A fast-response red shifted fluorescent probe for detection of H2S in living cells

Article abstract of DOI:10.3390/molecules25030437

Near-infrared (NIR) fluorescent probes are attractive tools for bioimaging applications because of their low auto-fluorescence interference, minimal damage to living samples, and deep tissue penetration. H₂S is a gaseous signaling molecule that is involved in redox homeostasis and numerous biological processes in vivo. To this end, we have developed a new red shifted fluorescent probe 1 to detect physiological H₂S in live cells. The probe 1 is based on a rhodamine derivative as the red shifted fluorophore and the thiolysis of 7-nitro 1,2,3-benzoxadiazole (NBD) amine as the H₂S receptor. The probe 1 displays fast fluorescent enhancement at 660 nm (about 10-fold turn-ons, k₂ = 29.8 M^?1s^?1) after reacting with H₂S in buffer (pH 7.4), and the fluorescence quantum yield of the activated red shifted product can reach 0.29. The probe 1 also exhibits high selectivity and sensitivity towards H₂S. Moreover, 1 is cell-membrane-permeable and mitochondria-targeting, and can be used for imaging of endogenous H₂S in living cells. We believe that this red shifted fluorescent probe can be a useful tool for studies of H₂S biology.

Full text of DOI:10.3390/molecules25030437

molecules
Article
A Fast-Response Red Shifted Fluorescent Probe for
Detection of H2S in Living Cells
Ismail Ismail 1 , Zhuoyue Chen 2, Xiuru Ji 3, Lu Sun 3, Long Yi 2,4,* and Zhen Xi 1,4,
*
1
State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, National
Engineering Research Center of Pesticide (Tianjin), College of Chemistry, Nankai University, Tianjin 300071,
China; ismailics86@gmail.com
2
3
Beijing Key Laboratory of Bioprocess and College of Chemical Engineering, Beijing University of Chemical
Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China;
2018210060@mail.buct.edu.cn
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and
Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China;
jxry1217@tmu.edu.cn (X.J.); sunlu@tmu.edu.cn (L.S.)
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China
Correspondence: yilong@mail.buct.edu.cn (L.Y.); zhenxi@nankai.edu.cn (Z.X.); Tel.: +86 022-23504782 (Z.X.);
Fax: +86 022-23500952 (Z.X.)
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Maged Henary
Received: 11 November 2019; Accepted: 27 December 2019; Published: 21 January 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Near-infrared (NIR) fluorescent probes are attractive tools for bioimaging applications
because of their low auto-fluorescence interference, minimal damage to living samples, and deep
tissue penetration. H2S is a gaseous signaling molecule that is involved in redox homeostasis and
numerous biological processes in vivo. To this end, we have developed a new red shifted fluorescent
probe
as the red shifted fluorophore and the thiolysis of 7-nitro 1,2,3-benzoxadiazole (NBD) amine as the
H2S receptor. The probe displays fast fluorescent enhancement at 660 nm (about 10-fold turn-ons,
k2 = 29.8 M−1s−1) after reacting with H2S in buffer (pH 7.4), and the fluorescence quantum yield of the
activated red shifted product can reach 0.29. The probe also exhibits high selectivity and sensitivity
towards H2S. Moreover, is cell-membrane-permeable and mitochondria-targeting, and can be used
1 to detect physiological H2S in live cells. The probe 1 is based on a rhodamine derivative
1
1
1
for imaging of endogenous H2S in living cells. We believe that this red shifted fluorescent probe can
be a useful tool for studies of H2S biology.
Keywords: fluorescent probe; red shifted; H2S; bioimaging
1. Introduction
Recently biological reports have demonstrated the important role of H2S, and the results suggested
that endogenously-produced hydrogen sulfide (H2S) has been marked as gasotransmitter, allowing the
regulation of numerous important physiological functions including; cardiovascular, gastrointestinal,
endocrine, nervous, and immune systems [
enzymatically from L-cysteine (Cys) by means of three distinctive enzymatic pathways; cystathionine
-lyase (CSE), cystathionine -synthetase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST) [ ].
