A smart mitochondria-targeting TP-NIR fluorescent probe for the selective and sensitive sensing of H2S in living cells and mice

Article abstract of DOI:10.1039/d1nj00840d

Hydrogen sulfide (H₂S) is one of the important gaseous signalling molecules, which plays key roles in various critical biological processes. In this work, we report a novel two-photon near-infrared (TP-NIR) fluorescent probe (MNIR-H₂S) for the sensing of mitochondrial H₂S in living cells, tissues, zebrafish and nude mice. Using a xanthene dye as a TP-excitation (810 nm) NIR-emission (668 nm) fluorophore, a well-known H₂S response moiety with a robust intramolecular charger transfer (ICT) effect can quickly respond to H₂S and effectively quench the fluorescence intensity, and an oxonium cation structure serves as a mitochondria-targeting site. Notably,MNIR-H₂Sdisplays high sensitivity, high selectivity, low cytotoxicity, powerful mitochondrial localization ability, deep tissue penetration, and a very minimal imaging background. Biological imaging results show the biological application ofMNIR-H₂Sas a robust new tool for use in H₂S imaging in biological systems.

Full text of DOI:10.1039/d1nj00840d

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A smart mitochondria-targeting TP-NIR
fluorescent probe for the selective and sensitive
Cite this: New J. Chem., 2021,
4
5, 7315
sensing of H S in living cells and mice†
2
a
a
a
a
a
Qiaomei Yang, Liyi Zhou, * Longpeng Peng, Gangqiang Yuan, Haiyuan Ding,
a
b
Libin Tan and Yizhuang Zhou*
Hydrogen sulfide (H
various critical biological processes. In this work, we report a novel two-photon near-infrared (TP-NIR)
fluorescent probe (MNIR-H S) for the sensing of mitochondrial H S in living cells, tissues, zebrafish and
nude mice. Using a xanthene dye as a TP-excitation (810 nm) NIR-emission (668 nm) fluorophore, a
2
S) is one of the important gaseous signalling molecules, which plays key roles in
2
2
2
well-known H S response moiety with a robust intramolecular charger transfer (ICT) effect can quickly
Received 19th February 2021,
Accepted 17th March 2021
respond to H
2
S and effectively quench the fluorescence intensity, and an oxonium cation structure
serves as a mitochondria-targeting site. Notably, MNIR-H
cytotoxicity, powerful mitochondrial localization ability, deep tissue penetration, and a very minimal
imaging background. Biological imaging results show the biological application of MNIR-H S as a robust
new tool for use in H S imaging in biological systems.
2
S displays high sensitivity, high selectivity, low
DOI: 10.1039/d1nj00840d
2
rsc.li/njc
2
unclear. Therefore, the development of accurate and reliable
methods to understand the biological roles of H S and its high
S) is notorious for having a strong smell that biocompatibility and site-specificity is urgently required.
resembles rotten eggs, and has traditionally been misunderstood Nowadays, several analytical methods (such as colorimetry
to be just a toxic gas. However, a number of current studies have and electrochemical analysis, gas chromatography and sulfide
indicated that H S has certain biological functions due to its precipitation) are used to detect H S, but these usually require
Introduction
2
Hydrogen sulfide (H
2
2
2
typical concentration in the blood being the range of 10–100 mM. complex pretreatment of samples, so they are often difficult to
7
Indeed, current studies regard H S as the third gaseous signalling use for in vivo detecting and imaging. Compared with the
2
molecule that is present in living biological systems, followed by above analytical methods, fluorescence imaging is considered a
1
carbon monoxide (CO) and nitric oxide (NO).
