Development of a potential method based on microchip electrophoresis with fluorescence detection for the sensitive determination of intracellular thiols in RAW264.7 cells.

ABSTRACT

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.