A novel FRET-based fluorescent probe for the selective detection of hydrogen sulfide (H2S) and its application for bioimaging

Article abstract of DOI:10.1039/c8tb01070f

Hydrogen sulfide (H₂S) is an endogenously produced gaseous signaling molecule with multiple functions in many physiological and pathological processes. Herein, our group developed a novel fluorescent probe (Flu-N₃) for H₂S

Full text of DOI:10.1039/c8tb01070f

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A novel FRET-based fluorescent probe for the selective
detection of hydrogen sulfide (H2S) and its application for
bioimaging
Received 00th January 20xx,
Accepted 00th January 20xx
Qi Zhao,a Fangjun Huo,b Jin Kang,a Yongbin Zhanga and Caixia Yin*a
DOI: 10.1039/x0xx00000x
Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule with multiple functions in
many physiological and pathological processes. Herein, our group developed a novel fluorescent probe (Flu-
N3) for H2S on the basis of 7-amino-4-methylcoumarin and fluorescein FRET system. Adding H2S to the
solution of Flu-N3 resulted in a clearly observable fluorescence enhancement (50-fold). This probe showed
high sensitivity and selectivity towards H2S over other reactive species with a detection limit (0.031 μM), fast
response time (within 8 s). Moreover, Flu-N3 displayed little toxicity to HepG-2 cells and were successfully
applied to image exogenous and endogenous H2S in living cells and nude mouse.
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Introduction
fluorescent probes to detect H2S sensitively and selectively in living
systems precedes other traditional methods including colorimetric,
Hydrogen sulfide (H2S) is proved to be third most attractive gas
signaling molecules in biology, with extensive biological effects.
Endogenous H2S mainly generated by some enzymes including
cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-
mercaptopyruvate sulphur-transferase (MST) from sulfur-
containing substrates, then H2S as a final product have negative
feedback regulation on these enzymes.1-5 The scope of
physiologically relevant H2S concentration is from nanomolar to
millimolar in human body, a third of it exists in the presence of gas
molecules and two-thirds exists in the form of NaHS, which not only
ensures the stability of the H2S in the body, but also maintains the
pH level of the internal environment. Along with nitric oxide (NO)
and carbon monoxide (CO), endogenous H2S acts as a gaseous
mediator for regulating endocrine, neuronal, cardiovascular,
immune and gastrointestinal systems.6-10 The change in
concentration of H2S also has been influenced many diseases
including Alzherimer’s disease, Down’s syndrome, diabetes, and
liver cirFlusis.11-15 In short, H2S and NO, CO may together form a
unique network of gas signals, which are independent of each other
and regulate the way the network to participate in the regulation of
the disease.
electrochemical and gas chromatographic (GC) techniques.16-19 To
date, fluorescence method has attracted our interesting in their
non-destructive, non-invasion, simple operation, as well as the
capacity of rapid detection target analyte with high spatial and
temporal resolution, it is an effective tool for detecting and imaging
H2S in biological systems.20-29 Although the research of fluorescent
probes for H2S has achieved great success, most of them showed a
delayed response with H2S.30-37 Thus, we intended to develop fast
response H2S probes based on the reduction of aromatic azides by
H2S. Based on our interesting in developing fluorescent probes that
were more effective and fast detection of H2S in a solution or cell,
we also undertook the effort of constructing a fluorescence
resonance energy transfer (FRET) system. The FRET system is a
common way since fluorescent probes on the basis of FRET strategy
are capable of reducing auto-fluorescence and scattering within
biological samples.38-39 However, the prerequisite for an efficient
FRET process is substantial overlap between acceptor absorption
and the donor emission, which would limit the development of
FRET-based probes. Therefore, it is of great significance to explore
the new fabrication of H2S fluorescence probes based on the FRET
mechanism. That is, the introduction of fluorescein based on
With the development of molecular tools, to use responsive commercial dyes 7-amino-4-methylcoumarin to construct a large
conjugated structure and an FRET system. The emission of the
coumarin fluorophore is centered in the blue region. We planted to
use FRET mechanism to achieve longer-wavelength emission of
novel H2S probe. As shown in Scheme 2, a traditional strategy for
H2S probes could be based on
comprised of a reaction-site-containing FRET acceptor and a FRET
donor (here azido-containing coumarin) linked by rigid
a two fluorophore cassette
a
piperazidine unit. After removing the reaction site in the FRET
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donor then FRET occurs, which results in a significant fluorescence spectrometer, respectively (Bruker, Billerica, MA). Coupling
enhancement of the FRET acceptor.
constants (J values) are reported in hertz. ESI determinations were
carried out on AB Triple TOF 5600plus System (AB SCIEX,
Framingham, USA). Imaging of cells was measured by a Zeiss
LSM880 Airyscan confocal laser scanning microscope. ELX808
atomatic enzymatic marker was used to measure cytotoxicity
experiments.
