A highly selective turn-on near-infrared fluorescent probe for hydrogen sulfide detection and imaging in living cells

Article abstract of DOI:10.1039/c2cc36088h

We have described a turn-on near-infrared fluorescent probe Cy-NO ₂ based on nitro group reduction for intracellular H₂S detection. The probe employs cyanine dye as a fluorophore, and is equipped with a nitro group as a fluorescent modulator. It is readily employed for assessing intracellular H₂S level changes, and confocal imaging is achieved successfully.

Full text of DOI:10.1039/c2cc36088h

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Highly selective turn-on near-infrared fluorescent probe for hydrogen
sulfide detection and imaging in living cells
Rui Wang, Fabiao Yu, Lingxin Chen*, Hao Chen, Linjie Wang and Weiwei Zhang
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
autoꢀfluorescence in living systems.14 Therefore, it is necessary to
We have described a turn-on near-infrared fluorescent probe
50 develop new type fluorescent probes that can be used for H2S
detection under physiological conditions, preferably with
emission locates in the NIR region. On the other hand, it is
reported that nitro group can be reduced by Na2S to produce the
corresponding amino group under mild conditions,15 which
55 inspires us to design and synthesize a new type fluorescent probe
containing a nitro group for H2S detection in cells. Herein, we
present a new NIR fluorescent probe CyꢀNO2 for detection of
H2S in living cells.
As an overall strategy, the heptamethine cyanine was
60 particularly chosen as fluorophore, because of its high molar
absorption coefficient and NIR emission, along with other
members of the cyanine family, which have been used for a
variety of fluorescence sensing applications. It is speculated that
the fluorescence properties of heptamethine cyanine dye could be
65 modulated via a photoinduced electron transfer (PET) process
from the excited fluorophore to a strong electronꢀwithdrawing
group (donorꢀexcited PET; dꢀPET).16 Therefore, mꢀnitrophenol
was selected as modulator for probe CyꢀNO2. After being
equipped with mꢀnitrophenol, the fluorescence of the cyanine
70 platform was quenched by electron transfer process between the
modulator and the fluorophore. As expected, the nitro group
could be reduced to amino group under physiological conditions,
triggering an increase of the fluorescence emission, thereby
allowing the formation of a ‘‘turnꢀon’’ fluorescent probe for H2S
75 detection. The structure and the proposed mechanism of
fluorescence probe CyꢀNO2 for H2S detection are shown in
Scheme 1.
Cy-NO2 based on nitro group reduction for intracellular H2S
detection. The probe employs cyanine dye as fluorophore, and
is equipped with nitro group as fluorescent modulator. It is
10 readily employed for assessing intracellular H2S level
changes, and confocal imaging is achieved successfully.
Hydrogen sulfide (H2S) is well known for its unpleasant rotten
eggs smell, which was considered as a toxic gas during a long
time. However, many studies on endogenous H2S have
15 demonstrated that hydrogen sulfide is the most recent endogenous
gasotransmitter along with nitric oxide (NO) and carbon
monoxide (CO).1 Endogenous hydrogen sulfide is biosynthesized
by at least three separate enzymes: cystathionineꢀβꢀsythase
(CBS),2 cystathionineꢀγꢀlyase (CSE),3 and 3ꢀmercaptopyruvate
20 sulfur transferase (3ꢀMST).4 It is reported that mitochondrial
sulfide quinoneoxidoreductase (SQR) and persulfidedioxygenase
(ETHEI) are involved in the consumption of H2S.5 H2S also
appears to be the regulation of inflammation.6 Furthermore, some
researches also have indicated that the hydrogen sulfide levels are
25 related with the Down syndrome and Alzheimer’s disease.7
The interests in understanding the physiological and
pathological functions of hydrogen sulfide have been increasing
steadily. A number of sensitive and selective methods have been
developed for the detection of H2S including colorimetry,8
30 electrochemical analysis,9 and gas chromatography.10 However,
these techniques often require postꢀmortem processing and/or
destruction of tissues or cells,11 and hence not suitable for the
analysis of endogenous H2S in realꢀtime. Compared with these
biological detection technologies, fluorescence method provides
35 greater sensitivity, less invasiveness, and more convenience, and
offers high sensitivity as well as realꢀtime imaging.12 Recently,
there has been an explosive increase in the number of fluorescent
probes for H2S.13 In general, these probes are divided into three
types: exploiting a unique reduction reaction between azide group
40 and H2S;13aꢀc,e,15c taking advantage of the double nucleophilic
character of H2S;13f,g,i utilizing copperꢀcentered coordination
complex in which Cu2+ can be released by binding H2S.13h,j
However, most of these fluorescent probes emit in the ultraviolet
or visible region, which can be interfered by cell autoꢀ
45 fluorescence easily. In contrast, long wavelength probes with
emission in the nearꢀinfrared (NIR) region are optimal for
biological imaging applications due to minimal photo damage to
biological samples and minimum interference from background
To verify whether the probe was suitable for the physiological
detection, we evaluated the effect of pH on the fluorescence of
80 the probe.17 As shown in Fig. S1, the pH of the medium had
hardly any effect on the fluorescence of CyꢀNO2 and its reduced
product CyꢀNH2 over the pH range of 4.2–8.2. Therefore, the
probe was expected to work well under physiological conditions
(40 mM HEPES, pH 7.4).