2
H S has been robust tool for sensing and monitoring some biomolecules
thought to be related to various physiological processes, such as in vivo due to its outstanding performance, including high
2
vasodilation, antioxidation, anti-apoptosis, anti-inflammation.
sensitivity, good selectivity, fast response, less bio-damage,
S fluorescent probes
Moreover, it plays the role of an antioxidant and reactive oxygen and real-time imaging. To date, many H
2
species (ROS) scavenger in vivo. In addition, studies have indi- have been reported. However, most of them are one-photon
cated that its deregulation could be related to the development (OP) fluorescent probes, which use short-wavelength of
3
and occurrence of certain diseases, such as liver cirrhosis,
ultraviolet-visible light excitation and emission to cause photo-
4
5
6
diabetes, Down’s syndrome, and Alzheimer’s disease. Although bleaching and shallow tissue imaging. In addition, most of
S has been recognized to be related to various physiological and them do not have organelle targeting groups with which to
pathological functions, some potential molecular events remain target organelles to provide quantitative analytical information
of H S at the organelle level. Therefore, this limits their
application in vivo to investigate the links between H S and
H
2
2
a
2
Hunan Key Laboratory of Processed Food for Special Medical Purpose,
National Engineering Laboratory for Deep Process of Rice and Byproducts,
College of Food Science and Engineering, Central South University of Forestry and
Technology, Changsha, Hunan 410004, China. E-mail: zhouly0817@163.com
Guangxi Key Laboratory of Tumor Immunology and Microenvironmental
Regulation, Guilin Medical University, Guilin, Guangxi 541001, China.
E-mail: zhouyizhuang3@163.com
8
physiology and pathology. In contrast, two-photon near-infrared
(TP-NIR) fluorescent probes adopt two photons of NIR laser
pulse excitation and NIR emission, which have many advantages
in biological imaging, such as high spatial resolution, deep
imaging depth, good three-dimensional spatial localization
effects, decreasing of photodamage to biological samples, and
b
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
9
,10
d1nj00840d
the elimination of autofluorescence.
More importantly,
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Fig. 1 (A) Synthesis steps of the TP-NIR fluorescent probe MNIR-H
a, EtOH, piperidine, 80 1C, 4 h; b, concentrated sulfuric acid, 90 1C, 8 h;
c, 2,4-dinitrofluorobenzene, CH Cl , triethylamine, 3 h. (B) Response
2
S:
2
Scheme 1 (A) Proposed mechanism for the sensing of H S in mitochondria
2
2
when using the TP-NIR fluorescent probe MNIR-H
previously reported unstable (NIR-1, NIR-2) and stable (NIR-3, NIR-4) dyes to
construct the new stable TP-NIR H S fluorescent probe MNIR-H S.
2
S. (B) Modification of mechanism of the mitochondrial TP-NIR fluorescent probe toward
hydrogen sulfide.
2
2
1
3
according to the methods described in the literature or
compared with fluorescent probes without organelle targeting modifications of these procedures. For the synthesis of 2 : 313
groups, organelle-targeted fluorescent probes can accurately, mg (1 mmol) of 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic
quickly and conveniently obtain H
2
S information at the organelle acid, 204 mg (1 mmol) of 1, and 15 mL of concentrated sulfuric
level, and provide a theoretical basis and technical support for the acid were added into a single-neck round-bottom flask
early diagnosis and treatment of H S-related diseases. Therefore, equipped with a condenser, and the reaction temperature was
2
there is an urgent need to develop organelle-targeted TN-NIR slowly raised to 90 1C, the temperature at which the mixture
fluorescent probes for the detection and imaging of H
biological systems Scheme 1.
2
S in was stirred for 8 h. After being allowed to cool, the reaction
solution was poured into 100 mL of ice-water, and then 3 mL of
In order to overcome these challenging bottlenecks, in this perchloric acid (HClO
work, taking previously reported unstable TP excitation NIR The resulting purple-black solid was filtered off under reduced
) was added into the mixture solution.
