Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells.
This paper, for the first time, reported the development of a simple, rapid, and reliable method for the separation and sensitive determination of four thiol compounds including homocysteine, cysteine, glutathione, and N-acetylcysteine based on glass MCE with fluorescence detection using a highly reactive fluorogenic probe, 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), as the labeling reagent. TMPAB-o-M reacted selectively with thiols to produce highly fluorescent derivatives and the highest derivatization efficiency was achieved within 6 min in physiological conditions. After the optimization of separation conditions, a baseline separation of the four thiol compounds was achieved with the detection limits ranging from 2 nM for glutathione to 4 nM for cysteine (S/N = 3) and RSDs (n = 5) in the range of 3.2-3.8%. The proposed method was significantly sensitive compared to those using electrochemical or even LIF detection in MCE-based setup reported previously, and applied to the determination of intracellular thiols in macrophage RAW264.7 cells.
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, P. R. China.
Feng-Qin
FQ
Tu
Li-Yun
LY
Zhang
Xiao-Feng
XF
Guo
Hong
H
Wang
Hua-Shan
HS
Zhang
D000111:Acetylcysteine / Q000032:analysis*; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis*; D046209:Electrophoresis, Microchip / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005978:Glutathione / Q000032:analysis*; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry; D057230:Limit of Detection; D008264:Macrophages / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D013438:Sulfhydryl Compounds / Q000032:analysis*
C534661:1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D008301:Maleimides; D013438:Sulfhydryl Compounds; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine
Fluorescence detection; Glutathione; Microchip electrophoresis; Thiols/1,3,5,7-Tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene