Novel turn-on fluorescence sensor for detection and imaging of endogenous H2S induced by sodium nitroprusside

Article abstract of DOI:10.1016/j.saa.2020.118775

Currently, fluorescence analysis method has a good application in the detection and imaging of biomarkers and has become an important analytical method. Although there are many fluorescent probes for detecting hydrogen sulfide(H₂S), they are mo

Full text of DOI:10.1016/j.saa.2020.118775

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Novel turn-on fluorescence sensor for detection and imaging of
endogenous H2S induced by sodium nitroprusside
Li Wang, Wenge Yang, Yiyi Song, Yonghong Hu
PII:
S1386-1425(20)30754-X
https://doi.org/10.1016/j.saa.2020.118775
SAA 118775
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 22 April 2020
Revised date:
19 July 2020
1 July 2020
Accepted
date:
2
Please cite this article as: L. Wang, W. Yang, Y. Song, et al., Novel turn-on fluorescence
sensor for detection and imaging of endogenous H2S induced by sodium nitroprusside,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020),
https://doi.org/10.1016/j.saa.2020.118775
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Novel turn-on fluorescence sensor for detection and imaging of
endogenous H S induced by sodium nitroprusside
2
Li Wang ·Wenge Yang·Yiyi Song · Yonghong Hu*
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,
No. 30, South Puzhu Road, Nanjing 211816, China
Corresponding author: Yonghong Hu
The two authors contributed equally.
Tel: +86-25-58139393
Fax: +86-25-58139393
E-mail: yonghonghuyg@163.com
1

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Abstract
Currently, fluorescence analysis method has a good application in the detection
and imaging of biomarkers and has become an important analytical method. Although
there are many fluorescent probes for detecting hydrogen sulfide(H S), they are
2
mostly based on fluorophores which already existed, such as 1,8-naphthalimide,
coumarin, rhodamine and their derivatives. Here, a new type of fluorescent molecule
(
BOTD) was synthesized and applied to the detection of H S. The probe BOTD could
2
quickly and sensitively detect H S and turn on fluorescence. Moreover, the probe
2
BOTD was successfully applied to the detection of exogenous and endogenous H S in
2
living cells, and may be expected to become a research tool for studying H S-induced
2
drugs.
Keywords: Hydrogen sulfide; Fluorescent probe; H S-induced drug; Bioimaging
2
1
. Introduction
Hydrogen sulfide (H S), as one of the gas signal molecules, plays an important
2
role in many physiological processes, including: vasodilation, inflammation control,
[
1-6]
neuroprotection, delaying aging, regulating glucocorticoids and insulin, etc.
Endogenous H S has multiple production pathways in the body. L-cysteine ( L-Cys) is
2
used as a substrate in the cytoplasm of the cell. It is produced under the catalytic
action of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and cysteine
[
7-9]
transferase
. In mitochondria, β-mercaptopyruvic acid is used as a substrate, and
[10-11]
H S is produced under the action of mercaptopyruvate trans-sulphurase (MPST)
2
.
There are usually two forms of H S in the body, H S and sodium sulfide (NaHS). The
2
2
changes in the content of H S in the body have great potential in the prevention and
2
diagnosis of cardiovascular disease. Unfortunately, it is still difficult to track and
accurately quantify H S in biological systems, and the regulatory mechanism of H S
2
2
is still unclear. Therefore, it is necessary to develop an analytical method for selective
recognition and highly sensitive detection of H S in living organisms.
2
Fluorescence analysis has a wide range of applications in biological detection
2

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[
12-14]
.
The general methods for detecting H S are: reduction of azide to amine,
2
nucleophilic addition, precipitation of copper sulfide, reduction of selenium oxide to
[
15-19]
selenide
. Under normal physiological conditions, ideal probes should respond to
small concentration changes and show sensitive changes, so as to obtain more
accurate information while avoiding interference from other substances as much as
possible, especially organisms such as glutathione, cysteine, etc. So, the development
of fluorescent probes for the perfect detection of H S remains a challenging task.