85
Scheme 1 Structures of CyꢀNO2, and its reduced product CyꢀNH2, and the
proposed mechanism of fluorescence probe CyꢀNO2 for H2S detection.
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The absorption and fluorescence spectra of the probe were
examined under simulated physiological conditions (40 mM
To check whether the probe might turn on fluorescence upon
incubation with other biological species over time, the probe’s
HEPES, pH 7.4, 10 ꢁM CyꢀNO2). In the absence of H2S, CyꢀNO2 55 time courses with various species for 90 min were measured. As
exhibited λmax for absorption and emission at 755 nm and 809 nm,
respectively, both of which lie in the NIR region (Fig. 1). There is
significant decrease in the absorption spectrum upon addition of
H2S (Fig. 1a). We attributed the phenomenon of absorption
demonstrated in Fig. 2, CyꢀNO2 can selectively respond to H2S
and avoid the interference of other relevant species. The
experimental results displayed that the selective reduction of Cyꢀ
NO2 to CyꢀNH2 could be used for the detection of H2S under
5
decrease to the difference of molar absorptivity between CyꢀNO2 60 simulated physiological conditions.
and CyꢀNH2. We calculated that the molar absorptivity of Cyꢀ
a
5x105
4x105
3x105
2x105
5x105
4x105
3x105
2x105
10 NH2 (ε755nm = 3.4×104 Mꢀ1cmꢀ1) was much lower than that of Cyꢀ
NO2 (ε755nm = 8.1×104 Mꢀ1cmꢀ1). Therefore, the absorption
spectrum shows a significant decrease after addition of H2S.
Upon addition different concentrations of H2S to the buffer
solution which contained 10 ꢁM CyꢀNO2, the fluorescence
15 intensity increased by ~ 12.7 fold and quantum yield increased
from 0.05 to 0.11. Interestingly, the emission spectrum showed a
little blue shift in λmax from 809 nm to 789 nm (Fig. 1b). This
phenomenon was induced by the pushꢀpull electronic effect of the
functional moiety.18 Upon reduction by H2S, the probe contained
20 an electronꢀdonating group (PhꢀNH2 moiety) instead of electronꢀ
withdrawing group (PhꢀNO2 moiety). Therefore, the fluorescence
spectrum underwent a slight blue shift.
b
Cl-
I-
NO2-
Br-
N3-
HCO3-
HPO42-
HSO3-
OAc-
Lipoic acid
Citrate
NO
SCN-
H2O2
2-
SO4
2-
HOCl
S-nitroasoglutathione
2-ME
Cysteine
SO3
S2O32-
S2O52-
S2O4
2-
1x105
0
Glutathione
Na2S
1x105
0
Na2S
20
40
60
80
20
40
60
80
Time (min)
Time (min)
Fig. 2 Fluorescence responses and time courses of 10 ꢁM CyꢀNO2 to
biologically relevant species for 90 min. (a) Anions and RSS. (b) RNS
and ROS. Data shown: Na2S, HSO3 , SO32ꢀ, S2O32ꢀ, S2O42ꢀ, S2O52ꢀ, NO,
ꢀ
65 H2O2 at 300 ꢁM. Clꢀ, Brꢀ, Iꢀ, N3 , NO2 , OAcꢀ, Lipoic acid, citrate,
glutathione, cysteine, Sꢀnitrosoglutathione and 2ꢀmercaptoethanol (2ꢀME)
at 1 mM.