4
11
emission fluorescent dyes, we rationally modified their structure pressure, washed with cooled water (3 Â 10 mL), and dried to
to obtain a new TP-excited (810 nm) NIR emission (668 nm) obtain the crude product. Finally, the crude product was further
mitochondria-targeting fluorescent probe MNIR-H S for the purified by column chromatography (CH
2 2 3
Cl /CH OH = 50: 1, v/v)
2
sensing of H S in living cells, tissues and nude mice. In this to obtain the target compound as a black solid in 85.3% yield.
2
1
new scaffold, an o-carboxyphenyl group was employed to occupy
H NMR (400MHz, d6-DMSO): 11.85 (s, 1H), 8.89 (s, 1H), 8.13
the para-position of the oxonium cation and prevent a Michael (s, 1H), 7.95–7.93 (t, J = 4.00Hz, 1H), 7.79–7.69 (d, J = 20.00Hz,
addition reaction, and also prevent the pH-triggered oxonium 3H), 7.38–7.37 (d, J = 4.00Hz, 1H), 6.71 (s, 1H), 6.51 (s, 3H), 6.41
cation ring-opening reaction. Notably, in this new fluorescent (s, 1H), 4.36 (s, 1H), 1.93–1.90 (q, J = 3.00Hz, 4H), 1.08–1.04 (t, J =
probe, the oxonium cation serves as a mitochondria-targeting 8.00Hz, 6H); 13C NMR (100MHz, d6-DMSO) d(ppm): 172.59,
moiety, and the well-known H S response moiety 2,4-dinitrophenyl 169.19, 158.68, 150.02, 149.66, 135.93, 128.55, 127.73, 126.77,
2
12
ether (DBN) has a strong ICT effect to quench NIR fluorescence 125.28, 124.61, 110.41, 103.84, 97.72, 56.49, 44.27, 21.58, 12.87;
+
intensity at 668 nm. In the presence of H S, the TP-NIR fluorophore LC-MS: m/z [C29
H
24NO
6
] calcd 482.51, found 482.52.
2
shows a ‘‘turn-on’’ fluorescence intensity at 668 nm, and the
fluorescence intensity increases by B150 fold. This new fluorescent
probe exhibits outstanding selectivity and sensitivity for the sensing A mixture of 582 mg (1 mmol) of ([C H NO ] + ClO )2, 186 mg
One-pot synthesis of the H
2
2
S fluorescent probe MNIR-H S (3):
À
2
9
24
6
4
2
of H S both in vitro and in vivo. Impressively, the biological images (1 mmol) of 2,4-dinitrofluorobenzene, 3 mL of triethylamine,
showed that this new probe can be successfully applied for the and 50 mL of CH Cl were added into a single-neck round-
2
2
sensing of H S in living HeLa cells, onion tissue, zebrafish and bottom flask equipped with a condenser, and the reaction
2
nude mice.
temperature was slowly raised to 45 1C, at which temperature
the reaction was stirred for 3 h. After the completion of the
reaction, the mixture was allowed to cool to room temperature,
and the solvent was evaporated in vacuo using a rotary evaporator
to give the crude product as a black solid. Finally, the crude
product was further purified by column chromatography
Experimental section
Materials and apparatus
The materials and equipment used are described in the ESI.†
(
CH Cl :CH OH = 50 : 1, v/v) to obtain the probe MNIR-H S as
2
2
3
2
Synthesis and characterization of MNIR-H
2
S
1
a black solid in 85.8% yield. H NMR (400MHz, d6-DMSO): 9.08
The synthesis of the fluorescent H S probe MNIR-H
2
2
S is shown (s,1H), 8.87–8.86 (d, J = 4.00Hz, 1H), 8.50 (s, 1H), 8.44–8.40 (q, J =
in Fig. 1A. The classical ssyntheses of 1 and 2 were carried out 4.00Hz, 1H), 7.96–7.94 (d, J = 8.00Hz, 1H), 7.79–7.68 (m, 2H), 7.47
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(s, 1H), 7.40–7.38 (d, J = 8.00Hz, 1H), 7.10–7.07 (d, J = 12.00Hz, group, mice were injected with 5 mM of the resolution probe
1
3
H), 6.74 (s, 1H), 6.51–6.48 (d, J = 12.00Hz, 4H), 3.40–3.37 (q, J = only, and NaHS was not used to resolve the images. In contrast,
.00Hz, 4H), 1.16–1.12 (t, J = 4.00Hz, 6H); 13C NMR (100MHz, in the experimental group, the mice were injected with 5 mM of
d6-DMSO) d(ppm): 169.17, 157.33, 156.18, 154.53, 153.56, MNIR-H S resolution, and 40 mM of NaHS resolution was
2
1
1
4
52.48, 149.57, 146.19, 141.60, 139.43, 137.97, 130.67, 129.59, further used to resolve the images by a PhotoIMAGERTMRT
26.00, 121.96, 118.22, 116.87, 110.67, 104.45, 83.21, 46.04, imager with a 622 nm excitation filter and a 668 nm emission
+
26 3 10
4.24, 19.03, 12.87; LC-MS: m/z [C35H N O ]
calcd 648.60, filter.