Herein, our group developed a new fluorescent probe named Flu-
N3 (Scheme 1) for H2S on the basis of 7-amino-4-methylcoumarin
(AMC) and fluorescein (termed ‘Fludol’) FRET system. In this design,
four factors were considered: (1) coumarin and Fludol were
selected as two fluorophores due to their excellent solubility, larger
molar extinction coefficient, high quantum yields, and well- Solution preparation and optical measurement.
separated maximum emission wavelengths (ca. 445 nm for
Stock solutions of probe Flu-N3 were prepared in DMSO. The stock
coumarin and ca. 542 nm for Fludol). (2) In addition, this single part
7-Azido-4-methylcoumarin (AzMC) is also a fluorescent probe useful
for the detection of H2S. Thus, the azide part of the probe provide
selective reaction sites for H2S. (3) Two fluorophores were linked by
a rigid piperazine and provided an advantage for Förster resonance
energy transfer (FRET) in the coumarin-Fludol scaffold, which
reducing the π-π stacking between dyes. The piperazidine linker
also can improve the cell permeability of the probe. (4) Azidation of
coumarin and Fludol could effectively quench the fluorescence of
the probe via the intramolecular spirocyclization and intramolecular
charge transfer (ICT) effect, respectively.40-44 The probe should bear
very low background fluorescence that is favorable for high
solution of H2S (2 mM) was prepared in deionized water, sodium
hydrosulfide solid was added to aqueous solution to prepare a H2S
solution. Reagents with analytical grades and demineralized water
were used for preparing the solutions. Stock solutions (2 mM) of
-
3-
Cys, Hcy, GSH, F-, Cl-, Br-, I-, SCN-, SO32-, SO42-, S2O32-, ClO4 , PO4
,
-
-
-
NO3 , NO2 , AcO-, HCO3 , CO32-, were prepared by direct dissolution
of proper amounts of sodium salts. All other chemicals used were of
analytical grade. UV-vis and fluorescence spectra were detected in
MeCN: PBS (9:1 v/v, PBS buffer, pH 7.4) solutions. Fluorescence
measurements were carried out with a slit width of 5 nm/2.5 nm
(λex = 470 nm).
sensitivity. Therefore, the aromatic azide moiety is selectively Cell culture and Imaging.
reduced in the presence of H2S, amine-group compounds would
HepG-2 cells were cultured at 37°C incubator with 5% CO2 and 95%
also be formed in this process, which should exhibit green
fluorescence of Fludol because of FRET. Flu-N3 exhibited desirable
properties such as high selectivity, fast response for H2S as well as
low cytotoxicity. The probe was also applied for fluorescence
imaging of H2S in HepG-2 cells and live mice model.
air and grown in Dulbecco’s Modified Eagle’s medium
supplemented with 12% Fetal Bovine Serum and 1% antibiotics at in
humidified environment. The cells were stained with Flu-N3 (5 μM)
for 15 min. Then the stained cells were incubated with H2S for
another 15 min at 37°C. Before imaging, the stained cells were
washed one time with PBS. Fluorescence imaging studies were
performed on Zeiss LSM880 Airyscan confocal laser scanning
microscope. To evaluate the cytotoxicity of Flu-N3, the cell viability
was determined by Cell Counting Kit-8 (CCK-8), and the absorbance
at 450 nm was measured to explicate the cells viability. HepG-2 cells
were seeded on a 96-well microtiter to a total volume of 100
μL/well, then the cells were incubated at 37℃ in a 5% CO2
incubator for 24 h. Different concentrations of Flu-N3 (0, 1, 2.5, 5,
10, 20, 30 and 50 μM) were then added to the wells. After
incubation for 5 or 10 h, CCK-8 (10% in serum free culture medium)
was added to each well, and then the cells continue to be incubated
for another 1 h. The absorbance of each well was measured at 450
nm by a microplate reader.
