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A mitochondria-targeted red-emitting probe for

Cite this: Org. Biomol. Chem., 2019,
17, 3389
imaging hydrogen sulfide in living cells and
zebrafish†
Ismail Ismail, ‡a Dan Wang,‡a Dawei Wang,a Cuili Niu,a Haojie Huang,b
Long Yi *b,c and Zhen Xi*a,c

Received 30th December 2018,
Accepted 1st March 2019
DOI: 10.1039/c8ob03219j
rsc.li/obc
Hydrogen sulfide (H2S) is an important endogenous signaling molecule with multiple biological functions.
Based on the thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amine, herein we rationally design and syn-
thesize a new fluorescent probe 1 for the detection of mitochondrial H2S in living cells. Probe 1 displays
bright red-emitting fluorescence after H2S activation (ϕ, 0.77). 1 shows higher selectivity and sensitivity for
H2S than the red-emitting probe Rho-NBD (ChemBioChem, 2016, 17, 962). Moreover, 1 is water-soluble,
cell-membrane-permeable, of low cytotoxicity and mitochondria-targeting, and can be employed for
monitoring mitochondrial H2S in living cells.

Introduction nate the reactivity of millimolar biothiols and micromolar H2S.

Hydrogen sulfide (H2S) is an important endogenous signaling
molecule with multiple biological functions.1–6 H2S could be
enzymatically produced in vivo by three distinctive pathways
including cystathionine β-synthase (CBS), cystathionine γ-lyase
(CSE) and 3-mercaptopyruvate sulfur transferase (3-MPST)/
cysteine aminotransferase (CAT) in different organs and
tissues.7–10 Recent studies have shown that the H2S level
in vivo is correlated with numerous diseases, including the
symptoms of Alzheimer’s disease, Down’s syndrome, diabetes
and liver cirrhosis.1,11,12 Although H2S has been recognized to
be linked to numerous physiological and pathological pro-
cesses, many of its underlying molecular events in vivo remain
largely unknown and could be further explored. Therefore,
developing efficient methods for in situ and real-time detection
of biological H2S is of significant research value.
One major challenge in the development of H2S probes is
the development of a chemical reaction to effectively discrimi-
To address this challenge, H2S-triggered chemical reac-
tions,13,14 including nucleophilic addition,15–19 reduction of
azide or nitro to amine,20–25 copper precipitation,26 thiolysis of
dinitrophenyl ether,27,28 thiolysis of NBD dyes,29–43 and clea-
vage of CvC bonds,44 have been successfully employed to
develop fluorescent and/or colorimetric probes for the detec-
tion of biological H2S. Among these, the thiolysis of NBD
amine is a successful reaction for the development of H2S-
specific fluorescent probes.29,30 In such a probe, the fluo-
rescence of the fluorophore can be quenched by the NBD
moiety in aqueous solution, which will be released after the
thiolysis reaction (Scheme 1). To this end, a series of NBD-
based H2S probes were developed by our groups and
others,31–39,45 which showed emissions from the blue to near-
infrared range (Table S1†).

a
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.
E-mail: zhenxi@nankai.edu.cn
b
State Key Laboratory of Organic-Inorganic Composites and Beijing Key Laboratory of
Bioprocess, Beijing University of Chemical Technology (BUCT), Beijing 100029,
China. E-mail: yilong@mail.buct.edu.cn
c
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Tianjin 300071, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob03219j Scheme 1 (A) Thiolysis of NBD amine for fluorescence sensing of H2S.
‡ These authors contributed equally to this work. (B) Chemical structures of three NBD-based H2S probes.

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On the other hand, Grimm et al. showed that replacing the
N,N-dialkyl group with the azetidine group can greatly improve
the brightness and quantum yield of the fluorophores.46 Yang
et al. reported that blocking the twisting N,N-diethylamino
group of coumarin can improve the fluorescence properties of
the fluorophore and, in turn, enhance the sensing perform-
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ance.47,48 We find that compared with Cou-NBD, the probe
Cyclo-Cou-NBD showed significantly improved performance
with respect to response time, detection limit and fluorescence
enhancement.49 In this work, we envision that rhodamine
with the twisting-blockage of the N,N-diethylamino group can
be used to develop H2S probes with better properties than
Rho-NBD, which showed only 4.5-fold turn-on fluorescence
during H2S activation.33 Herein, we rationally design and syn-
thesize a new azetidinyl-rhodamine-NBD dyad 1 for fast
sensing of H2S in the red light range. To our delight, 1 displays
19-fold fluorescence enhancement in the presence of H2S.
