A bifunctional fluorescent probe for simultaneous detection of GSH and H2Sn (n?>?1) from different channels with long-wavelength emission

Article abstract of DOI:10.1016/j.saa.2021.119789

In this work, we presented a long-wavelength emission fluorescent probe DCM-Cou-SePh that can discriminatively detect glutathione (GSH) and hydrogen polysulfides (H₂S_n, n > 1) from green and red emission channels, respectively. With the addition of GSH, probe DCM-Cou-SePh displayed green fluorescence emission (λ_ex/em = 430/530 nm). In the presence of H₂S_n, the probe exhibited a significant fluorescence enhancement in red channel (λ_ex/em = 560/680 nm). We also demonstrated that this probe was suitable to quantitatively detect GSH and H₂S_n with low detection limits (0.12 μM for GSH, 0.19 μM for H₂S_n). Furthermore, DCM-Cou-SePh can be used for sensing endogenous GSH and H₂S_n in living cells by dual-color fluorescence imaging.

Full text of DOI:10.1016/j.saa.2021.119789

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A bifunctional fluorescent probe for simultaneous detection of GSH and
H2Sn (n > 1) from different channels with long-wavelength emission
Peixin Niu a, Yifan Rong a, Yuyue Wang a, Huijie Ni a, Minghui Zhu a, Wenqiang Chen c, Xingjiang Liu a,
,
⇑
Liuhe Wei a, Xiangzhi Song b
a Green Catalysis Center, College of Chemistry, Zhengzhou University, Zhengzhou 450001, Henan Province, China
b College of Chemistry & Chemical Engineering, Central South University, Changsha 410083, Hunan Province, China
c Guangxi Key Laboratory of Natural Polymer Chemistry and Physics, Nanning Normal University, Nanning 530001, Guangxi Province, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
A bifunctional fluorescent probe,
DCM-Cou-SePh, for simultaneous
detection of GSH and H2Sn (n > 1)
from different channels were
developed.
ꢀ
This probe displayed long-
wavelength emissions in detection of
GSH and H2Sn (GSH: kex/
em = 430/530 nm, H2Sn: kex/
em = 560/680 nm).
ꢀ
ꢀ
This probe exhibited low detection
limits for the detection of GSH and
H2Sn (0.12
H2Sn).
lM for GSH, 0.19 lM for
This probe was capable of
distinguishing endogenous GSH and
H2Sn (n > 1) in living cells.
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work, we presented a long-wavelength emission fluorescent probe DCM-Cou-SePh that can dis-
criminatively detect glutathione (GSH) and hydrogen polysulfides (H2Sn, n > 1) from green and red emis-
sion channels, respectively. With the addition of GSH, probe DCM-Cou-SePh displayed green
fluorescence emission (kex/em = 430/530 nm). In the presence of H2Sn, the probe exhibited a significant
fluorescence enhancement in red channel (kex/em = 560/680 nm). We also demonstrated that this probe
Received 25 January 2021
Received in revised form 2 April 2021
Accepted 2 April 2021
Available online 08 April 2021
was suitable to quantitatively detect GSH and H2Sn with low detection limits (0.12
lM for GSH,
Keywords:
Glutathione
Polysulfide
Long-wavelength emission
Cell imaging
0.19 M for H2Sn). Furthermore, DCM-Cou-SePh can be used for sensing endogenous GSH and H2Sn in liv-
l
ing cells by dual-color fluorescence imaging.
Ó 2021 Elsevier B.V. All rights reserved.
Fluorescent probe
1. Introduction
Reactive sulfur species (RSS), a group of sulfhydryl-containing
compounds, generally exist in biological systems [1–3]. These
thiol-containing molecules coordinately adjust various physiologi-
cal and pathological processes to maintain the normal function of
⇑
Corresponding author.
E-mail address: xingjiangliu@zzu.edu.cn (X. Liu).