Scheme 1 Synthesis of the probe.
Experimental
Materials and Instruments.
All reagents and solvents were purchased from commercial
suppliers and used without further purification. Deionized water
was used throughout all experiments. The solutions of anions were
prepared from their sodium salts. The stock solutions of probe Flu-
N3 were prepared in DMSO. SNP solution (20 mM) was prepared by
dissolving 8.8 g SNP in 2 mL DMSO. Chromatography was carried
out on silica gel using silica gel GF254 plates with a thickness of
0.20-0.25 mm. A pH meter (Mettler Toledo, Switzerland) was used
to determine the pH. Ultraviolet-visible (UV-vis) spectra were
In vivo imaging.
Balb/c nude mice were purchased from Beijing Vital River
Laboratory Animal Technology Co., Ltd. Experiments were
conducted according to the National Institute of Health Guide for
the Care and Use of Laboratory Animals. The experimental
procedures were approved by the Institute of Pharmacology and
Toxicology Academy of Military Medical Sciences PLA, Peop. Rep.
China ethical committee on animal care and use, and all efforts
were made to minimize animal suffering and reduce the number of
animals used for the experiments. Imaging procedures were
conducted with adult nude mice under general anesthesia by
injection of sodium pentobarbital (0.5 mL/0.03 %). Then the mice
will be injected in the abdomen with 50 μM of Flu-N3, H2S was
carefully injected to the same location. Then, the fluorescence
recorded on
a Cary 50 Bio UV-visible spectrophotometer.
Fluorescence spectra were measured on Hitachi F-7000
fluorescence spectrophotometer. All fluorescence and UV-vis
spectra data were recorded at 2 min after the analytes addition. A
PO-120 quartz cuvette (1 cm) was purchased from Shanhai Huamei
Experiment Instrument Plants, China. 1H NMR and 13C NMR
experiments were performed with a Bruker AVANCE-600 MHz NMR
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images were recorded at different period of time (0, 5, 15, 30, 45, Results and Discussion
60 min).Images was taken using an excitation laser of 475 nm and
The absorption and fluorescence spectra of Flu-N3 were
investigated in MeCN: PBS (9:1 v/v, PBS buffer, pH 7.4). Fig. S1
shows the change of Flu-N3 (5 μM) in the UV-vis spectrum upon
addition of H2S (0-110 μM), the absorption peak of the probe at 508
nm is increased gradually. With the increase of H2S concentration,
the absorption peak at 508 nm was almost unchanged, and it was
speculated that the reduced product was stable in the presence of
H2S.
emission was collected 520±5 nm filter.
Preparation and characterization of compounds.
Compound 1: Fluorescein (1.7 g, 5.12 mmol) was dissolved in 12 mL
of 50 % NaOH solution (w/v) and heated at 160 ℃ in an oil bath for
1 h. After cooling to room temperature, the mixture was poured
into 80 mL of ice water, acidified with concentrated HCl, and
allowed to stand at room temperature for 2 h. The precipitate was
filtered and dried to afford the desired product as a beige powder
Subsequently, the fluorescence spectra response of control
(0.98 g, 74.1 % yield). m.p. 201.3-202.6℃. Elemental Analysis: probe Flu-N3 (5 μM) toward H2S (0-140 μM) was examined under
Found C, 64.9; H, 3.4%. Molecular formula: C14H10O5, requires: C, the same condition. On treatment of Flu-N3 with H2S (Fig. 1a), about
65.2; H, 3.9%. 1H NMR (600 MHz, MeOD): δ 8.11 (dt, J1 = 13.7, J2 = 50-fold remarkable fluorescence enhancement was obtained, which
6.9 Hz, 1H), 7.75-7.70 (m, 1H), 7.67-7.61 (m, 1H), 7.39 (dt, J1 = 14.4, implies that azide moieties of the probe were reduced by H2S to
J2 =7.2 Hz, 1H), 6.98-6.92 (m, 1H), 6.34 (d, J = 2.3 Hz, 1H), 6.27- 6.22 produce strongly fluorescent. The obvious green fluorescence
(m, 1H) 13C NMR (151 MHz, DMSO-d6): δ 200.9, 167.1, 165.4, 164.8, change of Flu-N3 before and after reaction would facilitate sensitive
140.4, 135.1, 132.7, 130.4, 130.1, 127.8, 113.7, 108.7, 102.9 (Fig. detection. Furthermore, a plot of fluorescence intensity at 538 nm
S1).
versus the concentrations of H2S displayed a good linearity (Fig. 1b).
