A highly sensitive endoplasmic reticulum-targeting fluorescent probe for the imaging of endogenous H2S in live cells

Article abstract of DOI:10.1016/j.saa.2020.118578

Hydrogen sulfide (H₂S) as an important signaling biomolecule participates in a series of complex physiological and pathological processes. In situ and rapid detection of H₂S levels in endoplasmic reticulum (ER) is of great importance for the in-depth study of its virtual functional roles. However, the ER-targeting fluorescent probe for the detection of H₂S in live cells is still quite rare. Herein, a new ER-targeting fluorescent probe (FER-H₂S) for detecting H₂S in live cells was characterized in the present study. This probe FER-H₂S was built from the hybridization of three parts, including fluorescein-based skeleton, p-toluenesulfonamide as ER-specific group, and 2,4-nitrobenzene sulfonate as a response site for H₂S. The response mechanism of the probe FER-H₂S to H₂S is on the basis of the ring-opening and ring-closing processes in fluorescein moiety. Moreover, the probe FER-H₂S was successfully used for the imaging of exogenous and endogenous H₂S in ER of live cells.

Full text of DOI:10.1016/j.saa.2020.118578

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A highly sensitive endoplasmic reticulum-targeting fluorescent
probe for the imaging of endogenous H2S in live cells
Lei Zhou, Zhi-Qiang Cheng, Ning Li, Yong-Xi Ge, Hong-Xu Xie,
Kongkai Zhu, Aiqin Zhou, Juan Zhang, Kai-Ming Wang, Cheng-
Shi Jiang
PII:
S1386-1425(20)30557-6
https://doi.org/10.1016/j.saa.2020.118578
SAA 118578
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 28 April 2020
Revised date:
30 May 2020
1 June 2020
Accepted
date:
Please cite this article as: L. Zhou, Z.-Q. Cheng, N. Li, et al., A highly sensitive
endoplasmic reticulum-targeting fluorescent probe for the imaging of endogenous H2S in
live cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2020),
https://doi.org/10.1016/j.saa.2020.118578
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A highly sensitive endoplasmic reticulum-targeting fluorescent probe for the
imaging of endogenous H2S in live cells
Lei Zhoua, Zhi-Qiang Chenga, Ning Lia, Yong-Xi Gea, Hong-Xu Xiea, Kongkai Zhub,
Aiqin Zhouc, Juan Zhang*,a, Kai-Ming Wang*,a, Cheng-Shi Jiang*,a
a School of Biological Science and Technology, University of Jinan, Jinan 250022,
China
b College of Chemistry and Molecular Engineering, Qingdao University of Science
and Technology, Qingdao, 266042, China
c Collage of Horticulture, Qingdao Agricultural University, Qingdao, 266109, China
*Corresponding author
E-mail addresses: bio_zhangj@ujn.edu.cn (J. Zhang); bio_wangkm@ujn.edu.cn
(K.-M. Wang); jiangchengshi-20@163.com (C.-S. Jiang)
Tel/Fax: 0086-531-89736799
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ABSTRACT
Hydrogen sulfide (H2S) as an important signaling biomolecule participates in a
series of complex physiological and pathological processes. In situ and rapid detection
of H2S levels in endoplasmic reticulum (ER) is of great importance for the in-depth
study of its virtual functional roles. However, the ER-targeting fluorescent probe for
the detection of H2S in live cells is still quite rare. Herein, a new ER-targeting
fluorescent probe (FER-H2S) for detecting H2S in live cells was characterized in the
present study. This probe FER-H2S was built from the hybridization of three parts,
including fluorescein-based skeleton, p-toluenesulfonamide as ER-specific group, and
2,4-nitrobenzene sulfonate as a response site for H2S. The response mechanism of the
probe FER-H2S to H2S is on the basis of the ring-opening and ring-closing processes
in fluorescein moiety. Moreover, the probe FER-H2S was successfully used for the
imaging of exogenous and endogenous H2S in ER of live cells
Keywords: Endoplasmic reticulum; Hydrogen sulfide; Fluorescent probe; Xanthene;
Fluorescein
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1. Introduction
Hydrogen sulfide (H2S) is a critical signaling reactive biomolecule, and plays
important roles in numerous biological processes in living system [1-3]. H2S is
generally produced from L-cysteine or homocysteine by the catalysis of the enzymes
including cystathionine-β-synthase, cystathionine-γ-lyase and 3-mercapto-pyruvate
sulfurtransferase in the organs including liver, heart and kidney [4-6]. Recently,
emerging evidences reveal that H2S is implicated in a great number of pathological
processes, such as inflammation, vasodilation and nervous system [7-11]. Moreover,
H2S also plays important roles in the protection against cardiac ischemia-reperfusion
[12]. Even so, the endogenous H2S in abnormal level could result in numerous
malignant diseases including Alzheimer's disease, heart disease, diabetes, and
cardiovascular diseases [13-16]. Therefore, the real-time and in situ detection of H2S
level is highly important for the further study of its roles in numerous biological
processes.