2
According to previous reports, triphenylamine and its derivatives have good
[20-23]
mitochondrial localization selectivity and effective electron donor capabilities
In this work, we developed a triphenylamine -based fluorescent probe that reacts with
H2S and detects it through fluorescence activation. The probe
E)-6-(4-(diphenylamino)benzylidene)-5-oxo-5,6,7,8-tetrahydronaphthalen-2-yl
.
(
2
,4-dinitrobenzenesulfonate (BOTD) could not only detect H S sensitively and
2
specifically, but also has low toxicity. This provides an effective means for the
detection of exogenous and endogenous H S. This fluorescence analysis method can
2
discover that the H S induced by sodium nitroprusside in living cells, which also
2
helps to investigate the H S metabolism pathway in target cells.
2
2
. Experiments
2
.1. Materials and instruments
Both organic compounds and inorganic salts were delivered from Energy
Chemical (Shanghai, China), which were analytical grade. The pH was measured by
pH meter of model pH400 (Alalis, USA). A560PC UV-VIS spectrophotometer
(Xiangyi, China) was used for the UV absorption spectra. The fluorescence spectra
1
were taken on F97xp spectrophotometer (Lengguang Tech, China). All H NMR and
1
3
C NMR of the sample were obtained by Bruker Avance AVIII-400 MHz
spectrometer (Bruker Daltonics Corp., USA) at room temperature. High-resolution
electrospray ionization mass spectra (HR-ESI-MS) were involved a Q-TOF6510
spectrograph (Agilent). A Zeiss LSM 710 confocal laser scanning microscope (CLSM)
was utilized in cell fluorescence imaging experiments.
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2
2
.2. Synthesis of the probe BOTD
.2.1 Synthesis of compound 2
Triphenylamine (3 g, 12.22 mmol) was dissolved in anhydrous N,
N-dimethylformamide (20 mL) in a reaction flask and stirred at 0 ℃ for 2 h. Then, to
the abovementioned solution, phosphorus oxychloride (5.7 mL, 61 mmol) was added
dropwise at 0 ℃. After the addition was completed, the mixture was warmed up to
room temperature and the reaction mixture was stirred for 2 h at 45 ℃. The crude
product was poured into an ice-water mixture, neutralized with solid sodium
bicarbonate. The resulting precipitate was filtered and dried, the crude product was
further purified by column chromatography with petroleum ether/ dichloromethane
1
(
10:1 v/v) as the eluent to afford 2 as a pale yellow solid (2.70 g, 81%). H NMR (400
MHz, DMSO-d ) δ 9.77 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.42 (t, J = 7.9 Hz, 4H),
6
7
2
.22 (dd, J = 17.6, 7.4 Hz, 6H), 6.89 (d, J = 8.7 Hz, 2H).
.2.2 Synthesis of compound BOT
The compound BOT was prepared using an acid-catalyzed method which was
[24]
named
Claisen‐ Schmidt
condensation
method
.
6
-hydroxy-3,4-dihydronaphthalen-1(2H)-one (0.324 g, 2 mmol), the appropriate
compound 2 (0.547 g, 2 mmol) and hydrochloric acid (36%; 20 ml) were added in a
flask filled with methanol (15 ml), then refluxed for 5 hours. After the reaction was
completed, ice cold water (20 ml) was added, and the precipitate was collected by
filtration, dried and further purified by silica gel chromatography to obtain BOT (0.6 g,
1
7
0.2%). H NMR (400 MHz, DMSO-d ) δ 10.38 (s, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.58
6
(
s, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.38 – 7.28 (m, 4H), 7.19 – 7.01 (m, 6H), 6.95 (d, J
8.7 Hz, 2H), 6.77 (d, J = 10.9 Hz, 1H), 6.67 (s, 1H), 3.04 (t, J = 5.9 Hz, 2H), 2.83 (t,
J = 6.4 Hz, 2H).
