Capillary electrophoresis strategy to monitor the released and remaining nitric oxide from the same single cell using a specially designed water-soluble fluorescent probe.

Gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) have attracted more and more attention in the past decades due to their unique signaling and functions. However, as a fundamental issue in the investigations of gasotransmitters, the cell membrane permeability and release behavior of them is controversial in reports because of the lack of an efficient approach to determine gasotransmitters released out of and remaining in the same cells simultaneously. To solve such problem, taking NO as representative, a robust and facile strategy has been reported based on a completely water-soluble fluorescent probe and a commercially available capillary electrophoresis system. A specially designed boron-dipyrromethene (BODIPY)-based fluorescent probe with two sulfonate groups, disodium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl) difluoroboradiaza-s-indance (TMDSDAB), has been developed. As a turn-on fluorescent probe, TMDSDAB can react with NO promptly in aqueous media, and 540-fold enhancement of fluorescence is obtained. Using TMDSDAB, the trapping and quantification of NO released out of and remaining in the same single cell was achieved by capillary electrophoresis with laser-induced fluorescence detection. The limit of detection is 0.5 nM for NO. The proposed method has been applied to estimate the release behavior of single macrophages, and the results indicated that the cell membrane should be a barrier to NO diffusion.

Dual labeling for simultaneous determination of nitric oxide, glutathione and cysteine in macrophage RAW264.7 cells by microchip electrophoresis with fluorescence detection.

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.

Simultaneous analysis of plasma thiols by high-performance liquid chromatography with fluorescence detection using a new probe, 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido)difluoroboradiaza-s-indacene.

The design, synthesis and properties of a new derivatizing reagent, 1,3,5,7-tetramethyl-8-phenyl-(4-iodoacetamido)difluoroboradiaza-s-indacene (TMPAB-I), for thiol groups are presented. Using the derivatization of TMPAB-I with thiols, a new high-performance liquid chromatographic method for measuring low-molecular-weight thiol-containing compounds, including coenzyme A (CoA), glutathione, N-acetylcysteine, cysteine, homocysteine (HCys) and 6-mercaptopurine has been developed. The reaction of TMPAB-I with thiols is specific, fast and stable for both TMPAB-I and the derivatives. A baseline separation of all the six derivatives is achieved by isocratic elution on reversed-phase column within 20 min with detection wavelengths of 500 and 510 nm for the excitation and emission, respectively, and the limits of detection (signal-to-noise ratio=3) are from 1.8 fmol (CoA) to 14.0 fmol (HCys), respectively, per 20 microL injection. The utility of the proposed method has been validated by measuring thiol-containing compounds in human plasma samples from healthy persons and patients with hypertension, with recoveries of 94.2-106.8%.