Endoplasmic reticulum (ER), as one of the vital organelles in nearly all the
eukaryotic cells, performs numerous critical physiological functions in the
biosynthesis of protein, lipid manufacture and metabolism, cellular Ca2+ concentration
stabilization, and detoxification [17-21]. Recently, a great variety of studies suggest
that H2S plays critical roles in the modulation of ER functions. The decrease of H2S
level in ER affects the phosphorylation of tyrosine-619 in PERK, and reduces the
activation of tyrosine-619 under ER stress [22]. H2S can result in the apoptosis of the
insulin-secreting β cells by the increase of ER stress through P38 MAPK activation
[23]. In spite of the remarkable advances in the study of H2S in ER, many virtual
functional roles of H2S in ER still remain unknown. Thereinto, one of the crucial
obstacles is the lack of effective methods for the real-time and in situ detection of H2S
level in ER, which is naturally in urgent need to be overcome.
Up to date, many available methods, including methylene blue colorimetric,
electrochemical and chromatographic analytic methods, have been developed for the
detection of H2S [24-28]. However, these are generally not suitable for real-time and
in situ detecting H2S levels in the sophisticated live systems. In comparison with these
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traditional methods, fluorescence imaging by molecular probes has become a more
powerful tool due to its significant advantages in simple operation, real-time and in
situ detection [29-35]. To date, a great many of fluorescent H2S probes have been
constructed on the basis of the reducibility of H2S and the nucleophilicity of HS-
[36-43]. However, to the best of our knowledge, ER-targeting fluorescence probes for
H2S are still quite rare [44,45]. Therefore, the development of the ER-targeting
fluorescent H2S probes is still in urgent need.
Herein, a new ER-targeting fluorescein-based fluorescent probe (FER-H2S) (Fig.
1) was designed and synthesized for the detection of exogenous and endogenous H2S
in live cells. The probe FER-H2S was smoothly constructed from three pivotal
functional fragments, including fluorescein-based potential fluorophore,
p-toluenesulfonamide as the ER-targeting moiety, and 2,4-nitrobenzenesulfonate as a
sensing site for H2S. Before interacting with H2S, FER-H2S showed nearly no
fluorescence because of ring-closing form of the spirolactone in the fluorescein
moiety. Once in response to H2S, the 2,4-nitrobenzene sulfonate unit was cleaved off
from FER-H2S, and the ring-opening response product generated simultaneously was
strongly emissive. More importantly, the imaging experiments demonstrated that
FER-H2S could be successfully applied to the detection of the exogenous and
endogenous H2S in ER of live cells.
Fig. 1. The structure of FER-H2S
2. Material and methods
2.1. Synthesis
Scheme 1 shows the synthetic route of FER-H2S.
2.1.1. Synthesis of 1
To the mixture of
β
-alanine (446 mg, 5.0 mmol) in 4 mL distilled water was
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added aqueous NaOH (2 M, 3.0 mL) and tosyl chloride (953 mg, 5.0 mmol). The
reaction mixture was stirred at 35°C, and 2M aqueous NaOH was added to keep the
pH value of the reaction system around 9. When the TLC indicated that the reaction
was completed, the solution was filtered, and the organic phase was acidified to pH ~
2 by addition of 0.5 M aqueous HCl at 0 °C. The obtained crystals were washed with
1
cold water and dried to produce 1 as white solid (496 mg, 40.7%). H NMR (600
MHz, DMSO-d6) δ 12.27 (brs, 1H, CO2H), 7.67 (d, J = 8.2 Hz, 2H), 7.58 (t, J = 5.8
Hz, 1H, NH), 7.40 (d, J = 8.2 Hz, 2H), 2.92–2.87 (m, 2H), 2.38 (s, 3H), 2.34 (t, J =
7.1 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 172.3, 142.7, 137.3, 129.7, 126.6,
38.6, 34.1, 21.0. ESI-MS: [M+H]+ calcd for C10H14NO4S+ 244.0, found 244.0.