found 648.61.
Results and discussion
Spectrophotometric measurements
Fluorescence spectroscopy measurements were carried out in In order to rationally design a TP-NIR fluorescent probe for
phosphate-buffered solution (PBS, 10 mM) in dimethyl sulfoxide sensing H S with organelle targeting properties, better biological
2
(
DMSO) as a co-solvent solution (H O/DMSO = 99 : 1, v/v). The pH imaging resolution, and deeper penetration depth, in this work, a
2
value of the PBS solution used was from 3.0 to 8.0, which was TP excitation NIR emission mitochondria-targeting fluorescent
achieved by adding minimal volumes of HCl or NaOH solutions. probe MNIR-H S was constructed for sensing H S in living cells,
2
2
Fluorescence emission spectra were recorded at an excitation onion tissue, zebrafish and nude mice. Its structure is shown in
wavelength of 622 nm in an emission wavelength range from Fig. 1. Notably, an oxonium cation moiety serves as a mitochondrial
2
630 to 800 nm. A 1 mM stock solution of the MNIR-H S probe targeting-group, an o-carboxyphenyl group is employed to occupy
was prepared by dissolving MNIR-H S in DMSO. The procedure the 4-position of the oxonium cation to prevent the Michael
2
for the calibration measurements of MNIR-H S in the buffer with addition reaction, and a well-known H S response site of DBN
2
2
different H S concentrations was as follows: 2 mL of a stock moiety acts as the H S ‘‘turn-on’’ moiety and the fluorescence
2
2
solution of MNIR-H
mL of different H S concentration solutions were combined to probe with some excellent properties, such as TP excitation,
afford test solutions of 1 mM of MNIR-H S. Solutions of the NIR emission, mitochondrial localization, and so on. The route
various test species were prepared from KCl, CaCl , NaCl, MgCl to synthesize MNIR-H S is shown in Fig. 1. Firstly, 2,4-dihydro-
ZnCl , FeCl , CuSO , Hg(NO , PbCl , glutathione (GSH), xybenzaldehyde is reacted with methyl acetoacetate in EtOH to
2 2
S and 1996 mL of PBS buffer solution with intensity quenching group. Such a design affords an MNIR-H S
2
2
2
2
2
,
2
2
3
4
)
3 2
2
cysteine (Cys), and glutamate (Glu), using twice-distilled water, obtain 1 using piperidine as a catalyst; secondly, 2-(4-diethylamino-
with final concentrations of 100 mM. The procedure for the 2-hydroxybenzoyl) benzoic acid is reacted with 1 in 98% H SO at
2
4
selectivity experiments was as follows. For cations or anions, 90 1C to give 2; finally, 2 is reacted with 2,4-dinitrofluorobenzene in
mL of a stock solution of MNIR-H S, 1798 mL of PBS solution dry CH Cl using triethylamine as a catalyst to give 3 (MNIR-H S).
pH 7.4) and 200 mL of solutions of each relevant species were All compounds were characterized by NMR spectroscopy and mass
combined to afford test solutions, containing 1 mM of MNIR-H spectrometry (see the ESI†).