ꢀ
ꢀ
4x105
Buffer slution
Bovine serum
1.0
3x105
5x105
4x105
3x105
2x105
b
M
a
350
µ
0.8
0.6
0.4
0.2
0.0
H
S
2
2x105
M
M
0
µ
0
µ
H
S
M
2
350
µ
1x105
0
1x105
0
50 100 150 200 250 300 350
[H S]
µ
M
0
750
2
775
800
825
850
600
650
700
750
800
Wavelength (nm)
Fig. 3 The relationship between fluorescence intensity and H2S conꢀ
70 centration in buffer solution (40 mM HEPES, pH 7.4) (blue) and
commercial fetal bovine serum (red). The concentration of fetal bovine
serum in the solution is 15% (v/v). 10 ꢁM CyꢀNO2 H2S, 0–350 ꢁM. The
fluorescence intensity were acquired at 37 oC after incubation of the probe
with H2S for 60 min (λex = 755 nm, λem = 789 nm).
Wavelength (nm)
Fig. 1 (a) Absorption and (b) Fluorescence spectra of probe CyꢀNO2 (10
25 ꢁM) treated with various concentrations of H2S: 0, 50, 100, 150, 200, 250,
300, 350 ꢁM. Spectra were acquired in 40 mM HEPES, pH 7.4, at 37 C
after incubation of the probe with H2S for 60 min with excitation at 755
nm and emission ranging from 755 to 900 nm.
o
75
We explored the ability of the probe to quantify H2S in buffer
solution and fetal bovine serum sample. To evaluate the ability of
CyꢀNO2 in the detection of H2S concentration, the probe was
treated with different concentrations of H2S (0–350 ꢁM). The
final concentration of the probe was maintained at 10 ꢁM. For the
During the experiments, interestingly, we found that the
30 reaction rate between CyꢀNO2 and H2S was severely affected by
the reaction temperature. In order to optimize the reaction
temperature, we tested the effect of temperature on the reaction
rate. The probe (10 ꢁM) and 350 ꢁM Na2S were equilibrated in
different temperature, and the fluorescence intensity was acquired
35 in 40 mM HEPES (pH 7.4) with emission at 789 nm. As shown
in Fig. S2, the reaction was very slow under room temperature.
The fluorescence intensity could reach saturation within 40 min
80 purpose of accurate analysis, a linear relationship is always
necessary. Herein, we obtained a calibration curve between
fluorescence emission intensity and H2S concentration. As shown
in Fig. 3, the fluorescence signal was linearly related to the
concentration of H2S under the given concentration range. The
85 regression equation was F789nm = 1111×[H2S] + 19632, with r =
0.992. Next, we used fetal bovine serum to investigate whether
our probe could detect H2S in complex biological samples. We
prepared the fetal bovine serum samples containing H2S in
different concentration (0ꢀ350 ꢁM). Following the above method,
90 we got another calibration curve between fluorescence emission
intensity and H2S concentration. The regression equation was F789
nm= 1073× [H2S] + 18012, with r = 0.996. These results indicated
that CyꢀNO2 could qualitatively and quantitatively detect H2S in
complex biological systems.
o
under 60 C, but the temperature was too high to detect H2S in
living cells. Excitedly, the probe exhibited almost the same
o
40 reaction rate under 37 C and 45 oC conditions. The fluorescence
o
intensity could reach saturation within 60 min. Thus, 37 C was
chosen as the optimal reaction temperature.
To verify whether there is fluorescence response to other
biological analytes or not, CyꢀNO2 (10 ꢁM) was treated with
45 various biologically relevant analytes in HEPES buffer (40 mM,
pH 7.4). Overall 24 biological species were screened. As shown
in Fig. 2, CyꢀNO2 showed selective response for H2S over
reactive oxygen species (ROS), reactive nitrogen species (RNS)
and anions. Of all the tested reactive sulfide species (RSS), only
95
Having demonstrated the selectivity and sensitivity of CyꢀNO2
for H2S in vitro, we next established the ability of CyꢀNO2 to
track H2S level changes in living cells by using a RAW264.7 cell
model. RAW264.7 cells were incubated with 10 ꢁM CyꢀNO2 for
ꢀ
50 S2O3 , S2O42ꢀ, S2O52ꢀ, glutathione and cysteine gave limited
increase in the fluorescence intensity. However, the intensity of
the fluorescence increase was far weaker than that caused by H2S.