2.1.2. Synthesis of 2
To a solution of resorcinol (607 mg, 5.5 mmol) in 20 mL nitrobenzene was added
phthalic anhydride (896 mg, 6.1 mmol) and AlCl3 (2.2 g, 16.5 mmol). The solution
was stirred overnight, and then poured into a mixed solution of hexanes (20.0 mL)
and aqueous HCl (35.0 mL 0.5 M) for 2 h. The formed yellow precipitate was
1
crystallized from EtOAc/PE to produce 2 as yellow solid (1.2 g, 85.0%). H NMR
(600 MHz, DMSO-d6) δ 13.16 (brs, 1H, CO2H), 12.23 (s, 1H), 10.72 (s, 1H), 7.99 (d,
J = 7.5 Hz, 1H), 7.71 (td, J = 7.7, 7.5 Hz, 1H), 7.64 (dd, J = 7.7, 7.3 Hz, 1H), 7.42 (d,
J = 7.3 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.32 (d, J = 2.2 Hz, 1H), 6.28 (dd, J = 8.8,
2.2 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) δ 200.5, 166.7, 165.0, 164.4, 140.0,
134.7, 132.3, 130.0, 129.7, 129.4, 127.4, 113.2, 108.3, 102.5.
2.1.3. Synthesis of 3
A mixture of 3-aminophenol (655 mg, 6.0 mmol), diethylene glycolmonomethyl
ether (3 mL), and bis(2-chloroethyl) aminehydrochloride (1.1 g, 6.0 mmol) was
heated at 150 °C for 12 h in dry N2 atmosphere. Then the mixture was cooled to room
temperature, and poured into MeOH (5.0 mL) followed by addition of Et2O (150.0
mL). The generate precipitate was washed with Et2O to give compound 3 as brown
solid (662 mg, 62.0%). 1H NMR (600 MHz, DMSO-d6) δ 9.11 (brs, 1H, CO2H), 6.96
(dd, J = 8.1, 8.1 Hz, 1H), 6.34 (dd, J = 8.1, 2.1 Hz, 1H), 6.27 (dd, J = 2.2, 2.1 Hz, 1H),
6.18 (dd, J = 8.1, 2.2 Hz, 1H), 2.98–2.93 (m, 4H), 2.82–2.77 (m, 4H). 13C NMR (150
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MHz, DMSO-d6) δ 158.1, 153.1, 129.4, 106.5, 106.0, 102.3, 49.3, 45.6. ESI-MS:
[M+H]+ calcd for C10H15N2O+ 179.1, found 179.0.
2.1.4. Synthesis of 4
Compound 2 (1.1 g, 4.4 mmol) was dissolved in CF3CO2H (15 mL), and then
compound 3 (659 mg, 3.7 mmol) was added. The mixture was heated to reflux. After
36 h, the solution was concentrated, and the residues was purified by flash column
chromatography using CH3OH:DCM=1:6 to produce red solid 4 (800 mg, 54.0%). 1H
NMR (600 MHz, DMSO-d6) δ 10.20 (brs, 1H, CO2H), 8.00 (d, J = 7.4 Hz, 1H), 7.79
(dd, J = 7.4, 7.2 Hz, 1H), 7.72 (dd, J = 7.4, 7.2 Hz, 1H), 7.25 (d, J = 7.4 Hz, 1H), 6.91
(s, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.70 (s, 1H), 6.59 (d, J = 9.3 Hz,1H), 6.57–6.54 (m,
2H); 3.47 (brs, 4H), 3.24–3.17 (m, 5H). 13C NMR (150 MHz, DMSO-d6) δ 168.7,
159.6, 158.3, 158.2, 151.9, 151.8, 151.6, 135.6, 130.1, 129.98, 129.1, 128.6, 126.2,
124.7, 124.0, 112.1, 109.6, 109.4, 103.1, 102.0, 44.6, 42.4.