Moreover, 1 can be employed for imaging biological H2S in
living cells and zebrafish.
Results and discussion
Azetidinyl-rhodamine 3 was prepared from fluorescein in a
straightforward way with good yields (Fig. 1). Probe 1 was pre-
pared by the coupling of 3 and NBD-piperazine. The structure
of 1 was confirmed by 1H NMR, 13C NMR, and high-resolution
mass spectroscopy (HRMS). Such a facile and economic syn-
thesis is important for the wide use of the probe.
With the probe in hand, we firstly tested the absorbance
and fluorescence spectra of the reaction between 1 and H2S
(using Na2S as an equivalent) in PBS buffer (20 mM, pH 7.4).
As shown in Fig. 2, 1 exhibited absorbance peaks at 565 nm
and 530 nm, which should be assigned to the rhodamine and
NBD fluorophores, respectively. After the reaction with H2S,
the absorbance peaks appeared at 565 nm and 500 nm, which
might be due to the production of 4 and NBD-SH, respectively.
Such a thiolysis reaction was also characterized by HRMS for
the peak at 479.2446 (the calculated value for [4]+: 479.2442).
Fig. 1 Synthetic route to probe 1 and its reaction with H2S. Reagents
and conditions: (a) Trifluoromethanesulfonic anhydride, pyridine,
CH2Cl2, 0 °C to room temperature, 83%; (b) Cs2CO3, Pd2dba3, XPhos,
azitidine, dioxane, 95%; and (c) NBD-piperazine, HATU, DIPEA, DMF,
59%.
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Fig. 2 (A) Time-dependent absorbance spectra of 1 (5 µM) and Na2S
(100 µM). (B) Time-dependent fluorescence spectra (excitation =
565 nm) of 1 (5 µM) and Na2S (100 µM). (C) The time-dependent fluor-
escence intensities at 585 nm for 1 (5 µM) in the presence of different
concentrations of H2S. The solid lines represent the best single-expo-
nential fitting. (D) The linear relationship of kobs versus H2S
concentrations.
HPLC studies (Fig. S1†) further suggested the fast conversion
of 1 into 4 in the presence of H2S.
As shown in Fig. 2, we tested the fluorescence spectra of the
reaction between 1 and H2S. Probe 1 showed weak fluo-
rescence (quantum yield ϕ, 0.17) upon excitation at 565 nm,
implying that the fluorescence in 1 is efficiently quenched by
NBD via the PET effect. After the reaction with H2S, a large
fluorescence change for 1 was observed, with about 19-fold
intensity increase at 585 nm. The quantum yield of 4 was
determined as ϕ = 0.77. The H2S-triggered fluorescence
enhancement folds of 1 are improved significantly compared
with those of Rho-NBD (4.5 folds),33 implying that the twist-
ing-blockage of the N,N-diethylamino group in rhodamine B
can significantly improve the performance of azetidinyl-rhoda-
mine-NBD probe 1. These results encouraged us to further
check the sensitivity and selectivity of probe 1.
The reaction kinetics of the probe with H2S is an important
parameter to examine its biological applicability on account of
the rapid catabolism of H2S under physiological conditions.
To this end, time-dependent fluorescence intensities at
585 nm for the probe in the presence of H2S were acquired for
data analysis (Fig. 2c). The pseudo-first-order rate, kobs, was
obtained by fitting the data with a single exponential function.
The reaction rate k2 (27.8 M−1 s−1) was obtained by the linear
fitting of kobs versus H2S concentrations (Fig. 2d). The H2S-reac-
tion rate of 1 is about one order of magnitude faster than that
of the o-fluorinated-azido-based probe,8 implying that such
NBD-based probes can be employed for the fast detection of
H2S.
Because the biological H2S level normally exists in the
range of nanomolar to micromolar concentrations,1–3 we pro-
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Fig. 3 The fluorescence titration of probe 1 (5 µM) toward H2S
(0–200 µM). The solid line represents the best fitting using one-site
binding of pharmacology in Origin software.
ceeded to study the fluorescence response of 1 upon increasing
the concentrations of H2S in the micromolar range (Fig. 3,
S2†). The fluorescence intensity was linearly related to
1–20 μM H2S. Specifically, the detection limit of 1 was deter-
mined to be 0.36 μM based on the 3σ/slope method, implying
that the probe is highly sensitive toward H2S. The complete
titration resulted in a formation constant of 7.1 × 104 M−1 for
H2S, implying a relatively good affinity of 1.
