Potent Method for the Simultaneous Determination of Glutathione and Hydrogen Peroxide in Mitochondrial Compartments of Apoptotic Cells with Microchip Electrophoresis-Laser Induced Fluorescence

摘要

The first application of microchip electrophoresis withnlaser-induced fluorescence (MCE-LIF) detection to simultaneouslyndetermine glutathione (GSH) and hydrogennperoxide (H2O2) in mitochondria was described. Organoseleniumnprobe Rh-Se-2 and bis(p-methylbenzenesulfonate)ndichlorofluorescein (FS) synthesized in our laboratorynwere utilized as fluorescent probes for GSH andnH2O2, respectively. Rh-Se-2, which is nonfluorescent,nreacts with GSH to produce rhodamine 110 (Rh110)nwith high quantum yield. Similarly, nonfluorescent FSnreacts with H2O2 and produces dichlorofluorescein (DCF)naccompanied by drastic fluorescence enhancement. Bothnprobes exhibit good sensitivity toward their respectiventarget molecule determination. Fast, simple, and sensitivendetermination of GSH and H2O2 was realized withinn37 s using a running buffer of 50 mM mannitol, 40 mMnHEPES (pH 7.4), and an electric field of 360 V/cm fornseparation. The linear ranges of the method were 3.3 ×n10-9-1.0 × 10-7 M/2.9 × 10-7-1.0 × 10-4Mand 2.7n× 10-9-4.0 × 10-7 M with detection limits (signal-tonoisenratio ) 3) of 1.3 nM (0.16 amol) and 1.0 nM (0.12namol) for GSH and H2O2, respectively. The relativenstandard deviations (RSDs) of migration time and peaknarea were less than 1.0% and 4.0%, respectively. ThenMCE-LIF assay was utilized to investigate the levels ofnGSH and H2O2 in mitochondria isolated from HepG2ncells and were found to be 2.01 ( 0.21mMand 5.36 (n0.45 μM, respectively. The method was further extendednto observe situations of the two species in mitochondrianof HepG2 cells experiencing cell apoptosis that were inducednby doxorubicin and photodynamic therapy (PDT).