2.1.5. Synthesis of 5
Compound 1 (486 mg, 2.0 mmol), HATU (912 mg, 2.4 mmol), and DIEA (505
μL, 4.0 mmol) were successively added to a solution of compound 4 (800 mg, 2.0
mmol) in 25 mL of DCM/DMF (v/v, 5:1). The mixture was stirred for 12 h at room
temperature, and then extracted using with mixed solution of DCM (30 mL) with
water (30 mL). The organic phase was dried over Na2SO4, and then concentrated. The
resulting restudies was subjected to flash column chromatography using
CH3OH/DCM (1/20, v/v) as the eluent to obtain compound 5 as a dark red solid (1.0 g,
81.0%).1H NMR (600 MHz, CDCl3) δ 8.23 (dd, J = 8.9, 0.9 Hz, 1H), 7.73 (ddd, J =
7.7, 7.5, 0.9 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H),7.66 (ddd, J = 8.0, 7.5, 1.0 Hz, 1H),
7.33–7.29 (m, 3H), 6.85 (d, J = 9.0 Hz, 1H), 6.83–6.80 (m, 2H), 6.67 (dd, J = 8.9, 2.1
Hz, 1H), 6.52 (dd, J = 8.9, 1.8 Hz, 1H), 6.44 (d, J = 1.4 Hz, 1H), 5.47 (t, J = 6.6 Hz,
1H, NH), 3.76 (t, J =5.1 Hz, 2H), 3.59 (t, J =5.4 Hz, 2H), 3.47–3.41 (m, 4H),
3.26–3.21 (m, 2H), 2.62 (t, J = 5.6 Hz, 2H), 2.42 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 169.9, 165.9, 159.1, 154.7, 153.8, 143.6, 137.2, 134.8, 132.7, 131.2, 130.7, 130.5,
130.3, 130.1, 130.0, 129.9, 129.7, 129.4, 129.1, 127.1, 116.9, 113.3, 111.7, 105.8,
100.4, 52.5, 47.0, 44.6, 41.0, 39.2, 33.3, 21.7. ESI-MS m/z 626.0 [M+H]+. HR-ESIMS:
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[M+H]+ calcd for C34H32N3O7S+ 626.1955, found 626.1950.
2.1.6. Synthesis of the probe FER-H2S
To a solution of 2,4-dinitrobenzenesulfonyl chloride (89 mg, 0.6 mmol) in dry
DCM (5.0 mL) was added compound 5 (74.5 mg, 0.12 mmol), and Et3N (151 μL).
The mixture was stirred at 0oC for 0.5 h, and then at room temperature for 12 h. The
solution was concentrated to obtain a crude material, which was extracted with
DCM/H2O. The organic phase was dried over Na2SO4, and concentrated. The
obtained residues was purified on silica gel column using CH3OH/DCM (1/20, v/v) to
yield FER-H2S as a yellow solid (50 mg, 49.1%). 1H NMR (600 MHz, CDCl3) δ 8.66
(d, J = 2.1 Hz, 1H), 8.52 (dd, J = 8.7, 2.1 Hz, 1H), 8.25 (d, J = 8.7 Hz, 1H), 8.02 (d, J
= 7.7 Hz, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.69 (dd, J = 7.5, 7.1 Hz, 1H), 7.64 (dd, J =
7.7, 7.1 Hz, 1H), 7.30 (d, J = 7.6 Hz, 2H), 7.18 (d, J = 2.2 Hz, 1H), 7.15 (d, J = 7.5 Hz,
1H), 6.87 (dd, J = 8.9, 2.1 Hz, 1H), 6.79 (d, J = 8.9 Hz, 1H), 6.67–6.63 (m, 2H), 6.60
(dd, J = 8.7, 2.4 Hz, 1H), 5.52 (t, J = 6.1 Hz, 1H, NH), 3.72 (t, J = 4.5 Hz, 2H), 3.54 (t,
13
J = 4.4 Hz, 2H), 3.25–3.19 (m, 6H), 2.59 (t, J = 5.3 Hz, 2H), 2.40 (s, 3H). C NMR
(150 MHz, CDCl3) δ 169.7, 169.2, 152.6, 152.6, 152.3, 152.0, 151.2, 149.5, 149.1,
143.5, 137.2, 135.5, 134.2, 133.4, 130.3, 130.2, 129.9, 129.0, 127.1, 126.8, 126.5,
125.4, 124.0, 120.6, 119.7, 117.2, 112.8, 111.1, 109.2, 102.4, 82.1, 48.2, 48.0, 44.8,
41.2, 39.2, 33.1, 21.6. ESI-MS m/z 856.0 [M+H]+. HR-ESIMS: [M+H]+ calcd for
+
C40H34N5O13S2 856.1589, found 856.1599.