=
2
.2.3 Synthesis of BOTD
As shown in Scheme.1, BOTD is synthesized from BOT and
,4-dinitrobenzenesulfonyl chloride in 10 ml of dichloromethane solution, and added
2
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with triethylamine and stirred at 0 °C for 1 h, and then stirred at room temperature
overnight. The crude product was purified by rotary evaporation and silica gel column
1
chromatography to obtain the pure product. H NMR (400 MHz, DMSO-d ) δ 9.13 (s,
6
1
H), 8.62 (d, J = 11.0 Hz, 1H), 8.32 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.67
(s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.43 – 7.32 (m, 4H), 7.30 (s, 1H), 7.22 – 7.04 (m,
13
7
H), 6.94 (d, J = 8.7 Hz, 2H), 3.09 (t, J = 5.9 Hz, 2H), 2.94 (t, J = 6.4 Hz, 2H). C
NMR (101 MHz, DMSO) δ 185.76, 152.04, 151.79, 148.65, 148.55, 146.84, 146.52,
1
1
36.97, 134.06, 133.15, 132.86, 132.27, 131.25, 130.59, 130.24, 128.42, 128.08,
25.61, 124.67, 122.07, 121.65, 121.21, 120.89, 60.22, 27.98, 26.88, 21.22, 14.55.
+
1
13
MS (ESI) m/z: calcd for [M+H] , 648.14; Found, 648.14. All H NMR, C NMR and
HRMS are shown in Fig.S1-5.
Scheme.1 Synthesis route of BOTD.
2
.3. Analytical procedures
The probe BOTD is prepared as a stock solution of 1 mM / L in DMSO.
Solutions for fluorescence and UV measurement is THF-PBS solvent (0.1 μM, pH 7.4,
3
1
0% THF, v/v) unless otherwise stated. Both excitation and emission of slit width was
−
−
−
−
-
−
−
3
2−
2-
−
2−
4
0 nm. And all analytes (F , Cl , Br , I , ClO , NO , HSO , SO , S , HS , SO ,
2
3
−
-
HSO , SCN , Cys, Hcy, GSH) were dissolved in double distilled water when used.
4
2
.4. Theoretical calculations
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Relevant theoretical calculations were performed using the Gaussian 09 program.
The geometry of BOT, DNS and BOTD was optimized using a three-parameter mixed
B3LYP density method and a base set of 6-31G. The excited states of all compounds
were studied by time-varying density functional theory (DFT).
2
.5. Cell culture and cytotoxicity assay
Hela cells were provided by Jiangsu Kaiji Biotechnology Co., Ltd. The cells
were cultured in a medium consisting of 90% MEM + 10% FBS, and putted in an
incubator at 37 ° C, 5% CO , and saturated humidity. The cytotoxic effects of the
2
[
25-27]
probe BOTD were tested by the MTT assay by the previous report
. Differently,
the cells were incubated with different concentrations of BOTD (0, 5, 10, 15, 20ꢀμM/L,
containing 0.5% DMSO) for 24ꢀh.
2
.6. Imaging of H S in living Cell
2
For fluorescence imaging experiments, the cells were divided into three samples;
one was incubated with the BOTD probe (10 μM/L) only as a control, the other group
was cultured with NaHS (100 μM/L) for 30 min and then the probe was added, and
the last group was cultured with 100 μM/L Sodium nitroprusside (SNP) for 60 min
and then with the probe, respectively. All cells were washed three times with PBS
buffer after incubation. The experiments of cell imaging were conducted by using a
laser scanning confocal fluorescence microscope (Zeiss LSM 710). Cell images were
obtained using 420 nm excitation and signals were collected from the green channel
(500–550 nm).
3
.Results and discussions
3
.1. The design of BOTD and properties of compound BOT
In order to better understand the role of H S at the mitochondrial level, a new
2
type of organelle-based fluorescent probe is urgently needed for detection and
imaging. Triphenylamine has good fluorescence characteristics, which is
mitochondrial targeted. And triphenylamine as a strong electron donor and
6
-hydroxy-3,4-dihydronaphthalen-1 (2H) -one conjugated to form a fluorescent probe
6

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precursor (BOT) and 2,4-dinitrobenzenesulfonyl (DNS) as the H S response site.
