title: Dual labeling for simultaneous determination of nitric oxide, glutathione and cysteine in macrophage RAW264.7 cells by microchip electrophoresis with fluorescence detection.
abstract: A simple, rapid and efficient method based on microchip electrophoresis coupled with fluorescence detection (MCE-FLD) was developed for simultaneous determination of nitric oxide (NO), glutathione (GSH) and cysteine (Cys) using dual labeling strategy. Two highly reactive fluorogenic probes, 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacene (DAMBO) and 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), were used for labeling NO and thiols, respectively, under physiological conditions. The rapid separation and sensitive detection of the derivatives were achieved on a glass microchip within 70s in a running buffer of 20mM H3Cit-Na2HPO4 solution (pH 7.4) containing 15% (v/v) acetonitrile at a separation voltage of 2400V. The limits of detection (S/N=3) for NO, GSH and Cys were 7.0, 3.0 and 2.0nM, respectively. The proposed method was validated by measuring intracellular levels of NO and biothiols in macrophage RAW264.7 cells.
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
authors: Feng-Qin
authors: FQ
authors: Tu
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
authors: Li-Yun
authors: LY
authors: Zhang
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
authors: Xiao-Feng
authors: XF
authors: Guo
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
authors: Zi-Xing
authors: ZX
authors: Zhang
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China. Electronic address: hongwang@whu.edu.cn.
authors: Hong
authors: H
authors: Wang
authors: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
authors: Hua-Shan
authors: HS
authors: Zhang
mesh_terms: D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000032:analysis* / Q000378:metabolism; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D005453:Fluorescence; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008264:Macrophages / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis* / Q000378:metabolism
chemical_list: D009569:Nitric Oxide; D005978:Glutathione; D003545:Cysteine
keywords: 1,3,5,7-Tetramethyl-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene; 1,3,5,7-Tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene; Dual labeling; Glutathione; Microchip electrophoresis; Nitric oxide