1–3]. Generally, endogenous H2S can be produced
γ
β
4
The interest in the molecular mechanisms of H2S associated with physiology and pathology was
sparked out of its recognition as a vital signaling molecule. However, the abnormal levels of H2S
production lead to number of different human diseases including; diabetes [
liver cirrhosis [ ], and the symptoms of Down’s syndrome [ 10]. As an important role played by H2S
in tumor biology, it is proposed that the both production and inhibition of H2S concentration beyond a
5], Alzheimer’s disease [6]
7
8–
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threshold level could exert anticancer effects [10
,11]. While in plants, the growth and development,
seed germination, and stress tolerance including cross-adaptation are regulated by H2S [12–14]. H2S
also plays important role in microorganisms [15]. Due to its wide distribution in all organisms,
the physiological characters of H2S and the precise mechanisms by which H2S may involve in vivo
still remain largely unexplored. Therefore, adequate tools (H2S probes or donors) are necessary to
further explore H2S biology [16
been successfully developed [22
still rare [16 18], especially for the imaging of H2S-related diseases including cancers [22]. Due to
–
21]. For cellular H2S detection, various fluorescence probes have
–
38]. However, H2S fluorescence probes for in vivo bioimaging are
–
the potential limitations of fluorescent probes in deep tissue penetration, photodamage to biological
samples, and background auto-fluorescence in living systems, it is crucial to develop probes associated
with long wavelength emission especially in the red shifted region [39–42].
Recently, numbers of red shifted and NIR fluorescent dyes have been developed [43
Consequently, various NIR fluorescent probes have been designed on the basis of different organic
reaction scenario [ 52 58]. Among them, we as well as others discovered a reaction of H2S-specifc
thiolysis of 7-nitro 1,2,3-benzoxadiazole (NBD) amines [33 58 59] to detect millimolar H2S in a long
–51].
8, –
,
,
range wavelength. Herein, this reaction strategy is further employed for the development of a new red
shifted fluorescent probe 1 (Scheme 1).
Scheme 1. (A) The thiolysis of 7-nitro 1,2,3-benzoxadiazole (NBD) amine reaction for H2S fluorescence
probe. (B) Chemical structures of selected NBD-based H2S probes.
NIR dyes, including cyanine (Cy), are considered the classic NIR fluorescent dyes [60]. However,
due to their flexible molecular structure, some of NIR fluorescent dyes accompanied some short
comings for example, small Stokes shift, limited fluorescence quantum yield, low photo-stability lying,
and high occupied molecular orbital (HOMO) energy levels [61
strongly affect the fluorescence signals due to the high background signal, which in turn result in
low contrast for bioimaging [63 66]. In 2017, we developed a Cy-NBD probe (Scheme 1) which has
,62]. Such photo-physical properties
–
the limitation of low quantum yield after H2S activation [58]. On the other side, classical rhodamine
dyes contributed much in the field of biomolecular detection and biomedical imaging because of its
magnificent photophysical and chemical properties [30,67–71]. Due to limited π-conjugated system of
xanthene core derivatives, such as rhodamine B, rhodamine 6G, and rhodamine 123 have their emission
wavelengths in the visible region (<600 nm). Recently, significant advancements have been made in the
improvement of rhodamines-based fluorescent dyes with extended the
long emission wavelength, high fluorescence quantum yield, and outstanding photostability [70
Herein, we report the development of an extended -conjugated rhodamine-NBD based probe
π-conjugated system possessing
–75].
π
1
for

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the highly selective imaging of endogenous H2S in a red shifted region [70
,
71]. The probe is high
selectivity towards H2S among other biothiols, with fluorescence emission in the red shifted region
(>660 nm) and high fluorescence quantum yield (0.29) after H2S activation. The probe is successfully
used for bioimaging of endogenous H2S in living cells.