With the MNIR-H S probe in hand, its spectral properties
toward H S were measured in 10 mM of PBS (1% DMSO, pH = 7.4),
as shown in Fig. 2. In this work, NaHS was chosen as the H2S
2
2
2
2
2
(
2
S
and 100 mM of the relevant species.
2
2
Cytotoxicity assays and imaging
HeLa cells at 1 Â 106 cells/well were seeded at varying density in source. In the absence of H S, the MNIR-H S probe alone shows a
2
2
a 96-well plate and incubated overnight. After that, they were characteristic absorption peak centered at 558 nm. While, in the
incubated with increasing concentrations of MNIR-H S (0–12 mM) presence of H S, the characteristic absorption band of MNIR-H
for 24 h. The MTT assay method was then used to assess the is red-shifted to 622 nm (a red shift of 64 nm), which matches well
cytotoxic effects of MNIR-H S. Before the fluorescence imaging, with the characteristic absorption band of fluorophore 2. For the
the cells were washed three times with 10 mM of PBS (pH7.4), purpose of measuring the time-dependent fluorescence spectra of
followed by incubating them with 1.0 mM of MNIR-H S for 30 min MNIR-H S toward 40 mM of NaHS in 10 mM of PBS (pH = 7.4, 1%
10 mM PBS, 1% DMSO) at 37 1C and they were then washed three DMSO) were used to study the sensitivity of the probe, and the
2
2
2
S
2
2
2
(
times with PBS before being fluorescence imaged using an fluorescence intensity was recorded at different times (0–10 min)
Olympus FV1000 laser confocal microscope (Japan). In the co- (Fig. S1, ESI†). Subsequently, we measured the fluorescence
staining experiments, 1.0 mM of commercialized MitoTracker response curve of MNIR-H
Green FM and 1.0 mM of MNIR-H S incubated together with cells in 10 mM of PBS (pH = 7.4), as shown in Fig. 2B. In the absence of
for 30 min, washed three times with 10 mM of PBS, and then S, the solution of MNIR-H S exhibited almost no fluorescence
0 mM of NaHS was added, and they were incubated for a further intensity signal at 668 nm. While in the presence of H S,
0 min to carry out colocalization imaging analysis. The green with increasing NaHS concentration (0–50 mM), the fluorescence
2 2
S and different concentrations of H S
2
H
2
2
4
3
2
channel was set to 500–560 nm, and the red channel was set to intensity rapidly and significantly enhanced at 668 nm, accompanied
40–680 nm, separately. The one-photon (OP) excitation wave- by a color change from yellow to blue (Fig. 1E and F). Therefore,
lengths were set to 488 and 635 nm, respectively, and the two- MNIR-H S displayed good colorimetric and fluorescence response to
6
2
photon (TP) excitation wavelength was set to 810 nm.
Nude mice aged 6–8 weeks were injected subcutaneously ratio I
with 5 mM of MNIR-H S resolution (1% DMSO). In the control obtained (Fig. 2D), and the detection limit of MNIR-H
H
2
S. Notably, a linear correlation between the fluorescence intensity
/I668 and H S concentrations in the range of 0–5 mM was
S response to
0
2
2
2
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influence can be ignored. The results suggest that MNIR-H
2
S
shows excellent selectivity and a specific response toward H
2
S
over other analyzed species. Following this, fluorescence
responses of MNIR-H S (1 mM) to H S at different pH values
2
2
were also studied (Fig. 3B). The experimental results exhibited
that in the absence of hydrogen sulfide, the pH of the solution
changed from 3.0 to 8.0, with almost no change in fluorescence
2
intensity at 668 nm being observed, and when MNIR-H S was
incubated with hydrogen sulfide in solutions of different pH
values (3.0–8.0), the fluorescent intensity showed significant
changes at 668 nm. Moreover, according to the experimental
results, the optimal pH range for the reaction of MNIR-H S with
2
hydrogen sulfide was determined as 6.0–8.0, showing that
MNIR-H S is suitable for use in biological applications at
2
physiological pH. Thus, 10 mM of pH 7.4 PBS solution was
used in all of the experiments.
