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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• 作者：Zhenzhen Chen ; Qingling Li ; Xu Wang ; Zhiyuan Wang ; Ruirui Zhang ; Miao Yin ; Lingling Yin ; Kehua Xu ; Bo Tang
• 刊名：Analytical Chemistry
• 出版年：2010
• 出版时间：March 1, 2010
• 年：2010
• 卷：82
• 期：5
• 页码：2006-2012
• 全文大小：261K
• 年卷期：v.82,no.5(March 1, 2010)
• ISSN：1520-6882

文摘

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. The linear ranges of the method were 3.3 × 10⁻⁹−1.0 × 10⁻⁷ M/2.9 × 10⁻⁷−1.0 × 10⁻⁴ M and 2.7 × 10⁻⁹−4.0 × 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 μM, 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).