2. Results and Discussion
2.1. Synthesis of 1
Probe
procedure described in the literature [76], 6-(dimethylamino)-3,4-dihydronaphthalen-1 (2H)-one
first synthesized from commercially available 6-amino-3,4-dihydronaphthalen-1(2H)-one, which was
then transformed to . Finally, the probe in 79% yield, was prepared by the coupling of compound
with NBD-piperazine. The facile and economic synthesis is important for the wide use of the probe.
The structure of compound
was confirmed by 1H NMR, 13C NMR, and high resolution mass spectrum
(HRMS). The spectra (Figure S8) are included in the Supplemental Information.
1
was constructed using a three-step route with a good yield (Scheme 2). By using the
2
was
3
1
3
1
Scheme 2. Synthetic route for probe
1
and its reaction with H2S. Reagents and conditions:
) 2-(4-diethylamino-2-hydroxybenzoyl)-benzoic
) NBD-piperazine, HATU, DIPEA, DMF, 79%.
a)
6-aminotetralone, CH3I, K2CO3, DMF, 0 ◦C, 24 h, 53%.
b
c
acid, N-substituent ketone, H2SO4 0–100 ◦C, 2 h, 80%.
2.2. UV-Vis and Fluorescence Response of 1 towards H2S
With the probe in hand, we first tested the solubility of
verified its good solubility up to 20 M (Figure S1). Further, we tested the optical properties of
the absorbance and emission profiles are illustrated in Figure 1. As shown in Figure 1A,
1
in buffer solution. The linearity of
1
µ
1, and
1
displayed UV
absorbance maxima at 620 nm and 500 nm, which are assigned to the rhodamine and NBD absorbance
respectively. Since Na2S is a well-known inorganic H2S donor that is widely employed in the study of
H2S effects on physiology, we used it as a H2S equivalent [77]. When reacted with H2S, the increase
in intensity of absorbance peaks appeared between 600 nm and 520 nm, which could be assigned to
the yielding of
4 and NBD-SH. The reaction between 1 and 500 µM H2S in PBS buffer (50 mM, pH
7.4) finished within 5 min. Furthermore, such thiolysis reaction was characterized by NMR with the
formation of NBD-SH peaks (Figure S7) and HRMS with the production of peak at 535.3070 (calculated
value for [4]+: 535.3068) (Figure S2).

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Figure 1.
(
A) Time-dependent absorbance spectra of
1
(5
µM) and H2S (500
µM) in PBS (50 mM, pH
7.4, containing 10% DMSO). (
B) Time-dependent fluorescence spectra (excitation = 620 nm) of
1
(2 µM)
and H2S (200 µM).
The probe
indicating that fluorescence in
process (PET) effect from the NBD moiety [58]. When
1
showed weak fluorescence (quantum yield
could be mainly quenched by the photoinduced electron transfer
reacted with H2S, an excellent fluorescence
φ, 0.021) upon excitation at 620 nm,
1
1
change in a red shifted range with high brightness was observed (Figure 1B) with 10-fold turn-ons at
660 nm, and the quantum yield of the red shifted product was 0.29. The absorbance and emission
data suggested the stokes shift up to 40 nm in PBS. A large Stokes shift could reduce the risk of
background fluorescence and thus avoid self-quenching and backscattering effect upon excitation.
These preliminary studies suggest the extended π-conjugated system of rhodamines provides excellent
red shifted fluorescence probes for detection in long range with high brightness.
2.3. Kinetics Studies
Reaction kinetics, as an important parameter, was investigated for the probe
1 with H2S on
account of its biological applicability under physiological conditions. To this end, the time-dependent
fluorescence at 660 nm was recorded for data analysis (Figure 2A). The pseudo-first-order rate, kobs
,
was found by fitting the data with a single exponential function. Plotting log[H2S] versus log[kobs
]
confirmed a first-order dependence in H2S (Figure 2B). The reaction rate k2 (29.8 M−1s−1) was obtained
by linear fitting of the kobs versus H2S concentration (Figure 2C). The H2S-reaction rate of 1 is faster
than our previous Rh-NBD-based probe [30], implying that such NBD-based probes can be employed
for fast detection of H2S. On the other hand, HPLC was further employed to identify the fast reaction
of
1 with H2S (Figure S4). Furthermore, fluorescent titration (Figure 3) was performed to determine
the limit of detection (LOD) of 1 for H2S as 0.27 µM by using the 3σ/k method [58].