A major challenge for H2S detection in biological systems is
to develop a highly selective probe that exhibits notably dis-
tinctive response to micromolar H2S over millimolar biothiols
and other cellular molecules. To this end, 1 was incubated
with various biologically relevant species including reactive
sulfur species (biothiols, SO32−), reactive oxygen species (H2O2,
NaOCl, •OH and ONOO−), NaNO2, and ions. These results indi-
cated that fluorescence intensity enhancement for any tested
Fig. 4 Fluorescence intensity at 585 nm of probe 1 (5 µM) in the pres-
ence of various species (100 µM). Lane 1, GSH; 2, Hcy; 3, CH3COOH; 4,
Cys; 5, H2O2; 6, K2C2O4; 7, KF; 8, mercaptoethanol; 9, Na2SO3; 10,
NaHCO3; 11, NaI; 12, NaNO2; 13, NaOCl; 14, •OH; 15, ONOO−; 16, Na2S
in PBS ( pH 7.4) at room temperature for 30 min.
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molecule was nearly negligible except for H2S (Fig. 4), implying
that 1 can be used to selectively detect H2S in biological
samples. Additionally, we also investigated the fluorescence
response of 1 to H2S at different pH values. The results indi-
cated that the probe can be functionalized over pH ranging
from 7.0 to 9.0 (Fig. S3†).
Encouraged by the above results, we proceeded to study the
biological applications of 1. Firstly, the cytotoxicity of the
probe was evaluated using HEK293 cells by the detection of
adherent cell proliferation by varying the electrical current
(Fig. 5, S4†).50 The probe did not show obvious cytotoxicity in
the 0–10 µM range upon 24 h of incubation, implying that the
probe is biocompatible at these concentrations. An obvious
inhibition of cell proliferation was observed at 20 µM 1, and
therefore, 10 µM 1 was used in the following in vivo imaging.
We further examined whether the probe can be used to
detect H2S in living cells. HEK293A cells were treated with
probe 1 and then washed with PBS to remove excess 1. The 1-
loaded cells were incubated with H2S and subsequently
imaged using confocal fluorescence microscopy (Fig. 6). The
addition of both 1 and H2S resulted in obvious red fluo-
rescence while the cells treated with only probe 1 did not show
obvious fluorescence. Bright-field images show that cells
retained good morphology after incubation with the probe,
which further suggested the good biocompatibility of 1. To test
whether 1 could detect the endogenous production of H2S, the
cells were co-incubated with L-Cys and 1. After using L-Cys to
induce endogenous H2S production, significant fluorescence
enhancement could be observed (Fig. 6C). These preliminary
studies suggested that probe 1 could be used for the visualiza-
tion of H2S in cells efficiently and selectively.
Since the probe could react quickly with H2S, we also tested
the time-dependent L-Cys-induced H2S biogenesis in living
cells. The average fluorescence of the time-dependent images
(Fig. S5†) implies that H2S biogenesis from L-Cys occurs con-
tinuously at a relatively stable rate. Taken together, these
results indicate that probe 1 can be used for bioimaging
endogenous H2S in living cells.
Furthermore, the probe contains a positive charge, which
should be mitochondria-targeting. A fluorescence co-localiz-
Fig. 5 Concentration-dependent maximum cell index in the presence
of probe 1 (0–20 µM) for 24 h.
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Fig. 6 Fluorescence images for H2S detection in living cells using 1.
Fluorescence images of cells upon incubation with 1 (10 µM) for 30 min
(A); with 1 and Na2S (100 µM) (B); with 1 and L-Cys (100 µM) (C); (D–F) a
merged image of the bright-field and fluorescence images for (A–C),
respectively. Scale bars: 50 μm.
ation assay with Mito-Tracker Green FM (a well-known mito-
chondria-specific dye) and probe 1 with L-Cys was performed.
As shown in Fig. 7, the images in the red channel and green
channel merged very well, implying that probe 1 was well co-
localized with Mito-Tracker Green FM (Pearson’s coefficient
0.947) (Fig. 7D). The above data clarified that probe 1 is an
excellent tool for imaging mitochondrial H2S.