2
When reacting with H S, the electrons of the terminal hydroxyl group are recovered
2
and the fluorescence is released.
We first assessed the ultraviolet absorption and fluorescence spectrum of BOT
(10 μM/L) in various solvents including ethanol, acetonitrile, acetone, DMF, DMSO
and THF. It could be learned that the maximum ultraviolet absorption of BOT is
around 400 nm in all six solvents form Fig.1. with the excitation wavelength at 400
nm, the fluorescence intensity of BOT in DMSO solvent is the highest. This shows
that BOT can be utilized as a precursor for fluorescent probe.
0
0
.5
.4
8
7
6
5
4
3
2
1
000
000
000
000
000
000
000
000
0
Ethanol
Acetonitrile
acetone
DMF
DMSO
THF
0.3
0.2
b
a
0
.1
0.0
Ethanol
acetone
DMF
DMSO
THF
Acetonitrile
5
00
600
700
800
350
400
450
500
wavelength (nm)
wavelength (nm)
Fig.1 The fluorescence spectrum of BOT (A) and ultraviolet absorption of BOT (B) in
different solvents.
3
.2. Effects of pH
It is necessary to study the effect of pH on the probe in the environment for
determining the applicable environment and stability of the probe BOTD. So, we
-
evaluated the effect of pH on the probe BOTD in the absence and presence of HS in
various pH buffers. As illustrated in Fig.2, when only the probe is present, the
-
fluorescence of the solution hardly changes, while in the presence of HS , the
fluorescence intensity of the solution increases sharply at pH 6-8. Consequently, the
-
probe could stably detect HS under normal physiological conditions.
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500
400
300
200
100
0
BOTD+HS-
BOTD
3
4
5
6
7
8
9
10
11
pH
-
Fig.2 Fluorescence signal changes in the probe BOTD (10 μM/L) treated with HS
(100 μM/L) at different pH values( λex=420 nm, λem=550 nm).
-
3
.3. Sensing properties of probe BOTD for HS
Under normal physiological conditions, the fluorescence response and UV
absorption of the probe BOTD to sodium sulfide were recorded in Fig.3. The
maximum absorption peak of BOTD was about 420 nm. In Figure 3(a) and Fig.3(b),
the fluorescence intensity of BOTD at 550 nm (λex=420 nm) increases from a very
weak and significant increase as the concentration of sodium sulfide increases. The
fluorescence quantum yield (ΦF) of BOTD increased from 0.02 to 0.23 after reacting
with sodium hydrosulfide (Table.S1). And the UV absorption of the probe BOTD
shifted to 400 nm in Fig.3(c), indicating that the 2,4-dinitrobenzenesulfonate bond
was broken. Moreover, the fluorescence intensity of probe BOTD at 550 nm has a
good linear relationship with the concentration of sodium sulfide from 0 to 50 μM/L
2
(
R = 0.9947) (Fig. 3(c)). The corresponding detection limit was calculated to be 27.3
nmol/L with the equation of 3σ / k, where k is the slope and σ is the standard
[
28-33]
deviation of the blank sample, which was lower than other reported probes
Table.S2).
(
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5
4
3
2
1
00
00
00
00
00
0
5
4
3
2
1
00
00
00
00
00
0
b
a
5
00
600
700
800
0
10
20
30
40
50
Wavelength (nm)
C(HS-)
0
0
0
0
0
0
.5
.4
.3
.2
.1
.0
BOTD
BOTD+HS-
equation
y = a + b*x
4
00
50
00
intercep 30.10929 ± 6.91
slope
Pearson'
R^2
11.80657 ± 0.38
0.99737
0.99474
3
3
d
c
250
2
1
00
50
100
5
0
0
3
00
400
500
0
5
10
15
20
25
30
Wavelength (nm)
C(HS-)
Fig.3 (a) Fluorescence spectra of the probe BOTD (10 μM/L) in aqueous buff er in
PBS buff er (pH 7.4, 30% THF) response to NaHS (0–50 μM/L). λex=420 nm,
λem=550 nm. (b) The correspondence between fluorescence intensity of probe BOTD
and NaHS concentration. (c) The UV absorption of probe BOTD with NaHS (50
μM/L) in aqueous buff er. (d) The linear correlation between the concentration of H2S
(0–30 μM/L) and the fluorescence intensity of BOTD at 550 nm.