2
Fig. 2 (A) The normalized absorbance spectra of 1, 2, and MNIR-H S;
(
B) fluorescence response spectra of MNIR-H
concentrations of NaHS (0–50.0 mM), lex = 622 nm; (C) calibration curve
of MNIR-H S toward NaHS (0–50.0 mM); (D) linear response of 1.0 mM of
MNIR-H S and 0–5.0 mM of NaHS in 10 mM of PBS buffer (1% DMSO); (E, F)
the insets show the change in the color of the MNIR-H S probe before and
after adding 50.0 mM of H S into 1.0 mM of MNIR-H S, where the
photographs were obtained under visible light.
2
S (1.0 mM) with varied
Encouraged by the above results, we evaluated the biological
application of MNIR-H
2 2
S in the imaging of H S in living cells.
2
First, we investigated the cytotoxicity of MNIR-H S by MTT
2
2
(
Fig. S3, ESI†). When HeLa cells were incubated with 0–12 mM
of the probe, the cell viability did not decrease significantly,
indicating that MNIR-H S shows low cytotoxicity to living cells
2
2
2
2
under physiological conditions and is suitable for biological
application. Subsequently, we investigated the feasibility of
H
3
2
S was calculated to be as low as 14.8 nM (Fig. 2D) according to the using MNIR-H
s/slope. Therefore, MNIR-H S can be used as a potential cells were incubated with MNIR-H
fluorescence probe with outstanding properties for the sensing in the red channel (Fig. 4A: a and b) upon excitation at 635 nm. The
2
S for the sensing H
2
S in living HeLa cells. HeLa
2
2
S and showed weak fluorescence
of H S in living biological systems.
HeLa cells were stained with 1 mM of MNIR-H S for 30 min, washed
2
2
To examine the selectivity, we incubated MNIR-H
2
S with three times with 10 mM of PBS to remove any excess probe, and
different types of cations, anions, common amino acids and were then further incubated with 40 mM of NaHS for 30 min to
biothiols at 100 mM, such as cysteine (Cys), glutathione (GSH), detect H S (Fig. 4A: c and d), where the fluorescence intensity was
Na , ascorbic acid (vitamin C), K , Ca , Mg , Fe , Zn , Cu , bright in the red channel. This result shows that MNIR-H S can be
2
+
+
2+
2+
3+
2+
2+
2
2
+
H
2
S, Glycine (Gly), Blank, Glucose (Glu), and Pb , respectively. applied in the imaging of H
2
S in living HeLa cells.
