Liu Xin, Li Qingling, Gong Xiaocong, Li Hongmin, Chen Zhenzhen, Tong Lili, Tang Bo
College of Chemistry, Chemical Engineering and Materials Science, Ministry of Education, Shandong Normal University, Jinan, P. R. China.
Electrophoresis. 2009 Mar;30(6):1077-83. doi: 10.1002/elps.200800421.
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.
建立了一种基于微芯片电泳结合激光诱导荧光检测超氧阴离子自由基（O(2) (-)）的方法。采用本实验室合成的荧光试剂2-氯-1,3-二苯并噻唑啉环己烯作为标记试剂，在20 mM HEPES缓冲液（pH 7.4）中于37℃反应10分钟可获得最高衍生化效率。在玻璃微芯片上，使用50 mM HEPES缓冲液（pH 7.4）可实现对O(2) (-)的最佳检测。在优化条件下，响应线性范围为4.0x10(-7)-1.0x10(-5) M，检测限（S/N = 3）为0.15 microM，迁移时间和峰面积的相对标准偏差分别为2.6%和3.8%。进行了干扰实验，结果表明在复杂体系中，1000倍摩尔过量的过氧化氢不干扰O(2) (-)的测定。最后，该方法已成功应用于测定肝癌细胞以及佛波酯刺激的RAW264.7巨噬细胞中的O(2) (-)，平均回收率分别为97.3%和98.6%。