3
.4. Selective response of BOTD to H2S
Subsequently, the selectivity of the probe BOTD for H S was examined with
2
−
−
−
−
-
−
−
2−
various analytes in PBS buffer，including F , Cl , Br , I , ClO , NO , HSO , SO ,
2
3
3
-
2−
2−
−
4
-
HS , S , SO , HSO , SCN , Cys, Hcy, GSH. As illustrated in Fig.4, the
4
−
2-
fluorescence of probe BOTD was turn on when detecting HS and S . While the
other analytes showed no significant change in fluorescence and UV absorption
(
Fig.S6). These results indicated that probe BOTD could detect H S with high
2
selectivity.
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500
400
300
200
100
0
−
−
Cl
−
Br
−
−
−
−
−
−
F
I
−
S2
HS-
Cys Hcy GSH
ClO- NO2
SCN-
HSO3 SO32
SO42 HSO4
Fig.4 Fluorescence responses of probe BOTD (10ꢀμM/L) towards various analytes
1ꢀmM/L)
(
3
.5. Response time
To obtain the response time of the probe BOTD to H S, the data of the
2
fluorescence intensity at 550 nm was measured and plotted in Fig.5. As shown by the
results, the fluorescence intensity of free BOTD in PBS buffer is almost unchanged,
but only a weak fluorescence is maintained. Conversely, the fluorescence intensity of
-
BOTD gradually increased and reached a peak within 30s after HS was added.
Because the probe BOTD has extremely short response time to H S, it shows
2
excellent potential for real-time detection of H S in biological systems.
2
1
0

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1000
800
600
400
200
0
0
10
20
30
40
50
60
70
80
Time (s)
Fig.5 The fluorescence intensity of the probe BOTD (λex = 420 nm, λem = 550 nm)
in the presence of H S depending on time
2
3
.6. Molecular orbital and mechanism study
To obtained full comprehension of the changes in the photophysical properties of
compound BOT and BOTD, the theoretical calculations and geometric optimizations
based on DFT were carried out. The spatial distributions and orbital energies of the
highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO) are shown in Fig. 6. The HOMO energy (-8.34 eV) of DNS is
between the HOMO energy (-5.76 eV) and LUMO energy (-8.43 eV) of BOT, and it
is lower than the HOMO energy (-8.10 eV) of BOTD, which prevents probe BOTD
from emitting fluorescence. Therefore, the molecular orbital energy diagram clearly
indicates a PET process.
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Fig.6 Frontier molecular orbitals of BOT, DNS and BOTD.
The putative detection mechanism was shown in Scheme.2. By analyzing the
-
reaction products of the probe BOTD and HS , we found that a peak of m / z 418.18
+
[
(
BOT + H] , which was assigned to BOT (calculated as 417.17 for C H NO )
29
23
2
1
-
Fig.S7). The H spectrum (Fig.S8) of the BOTD-HS product is basically consistent
with BOTD, which also proved that the detection mechanism is reasonable.
-
Scheme.2 Schematic illustration of BOTD's sensing towards HS cations.
3
.7. Toxicity of probe BOTD and cell imaging
Before applying cell imaging to live cells, the MTT assay was performed to
verify the low acute toxicity of BOTD to living HeLa cells. It could be seen that even
with the probe BOTD concentration of 20 μM/L, the survival rate of HeLa cells is still
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2

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8
5% after 24 h of incubation (Fig.S9). This shows that the probe BOTD has excellent
viability and biocompatibility.
Then BOTD is exploited for H S detection and imaging in living cell. As
2
-
described in Fig.7, the probe BOTD has no fluorescent signal without HS (Fig.7(a-c)).
