A new route for simple and rapid determination of hydrogen peroxide in RAW264.7 macrophages by microchip electrophoresis
A new route for simple and rapid determination of hydrogen peroxide in RAW264.7 macrophages by microchip electrophoresis
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微芯片电泳简单快速测定RAW264.7巨噬细胞中过氧化氢的新途径
DOI：
10.1002/elps.200800635
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发表时间：
2009-06-01
期刊：
影响因子：
2.9
通讯作者：
Zhong, Hongbo
中科院分区：
文献类型：
--
作者：
Gong, Xiaocong;Li, Qingling;Zhong, Hongbo
建立了微波毛细管电泳-激光诱导荧光法测定过氧化氢的新方法。以本实验室合成的新荧光试剂双（对甲基苯磺酰基）二氯荧光素为标记试剂，在0.10MHEPES缓冲液（pH7.4）中于37 ℃衍生化30 min。使用40 mM HEPES缓冲液、20%甘露醇（pH 7.4）在玻璃微芯片上在55 s内完成H2 O2的检测。迁移时间和峰面积的RSD分别为1.8%和3.7%。方法验证显示线性响应范围为0.50 - 50 μ M，LOD（S/N = 3）为0.20 μ M（19.1阿莫尔）。该方法用于测定佛波酯刺激的RAW264.7巨噬细胞中H2 O2的浓度为1.86 +/- 0.05 μ M;巨噬细胞样品的回收率为96.7 - 97.8%，日内和日间准确度分别为4.5%（n = 5）和6.8%（n = 5）。
A new method using MCE with LIF detection was developed for the determination of hydrogen peroxide (H2O2). Bis(p-methylbenzenesulfonyl)dichlorofluorescein, a new fluorogenic reagent synthesized by our laboratory was employed as a labeling reagent, the derivatization reaction was performed in 0.10 M HEPES buffer (pH 7.4) for 30 min at 37 degrees C. The detection of H2O2 was accomplished in 55 s, using a 40 mM HEPES buffer, 20% mannitol, pH 7.4, on a glass microchip. The RSDs of migration time and peak area were 1.8 and 3.7%, respectively. Method validation showed the linear response ranging from 0.50 to 50 mu M with an LOD (S/N = 3) of 0.20 mu M (19.1 amol). The proposed method was applied to determine H2O2 in phorbol myristate acetate-stimulated RAW264.7 macrophages, the concentration of H2O2 was found to be 1.86 +/- 0.05 mu M; recoveries for macrophage samples were from 96.7 to 97.8%, within-days and between-days accuracies were 4.5% (n = 5) and 6.8% (n = 5), respectively.