To further demonstrate the biological applicability of the
NBD-based probe, we examined whether it can be used to
detect H2S in living animals, and zebrafish larvae were selected
as the biological model. Firstly, the zebrafish was incubated
with 1 for 1 h, washed, and then treated with Na2S (200 μM)
Fig. 7 Confocal microscopy images of the mitochondrion in living
cells. Cells were co-stained with 1 (10 μM), L-Cys (100 μM) and commer-
cial MitoTracker® Green FM (2.5 μM). (A) Red channel (555–655 nm,
excitation at 543 nm); (B) green channel (500–530 nm, excitation at
488 nm); (C) a merged graph from fluorescence images of 1 and
MitoTracker® Green FM. Scale bars: 50 μm. (D) Intensity correlation plots
of the green channel and the red channel. Pearson’s coefficient: 0.947.
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Fig. 8 Confocal microscopy images of exogenous and L-Cys-induced
endogenous H2S detection in zebrafish larvae. (A) The zebrafish was
incubated with probe 1 (10 μM) for 1 h and imaged in the red field (left)
and bright field (right). (B) The zebrafish was incubated with probe 1
(10 μM) for 1 h, then with Na2S (200 μM) for 3 h and imaged in the red
field (left) and bright field (right). (C) The zebrafish was incubated with
probe 1 (10 μM) for 1 h, then with L-Cys (200 μM) for another 3 h, and
imaged in the red field (left) and bright field (right).
for another 3 h. The zebrafish stained with 1 alone was used as
the control group. No obvious fluorescence was observed in
the 1-loaded control group (Fig. 8A), whereas strong red fluo-
rescence was seen in the H2S-treated zebrafish (Fig. 8B). A sig-
nificant red fluorescence enhancement was also observed in
the Cys-treated zebrafish (Fig. 8C). These results imply that
probe 1 can be used to visualize H2S in vivo.
Conclusions
A new NBD-based fluorescent turn-on probe is developed for
H2S detection in aqueous buffer and in living cells. The probe
is based on the fast and selective thiolysis of NBD amine,
which can be a general design strategy for H2S probes.52 Probe
1 shows 19-fold turn-on fluorescence at 585 nm upon H2S acti-
vation. 1 contains a positive charge, which is useful for fast
sensing of H2S (k2 = 27.8 M−1 s−1) and mitochondria-targeting.
1 exhibits excellent selectivity toward H2S over millimolar
biothiols in physiological buffer. Moreover, 1 is successfully
applied in the bioimaging of H2S in living cells and zebrafish.
This probe could be a useful tool for potential applications in
H2S biology.
Experimental
Materials and methods
All chemicals and solvents used for the synthesis were pur-
chased 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
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Organic & Biomolecular Chemistry
(Merck 60F-254, 250 µm in thickness), and spots were visual-
ized by basic KMnO4, UV light or iodine. Merck silica gel 60
(100–200 mesh) was used for general column chromatography
purification. 1H NMR and 13C NMR spectra were recorded on a
Bruker 400 spectrometer. Chemical shifts are reported in parts
per million with respect to the internal standard tetramethyl-
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silane (Si(CH3)4 = 0.00 ppm) or residual solvent peaks (CD2Cl2
= 5.32 ppm; CDCl3 = 7.26 ppm; DMSO-d6 = 2.5 ppm). 1H NMR
coupling constants ( J) are reported in hertz (Hz), and multi-
plicity is indicated as the following: s (singlet), d (doublet),
t (triplet), dd (doublet of doublets), m (multiple). High-resolu-
tion 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). Fluorescence
study was carried out using an F-280 spectrophotometer
(Tianjin Gangdong Sci & Tech., Development. Co., Ltd).
3-Oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-3′,6′-diyl bis
(trifluoromethanesulfonate) 2
A two-necked round-bottom flask was charged with fluorescein
sodium (5 g, 15.04 mmol) in anhydrous CH2Cl2 (60 mL) and
cooled to 0 °C. Pyridine (9.74 ml, 12.04 mmol, 8.0 eq.) and tri-
fluoromethanesulfonic anhydride (10.12 ml, 60.2 mmol,
4.0 eq.) were added slowly into the mixture under an inert
atmosphere. The reaction mixture was stirred at room tempera-
ture for 4 h. The reaction was quenched with water, and the
reaction mixture extracted with CH2Cl2, washed with saturated
brine, and dried over anhydrous sodium sulfate. After the
solvent was removed in vacuo, recrystallization from ethyl
acetate and hexanes afforded the pure product as an off-white
solid (7.4 g) with 83% yield. Rf = 0.7 (ethyl acetate/hexanes = 3/
10); 1H NMR (400 MHz, DMSO-d6) δ 8.09 (d, J = 7.2 Hz, 1H),
7.87–7.76 (m, 4H), 7.50 (d, J = 7.6 Hz, 1H), 7.32 (dd, J = 8.8, 2.4
Hz, 2H), 7.12 (d, J = 8.8 Hz, 2H); 13C NMR (101 MHz, DMSO-
d6) δ 168.12, 151.85, 150.74, 149.82, 136.24, 130.89, 130.62,
125.28, 125.02, 124.20, 119.80, 119.38, 118.01, 116.61, 110.84,
79.66; HRMS (ESI): m/z [M]+ calcd for C22H10F6O9S2: 595.9670,
found: 596.9746.