2.2. Fluorescence imaging of H2S in live cells
HepG2 cells were incubated in Eagle medium (10% fetal bovine serum and 1%
antibiotic) under the environment with 5% CO2 and 95% air at 37 °C. Subsequently,
the HepG2 cells were transferred to glass-bottom culture dishes and then cultured
overnight. All the fluorescent images in this work were acquired using a Nikon A1R
confocal microscope.
For the imaging of the exogenous H2S: HepG2 cells were pre-incubated with 5
μM FER-H2S for 30 min and then washed by PBS buffer three times. Subsequently,
the HepG2 cells were incubated with 100 μM or 200 μM Na2S in PBS solution for
another 30 min.
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For the colocalization experiment of the probe FER-H2S in live cells: After the
pre-treatment of the HepG2 cells with 5 μM FER-H2S for 30 min and 100 μM Na2S
in PBS solution for another 30 min, the HepG2 cells were stained with 1 μM
ER-Tracker (a commercial ER tracker) for 5 min.
For the imaging of the endogenous H2S: HepG2 cells were treated with 1 mM
DL-propargylglycine (PAG) for 1 h, and then washed with PBS three times. Then,
these cells were further stained with 5 μM FER-H2S for 30 min. Finally, 200 μM
Na2S in PBS solution was added and the cells were incubated for another 30 min.
3. Results and discussion
3.1. Design and synthesis of FER-H2S
Fluorescein is well-known as an excellent and versatile fluorescent dye because
of its high-profile physicochemical advantages, such as the brilliant photostability and
the excellent fluorescence quantum yield. Meanwhile, the fluorescence property of the
fluorescein could be conveniently controlled by regulating the ring opening/closing
form of the fluorescein moiety in different response environments. Literature survey
indicated that p-toluenesulfonamide could serve as an ER-specific unit, and thus it
was embedded into the fluorescein moiety to endow the hybrid molecule with
ER-targeting property. Besides, 2,4-nitrobenzenesulfonate could function as an
excellent response site for H2S. With these considerations in mind, the
above-mentioned three fragments were fused into one molecule to construct a new
ER-targeting fluorescein-based fluorescent probe (FER-H2S), which can be employed
for the real-time detection of H2S in live cells.
The synthesis of the probe FER-H2S was shown in Scheme 1. Briefly, treatment
of β-alanine with tosyl chloride afforded
obtained from the starting materials, resorcinol/phthalic anhydride and
3-aminophenol/bis(2-chloroethyl) aminehydrochloride, respectively. The
1. Compounds 2 and 3 could be easily
hybridization of 2 and 3 provided the fluorescein block 4, which was then reacted
with 1 to yield the key intermediate 5. The target compound FER-H2S was achieved
through the esterification reaction of 5 and 2,4-dinitrobenzenesulfonyl chloride in
the presence of Et3N. The structures of these synthetic compounds were confirmed
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by 1H NMR, 13C NMR and MS data (ESI†).
Scheme 1 Synthesis route of the probe FER-H2S. (a) β-alanine, tosyl chloride,
aqueous NaOH, r.t., overnight; (b) pathalic anhydride, AlCl3, nitrobenzene, r.t.,
overnight;
(c)
bis(2-chloroethyl)
aminehydrochloride,
diethylene
, HATU,
glycolmonomethyl ether, 150°C, 12 h; (d) CF3CO2H, reflux, 36 h; (e)
1
DIEA, DCM/DMF (5:1), r.t., 12 h; (f) 2,4-dinitrobenzenesulfonyl chloride,
Et3N, DCM, r.t., 12 h.