And the probe BOTD has a yellow-green fluorescence emission after incubation with
exogenous H S in Fig.7(d-f). More importantly, the HeLa cells incubated with sodium
2
nitroprusside also exhibited fluorescence emission (Fig.7(g-i)), indicating that sodium
nitroprusside could induce H S production in cells. Sodium nitroprusside is a
2
commonly used drug to regulate blood pressure, but its mechanism of action remains
uncertain. Therefore, the probe BOTD can not only detect H2S in cells, but also
provide help for studying the causes of H S.
2
Fig.7 Fluorescence images of HeLaꢀcells (λex = 420nm，λem= 500-550nm). (left) dark
field; (middle) bright field and (right) overlay image. (a–c) images of HeLa cells
1
3

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incubated with BOTD (10ꢀμM/L) for 30 min; (d–f) fluorescence images of cells
pre-treated with NaHS(100 μM/L) for 1 h and then incubated with BOTD (10ꢀμM/L)
for 30 min; (g–i) images of cells pre-treated with 100 μM/L SNP for 1 h, and then
incubated with BOTD (10ꢀμM/L) for 30 min; scale bar was 20 μm.
4
. Conclusion
In general, we have successfully constructed a new type of probe BOTD based
-
on PET theory. The probe BOTD is non-fluorescent, but the reaction product with HS
has fluorescent emission, which is due to blocking the process of PET. Further, the
probe could quickly detect H S, and has good selectivity and biocompatibility, and
2
detection limits as low as 27.3 nmol/L. Finally, BOTD could detect exogenous and
drug-induced endogenous H S in living cells. Therefore, the probe BOTD could be a
2
powerful tool for sensitive, real-time detection of H S in living cells.
2
Acknowledgment
This work was financially supported by The Jiangsu Province Agricultural
Science and Technology Independent Innovation Fund Project (CX(17)3101), 2019
State Key Laboratory of Materials Chemistry Independent Project (ZK201904),
Special funds for the transformation of scientific and technological achievements
(SBA2019030143).
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References
1] J. Te Winkel, Q.E. John, B.D. Hosfield, N.A. Drucker, A. Das, K.R. Olson, et al.,
[
Mesenchymal stem cells promote mesenteric vasodilation through H2S and
endothelial nitric oxide, Am J Physiol-Gastr L, 317(2019) G441-G6.
[
2] C.M. Levinn, M.M. Cerda, M.D. Pluth, Development and Application of Carbonyl
Sulfide-Based Donors for H2S Delivery, Accounts Chem Res, 52(2019) 2723-31.
3] E. Donnarumma, R.K. Trivedi, D.J. Lefer, Protective Actions of H2S in Acute
Myocardial Infarction and Heart Failure, Compr Physiol, 7(2017) 583-602.
4] L.T. Sun, L. Chen, F.G. Wang, X. Zheng, C.F. Yuan, Q.G. Niu, et al., Exogenous
[
[
H2S prevents lipopolysaccharide-induced inflammation by blocking the
TLR4/NF-kappa B pathway in MAC-T cells, Gene, 710(2019) 114-21.
[
5] A.V. Do, R. Smith, P. Tobias, D. Carlsen, E. Pham, N.B. Bowden, et al.,
Sustained Release of H2S (H2S) from Poly(Lactic Acid) Functionalized
-Hydroxythiobenzamide Microparticles to Protect Against Oxidative Damage, Ann
Biomed Eng, 47(2019) 1691-700.
6] S.E. Ohia, J. Robinson, L. Mitchell, K.K. Ngele, S. Heruye, C.A. Opere, et al.,
4
[
Regulation of Aqueous Humor Dynamics by H2S: Potential Role in Glaucoma
Pharmacotherapy, J Ocul Pharmacol Ther, 34(2018) 61-9.
[
7] M.J. Zhang, X.W. Wu, Y.X. Xu, M.J. He, J.Y. Yang, J. Li, et al., The
cystathionine beta-synthase/H2S pathway contributes to microglia-mediated
neuroinflammation following cerebral ischemia, Brain Behav Immun, 66(2017)
3
32-46.