https://doi.org/10.1016/j.saa.2021.119789
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

P. Niu, Y. Rong, Y. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
body [4–6]. The abnormal change of RSS may be a signal of certain
diseases [1,5,7–13], such as Alzheimer’s disease, hypertension, car-
diovascular disease and liver cirrhosis. The typical RSS, GSH and
H2Sn, play essential roles in redox biology [14–17] and pathological
processes [5,18–30]. In recent years, increasing numbers of studies
have reported that these two species are deeply related in lots of
physiological processes [31–34] and endogenous H2Sn can be
biosynthesized from GSH (sulfur source) through cystarhionine-
used without further purification. The distilled water was used in
synthesis and fluorescent measurements. Use Bruker 400 NMR to
record 1H NMR and 13C NMR spectra by TMS as an internal stan-
dard. The solution with different pH values were prepared by a
Leici PHS-3C meter. HRMS spectra were obtained by Aglient 7250
and Waters UPLC G2-XS. The emission spectra were recorded on a
Hitachi F-4600 fluorescence spectrometer. Uv–vis spectrometer
(Puxi TU-1901, Beijing) was used to record absorption spectra. Cell
imaging experiments were carried out on Olympus FV1000 confo-
cal microscope.
c-lyase (CSE) [9,35]. In order to reveal the interaction between
GSH and H2Sn in cellular activity, it is highly valuable to develop
methods that can simultaneously differentiate GSH and H2Sn in
the same situation. However, it is challenging to simultaneously
sense GSH and H2Sn because of their similar structure and reaction
reactivity.
2.2. Synthesis of 2, 4, DCM and Cou-SePh
Based on the reported literature procedures [63,64], compound
2 and DCM were prepared. According to the literature methods
[65], we synthesized compound 4 and Cou-SePh.
Fluorescent probe, for its unique functions and features such as
noninvasive, high-resolution, highly sensitive and real-time detec-
tion, has been widely developed and employed in monitoring and
imaging all kinds of analytes [36–43]. In recent years, a lot of fluo-
rescent sensors or probes for sensing of GSH or H2Sn have been pre-
pared and reported [44–56]. But most of them only can response to
one analyte at a time from single-channel. Such probes fail to
simultaneously detect and image GSH and H2Sn under the same
condition. One simple way gets around this problem is to use
two specific probes at the same time [57,58]. However, such
method not only has the flaw of larger invasive effects, but also
brings potential interference between the two selected probes
[59–62]. Thus, it is desired to develop a single-molecule fluores-
cent probe for simultaneously detecting GSH and H2Sn from differ-
ent channels. Especially, the bifunctional fluorescent probes with
long-wavelength emission are more expect, because the long-
wavelength emission has strong tissues penetration force and
can also reduce the interference of auto-fluorescence aroused by
cell components.
In this work, we designed and synthesized a dual-detection
probe DCM-Cou-SePh with long-wavelength emission. This probe
was constructed by connecting coumarin derivative (Cou) with
phenylselenide moiety (-SePh), recognition site 1 and dye DCM
through ester bond serves as site 2 (synthetic route shown in
Scheme 1). We expected this probe exhibited green fluorescence
in the presence of GSH and red fluorescence when treated with
H2Sn. We also demonstrated that probe DCM-Cou-SePh can be
used as a detector to simultaneously image intracellular GSH and
H2Sn, and this probe was also capable of detecting endogenously
produced H2Sn.
2.3. Synthesis of DCM-Cou-SePh
Dye DCM (0.687 g, 2.2 mmol), coumpound Cou-SePh (0.835 g,
2.0 mmol), EDC (0.421 g, 2.2 mmol) and DMAP (0.269 g, 2.2 mmol)
were dissolved in 5.0 mL anhydrous dichloromethane. The result-
ing reaction mixture was stirred for 5 h at room temperature under
nitrogen atmosphere. After removal of organic solvent, using silica
gel chromatography to purify the aimed product from the crude
product using dichloromethane (DCM) / ethyl acetate (EA) as elu-
ent (v/v, 10:1). Finally we obtained probe DCM-Cou-SePh, as an
orange solid (0.745 g, yield 52.4%). 1H NMR (400 MHz, CDCl3) dH
8.95 (d, J = 8.4 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.71 (d, J = 9.1 Hz,
1H), 7.68–7.61 (m, 4H), 7.60–7.55 (m, 2H), 7.49 (t, J = 7.8 Hz, 1H),
7.32 (s, 1H), 7.30 (t, J = 3.1 Hz, 3H), 7.29 (s, 1H), 6.91 (s, 1H), 6.82
(d, J = 16.0 Hz, 1H), 6.51 (d, J = 9.1 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H),
3.43 (q, J = 7.1 Hz, 4H), 1.23 (t, J = 7.1 Hz, 6H).13C NMR (100 MHz,
CDCl3) dC 163.4, 157.6, 157.3, 155.5, 152.9, 152.3, 151.9, 137.9,
134.7, 132.6, 131.3, 129.8, 129.6, 128.1, 126.0, 125.8, 122.6,
119.8, 119.0, 118.9, 118.7, 117.8, 116.8, 115.7, 109.5, 108.5,
107.1, 97.1, 63.0, 45.0, 12.5. HRMS (ESI) m/z: calcd. for C40H30N3O5-
Se [M + H]+ 712.1351, found 712.1350.