3.2. Optical response of FER-H2S to H2S
Initially, the absorption and fluorescence spectra of FER-H2S in the absence and
presence of Na2S were tested to assess the optical response of FER-H2S to H2S. In
PBS buffer (20 mM, pH = 7.4) containing 10% EtOH, the probe FER-H2S displayed
nearly no obvious absorption in the visible region (Fig. S1, ESI†). After the treatment
of the probe FER-H2S with Na2S at different concentrations, the absorption peak at
512 nm appeared and showed continuous increase with the increase of Na2S
concentrations, suggesting that the reaction between FER-H2S and Na2S occurs.
FER-H2S was nearly no emissive with the extremely low fluorescence quantum yield
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(Ф < 0.0001) under excitation at 500 nm, due to the ring closing form of the xanthene
moiety (Fig. 2A). When Na2S was introduced into the solution of the probe FER-H2S,
a significant enhancement in the emission at 556 nm was observed clearly (Fig. 2A).
The fluorescence intensity at 556 nm increased by about 100-fold after the response
of FER-H2S to 600 μM Na2S. Meanwhile, the fluorescence intensity at 556 nm
showed a desirable linear relationship with Na2S in the concentration of 0-20 μM, and
the calculated detection limit was 0. 081 μM (Fig. 2B). Furthermore, the
time-dependent fluorescence spectra of 5 μM FER-H2S to 100 μM Na2S were then
conducted to explore sensitivity of FER-H2S to H2S. The fluorescence intensity at
556 nm successively increased with time, and the fluorescence intensity at 556 nm
could reach the plateau within 8 min (Fig. 2C and 2D). Taken together, FER-H2S
could be potentially employed as a sensitive fluorescent probe for the imaging of H2S
in live systems.
(B) 600
(A) 6000
Y = 23.38 * X + 52.13
R2 = 0.9897
600 M
400
4000
0 M
200
0
2000
0
0
5
10
15
20
550
600
650
700
Concentration / M
Wavelength / nm
(C) 3200
2400
1600
800
3200
2400
1600
800
0
(D)
60 min
0 min
0
550
600
650
700
0
20
40
60
Wavelength / nm
Time / min
Fig. 2. (A) Fluorescence spectra of 5 μM FER-H2S upon the addition of 0-600 μM
Na2S in PBS buffer (10% EtOH, pH = 7.4) under excitation at 500 nm. (B) Linearity
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between the fluorescence intensity at 556 nm and Na2S concentration in the range of 0
-20 μM. (C) Time-dependent fluorescence spectra of 5 μM FER-H2S in the presence
of 100 μM Na2S under excitation at 500 nm in PBS (10% EtOH, pH = 7.4). (D)
Fluorescence intensity at 556 nm as a function of time.
To explore the sensing mechanism of FER-H2S to H2S, the HRMS and HPLC
tests were performed. HRMS data suggested that a peak at 626.1958 (Fig. S2, ESI†)
ascribed to compound 5 (Cald. [M+H]+ = 626.1955) can be found apparently after the
reaction of FER-H2S with Na2S by the cleavage of 2,4-nitrobenzene sulfonate unit.
Meanwhile, the HPLC data further suggested that the response product was the
compound 5 after the treatment of FER-H2S with Na2S (Fig. S3, ESI†). Namely, the
sensing mechanism of FER-H2S to H2S is on the basis of the cleavage of
2,4-nitrobenzene sulfonate unit (Scheme 2).
Scheme 2 Proposed response mechanism of FER-H2S to H2S.
3.3. Selectivity of FER-H2S towards H2S
To assess the selectivity of FER-H2S to H2S, the fluorescence spectra of
FER-H2S in the presence of a great variety of competing analytes such as various
2-
ions (Al3+, Ca2+, Zn2+, F-, I-, SO3 ), biothiols (Cys, GSH, Hcy), ROS (H2O2, ClO-, etc)
and other biological species were conducted. As shown in the Fig. 3, the fluorescence
intensity at 556 nm obviously increased after the reaction with Na2S, whereas the
other species lead to nearly no fluorescence response. These results suggest that
FER-H2S shows high selectivity to H2S in comparison with other competing analytes
and could be employed as a specific probe for the detection of H2S in biological
systems.