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Figure 2.
of different concentrations of H2S in PBS (50 mM, pH 7.4, containing 10% DMSO). (
relationship of log(kobs) versus log([H2S]). ( ) The linear relationship of kobs versus H2S concentrations.
(A
) The time-dependent fluorescence intensities at 660 nm for
1
(2
µM) in the presence
B
) The linear
C
Figure 3. (A) The fluorescence changes of probe
1
(2
µM) responses to various concentrations of
H2S (0–50 M) in PBS (50 mM, pH 7.4, containing 10% DMSO). (
µ
B) The linear relationship between
concentrations of H2S and emission at 660 nm. The detection limit was calculated to be 0.27 µM.
2.4. Selectivity and Co-Interference Studies
With above promising outcomes, we further investigated the selectivity and sensitivity of probe 1.
The fluorescent “off–on” response of
1 towards biothiols was measured. Probe 1 (2 µM) was treated
with Cys, Hcy, and GSH individually (each 1 mM). As shown in Figure 4, the results showed that

Molecules 2020, 25, 437
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fluorescence intensity enhancement for analytes was nearly negligible except H2S, suggesting that
can selectively sense H2S. In order to check the interference of biothiols with coexistent H2S, we also
tested with these analytes in the presence of H2S (Figure 4). These findings suggested that all analytes
did not interfere the H2S-specific thiolysis reaction. Furthermore, pH-dependent experiments were
carried out to check whether could sense at physiological pH (Figure S5). Obviously, the fluorescence
enhancement occurred at pH 7.0–9.0, implying that
1
1
1
1
could work efficiently at physiological conditions.
Figure 4. Fluorescence intensity at 660 nm of
1
(2
µM) upon reacting with biothiols (1 mM) or H2S
(200 µM) or biothiols with coexistent H2S species in PBS buffer (pH 7.4) for 30 min.
2.5. Imaging of Probe 1 in Living Cells
Encouraged by the above results, we moved forward to study the biological applications of 1. The
cytotoxicity of the
(HUVEC) line via a standard MTT assay (Figure S6). After 24 h incubation with a varied concentration
range of from 5 M, over 85% of the cells still remained viable, implying the relatively good
biocompatibility of 1.
To examine the application potential of
as the model biological system. Briefly, the cells were incubated with
1 was evaluated firstly by using the normal human umbilical vein endothelial cell
1
µ
1
for H2S detection in living cells, HeLa cells were chosen
alone or co-incubated with
and Na2S/D-Cys for 30 min. Then, all cells were examined via the confocal microscopy. Cells
1
1
with probe
1
treatment displayed faint fluorescence (Figure 5E), while cells displayed remarkable red
and Na2S (Figure 5F). These results demonstrated that could be
used for selective imaging of exogenous H2S. For detection of endogenous production of H2S, cells
were co-incubated with D-Cys and , as D-Cys can induce H2S biosynthesis via the 3-MST pathway [ ].
Strong fluorescence was observed in cells (Figure 5G), which revealed that the endogenous production
of H2S from D-Cys could be detected by . To further confirm the detection of endogenous production of
H2S from D-Cys by , an inhibitor (aminooxyacetic acid, AOAA) was introduced to block the pathway
for H2S production from D-Cys [ ]. No obvious fluorescence was detected in the AOAA-treated cells
fluorescence in the presence of
1
1
1
4
1
1
4
(Figure 5H). These preliminary studies suggested that probe 1 could be used for visualization of H2S in
cells efficiently and selectively.