2.4. Cell culture and imaging
MGC-803 cells and RAW264.7 cells were cultured in CMEM
medium containing 1% penicillin and 10% fetal bovine serum
(FBS). The cultivation conditions of cells were as follows: culture
temperature 37 °C, atmosphere containing 5% CO2 and culture time
24 h. Before imaging experiments, the cells were washed by PBS
buffer for three times. For the purpose of investigating the capabil-
ity of the probe to image intracellular GSH and Na2S2, three group
experiments were carried out using MGC-803 cells: A) For imaging
GSH, the cells were incubated with probe DCM-Cou-SePh (10.0
2. Experimental
2.1. Instruments and materials
The reagents used in the experiment were purchased from com-
mercial suppliers. Unless otherwise specified, all reagents were
lΜ) for 30 min. B) For imaging Na2S2, the cells were pre-cultured
Scheme 1. The synthetic route of probe DCM-Cou-SePh. (a) malononitrile, acetic anhydride, reflux 10 h, yield 39.3%; (b) 4-hydroxybenzaldehyde, piperazine, acetic acid,
methylbenzene, reflux 6 h, yield 42.7%; (c) resorcinol, NaClO2, NaH2PO4, H2O, 0 °C, 30 min, yield 76.2%; (d) phenylselenol, DMF, Et3N, 25 °C, 20 min, yield 98.0%; (e) EDC,
DMAP, DCM, rt, 5 h, yield 52.4%.
2

P. Niu, Y. Rong, Y. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
with Na2S2 (220.0
probe (10.0 Μ) for 30 min. C) As a control experiment, the cells
were pretreated by N-ethylmaleimide (NEM, 1.0 mM) for 15 min
and then treated with the probe (10.0 Μ) for 30 min. In the imag-
ing experiments of endogenous Na2S2, we conducted two group of
experiments: A) After pretreated with 1.0 g/mL lipopolysaccharide
(LPS), the RAW264.7 cells were treated with NEM (1.0 mM) for
l
Μ) for 15 min and then incubated with the
deep red fluorescence with emission band centered at 680 nm. The
emission spectra of them were well-separated (Cou: kem = 515 nm,
DCM: kem = 680 nm) (Fig. S1), which can enable to provide differ-
ent fluorescence signal patterns to sense GSH and H2Sn from differ-
ent emission channels. There are two recognition sites in DCM-
Cou-SePh. For site 1, the benzeneselenol moiety not only acts as
an effective group to quench fluorescence of this probe via
photo-induced electron transfer (PET) process [66,67], but also
plays the action of leaving group through SNAr reaction. Moreover,
the ester bond (site 2) between DCM and Cou functions as another
recognition factor to realize the distinguishing of H2Sn over other
RSS. When DCM-Cou-SePh was treated with GSH, thiol group of
GSH as a nucleophile agent, would directly attack benzeneselenol
moiety to generate the green-emitting sulfur-substitued DCM-
Cou-S-GSH via SNAr substitution reaction. Similarly, when this
probe was incubated with Cys/Hcy, site 1 can be cut off to give
DCM-Cou-S-Cys/Hcy by SNAr substitution reaction. However, the
intermediate DCM-Cou-S-Cys/Hcy can be converted to corre-
sponding nonfluorescent amino-substituted derivatives DCM-
Cou-N-Cys/Hcy through intermolecular rearrangement. Treatment
of DCM-Cou-SePh with H2Sn was expected to rapidly replace the
phenylselenide moiety via a SNAr displacement to obtain DCM-
Cou-SSH. Subsequently, the initially formed DCM-Cou-SSH can
undergo a further intramolecular cyclization between the the ester
group (site 2) and thiol group to release the red-emitting dye DCM
and the nonfluorescent coumarindithiolone (Cou-SS). H2S may
react with this probe to generate a nonfluorescent compound,
DCM-Cou-SH by SNAr mechanism. Therefore, this probe could
selectively detect GSH and H2Sn over other RSS from different
channels.
l
l
15 min. Next, the cells were incubated with cystine (200.0
lΜ)
for 30 min and finally treated with this probe (10.0 Μ) for another
l
30 min. B) As a control experiment, the RAW264.7 cells were pre-
treated with NEM (1.0 mM) for 15 min and then incubated with
cystine (200.0 lΜ) for another 30 min. Finally, the cells were trea-
ted with probe DCM-Cou-SePh (10.0 lΜ) for 30 min.