2
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o
10 min at 37 C, and then washed with physiological saline to
aqueous solution and living cells. Our probe CyꢀNO2 shows
remarkable turn on fluorescence for H2S compared to other
remove excess CyꢀNO2. The treated cells were incubated with
buffer containing different concentrations of Na2S (50, 150, 250 50 biologically relevant species. Confocal microscopy images
and 350 ꢁM). After incubation for 30 min in RPMI 1640 Medium
at 37 oC, the cells were washed with physiological saline to
remove the excess Na2S, and then the cells were imaged by a
confocal fluorescence microscope. The fluorescence was
collected at 650–800 nm on excitation at 635 nm. As a control,
the cells not treated with Na2S were also imaged. The control
10 experiments showed faint fluorescence (Fig. 4a), but those treated
with various concentrations of Na2S displayed different
fluorescence intensities. The confocal fluorescence images grew
brighter as the concentrations of Na2S increasing from 50 to 350
ꢁM (Fig. 4bꢀe). The cell body regions in the visual field (Fig. 4a–
15 e) were selected as the region of interest (ROI), and the average
fluorescence intensity was determined via confocal laserꢀ
scanning microscopy with various H2S concentrations (Fig.
4f).The results suggested that CyꢀNO2 had good membrane
permeability, and these data also established that CyꢀNO2 could
20 respond to intracellular H2S level changes within living cells.
indicate that our probe can detect the level changes of H2S in
living cells. We anticipate that the fluorescent probe will be of
great benefit for biomedical researchers to investigate the effects
of H2S in biological systems.
This work was financially supported by the National Natural
Science Foundation of China (21275158, 31200041), the
Innovation Projects of the Chinese Academy of Sciences
(KZCX2ꢀEWꢀ206), and the 100 Talents Program of the Chinese
Academy of Sciences.
5
55
60 Notes and references
Key Laboratory of Coastal Zone Environmental Processes, Yantai
Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai
264003, P. R. China Fax: (+ 86) 535ꢀ2109130 Eꢀmail: lxchen@yic.ac.cn
†Electronic Supplementary Information (ESI) available: general methods,
65 synthesis and characterization of compounds, effect of pH and
temperature, MTT and brightꢀfield confocal images. See
DOI: 10.1039/b000000x/
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Fig. 4 Confocal fluorescence images of living RAW264.7 cells incubated
with various concentrations of Na2S. RAW264.7 cells loaded with 10 ꢁM
CyꢀNO2 and Na2S for 30 min. (a) Control, (b) 50 ꢁM, (c) 150 ꢁM, (d) 250
25 ꢁM, and (e) 350 ꢁM Na2S. Scale bar is 20 ꢁm. Incubation was performed
at 37 oC under a humidified atmosphere containing 5% CO2. The
fluorescence was collected at 650–800 nm on excitation at 635 nm. (f)
The relationship between average fluorescence intensity and added
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35
We also applied CyꢀNO2 to the subcellular locations of H2S in
the RAW264.7 cells using confocal fluorescence microscopy.
The cells with the same conditions used in Fig. 4e, were coꢀ
staining with CyꢀNO2 (10 ꢁM) and Janus Green B (JGB, 1 ꢁM)
for 15 min. Fig.S5 further revealed the location of the probe in
100
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40 the cytoplasm of these living RAW264.7 cells. We also employed
the Pearson correlation coefficient (r) which was used to quantify
the degree of colocalization between fluorophores19 to further
reveal the subcellular locations of CyꢀNO2. By using Olympus
software, we obtained the value of CyꢀNO2 with JGB r = 0.85,
45 revealing that CyꢀNO2 primarily locates in the cytoplasm.
In summary, we have developed a new NIR fluorescent probe
that exhibits high selectivity and sensitivity for H2S both in
105
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