As shown in Fig. 3A and Fig. S2 (ESI†), in the presence of H S,
2
when excited with an excitation wavelength of 622 nm, H S can
2
induce a robust fluorescence intensity enhancement at 668 nm,
while in the presence of100 mM of the other analyzed species
only slight changes can be observed, meaning that their
Fig. 4 (A) Confocal images of HeLa cells treated with MNIR-H
2
S for
detecting H S: (a) HeLa cells incubated with only MNIR-H S in the
2
2
fluorescence image of the red channel excited at 635 nm; (b) an overlay
image of (a) and bright field imaging; (c) HeLa cells treated with MNIR-H2S,
following treatment with NaHS (40 mM) for red channel imaging excited at
635 nm; (d) an overlay image of (c) and bright field imaging. (B) Mitochondria
Fig. 3 (A) Selectivity experiments: 1 mM of MNIR-H
various analyzed species/NaHS (100 mM of other species and 40 mM of
NaHS) in PBS buffer (10 mM, pH = 7.4, water/DMSO = 99/1, v/v), lex
2
S incubated with
=
2 2
co-localization images of MNIR-H S in HeLa cells: (a) MNIR-H S (1 mM, red
channel, lex = 635 nm, lem = 640–680 nm) for 30 min, following treatment
with NaHS (40 mM) for 30 min for the red channel imaging; (b) cells
+
2+
+
6
22 nm, legend: (1) GSH, (2) Cys, (3) Vitamin C, (4) K , (5) Ca , (6) Na , (7)
2+
2+
3+
2+
2+
Mg , (8) Zn , (9) Fe , (10) Gly, (11) Cu , (12) Blank, (13) H
15) Glu; (B) The effect of different pH values (3.0–8.0) on the response of 1
mM of MNIR-H S and hydrogen sulfide, blue columns: in the presence of
2
S, (14) Pb ,
(
incubated with MitoTracker Green FM (1 mM, green channel, lex
=
2
488 nm, lem = 500–560 nm) for the green channel imaging; (c) overlay
NaHS (40 mM), blue-green columns: in the absence of NaHS. Each
experiment was repeated three times, lex = 622 nm.
image of (a), (b) and the bright field imaging; (d) the correlation of MNIR-H
and MitoTracker Green FM intensities. Scale bar: 20 mm.
2
S
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For the purpose of verifying that MNIR-H
2
S can target the
mitochondria for the sensing of H S, HeLa cells were co-stained
2
with 1 mM of MNIR-H S and 1 mM of MitoTracker Green (FM)
2
for 30 min, and then washed three times with 10 mM of PBS
(
pH7.4) to remove the excess MNIR-H S and FM, and were then
2
further incubated with 40 mM of NaHS for 30 min to carry out
fluorescence imaging. As shown in Fig. 4B, the confocal laser
scanning microscopy results show that MNIR-H S exhibits red
2
fluorescence intensity around the perinuclear areas in the
cytoplasm upon excitation at 635 nm. The co-localization and
colorimetric assays with MitoTracker Green FM were observed
with a high correlation coefficient of 0.95 (Fig. 4B: a), indicating
Fig. 6 Representative fluorescence images (rainbow color) for anesthetized
nude mice subcutaneously injected with MNIR-H
mM) and NaHS (40 mM). (A) Control experiments: injected with MNIR-H
S (5 mM) and NaHS (40 mM) 10 min for
imaging; (C) after injecting with MNIR-H S (1 mM) and NaHS (40 mM) 10 min
2 2
S (1 mM) or MNIR-H S (1 or
5
2
S
that MNIR-H S is membrane permeable and capable of the (1 mM); (B) after injecting with MNIR-H
2
2
fluorescence imaging of mitochondrial H
We further evaluated the biological application of MNIR-H
the imaging of H S in living tissues and zebrafish from TP images
TPA = 40 GM, lex = 810 nm, Table S1, ESI†). First, MNIR-H S was
applied to onion tissue slices. The onion tissue slices were stained
2
S in living cells.
2
2
for imaging; (D) after injecting with MNIR-H S (1 mM) and NaHS (40 mM) 10
min for simultaneous 4-view imaging.
2
S in
2
(d
2
showed dim fluorescence intensity in the red channel (Fig. 5C).
with 1 mM MNIR-H S for 30 min, and subsequently stimulated
2
While, for the experimental group, in the presence of 40 mM NaHS
without or with 40 mM NaHS for 30 min to carry out fluorescence
imaging, where the red fluorescence intensity in the tissues was
observed by multiphoton confocal microscopy (Olympus, FV1000)
2
for 30 min, and then subsequently being incubated with MNIR-H S
for 30 min, the fluorescence intensity was obviously enhanced in
the red channel (Fig. 5D), which exhibited that the MNIR-H S probe
2
in z-scan mode, which showed that MNIR-H
imaging at depths of 90 and 95 mm (Fig. 5A and B) by 2PFM
imaging, and possesses good tissue penetration. Next, MNIR-H
was used to carry out fluorescence imaging in a zebrafish model. 5
Day-old vertebrate zebrafish were chosen as the experimental
samples, which were divided into control and experimental groups.