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(A) 3200
2400
1600
800
(B) 3200
FER-H2S
2400
FER-H
2
S or
S + other analyte
1600
800
0
FER-H
2
0
550
600
650
700
1
3
5
7
9
11 13 15 17 19 21
Wavelength / nm
Fig. 3. Fluorescence spectra (A) and fluorescence intensity at 556 nm (B) of 5 μM
FER-H2S in the presence of various species in PBS buffer (10% EtOH, pH = 7.4)
under excitation at 500 nm. 1, blank; 2, KI; 3, CaCl2; 4, FeSO4; 5, Cys; 6, CoCl2; 7,
MgCl2; 8, NaBr; 9, NaF; 10, CuSO4; 11, GSH; 12, H2O2; 13, TBHP; 14, DBTP; 15,
Hcy; 16, ZnCl2; 17,VC; 18, NaNO2; 19, NaClO; 20, Na2SO3; 21,Na2S. Concentration:
GSH, 10 mM; other analytes, 100 μM.
4000
FER-H2S
FER-H2S + H2S
3000
Normal physiological
pH (~7.4)
2000
1000
0
4
5
6
8
9
pH 7
Fig. 4. The fluorescence responses of 5 μM FER-H2S at 556 nm in the absence and
presence of 100 μM Na2S at diff erent pH values.
The pH impact on the fluorescence spectra of FER-H2S treated without or with
Na2S was then investigated to examine if FER-H2S could work efficiently under
physiological conditions. Before the reaction with Na2S, the fluorescence spectra of
FER-H2S showed that the fluorescence was very weak in the pH range 4.0-9.0,
suggesting that FER-H2S exhibited desirable stability to different pH circumstance.
After the addition of Na2S to the solution of the probe, FER-H2S could show
desirable response to Na2S in the pH range 5.5-9.0 (Fig. 4 and Fig. S4, ESI†). Taking
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into consideration of the normal physiological pH at about 7.4, FER-H2S could serve
as an available probe for monitoring H2S in live systems.
3.4. Fluorescence imaging in live cells
The feasibility of FER-H2S to image cellular H2S was subsequently investigated
using live HepG2 cells. Firstly, MTT assay was performed to test the cytotoxicity of
FER-H2S with different concentrations to live HepG2 cells (Fig. S5, ESI†). The MTT
data showed that FER-H2S possessed no significant cytotoxicity below the
concentration of 20 μM and was available for the fluorescence imaging of cellular
H2S. As depicted in Fig. 5, when the live cells were firstly stained with 5 μM
FER-H2S for 30 min, there was very weak fluorescence in green channel. However,
after the further treatment of the HepG2 cells with 100 μM or 200 μM Na2S, the
strong fluorescence in green channel appeared clearly, and the fluorescence intensity
obviously enhanced with the Na2S level changed from 100 μM to 200 μM. It suggests
that the probe FER-H2S could be employed to image exogenous H2S in live cells.
(B)
(A)
Bright field Green channel
Merged
3600
2700
1800
900
0
A1
A2
B2
C2
A3
B3
C3
B1
C1
Fig. 5. (A) A1-A3: Images of HepG2 cells treated with 5 μM FER-H2S for 30 min;
B1-B3: Images of HepG2 cells treated with 5 μM FER-H2S for 30 min and the
treated with 100 μM Na2S for another 30 min; C1-C3: Images of HepG2 cells treated
with 5 μM FER-H2S for 30 min and the treated with 200 μM Na2S for another 30 min.
λem = 500-550 nm, λex = 488 nm. Scale bar = 20 μm. (B) Quantified fluorescence
intensity of a single cell in Green channel analyzed by ImageJ software.
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We then assessed the feasibility of FER-H2S to selectively visualize H2S in ER
of live cells. The colocalization experiments were performed to check the main
distribution of FER-H2S at subcellular level by using live HepG2 cells and a
commercial ER tracker. The cells were firstly stained with 5 μM FER-H2S for 30 min
and treated with 100 μM Na2S for 30 min, and then incubated with 1 μM ER-Tracker
for 5 min. As depicted in Fig. 6, the fluorescence in green channel was generated from
the response FER-H2S to cellular Na2S, and displayed prominent overlap with the
fluorescence in blue green that produced from ER-tracker. The calculated Pearson’s
colocalization coeffcient (R) value of the fluorescence in blue and green channels was
0.93. Therefore, the probe FER-H2S was mainly distributed in ER and can be
employed as an ER-specific probe.
