Nitric oxide (NO) is an intracellular and intercellular messenger involved in numerous physiological and pathophysiological processes. Small-molecule fluorescent probes coupled with fluorescence microscopy provide excellent tools for real-time detection of NO in situ. However, most probes are designed for imaging intracellular NO, which cannot reflect the release behavior of endogenously produced NO. In order to visualize extracellular NO released from living cells, we report herein a particularly designed amphiphilic fluorescent probe, disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), in which hydrophilic groups are introduced to keep the fluorophore and recognition domain outside the cell and a hydrophobic C16 alkyl chain acts as the membrane anchor. Based on this design, NO released out of the cells has been visualized on the outer surface of the plasma membrane. Using RAW 264.7 cells and ECV-304 cells as models, the diffusion of NO across the plasma membrane has been directly observed. The amphiphilic design strategy of fluorescent probes holds great promise for developing fluorescent imaging probes to study the release behaviors of other endogenous gasotransmitters.
Single-cell
analysis contributes to the understanding of cellular
heterogeneity and behaviors. Nitric oxide (NO) is an important intracellular
and intercellular signaling molecule, and the functions of NO are
closely related to the balance between intra- and extracellular NO
levels. In this manuscript, a convenient and reliable method based
on a dual-labeling strategy using capillary electrophoresis (CE) separation
with laser-induced fluorescence (LIF) detection has been presented
for quantifying intra- and extracellular NO simultaneously in single
cells. Followed by single-cell injection, a plug of HEPES buffer containing
1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene and disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4′-difluoro-4-bora-3a,4a-diaza-s-indacene as the labeling reagents for intra- and extracellular
NO, respectively, was aspirated from the inlet of the capillary. The
on-line derivatization was carried out on the tip of the capillary
at room temperature for 20 min. Then, the cell was lysed and NO derivatives
were well separated within 14 min, producing mass detection limits
(S/N = 3) of 2.4 and 8.1 amol for intra- and extracellular NO, respectively.
The proposed method was validated by simultaneous analysis of intra-
and extracellular NO in single macrophage cells. The dual labeling-based
CE–LIF method holds great promise for research on the functions
of NO as well as other bioactive molecules at the single-cell level.
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