2-(3-(Azetidin-1-ium-1-ylidene)-6-(azetidin-1-yl)-3H-xanthen-
9-yl)benzoate 3
A 25 ml two-necked round bottom flask was charged with
fluorescein ditriflate (0.15 g, 25.2 mmol), 2-dicyclohexyl-
phosphino-2′,4′,6′-triisopropylbiphenyl (Xphos, 0.036 g,
75.4 μmol. 0.3 eq.), Cs2CO3 (0.23 g, 704 μmol, 2.8 eq.) and tris
(dibenzylideneacetone)dipalladium(0) (Pd2dba3, 0.023 g,
25.2 mmol. 0.1 eq.) in dioxane (2 mL). Argon was fluxed before
adding azetidine (40.6 μL, 604 μmol, 2.4 eq.), and the reaction
mixture was stirred at 100 °C under an argon atmosphere for
18 h. After completion the reaction system was cooled to room
temperature, diluted with CH3OH, filtered with Celite, and
concentrated in vacuo. Purification by silica gel chromato-
graphy using a solvent system (0–7% CH3OH (2 M NH3)/
CH2CI2) afforded a purple solid (0.098 g, 95%). Rf = 0.6
(CH3OH/CH2Cl2/NH3 = 2/10/0.5); 1H NMR (400 MHz, CD2Cl2)
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δ 8.00 (d, J = 7.6 Hz, 1H), 7.64 (dt, J = 22.4, 7.2 Hz, 2H), 7.15 (d,
J = 7.2 Hz, 1H), 6.60 (d, J = 8.4 Hz, 2H), 6.22 (s, 2H), 6.14 (d, J =
8.4 Hz, 2H), 3.94 (t, J = 7.2 Hz, 8H), 2.48–2.31 (m, 4H); 13C
NMR (101 MHz, CD2Cl2) δ 169.62, 154.26, 153.33, 152.49,
134.87, 129.77, 129.16, 128.18, 125.34, 124.61, 52.50, 17.01.
HRMS (ESI): m/z [M + H]+ calcd for C26H22N2O3: 410.1630,
found: 411.1706.
Azetidinyl-rhodamine-NBD dyad 1
2-(3-(Azetidin-1-ium-1-ylidene)-6-(azetidin-1-yl)-3H-xanthen-9-yl)
benzoate (0.134 g, 0.33 mmol) was dissolved in 8 ml DMF, fol-
lowed by the addition of HATU (0.144 g, 0.38 mmol) and
DIPEA (148 μl, 0.75 mmol). The solution was stirred for 5 min
and NBD-piperazine33,51 (0.068 g, 2.83 mmol) was added to
the solution; stirring was continued for 12 h at room tempera-
ture. After completion of the reaction DMF was removed
in vacuo. The residue was purified by silica gel column chrom-
atography to yield dark-red solid 1 (100 mg, 59%). Rf = 0.7
(CH3OH/CH2Cl2/NH3 = 2/10/0.5). 1H NMR (400 MHz, DMSO-
d6) δ 8.53 (d, J = 9.0 Hz, 1H), 7.77 (s, 3H), 7.53 (s, 1H), 7.14 (d,
J = 9.2 Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 6.59–6.46 (m, 3H), 4.25
(t, J = 7.2 Hz, 8H), 4.01 (s, 4H), 3.71 (s, 2H), 3.54 (s, 2H),
2.48–2.36 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 166.80,
156.46, 155.92, 155.69, 145.26, 144.73, 136.29, 135.05, 131.71,
130.82, 130.37, 129.94, 127.54, 121.49, 113.14, 112.47, 103.38,
93.97, 51.79, 15.41. HRMS (ESI): calcd for C36H32N7O5+:
642.2459, found m/z [M]+: 642.2462.