2.5. Detection limit
The detection limit was calculated according to the following
equation:
Detection limit = 3
r/k
where is the standard deviation of blank measurement, k is the
r
slope between fluorescence intensity versus GHS/Na2S2
concentration.
3. Results and discussion
3.1. Molecular design of DCM-Cou-SePh
For the above-mentioned considerations, we developed a fluo-
rescent probe, DCM-Cou-SePh, for simultaneous detection of
GSH and H2Sn. The sensing mechanism of probe DCM-Cou-SePh
for distinguishing RSS was shown in Scheme 2. This probe contains
two dyes connected with an ester bond: dye 7-
diethylaminocoumarin (Cou) and (2-(4-hydroxystyryl)-4H-chro
men-4-ylidene) malononitrile (DCM). Both of them displayed
excellent optical properties [63,65]. Particularlu, dye DCM emitted
3.2. Sensitivity study
To investigate the sensitivity of DCM-Cou-SePh toward GSH
and Na2S2, the fluorescence titration spectra of this probe were
studied in PBS buffer (10.0 mM, pH = 7.4, containing 1.0 mM CTAB)
Scheme 2. The sensing mechanism of probe DCM-Cou-SePh for distinguishing RSS.
3

P. Niu, Y. Rong, Y. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
3.4. Selectivity study
at 25 °C. Firstly, the fluorescence quantum yield of this probe was
determined in the test system, and as expected the solution of this
probe exhibited a low quantum yield (0.17%). The solution of this
To investigate the selectivity of probe DCM-Cou-SePh, the fluo-
probe (10.0
l
M) subjected to an obvious emission enhancement
rescence behaviors of the probe (10.0 lM) responding to various
with kem = 530 nm upon gradual addition of GSH (Fig. 1A1), and
fluorescence intensity at 530 nm (I530 nm) enhanced with the
increasing amount of GSH and reached the maximum when 1.4
equiv. of GSH was added. I530 nm was linear with the concentration
potential analytes (NaCl, KCl, CaCl2, ZnCl2, MgCl2, NaClO, Na2S2O3,
NaSO3, NaHSO3, Na2SO4, Na2S, H2O2, L-Pro, L-Ser, L-Glu, DL-Tyr,
Cys, Hcy, GSH and Na2S2) were examined in PBS buffer (10.0 mM,
pH = 7.4, 1.0 mM CTAB). As shown in Fig. 3, comparison of fluores-
cence emission spectra before and after treatment with analytes
showed that only GSH and Na2S2 can generate significant fluores-
cence enhancement. Overall, the results confirmed that probe
DCM-Cou-SePh exhibited high selectivity to GSH and H2Sn over
other potential interfering species.
of GSH in the range of 0.0–4.0
coefficient (R = 0.9994). To our delight, this probe displayed low
detection limit for GSH (0.12 M) when the ratio of signal to noise
lM with good linear correlation
l
is 3 (Fig. 1A2). In the case of Na2S2, an obvious emission band cen-
tered at 680 nm can be observed and the emission intensity
enhanced with the rising of concentration of Na2S2 (Fig. 1B1).
The intensity can reach equilibrium when 22.0 equiv. of Na2S2
was added. Furthermore, a good linear relationship (R = 0.9945)
3.5. Effect of pH
between I680
and the concentrations of Na2S2 was found in
nm
In order to evaluate whether probe DCM-Cou-SePh can work
under physiological environment, the fluorescence patterns of
the probe (10.0 lM) with GSH/Na2S2 were studied in different pH
the range of 0.0–220.0 lM with a 0.19 lM detection limit based
on S/N = 3 (Fig. 1B2). As consequence, probe DCM-Cou-SePh was
able to distinguish GSH and Na2S2 from green and red emission
channels with good sensitivity.
buffer solutions. The measurements were depicted in Fig. S2.
According to the study, we found that the fluorescence change at
530 nm and 680 nm of this probe can be neglected in pH range
from 2.0 to 10.0, suggesting that this probe can keep stable in a
wide pH range. Upon treatment with GSH, the fluorescence signal
at 530 nm displayed remarkable enhancement in the pH range
from 7.0 to 12.0. In the case of Na2S2, we observed the fluorescence
value at 680 nm enhanced significantly in the pH range of 7.0–10.0.
