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BODIPY-based probes have excellent fluorescence properties. However, small Stokes shifts approximately 5-15 nm greatly affect their detection sensitivity. In this study, we compared the Stokes shifts of reported BODIPY-based probes with various of substituents, and found that the phenyl groups on the specific position of BODIPY core could expand the Stokes shift of BODIPY-based probes, and methoxy groups on these phenyl substituents could enhance such effects. Then, by quantum chemical calculations, we found that the number of methoxy groups might also have obvious effect on the Stokes shift of BODIPY. Taking nitric oxide (NO) as analyte, 4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(2,4-dimethoxyphenyl)-4-bora-3a,4a-diaza-s-indancene (DMOPB) with diaminophenyl substituents has been designed and synthesized. Compared with monomethoxy-phenyl substituted BODIPY-based probes (MOPBs) in our previous work, Stokes shift of DMOPB was expanded by 10 nm when using dimethoxyphenyl instead of monomethoxyphenyl, which is basically consistent with the quantum chemistry calculation of 11 nm. DMOPB can react with NO in only 2 min to form the triazole DMOPB-T with a fluorescence quantum yield of 0.32. An excellent linear relationship was observed in the range of NO concentration from 0.5 μM to 4 μM and the detection limit was 1 nM. The experimental results indicate that DMOPB with high sensitivity, excellent selectivity, low toxicity and dark background can be a great candidate for imaging NO in cells and tissues. Considering the lack of practical way to increase Stokes shift of small-molecule fluorescent probes based on specific fluorophore, the proposed strategy has great potential for the designing of probes with large Stokes shift., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hydrogen sulfide (H 2 S) is a new endogenously generated gasotransmitter and has implicated in many physiologies and pathologies closely related to its intracellular and intercellular signaling transduction. Although many fluorescent probes have been exploited to track and quantify H 2 S in living systems, none of them could be used for monitoring intercellular transmission of H 2 S. Herein, we developed a cell surface specific H 2 S probe, 4-azido-6-sulfo-N-hexadecyl-1,8-naphthalimide, sodium salt (ASNHN-N 3 ), trying to investigate the behaviors of extracellular release of H 2 S. ASNHN-N 3 is week fluorescent and could react with H 2 S at 37 °C in pH 7.4 buffer solutions to form product ASNHN-NH 2 with strong fluorescence (Φ = 0.22). Using ASNHN-N 3 as H 2 S probe, excellent linear correlation versus the concentration of NaHS was obtained ranging from 0 to 10 μM and the detection limit was 0.75 μM. With the lipid anchor and the hydrophilic sulfonic group introduced into the 1,8-naphthalimide (a skeleton of two-photon fluorescent probe), the amphiphilic probe is located at the surface of living cells which can record H 2 S efflux from the cell diffusing across the plasma membrane in living cells and deep-tissue by using two-photon microscopy. Thus we present a new strategy for further studying the mechanism of signaling molecules in cell communication and signal pathways., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A dual-color fluorescence imaging method for simultaneous monitoring of intra- and extracellular nitric oxide (NO) was developed. Assisted by confocal laser scanning microscope, the intra- and extracellular NO can be successfully visualized by using two selected probes, 4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene (p-MOPB) and disodium 2,6-disulfonate-1,3-dimethyl-5-hexadecyl-8-(3,4-diaminophenyl)-4,4'-difluoro-4-bora-3a,4a-diaza-s-indacene (DSDMHDAB), which display distinct membrane permeability and show different colors of fluorescence after reaction with NO. Results indicated that intra- and extracellular NO could be fluorometrically detected without mutual interference. The applicability of the proposed method was validated by dual-color imaging of NO on both sides of the plasma membrane in RAW 264.7 murine macrophages and human vascular endothelial (ECV-304) cells. This multi-labeling approach using multi-laser excitation and multi-color fluorescence detection holds great promise for simultaneous analysis of NO as well as other gasotransmitters in living cells with subcellular resolution., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.