The detection limit was calculated to be 0.031 μM with the
definition: Detection limit = 3σ/k, which is highly sensitive among
the existing H2S fluorescent probes.
Compound 2: To compound 1 (0.87 g, 3.37 mM) in 20 mL of TFA
was added 1-(3-hydroxyphenyl)-piperazine (0.5 g, 2.80 mM), then
heated to reflux and stirred for 36 h. The solvent was removed by
rotavapor, and the left residue was dissolved in 10 mL of H2O. The
In order to determine the effect of different pH on probe
crude product was extracted with EtOAc (25 mL × 3), and dried with function, the influence of pH on the fluorescence behavior of Flu-N3
Na2SO4. The product was further purified by column and its response to H2S were investigated. In the absence of H2S,
chromatograph, and obtained as a red solid (0.8 g, 71%). m.p. the part of fluorescein in the probe showed an open-loop state with
210.7-212.0℃. Elemental Analysis: Found C, 71.9; H, 4.8; N, 6.8%. the enhancement of alkalinity and be accompanied by an increase
Molecular formula: C24H20N2O4, requires: C, 72.0; H, 5.0; N, 7.0%. 1H in auto-fluorescence. Therefore, under the alkaline conditions, the
NMR (600 MHz, DMSO-d6) : δ 8.88 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), fluorescent enhancement effect of the probe is weaker as the
7.80 (t, J = 7.5 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.26 (d, J = 7.6 Hz, hydrogen sulfide increases. The results (Fig. 1c) indicated that the
1H), 6.78 (d, J = 8.9 Hz, 1H), 6.69 (s, 1H), 6.60-6.54 (m, 1H), 3.52- probe can work effectively under the reaction conditions of pH 7.4.
3.39 (m, 2H), 3.23 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 169.1, Next, we further examined the time courses of the fluorescence
162.8, 160.1, 158.9, 158.7, 158.5, 152.7, 152.4, 152.3, 152.0, 136.0, intensities of Flu-N3 in the presence of 10 equiv. HS- in MeCN: PBS
130.5, 129.5, 129.0, 126.6, 125.1, 124.4, 118.2, 116.2, 113.1, 112.5, (9:1 v/v, PBS buffer, pH 7.4). As Fig. 1d depicted, upon the addition
109.9, 109.8,102.6,102.4, 45.0, 42.8 (Fig. S2).
of H2S, the fluorescence intensity increased rapidly and reached a
plateau within 8 s.
Compound
Flu-N3:
To
a
mixture
of
7-azido-4-
carbamoylmethylcoumarin45 (49 mg, 0.2 mmol), compound 2 (0.2
To explore selectivity and special recognition ability of Flu-N3
mmol), EDC (38 mg, 0.2 mmol) and DMAP (2.44 mg, 0.02 mmol) toward H2S, the influence of various analytes was investigated in
was added CH2Cl2/DMF (v:v 5:1, 15 mL) at room temperature. The MeCN: PBS (9:1 v/v, PBS buffer, pH 7.4) including S2O32-, SO32-, ClO-,
mixture was stirred for 24 hours. Then solvent was evaporated ClO2-, ClO4-, F-, HSO3-, NO2-, S2O42-, S2O52-, SCN-, Cys, GSH, Hcy. As
under reduced pressure and resulted residue was subjected to falsh shown in Fig. S2, obvious change to the fluorescence intensity of
column chromatography for purification. Compound Flu-N3 was Flu-N3 solution was only observed upon reaction with H2S, the
obtained as a red solid in 50% yield. m.p. 116.7-118.5℃. Elemental addition of other relevant analytes just caused slight fluorescence
Analysis: Found C, 66.8; H, 4.6; N, 11.1%. Molecular formula: change to the Flu-N3 solution. It is a bonus that the probe Flu-N3
C
35H25N5O7, requires: C, 66.9; H, 4.1; N, 11.2%. 1H NMR (600 MHz, exhibits excellent selectivity toward H2S (140 μM) over Hcy (1.0
DMSO-d6): δ 10.12 (s, 1H), 8.00 (d, J = 7.2 Hz, 1H), 7.82-7.67 (m, 3H), mM), Cys (1.0 mM), and GSH (1.0 mM). All these experimental
7.22 (dd, J1 = 60.9, J2=23.6 Hz, 3H), 6.85 (s, 1H), 6.77 (s, 1H), 6.69 (s, results indicated that Flu-N3 was highly selective for H2S among the
1H), 6.55 (d, J = 16.8 Hz, 3H), 6.36 (s, 1H), 4.05 (d, J = 49.9 Hz, 2H), various analytes in abiotic systems.