8] E.K. Choi, S.H. Park, J.A. Lim, S.W. Hong, K.H. Kwak, S.S. Park, et al.,
Beneficial Role of H2S in Renal Ischemia Reperfusion Injury in Rats, Yonsei Med J,
9(2018) 960-7.
9] A. Asimakopoulou, P. Panopoulos, C.T. Chasapis, C. Coletta, Z.M. Zhou, G.
[
5
[
Cirino, et al., Selectivity of commonly used pharmacological inhibitors for
cystathionine synthase (CBS) and cystathionine lyase (CSE), Brit J Pharmacol,
1
69(2013) 922-32.
10] K.R. Olson, H2S and polysulfide metabolism: Conventional and unconventional
pathways, Biochem Pharmacol, 149(2018) 77-90.
11] V. Citi, E. Piragine, L. Testai, M.C. Breschi, V. Calderone, A. Martelli, The Role
[
[
of H2S and H2S-donors in Myocardial Protection Against Ischemia/Reperfusion
Injury, Curr Med Chem, 25(2018) 4380-401.
[
12] Bhaskar R, Vijayakumar V, Srinivasadesikan V, Lee S-L, Sarveswari S.
Rationally designed imidazole derivative as colorimetric and fluorometric sensor for
selective, qualitative and quantitative cyanide ion detection in real time samples.
Spectrochimica Acta, Part A: Molecular and Biomolecular Spectroscopy.
2
020;234:118212.
13] F. Ghorbani, H. Abbaszadeh, A. Mehdizadeh, M. Ebrahimi-Warkiani, M.R.
Rashidi, M. Yousefi, Biosensors and nanobiosensors for rapid detection of
[
1
5

Journal Pre-proof
autoimmune diseases: a review, Mikrochim Acta, 186(2019) 838.
[
14] Erdemir S, Malkondu S, Kararkurt S. Synthesis and cell imaging studies of an
unusual "OFF-ON" fluorescent sensor containing a triazole unit for Al(3+) detection
via selective imine hydrolysis. The Analyst. 2020.
[
15] D.A. Jose, N. Sharma, R. Sakla, R. Kaushik, S. Gadiyaram, Fluorescent
nanoprobes for the sensing of gasotransmitters H2S (H2S), nitric oxide (NO) and
carbon monoxide (CO), Methods, 168(2019) 62-75.
[
16] Y.N. Luo, C.Z. Zhu, D. Du, Y.H. Lin, A review of optical probes based on
nanomaterials for the detection of H2S in biosystems, Analytica Chimica Acta,
061(2019) 1-12.
17] Y. Takano, H. Echizen, K. Hanaoka, Fluorescent Probes and Selective Inhibitors
1
[
for Biological Studies of H2S- and Polysulfide-Mediated Signaling, Antioxid Redox
Sign, 27(2017) 669-83.
[
18] T.T. Zhou, Y.T. Yang, K.Y. Zhou, M. Jin, M.N. Han, W. Li, et al., Efficiently
mitochondrial targeting fluorescent imaging of H2S invivo based on a
conjugate-lengthened cyanine NIR fluorescent probe, Sensor Actuat B-Chem,
3
01(2019).
[
19] X.Y. Jing, F.Q. Yu, W.Y. Lin, A PET-based lysosome-targeted turn-on
fluorescent probe for the detection of H2S and its bioimaging application in living
cells and zebrafish, New J Chem, 43(2019) 16796-800.
[
20] Y.L. He, X.M. Wang, K. Wang, L.M. Wang, A triarylamine-based fluorescent
covalent organic framework for efficient detection and removal of Mercury(II) ion,
Dyes Pigments, 173(2020).
[
21] F. Hammerer, F. Poyer, L. Fourmois, S. Chen, G. Garcia, M.P. Teulade-Fichou,
et al., Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced
two-photon photodynamic therapy, Bioorgan Med Chem, 26(2018) 107-18.