These experiment results proved that this probe had the potential
to be used for simultaneously sensing GSH and H2Sn under physi-
ological environment.
3.3. Kinetic study
Time-dependent fluorescence experiments of probe DCM-Cou-
SePh toward GSH/Na2S2 were performed in PBS buffer (10.0 mM,
pH = 7.4, containing 1.0 mM CTAB) at 25 °C. When the probe
(10.0 lM) was treated with 1.4 equiv. of GSH, the fluorescence
emission at 530 nm increased immediately and achieved the bal-
ance within 9 min (Fig. 2a). After addition of 22.0 equiv. of Na2S2
to the solution of the probe (10.0 lM), the fluorescence intensity
at 680 nm increased with time and reached the peak value at
30 min (Fig. 2b). The intensity at 530 nm and 680 nm of the free
probe had no significant changes under the same experiment con-
dition. These experiment results clearly stated that probe DCM-
Cou-SePh possessed a good stability and could rapidly response
to GSH and H2Sn.
3.6. Responding mechanism study
In order to verify the response mechanism of the probe, HRMS
analysis was carried out on the reaction mixtures of DCM-Cou-
SePh with GSH and Na2S2. As shown in Fig. S6, the mixture of
DCM-Cou-SePh with GSH displayed an obvious peak at
Fig. 1. (A1-B1) Fluorescence spectra of DCM-Cou-SePh (10.0
lM) was treated with different concentrations of GSH (0.0–4.0 equiv.) and Na2S2 (0.0–32.0 equiv.). (A2-B2) Linear
correlation between concentrations of GSH/Na2S2 and fluorescence intensity (530 nm and 680 nm). (B3-B3) Fluorescence spectra changes of DCM-Cou-SePh (10.0
l
M) upon
addition GSH (0.0–4.0 equiv.) and Na2S2 (0.0–32.0 equiv.). Data were recorded in PBS buffer (10.0 mM, pH = 7.4, 1.0 mM CTAB) at 25 °C. (A1-A2) Excited at 430 nm, (B1-B2)
Excited at 560 nm.
4

P. Niu, Y. Rong, Y. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
Fig. 2. Time-dependent fluorescence intensitiy at 530 nm and 680 nm for probe DCM-Cou-SePh (10.0
lM) with (a) GSH (1.4 equiv.) and (b) Na2S2 (22.0 equiv.) respectively.
Data were recorded in PBS buffer (10.0 mM, pH = 7.4, 1.0 mM CTAB) at 25 °C. (a) Excited at 430 nm, (b) Excited at 560 nm.
Fig. 3. The ratio of fluorescence intensity of probe DCM-Cou-SePh (10.0
Data were recorded in PBS buffer (10.0 mM, pH = 7.4, 1.0 mM CTAB) at 25 °C. (a) Excited at 430 nm, (b) Excited at 560 nm. The concentration of each analyte: 14.0
experiment for GSH, 220.0 M in the experiment for Na2S2.
l
M) at 530 nm for GSH and 680 nm for Na2S2 upon treatment with various analytes respectively.
l
M in the
l
861.2547, which was practically identical to the mass weight of
DCM-Cou-S-GSH (calcd. for 44H41N6O11
[M+H]+, m/
pretreated with Na2S2 (220.0
with the probe (10.0 M) for 30 min (Fig. 4B1-B4). For the control
experiment, very weak fluorescence was observed in two emission
channels when this probe (10.0 M) was treated with the NEM-
pretreated cells for 30 min (Fig. 4C1-C4). These cell experiments
studies showed that this probe could be applied in the discrimina-
tion of intracellular GSH and H2Sn in living cells.
lM) for 15 min and futher treated
C
S
l
z = 861.2554). As shown in Fig. S7, for Na2S2, two peaks at
308.0423 and 313.0968 were found, corresponding to Cou-SS
(calcd. for C14H14NO3S2 [M+H]+, m/z = 308.0415) and DCM (calcd.
for C20H13N2O2 [M+H]+, m/z = 313.0977). The experimental results
clearly and convincingly supported the proposed sensing
mchanism.