3.72 (s, 2H), 3.62 (s, 2H), 3.40 (s, 2H), 3.30-3.23 (m, 2H). 13C NMR
(151 MHz, DMSO-d6): δ 169.2, 167.3, 160.0，159.9, 152.9, 152.6,
152.4, 152.3, 151.9, 143.6, 136.1, 130.6, 129.6, 128.9, 128.1, 125.1,
124.5, 117.1, 116.0, 115.2, 113.1, 112.4, 112.3, 110.1, 109.4, 107.3,
102.7, 101.8, 83.7, 47.8, 47.5, 45.2, 41.3, 40.9, 36.53 (Fig. S3). MS
(ESI): Calcd for [C35H25N5O7+H]+: 628.1754, found: m/z 628.1851
(Fig. S4).
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Scheme 2 Possible reactions of Flu-N3 with H2S
advantage for Förster resonance energy transfer (FRET) in the
coumarin-Fludol scaffold, which reducing the π-π stacking between
dyes. When the probe is treated with H2S, as we expected Flu-N3
would be converted into Flu-NH2, H2S would exactly react with the
azide moiety to produce amino group and release green
fluorescence of fluorescein. The results above also agreed with the
mass spectral peak of the reaction product at m/z 602.1936
correspond to [M+H]+ for compound Flu-NH2 (Fig. S6)
Proposed mechanism
The fluorescence turn-on mechanism of Flu-N3 is also described in
Scheme 2. Flu-N3 is non-fluorescent due to an effective
intramolecular spirocyclization and intramolecular charge transfer
(ICT) effect between Azidation of coumarin and Fludol. Meanwhile,
two fluorophores were linked by a rigid piperazine and provided an
Fig. 1 (a) Fluorescence response of Flu-N3 (5 μM) upon the addition of H2S (0-140 μM) in MeCN: PBS (9:1 v/v, PBS buffer, pH 7.4) solution.
Inset: The color changes of Flu-N3 without and with addition of H2S under a 365 nm lamp illumination; (b) Dependence of fluorescence
intensity at 538 nm on H2S concentration; (c) Effect of pH on the fluorescence intensity of Flu-N3 (5 μM) at 538 nm in MeCN: PBS (9:1 v/v,
PBS buffer, pH 7.4) solution with or without H2S (140 μM). λex =470 nm. Slit width=5 nm/2.5 nm; (d) Time profile of Flu-N3 (5 μM) in
presence of H2S (10.0 equiv.) at 538 nm in MeCN: PBS (9:1 v/v, PBS buffer, pH 7.4) solution.
treated with 100 μM H2S for 30 min and incubated with probe Flu-
Cell experiments
N3 (5.0 μM) for another 15 min, an obvious fluorescence increase in
We proceeded to perform the cellular imaging experiments to the green channel (Fig. 2c) were observed. To detect endogenously
further explore whether the probe could detect endogenous and generated H2S in living cells, the cells were stimulated with sodium
exogenous H2S. Here, HepG-2 cells were chosen for intracellular nitroprusside (SNP) for 30 min. SNP stimulated cells were then
fluorescence imaging experiments. As shown in Fig. 2b, we incubated with Flu -N3 for 20 min and cell imaging was performed
incubated HepG-2 cells with Flu-N3 (5.0 μM) for 30 min at 37℃ and (Fig. 2a). The cell imaging results showed an enhancement in
observed a weak fluorescence signal. Furthermore, the cells were fluorescence intensity is due to the fact that SNP treatment
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37
℃. (c): cells were treated with Flu-N3 (5.0 μM) for 20 min and
stimulated cells to generate H2S. In short, endogenous H2S
concentration was not large enough to enhance fluorescence when
untreated with SNP. From the above results, we propose that Flu-
N3 could be a good candidate for detecting H2S in living cells.
then incubated with H2S (100 μM) for 20 min. Images were taken
λex= 488 nm and λem range 500-550 nm.