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Figure 5. Fluorescence images of probe
images of cells upon incubation with
1
(5
for H2S detection in living cells. Bright-field and fluorescence
M) for 30 min ( ); with and Na2S (100 M) ( ); with 1
1
µ
A,
E
1
µ
B
,
F
and D-Cys (100 µM) (C,G); with inhibitor (AOAA), 1 and D-Cys (100 µM) (D,H). Scale bars: 50 µm.
The probe
fluorescent co-localization assay with Mito-Tracker Green FM (a well-known mitochondria specific
dye) and probe with D-Cys was carried out. As shown in Figure 6, the green fluorescence signal
produced by Mito-Tracker Green FM and the red fluorescence signal from probe merged well in the
is a promising
1 contains a positive charge, which might be mitochondria-targeting [33]. To this end, a
1
1
cells (Figure 6C). The Pearson’s coefficient is 0.946. These data implied that the probe
1
tool for imaging of mitochondria H2S.
Figure 6. Confocal microscopy images of mitochondrion by
with (5 M), D-Cys (100
M) and commercial MitoTracker® Green FM (2.5
displayed by green channel (500–530 nm, excitation at 488 nm). ( ) The cell image displayed by
) Merged graph of fluorescence images of and
1
in living cells. Cells were co-stained
1
µ
µ
µ
M). ( ) The cell image
A
B
red channel (620–660 nm, excitation at 594 nm). (
MitoTracker® Green FM. Scale bars: 50 µm.
C
1

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3. Experimental
3.1. Materials and Methods
All chemicals and solvents used for the synthesis were purchased from commercial suppliers
and applied directly in the experiments without further purification. The progress of the reaction
was monitored by TLC on pre-coated silica plates (60F-254, 250 µm) in thickness (Merck, Darmstadt,
Germany), and spots were visualized by basic KMnO4, UV light or iodine. Merck silica gel 60
1
(100–200 mesh) was used for general column chromatography purification. H NMR and 13C NMR
spectra were recorded on a Bruker 400 spectrometer (Karlsruhe, Germany). Chemical shifts are reported
in parts per million with respect to the internal standard tetramethylsilane (Si(CH3)4 = 0.00 ppm)
1
or residual solvent peaks (CD2Cl2 = 5.32 ppm; CDCl3 = 7.26 ppm; DMSO-d6 = 2.5 ppm). H NMR
coupling constants (J) are reported in hertz (Hz), and multiplicity is indicated as the following:
s (singlet), d (doublet), t (triplet), dd (doublet of doublets), m (multiple). High-resolution mass
spectra (HRMS) were obtained on an Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS or Varian 7.0
T FTICR-MS. The UV-visible spectra were recorded on a UV-3600 UV-VIS-NIR spectrophotometer
(Shimadzu, Japan). The fluorescence study was carried out using an F-280 spectrophotometer (Tianjin
Gangdong Sci & Tech., Development. Co., Ltd. Tianjin, China).
3.2. Synthesis of 6-(dimethylamino)-3,4-Dihydronaphthalen-1(2H)-One
To a mixture of 6-aminotetralone (0.50 g, 3.1 mmol), CH3I (0.06 g, 4.6 mmol) and K2CO3 (1.3 g,
9.3 mmol) in DMF (5 mL) was stirred for 24 h at 40–45 ◦C. After completion of reaction, the mixture
was cooled to room temperature, water (10 mL) was added and the solution was extracted with EtOAc
(3
×
50 mL). The organic layers were combined, dried with anhydrous MgSO4 and the solvent was
removed under reduced pressure. The residue was purified by silica gel column chromatography
using PE (petroleum ether): EtOAc = 6:1 as the eluent to obtain pure compound (Yield: 53.0%, 0.31 g).