(A)
(B)
(C)
(D)
Fig. 6. Fluorescence images of HepG2 cells treated with 5 μM FER-H2S for 30 min
and the treated with 100 μM Na2S for another 30 min, and then treated with 1 μM
ER-tracker for 5 min. (A) Fluorescence image of ER-Tracker Blue collected at
425-475 nm at 405 nm excitation. (B) Fluorescence image of FER-H2S collected at
500-550 nm at 488 nm excitation. (C) Merged image of (A) and (B). (D) Correlation
plot of the fluorescence intensity in (A) and (B). Scale bar = 20 μm.
Finally, the capability of FER-H2S to detect endogenous H2S was investigated.
When the HepG2 cells were only stained with 5 μM FER-H2S for 30 min, the cells
showed very weak fluorescence under excitation at 488 nm. DL-propargylglycine
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(PAG), is an efficient inhibitor for H2S, which can reduce the endogenous H2S levels
in live systems by lowering the activity of L-cysteine desulfhydrase (LCD) and
D-cysteine desulfhydrase (DCD) [46,47]. When the HepG2 cells were stimulated by 1
mM PAG for 1 h and subsequently treated with 5 μM FER-H2S for another 30 min,
nearly no fluorescence could be observed (Fig. 7). It indicates that the endogenous
H2S level in live cells decreases under the stimulation of PAG. Meanwhile, the
fluorescence intensity in green channel of the cells treated with 1 mM PAG and 5 μM
FER-H2S was significantly enhanced after the further addition of 200 μM Na2S,
indicating that the probe FER-H2S shows fluorescence response to the exogenous
H2S (Fig. 7). Therefore, the probe FER-H2S could be applied to detect endogenous
H2S in ER of live cells.
(A)
Bright field Green channel
Merged
(B)
A1
A2
B2
C2
A3
B3
C3
4500
3000
1500
0
B1
C1
Fig. 7. (A) Fluorescence images of HepG2 cells treated with 5 μM FER-H2S for 30
min (A1-A3). HepG2 cells were pre-treated with 1 mM PAG for 1 h and then
incubated with 5 μM FER-H2S for 30 min (B1-B3), then incubated with 200 μM
Na2S for 30 min (C1-C3), λem = 500-550 nm, λex = 488 nm. Scale bar = 20 μm. (B)
Quantified fluorescence intensity of a single cell in Green channel analyzed by
ImageJ software.
4. Conclusions
In conclusion, a new fluorescent probe (FER-H2S) was constructed for the
detection of H2S in ER. This probe FER-H2S was built from the hybridization of
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three parts, including fluorescein-based skeleton, p-toluenesulfonamide as
ER-specific group and 2,4-nitrobenzene sulfonate as a response site for H2S. The
response mechanism of the probe FER-H2S to H2S is on the basis of the ring
opening/clsosing of the xanthene in fluorescein moiety. Moreover, the probe
FER-H2S was successfully used for the imaging of exogenous and endogenous H2S
in ER of live cells.
Declaration of competing interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper
Acknowledgements
This work was financially supported by the Agricultural Variety Improvement
Project of Shandong Province of China [No. 2019LZGC007], National Natural
Science Foundation of China [Nos. 21672082, 81603215], Natural Science
Foundation of Shandong Province [Nos. ZR2019YQ31, ZR2017BC101], Doctoral
Fund Project of University of Jinan [No. 301/160100394], Shandong Talents Team
Cultivation Plan of University Preponderant Discipline [No. 10027].
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Credit author statement
Cheng-Shi Jiang directed the research, and reviewed the paper.
Juan Zhang and Kai-Ming Wang designed and performed the experiments, analyzed
the data, and wrote the paper.
Lei Zhou performed the experiments and analyzed the data;
Zhi-Qiang Cheng, Ning Li, Yong-Xi Ge, Hong-Xu Xie, Kongkai Zhu, and Aiqin Zhou
helped with the experiments and analyzed the data.
All the authors discussed the results and commented on the manuscript.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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Graphic abstract
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Highlight
1. A novel ER-targeting fluorescent probe (FER-H2S) for H2S was designed.
2. The probe FER-H2S showed high sensitive and selective response to H2S.
3. The probe FER-H2S can be applied for the imaging of endogenous H2S in live
cells.
25