Limits...
Highly selective fluorescent and colorimetric probe for live-cell monitoring of sulphide based on bioorthogonal reaction.

Huo FJ, Kang J, Yin C, Chao J, Zhang Y - Sci Rep (2015)

Bottom Line: To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired.In the presence of H2S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H2S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H2O/CH3CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes.All these features are favorable for direct monitoring of H2S with satisfactory sensitivity, demonstrating its value of practical application.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Applied Chemistry, Shanxi University, Taiyuan. 030006, China.

ABSTRACT
H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H2S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H2O/CH3CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes. All these features are favorable for direct monitoring of H2S with satisfactory sensitivity, demonstrating its value of practical application.

No MeSH data available.


Related in: MedlinePlus

(a) The time dependence of confocal fluorescence images of exogenous sulphide in HepG2 cells. (h) and (i) Fluorescence image of HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C and then incubated with 4 μmol/L H2S for 15 min and 45 min at 37°C, respectively; (j), (k) were their brightfield images of (h), (i); (b) Confocal fluorescence images of endogenous H2S in living HepG2 cells with probe (2 μmol/L) upon excitation at 425 nm. Cells were prestimulated with SNP (100 μM) for 30 min, then incubated with probe (2 μmol/L) for 20 min (l, n). Cells were prestimulated with SNP (200 μmol/L) for 30 min, and then incubated with probe (2 μmol/L) for 20 min (m, o).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4355735&req=5

f6: (a) The time dependence of confocal fluorescence images of exogenous sulphide in HepG2 cells. (h) and (i) Fluorescence image of HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C and then incubated with 4 μmol/L H2S for 15 min and 45 min at 37°C, respectively; (j), (k) were their brightfield images of (h), (i); (b) Confocal fluorescence images of endogenous H2S in living HepG2 cells with probe (2 μmol/L) upon excitation at 425 nm. Cells were prestimulated with SNP (100 μM) for 30 min, then incubated with probe (2 μmol/L) for 20 min (l, n). Cells were prestimulated with SNP (200 μmol/L) for 30 min, and then incubated with probe (2 μmol/L) for 20 min (m, o).

Mentions: Most publications suggest that the average endogenous H2S level is in the μmol/L range313241, Since the detection limit of this probe was found to be 0.035 μmol/L, thus it become possible that the probe can detect H2S level in tissue imaging. The ability of probe to detect sulphide within living cells was also evaluated by laser confocal fluorescence imaging using a Leica TCS SP5 laser scanning microscope. Imaging of sulphide substrates in HeLa cells after 30 min incubation using probe (2 μmol/L) showed weak green fluorescence (Fig. 5b). HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C, and with 4 μmol/L exogenous H2S for another 30 min at 37°C, showed green fluorescence (Fig. 5c) (it is noted that 30 min was usually selected in cell imaging experiment). We also carried out time course experiment in the cell. Fig. 6 (left) indicated that a 15 min is enough for cell permeability (Fig. 6h) reaction and the cell can survive even if in a 45 min after H2S was added (Fig. 6i). In addition, according to the Qian's method42, we employed sodium nitroprusside (SNP, a NO donor) to stimulate the production of endogenous H2S in cells43. With the addition of probe into the culture of the SNP (100 μmol/L or 200 μmol/L)-loaded cells for 20 min, a drastic increase of emission intensity (Fig. 6l, 6m), indicating the generation of endogenous H2S within the cells. These results demonstrate that this probe is selective for sulphide and amenable for live-cell imaging.


Highly selective fluorescent and colorimetric probe for live-cell monitoring of sulphide based on bioorthogonal reaction.

Huo FJ, Kang J, Yin C, Chao J, Zhang Y - Sci Rep (2015)

(a) The time dependence of confocal fluorescence images of exogenous sulphide in HepG2 cells. (h) and (i) Fluorescence image of HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C and then incubated with 4 μmol/L H2S for 15 min and 45 min at 37°C, respectively; (j), (k) were their brightfield images of (h), (i); (b) Confocal fluorescence images of endogenous H2S in living HepG2 cells with probe (2 μmol/L) upon excitation at 425 nm. Cells were prestimulated with SNP (100 μM) for 30 min, then incubated with probe (2 μmol/L) for 20 min (l, n). Cells were prestimulated with SNP (200 μmol/L) for 30 min, and then incubated with probe (2 μmol/L) for 20 min (m, o).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355735&req=5

f6: (a) The time dependence of confocal fluorescence images of exogenous sulphide in HepG2 cells. (h) and (i) Fluorescence image of HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C and then incubated with 4 μmol/L H2S for 15 min and 45 min at 37°C, respectively; (j), (k) were their brightfield images of (h), (i); (b) Confocal fluorescence images of endogenous H2S in living HepG2 cells with probe (2 μmol/L) upon excitation at 425 nm. Cells were prestimulated with SNP (100 μM) for 30 min, then incubated with probe (2 μmol/L) for 20 min (l, n). Cells were prestimulated with SNP (200 μmol/L) for 30 min, and then incubated with probe (2 μmol/L) for 20 min (m, o).
Mentions: Most publications suggest that the average endogenous H2S level is in the μmol/L range313241, Since the detection limit of this probe was found to be 0.035 μmol/L, thus it become possible that the probe can detect H2S level in tissue imaging. The ability of probe to detect sulphide within living cells was also evaluated by laser confocal fluorescence imaging using a Leica TCS SP5 laser scanning microscope. Imaging of sulphide substrates in HeLa cells after 30 min incubation using probe (2 μmol/L) showed weak green fluorescence (Fig. 5b). HepG2 cells incubated with 2 μmol/L probe for 30 min at 37°C, and with 4 μmol/L exogenous H2S for another 30 min at 37°C, showed green fluorescence (Fig. 5c) (it is noted that 30 min was usually selected in cell imaging experiment). We also carried out time course experiment in the cell. Fig. 6 (left) indicated that a 15 min is enough for cell permeability (Fig. 6h) reaction and the cell can survive even if in a 45 min after H2S was added (Fig. 6i). In addition, according to the Qian's method42, we employed sodium nitroprusside (SNP, a NO donor) to stimulate the production of endogenous H2S in cells43. With the addition of probe into the culture of the SNP (100 μmol/L or 200 μmol/L)-loaded cells for 20 min, a drastic increase of emission intensity (Fig. 6l, 6m), indicating the generation of endogenous H2S within the cells. These results demonstrate that this probe is selective for sulphide and amenable for live-cell imaging.

Bottom Line: To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired.In the presence of H2S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H2S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H2O/CH3CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes.All these features are favorable for direct monitoring of H2S with satisfactory sensitivity, demonstrating its value of practical application.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Applied Chemistry, Shanxi University, Taiyuan. 030006, China.

ABSTRACT
H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H2S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H2O/CH3CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes. All these features are favorable for direct monitoring of H2S with satisfactory sensitivity, demonstrating its value of practical application.

No MeSH data available.


Related in: MedlinePlus