditions, linearity of response was obtained in the range of 4.0x10(-7)-1.0x10(-5) M, the detection limit (S/N=3) was 0.15 microM, the RSDs of migration time and peak area were 2.6 and 3.8%, respectively. Interference experiment was investigated and the result indicates that 1000-fold molar excess of hydrogen peroxide does not interfere with the determination of O(2) (-*) in complex system. Finally, the method has been successfully applied to determine O(2) (-*) in hepatocellular carcinoma cells as well as phorbol 12-myristate 13-acetate stimulated RAW264.7 macrophages. The average recoveries were 97.3 and 98.6%, respectively.