Moreover, the CCK-8 method with Flu-N3 was carried out to
evaluate its cytotoxicity. As illustrated in Fig. S6, the result of the
experiments showed Flu-N3 exhibited good biocompatibility and
low cytotoxicity of Flu-N3 towards HepG-2 cells at a concentration
of 50 µM (81.5 % viability).
Fluorescence imaging of H2S in live animal
Given the results that obtained on fluorescence detection of H2S in
solution and intercellular environment, we investigated the
fluorescence imaging applicability of the probe Flu-N3 for visualizing
H2S in mice in vivo. The balb/c mice were fed commercial mice
chow in individual stainless steel cages and left freely wandering in
their housing for two weeks with 12 h dark/light cycles for
Fig. 3 Fluorescence images of Flu-N3 responding to exogenous H2S
in nude mice. (a) Nude mice without any treatment, (b) 50 μM of
Flu-N3 was subcutaneously injected only, (c-h) 50 μM H2S were
subcutaneous injected for 0, 5, 15, 30, 45, 60 min respectively.
acclimatization before the experiment. Living animal imaging Images were taken using an excitation laser of 475 nm and an
emission filter of 520±5 nm.
system is used in mice imaging. After all the preparatory work has
been completed, the mice were in anesthesia by 100 μL
pentobarbital intraperitoneal injection. As the Fig. 3 shows, the
mice will be injected in the abdomen with 50 μM of Flu-N3, H2S was
carefully injected to the same location. Then, the fluorescence
images were recorded at different period of time (0, 5, 15, 30, 45,
60 min). Comparing with the fluorescence of Flu-N3, the fluorescent
intensity increased steadily in a time range of 30 min was observed
When H2S was added. Though the probe can be used to in vivo
imaging of exogenous H2S, near-infrared probes should be more
Conclusions
In summary, we have developed an FRET-based turn on probe Flu-
N3 for sensing H2S, which was composed of 7-amino-4-
methylcoumarin (energy donor) and fluorescein (energy acceptor).
The addition of H2S to the solution of Flu-N3 resulted in a clearly
observable enhancement (50-fold) of emission intensity at 538 nm
whereas the addition of Cys, Hcy and GSH induced weak
fluorescence enhancement. Flu-N3 exhibited outstanding properties,
such as high sensitivity and selectivity (detection limit of 0.031 μM),
fast response time (within 8 s) and low toxicity. Notably, Flu-N3 was
further used to image H2S in living cells and mice successfully. The
excellent properties of Flu-N3 make it have great potential in
biological applications.
favorable for endogenous H2S imaging in vivo45
.
Acknowledgements
We thank the National Natural Science Foundation of China
(No. 21672131, 21775096, 21705102), Talents Support Program
of Shanxi Province (2014401), Shanxi Province Foundation for
Returness (2017-026), Shanxi Province Science Foundation for
Youths (201701D221061) and Scientific Instrument Center of
Shanxi University, Institute of Pharmacology and Toxicology
Academy of Military Medical Sciences PLA, Peop. Rep. China. We
also thank Dr Y. S. Yao for his assistance in in vivo imaging.
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Fig. 2 Cell images of HepG-2 cell lines. (a): cells were stimulated
with SNP for 30 min at 37 and then treated with Flu-N3 (5.0 μM)
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℃
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Journal of Materials Chemistry B
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DOI: 10.1039/C8TB01070F
ARTICLE
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6 | J. Name., 2012, 00, 1-3
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Journal of Materials Chemistry B
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DOI: 10.1039/C8TB01070F
A novel FRET-based fluorescent probe for the selective
detection of hydrogen sulfide (H2S) and its application for
bioimaging
probe
probe+H2S 30 min
60 min
ABSTRACT: Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling
molecule with multiple functions in many physiological and pathological processes.
Herein, our group developed a novel fluorescent probe (Flu-N3) for H2S on the basis
of 7-amino-4-methylcoumarin and fluorescein FRET system. Adding H2S to the
solution of Flu-N3 resulted in a clearly observable fluorescence enhancement
(50-fold). This probe showed high sensitivity and selectivity towards H2S over other
reactive species and exhibits detection limit (0.031 μM), fast response time (within 8
s). Moreover, Flu-N3 displayed little toxicity to HepG-2 cells and were successfully
applied to image exogenous and endogenous H2S in living cells and nude mouse.