Potent Method for the Simultaneous Determination of Glutathione and Hydrogen Peroxide in Mitochondrial Compartments of Apoptotic Cells with Microchip Electrophoresis-Laser Induced Fluorescence
Potent Method for the Simultaneous Determination of Glutathione and Hydrogen Peroxide in Mitochondrial Compartments of Apoptotic Cells with Microchip Electrophoresis-Laser Induced Fluorescence
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微芯片电泳-激光诱导荧光同时测定凋亡细胞线粒体区室中谷胱甘肽和过氧化氢的有效方法
DOI：
10.1021/ac902741r
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发表时间：
2010-03-01
影响因子：
7.4
通讯作者：
Tang, Bo
中科院分区：
文献类型：
--
作者：
Chen, Zhenzhen;Li, Qingling;Tang, Bo
首次应用微芯片电泳激光诱导荧光 (MCE-LIF) 检测同时测定线粒体中的谷胱甘肽 (GSH) 和过氧化氢 (H2O2)。本实验室合成的有机硒探针Rh-Se-2和双(对甲基苯磺酸)二氯荧光素(FS)分别用作GSH和H2O2的荧光探针。 Rh-Se-2 是非荧光的，与 GSH 反应生成高量子产率的罗丹明 110 (Rh110)。类似地，非荧光 FS 与 H2O2 反应并产生二氯荧光素 (DCF)，并伴有显着的荧光增强。两种探针对其各自的目标分子测定都表现出良好的灵敏度。使用 50 mM 甘露醇、40 mM HEPES (pH 7.4) 的运行缓冲液和 360 V/cm 的电场进行分离，可在 37 秒内快速、简单、灵敏地测定 GSH 和 H2O2。该方法的线性范围为 3.3 x 10(-9)-1.0 x 10(-7) M/2.9 x 10(-7)-1.0 x 10(-4) M 和 2.7 x 10(-9)-4.0 x 10(-7) M，检测限（信噪比 = 3）为 1.3 nM (0.16 amol)， GSH 和 H2O2 为 1.0 nM (0.12 amol)， 分别。迁移时间和峰面积的相对标准偏差 (RSD) 分别小于 1.0% 和 4.0%。 MCE-LIF 测定用于研究从 HepG2 细胞分离的线粒体中 GSH 和 H2O2 的水平，发现分别为 2.01 +/- 0.21 mM 和 5.36 +/- 0.45 muM。该方法进一步扩展到观察阿霉素和光动力疗法（PDT）诱导的经历细胞凋亡的HepG2细胞线粒体中两种物质的情况。
The first application of microchip electrophoresis with laser-induced fluorescence (MCE-LIF) detection to simultaneously determine glutathione (GSH) and hydrogen peroxide (H2O2) in mitochondria was described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichlorofluorescein (FS) synthesized in our laboratory were utilized as fluorescent probes for GSH and H2O2, respectively. Rh-Se-2, which is nonfluorescent, reacts with GSH to produce rhodamine 110 (Rh110) with high quantum yield. Similarly, nonfluorescent FS reacts with H2O2 and produces dichlorofluorescein (DCF) accompanied by drastic fluorescence enhancement Both probes exhibit good sensitivity toward their respective target molecule determination. Fast, simple, and sensitive determination of GSH and H2O2 was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH 7.4), and an electric field of 360 V/cm for separation. Die linear ranges of the method were 3.3 x 10(-9)-1.0 x 10(-7) M/2.9 x 10(-7)-1.0 x 10(-4) M and 2.7 x 10(-9)-4.0 x 10(-7) M with detection limits (signal-to-noise ratio = 3) of 1.3 nM (0.16 amol) and 1.0 nM (0.12 amol) for GSH and H2O2, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 1.0% and 4.0%, respectively. The MCE-LIF assay was utilized to investigate the levels of GSH and H2O2 in mitochondria isolated from HepG2 cells and were found to be 2.01 +/- 0.21 mM and 5.36 +/- 0.45 mu M, respectively. The method was further extended to observe situations of the two species in mitochondria of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin and photodynamic therapy (PDT).