For the control group, in the absence of NaHS, the zebrafish
2
S is capable of tissue
has the capacity to image H S in living tissues and zebrafish.
2
Finally, the MNIR-H
-view fluorescence imaging of H
intraperitoneal injection of 1 mM or 5 mM of MNIR-H
2
S probe was further applied for the
S in living nude mice. After
S into the
2
S
4
2
2
living mice, 40 mM of NaHS solutions were further injected to
trigger fluorescence intensity, and a bright fluorescence
intensity was observed from the mice (Fig. 6B), and 1 mM of
probe MNIR-H S together with 40 mM of NaHS solution
2
triggered a relatively dim fluorescence intensity enhancement in
the mice (Fig. 6C). The images show that MNIR-H S exhibits high
2
resolution for bioimaging. In order to further eliminate false
positives, we adopted a 4-view mode for imaging (Fig. 6D). These
results demonstrate that the probe possesses high penetration
2
capability in animals for the fluorescence imaging of H S.
Conclusions
In summary, in this work, we have reported a new mitochondria-
targeting two-photon in near-infrared out probe MNIR-H S. In this
2
probe, an oxonium cation acts as a mitochondria-targeting group,
while a hydroxyl group is linked to a DNB group as a hydrogen
sulfide response moiety, which affords the MNIR-H
good selectivity and high sensitivity towards H S. The constructed
probe was successfully applied in ‘‘turn-on’’ fluorescence intensity
for the sensing of H S in HeLa cells, onion tissue, zebrafish, and
2
S probe with
2
2
nude mice. Therefore, we hope that MNIR-H S can be used as a
2
robust tool with potential biological applications for imaging of
the gaseous signalling molecule H S in biological systems.
2
Fig. 5 The fluorescence imaging of onion tissue and zebrafish incubated
with MNIR-H
treatment with NaHS (40 mM) for 30 min for imaging. (A) Treatment with
only MNIR-H S for onion tissue imaging; (B) treatment with MNIR-H S and
NaHS for onion tissue imaging; (C) treatment with only MNIR-H S for
zebrafish imaging; (D) treatment with MNIR-H S and NaHS for zebrafish
imaging. lex = 810 nm, lem = 640–680 nm, scale bar: 100 mm.
2
S (1 mM) for 30 min, and then without or with continued
Live subject statement
2
2
2
All animal procedures were performed in accordance with the
Guidelines for Care and Use of Laboratory Animals of Central
2
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2021
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Paper
NJC
South University of Forestry and Technology and approved by
the Animal Ethics Committee of Central South University of
Forestry and Technology.
297, 126773; (c) Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He,
Y. Jiao, W. He, L. Qiu, J. Cen and Z. Guo, Angew. Chem.,
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X. Qu and J. Hua, J. Mater. Chem. B, 2017, 5, 2172; (e) A. Ji,
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Conflicts of interest
3, 992; ( f ) Y. L. Pak, J. Li, K. C. Ko, G. Kim, J. Y. Lee and
There are no conflicts to declare.
J. Yoon, Anal. Chem., 2016, 88, 5476.
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M. H. Lee, J. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015,
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Acknowledgements
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This work was supported by the Natural Science Foundation of
China (NSFC, Grants 21605046, 32060144), the Hunan Provincial
Natural Science Foundation of China (No. 2017JJ3060), and the
Science and Technology Innovation Platform and Talent Project
of Hunan Province (2017TP1021, 2018JJ3117).
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