l
Then, we set out to perform the experiments of the imaging
endogenous H2Sn in living RAW264.7 cells. According to the
reported methods, cystathionine-c-lyase (CSE) can induce cells
3.7. Cell imaging
to produce endogenous H2Sn using cystine as sulfur source
[17,65,68]. In the experimental group, cells were pretreated
Firstly, we performed MTT assays on probe DCM-Cou-SePh to
for 8 h with 1.0 lg/mL lipopolysaccharide (LPS, stimulate the
evaluate its cytotoxicity to HeLa cells. As shown in Fig. S10, the sur-
overexpression of the CSE mRNA in cells), further treated with
vival rate for this probe at the concentration below 20.0 lM is up
N-ethylmaleimide (NEM, 1.0 mM) for 15 min, then incubated
to 93% after incubation for 24 h, indicating that this probe had a
low cytotoxicity to living cells. It was proved that the applied
potential of this probe in biological system.
with cystine (200.0
lM) for 30 min, and finally incubated with
this probe (10.0 M) for another 30 min. The imaging results
l
were shown in Fig. 5A1-A4, an obvious red fluorescent signal
and a faint green fluorescent signal can be observed. As for
the control group, RAW264.7 cells were cultured with NEM
To verify the capability of probe DCM-Cou-SePh to image intra-
cellular GSH and Na2S2, cell imaging experiments were conducted.
As shown in Fig. 4A1-A4, when MGC-803 cells were only treated
(1.0 mM) at first, and then incubated with cystine (200.0
lM)
with the probe (10.0 lM) for 30 min, we observed strong green flu-
and the probe (10.0 M), successively. Both the green and red
l
orescence and very weak red fluorescence from different channels,
which showed that this probe was responsive to intracellular GSH.
For the imaging of H2S2, red channel displayed strong fluorescence
and green channel emitted faint fluorescence when the cells were
channels emitted very weak fluorescence. These imaging exper-
iments confirmed that this probe could image endogenous H2Sn
in living cells.
5

P. Niu, Y. Rong, Y. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
Fig. 4. Fluorescence images of GSH and H2Sn in living MGC-803 cells. (A1-A4) Cells only incubated with DCM-Cou-SePh (10.0
Na2S2 (220.0 M) for 15 min and then incubated with DCM-Cou-SePh (10.0 M) for 30 min; (C1-C4) NEM-pretreated cells incubated with DCM-Cou-SePh (10.0
30 min. Green channel: kex = 405 nm, emissions were collected at 500–550 nm. Red channel: kex = 543 nm, emissions were collected at 650–700 nm.
l
M) for 30 min; (B1-B4) Cells treated with
l
l
lM) for
Fig. 5. Fluorescence images of endogenous H2Sn in RAW264.7 cells. (A1-A4) Cells were pretreated with 1
l
g/mL LPS for 8 h, NEM (1.0 mM) for 15 min, cystine (200.0
lM) for
30 min and then incubated with DCM-Cou-SePh (10.0 M) for 30 min, (B1-B4) Cells were pretreated with NEM (1.0 mM) for 15 min, cystine (200.0
l
lM) for 30 min and fianlly
treated with probe (10.0
700 nm.
lM) for 30 min. Green channel: kex = 405 nm, emissions were collected at 500–550 nm. Red channel: kex = 543 nm, emissions were collected at 650–
4. Conclusions
CRediT authorship contribution statement
In summary, a long-wavelength emission fluorescent probe,
DCM-Cou-SePh was developed for distinguish GSH and H2Sn
(n > 1) from different fluorescence emission channels (GSH:
kex/em = 430/530 nm, H2Sn: kex/em = 560/680 nm). This probe
displayed good stability, good selectivity and high sensitivity
during the simultaneous detection of GSH and H2Sn. This
probe also can quantitatively detect GSH and H2Sn with low
Peixin Niu: Writing - original draft, Investigation. Yifan Rong:
Writing - original draft, Investigation, Data curation. Yuyue Wang:
Investigation. Huijie Ni: Investigation. Minghui Zhu: Investigation.
Wenqiang Chen: Writing - review & editing, Formal analysis.
Xingjiang Liu: Conceptualization, Supervision, Funding acquisi-
tion. Liuhe Wei: Project administration, Resources. Xiangzhi Song:
Writing - review & editing.
detection limits, 0.12 lM for GSH and 0.19 lM for H2Sn
respectively. The capability of probe DCM-Cou-SePh for simul-
taneously sensing intracellular GSH and H2Sn in living cells
was successfully demonstrated by cell imaging experiments.
Moreover, this probe was capable of imaging endogenously
produced H2Sn.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
6

P. Niu, Y. Rong, Y. Wang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 257 (2021) 119789
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Acknowledgements
This research was supported by the National Natural Science
Foundation of China (No.22008222).
Appendix A. Supplementary material
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