1H NMR (400 MHz, CDCl3)
δ 7.95 (dd, J = 8.8, 1.6 Hz, 1H), 6.62 (d, J = 8.8 Hz, 1H), 6.43 (s, 1H), 3.06 (s,
6H), 2.88 (t, J = 5.2 Hz, 2H), 2.59–2.55 (m, 2H), 2.10–2.05 (m, 2H). 13C NMR (101 MHz, CDCl3)
δ 196.9,
153.5, 146.6, 129.4, 121.9, 110.3, 109.6, 40.2, 38.9, 30.7, 23.7. HRMS [C12H16NO]+: Calcd. for [M+H]+
190.1232; found: [M+H]+ 190.1228.
3.3. Synthesis of Intermediate 3
2-(4-diethylamino-2-hydroxybenzoyl)-benzoic
acid
(0.151
g,
0.48
mmol)
and
6-(dimethylamino)-3,4-dihydronaphthalen-1(2H)-one (0.091 g, 0.48 mmol) were added to concentrated
H2SO4 (10 mL) at 0 ◦C. The mixture was stirred at 100 ◦C for 2 h. After completion of reaction the
mixture was allowed to cool at room temperature and was poured onto ice (10 g). HClO4 (1 mL)
was gently added to the solution and the resulting precipitate was filtered off and washed with cold
water. After drying, the residue was purified by silica gel column chromatography using CH2Cl2:
methanol = 20:1 as the eluent to obtain pure compound as a purple-green solid (Yield: 80.0%, 0.17 g).
1H NMR (400 MHz, DMSO-d6)
δ 13.22 (s, 1H), 8.19 (dd, J = 7.2, 2.4 Hz, 2H), 7.86 (t, J = 7.2 Hz, 1H), 7.75
(t, J = 7.6 Hz, 1H), 7.41 (d, J = 7.2 Hz, 1H), 7.24 (d, J = 1.6 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 6.93 (dd,
J = 9.2, 2.0 Hz, 1H), 6.87 (d, J = 9.2 Hz, 1H), 6.75 (s, 1H), 3.58 (q, J = 6.4 Hz, 4H), 3.18 (s, 6H), 2.93–2.80
(m, 2H), 2.49–2.35 (m, 2H), 1.19 (t, J = 6.9 Hz, 6H). 13C NMR (101 MHz, DMSO-d6)
δ 166.5, 163.4, 156.1,
155.0, 153.1, 145.1, 134.4, 133.0, 130.9, 130.0, 129.2, 129.1, 128.6, 118.4, 115.2, 114.3, 113.0, 112.0, 110.7,
96.0, 44.8, 40.0, 26.9, 23.6, 12.4. HRMS [C30H31N2O3]+: Calcd. for [M]+ 467.2329; found: [M]+ 467.2331.
3.4. Synthesis of Probe 1
Dissolved compound
(0.122 g, 0.32 mmol) and DIPEA (102
2
(0.121 g, 0.2 mmol) in 5 mL DMF, followed by the addition of HATU
L, 0.75 mmol). Stirred the solution for 5 min, NBD-piperazine
µ
(0.064 g, 0.2 mmol) was added to the solution and continue the stirring for 12 h at room temperature.
After completion of reaction DMF was removed in vacuo. The residue was purified by silica gel

Molecules 2020, 25, 437
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1
column chromatography to give dark-red solid
1 (0.12 g, 79%). H NMR (400 MHz, DMSO-d6) δ 8.50
(d, J = 8.8 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.74 (bs, 3H), 7.48 (bs, 1H), 7.20 (bs, 1H), 7.07 (bs, 2H), 6.91
(d, J = 8.0 Hz, 1H), 6.72 (s, 1H), 6.54 (d, J = 9.2 Hz, 1H), 4.22–4.14 (m, 2H), 4.03–3.83 (m, 2H), 3.82–3.46
(m, 8H), 3.19 (s, 6H), 2.98–2.78 (m, 2H), 2.62–2.45 (m, 2H), 1.16 (s, 6H). 13C NMR (101 MHz, DMSO-d6)
δ 167.0, 163.8, 156.0, 155.2, 153.0, 145.5, 145.3, 144.7, 136.2, 134.2, 131.9, 130.2, 129.5, 129.3, 129.3, 129.2,
127.6, 121.5, 119.6, 114.9, 114.3, 113.0, 112.1, 110.7, 103.4, 96.1, 49.0, 48.1, 45.6, 44.8, 40.8, 27.0, 23.9, 12.4.