[
22] H.H. Zhang, G.J. Zhang, G. Liu, Y. Xia, M. Fang, X.J. Zhu, et al.,
Mitochondrion-targeted two-photon probes: Real-time monitoring endogenous GSH
via situ reaction in Hela cells, Dyes Pigments, 161(2019) 233-9.
[
23] R. Chennoufi, H. Bougherara, N. Gagey-Eilstein, B. Dumat, E. Henry, F. Subra,
et al., Mitochondria-targeted Triphenylamine Derivatives Activatable by Two-Photon
Excitation for Triggering and Imaging Cell Apoptosis, Sci Rep-Uk, 6(2016).
[
2
24] L.J. Legoabe, M.M. Van der Walt, G. Terre'Blanche, Evaluation of
-benzylidene-1-tetralone derivatives as antagonists of A(1) and A(2A) adenosine
receptors, Chem Biol Drug Des, 91(2018) 234-44.
25] X.H. Xu, C. Liu, Y. Mei, Q.H. Song, BODIPY-based selenides as fluorescent
[
probes for rapid, sensitive and mitochondria-specific detection of hypochlorous acid, J
Mater Chem B, 7(2019) 6861-7.
[
26] S.K. Samanta, K. Maiti, S.S. Ali, U.N. Guria, A. Ghosh, P. Datta, et al., A
solvent directed D-pi-A fluorescent chemodosimeter for selective detection of
hazardous hydrazine in real water sample and living cell, Dyes Pigments, 173(2020).
[
27] D. Pfeifer, A. Russegger, I. Klimant, S.M. Borisov, Green to red emitting
1
6

Journal Pre-proof
BODIPY dyes for fluorescent sensing and imaging of carbon dioxide, Sensor Actuat
B-Chem, 304(2020).
[
28] H. Zhang, W. Li, J. Chen, G. Li, X. Yue, L. Zhang, et al., Simultaneous detection
of Cys/Hcy and H2S through distinct fluorescence channels, Anal Chim Acta,
097(2020) 238-44.
29] D. Su, D. Cheng, Y. Lv, X. Ren, Q. Wu, L. Yuan, A unique off-on near-infrared
1
[
QCy7-derived probe for selective detection and imaging of H2S in cells and in vivo,
Spectrochim Acta A Mol Biomol Spectrosc, 226(2020) 117635.
[
30] J. Li, C.X. Yin, Y.B. Zhang, Y.K. Yue, J.B. Chao, F.J. Huo, High selective
distinguishable detection GSH and H2S based on steric configuration of molecular in
Vivo, Dyes Pigments, 172(2020).
[
31] C. Xie, C. Ma, X. Jia, X.Q. Zhang, C. Wei, P.Z. Zhang, et al., A Response Rate
Matching Dual-Reactable Probe for Fluorescent Recognition of H2S, Chinese J Org
Chem, 39(2019) 3277-82.
[
32] Z.Y. Chen, X.L. Mu, Z. Han, S.P. Yang, C.L. Zhang, Z.J. Guo, et al., An
Optical/Photoacoustic Dual-Modality Probe: Ratiometric in/ex Vivo Imaging for
Stimulated H2S Upregulation in Mice, J Am Chem Soc, 141(2019) 17973-7.
[
33] L.L. Mittapelli, G.N. Nawale, S.P. Gholap, O.P. Varghese, K.R. Gore, A turn-on
fluorescent GFP chromophore analog for highly selective and efficient detection of
H2S in aqueous and in living cells, Sensor Actuat B-Chem, 298(2019).
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Author Statement
Li Wang: Conceptualization, Methodology, Writing – review & editing. Wenge Yang:
Project administration, Resources. Yiyi Song: Visualization, Investigation. Yonghong
Hu: Funding acquisition, Investigation.
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Graphical abstract
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Hightlights
1
2
3
4
. A novel fluorescent sensor BOTD was developed for detecting H S.
2
. BOTD showed high sensitivity and selectivity to H S in ultrafast time.
2
. BOTD has low detection limits for H S.
2
. BOTD was successfully applied to the detection of exogenous and endogenous
H S in living cells
2
2
0