Solution preparation method for spectroscopic studies
All spectroscopic measurements were performed in PBS buffer
(20 mM, pH 7.4) in a 3 ml cuvette with 2 ml solution. 1 was
dissolved in DMSO to prepare the stock solutions with a con-
centration of 5 mM. 1–1000 mM stock solutions of Na2S in
degassed PBS buffer were used as the H2S source. The probe
was diluted in PBS buffer to achieve the final concentration of
2–5 µM. For the selectivity tests, different biologically relevant
species (100 mM) were prepared as stock solutions in PBS
buffer or DMSO. The •OH was generated by the Fenton reac-
tion in which ferrous chloride was added in the presence of
10 equiv. of H2O2. The ONOO− was generated by the reaction
of nitrous acid with hydrogen peroxide in acidic solution
(the concentration was determined by UV spectrophotometry
at 302 nm).53 All species were incubated at room temperature
for 30 min and then the spectra were recorded under excitation
at 565 nm.
Cell culture
Complete growth medium was used to culture human embryo-
nic kidney HEK-293A cells, which contained high glucose
DMEM (Gibco) supplemented with FBS (10%), penicillin (100
U ml−1), streptomycin (10 mg ml−1), and L-glutamine (4 mM).
The cells were maintained in exponential growth, and then
seeded in a 35 mm glass-bottom plate. The cells were passaged
every 2–3 days and used between passages 3 and 10.
Cytotoxicity assay. The cytotoxicity of 1 was determined
using a xCELLigence RTCA S16 (ACEA Biosciences),50 which is
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an instrument that uses microelectronic plates integrated with
gold microelectrode arrays on a glass substrate in the bottom
of the wells to measure the proliferation of adherent cells.
When cells replicate and grow, they can cover the electrosensi-
tive elements and cause higher impedance, and the electronic
current will be changed. The software connected to a computer
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can monitor these changes in real time, reflecting the prolifer-
ation of adherent cells. Briefly, HEK293A cells growing in the
log phase were seeded into a 16-well cell-culture plate (100 µL
per well). When the amount of the cells reached 5 × 104 cells
per well, probe 1 was added into each well with final concen-
trations of: 0, 1, 2, 5, 10, 15 and 20 µM. These cells were incu-
bated at 37 °C under 5% CO2, and the data were collected
every 15 min for the duration of the assay.
Cell bioimaging. An FV1000 inverted fluorescence confocal
microscope (Olympus, Japan) with a 40× objective lens was
used for imaging. For exogenous H2S, HEK293A cells were
incubated with the probe (2, 5 or 10 μM) for 30 min, washed,
and then incubated with Na2S (100 μM) for another 30 min.
The medium was washed and replaced by PBS, and the cells
were imaged via the red channels (Ex. = 543 nm, Em. =
555–655 nm). The control cells were treated with only the
probe (10 μM) for 30 min. The HEK293A cells were incubated
with 100 μM L-Cys for 30 min to induce endogenous H2S pro-
duction, washed with PBS, and then incubated with the probe
(5 or 10 μM) for another 30 min. The medium was washed and
replaced by PBS, and the cells were imaged via the red chan-
nels. The imaging results were analyzed using Olympus
FV1000-ASW software.
Co-localization. Living HEK293 cells were co-incubated with
L-Cys (100 μM) and the probe (10 μM) for 30 min and washed
with PBS; then Mito-Tracker Green FM (2.5 μM) was added for
another 30 min. The images were collected from band pass
555–655 nm upon excitation at 543 nm, from band pass
500–530 nm upon excitation at 488 nm, and bright-field.
In vivo imaging in zebrafish. Imaging in zebrafish was per-
formed according to the institutional ethical guidelines for
animal experiment. The accreditation number of the labora-
tory is SYXK (Jin) 2014-00003 promulgated by Tianjin Science
and Technology Commission. 7-day-old zebrafish was incu-
bated with the probe (10 μM) for 1 h, washed with culture
medium, then incubated with Na2S (200 μM) or L-Cys (200 μM)
for 3 h, and imaged with an Olympus fluorescence microscope
and imaged in the red channel.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the National Key R&D Program of
China (2017YFD0200500, 16JCYBJC20200) and the NSFC
(21702111, 21572019, 21877008).
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Organic & Biomolecular Chemistry
Notes and references
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