HRMS [C40H40N7O5]+: Calcd. for [M]+ 698.3085; found: [M]+ 698.3090.
3.5. Procedure for Spectroscopic Studies
All spectroscopic measurements were performed in phosphate-buffered saline buffer (PBS, 50 mM,
pH 7.4, containing 10% DMSO) at room temperature. Compounds were dissolved into DMSO to
prepare the stock solutions with a concentration of 5 mM. 1–500 mM Stock solutions of Na2S in
degassed (by bubbling N2 for 30 min) PBS buffer were used as H2S source. Probes were diluted in
PBS buffer (50 mM, pH 7.4, containing 10% DMSO) to afford the final concentration of 2–5 µM. For
the selectivity experiment, different biologically relevant molecules (100 mM) were prepared as stock
solutions in degassed PBS buffer. Appropriate amount of biologically relevant species was added to
separate portions of the probe solution and mixed thoroughly. All measurements were performed in a
3 mL corvette with 2 mL solution. The reaction mixture was shaken uniformly before emission spectra
were measured.
3.6. Cell Culture and Cytotoxicity Assay
The HUVEC and HeLa cell lines were purchased from the Cell Bank of the Chinese Academy of
Sciences (Shanghai, China). And the cells were cultured in RPMI 1640 medium with 10% fetal bovine
serum and 1% penicillin/streptomycin under standard cell culture conditions at 37 ◦C in a humidified
CO2 incubator. Before the cytotoxicity assay, the HUVEC cells were transferred to the 96-well plate and
cultured for one night. After that, the culture medium was replaced with a fresh one and the HUVEC
cells were pre-incubated with probe 1 with a concentration range of 5–25 µM for 24 h. The cell viability
was then measured by the standard MTT assay.
3.7. Cell Imaging
Glass bottom dishes were added into a 24-well plate for cell imaging before cells were seeded.
Then, the HeLa cells were transferred to the 24-well plate and cultured for one night before the
experiments. After that, the culture medium was replaced with the fresh one and the cells were treated
with the desired reagents. After incubation, the HeLa cells were quickly washed with PBS three
times, and then fixed with 4% paraformaldehyde solution for 10 min. Finally, the HeLa cells were
washed with PBS and imaged using a confocal microscope (Olympus FV1000) with a 40 objective lens.
×
Emission was collected at the green channel (500–530 nm, excitation at 488 nm) and the red channel
(620–660 nm, excitation at 594 nm).
4. Conclusions
In summary, we have developed a new, extended
fluorescence probe capable of detection H2S in live cells. The probe shows a relatively large Stokes
shift (40 nm), fast response (k = 29.8 M−1s−1), and good quantum yield (ø = 0.29) after H2S activation.
Moreover, was water-soluble, cell-membrane-permeable, and had high selectivity and sensitivity for
π-conjugation rhodamine-NBD a red shifted
1
1
H2S. We believe that this red shifted range probe
the future.
1 could be a useful tool for studies of H2S biology in
Supplementary Materials: Supplementary data associated with this article can be found in the online.

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Author Contributions: Formal preparation and writing—original draft, (I.I.); writing—review and editing (L.Y.,
L.S. & Z.C.); Bioimaging (I.I., X.J. & L.S.); Supervision and funding acquisition (L.Y. & Z.X.). All authors have read
and agreed to the published version of the manuscript.
Funding: This research was supported by the NFSC (21702111).
Acknowledgments: We thank the prospective project supported by the National Natural Science Foundation of
China (21702111).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1 are available from the authors.
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