Mitochondria-targeted reversible ratiometric fluorescent probe for monitoring SO2/HCHO in living cells

Article abstract of DOI:10.1016/j.saa.2020.118253

Sulfur dioxide (SO₂) maintains a certain steady state balance in the body, high concentration SO₂ will be harmful to human health. Seeking a suitable detection method to monitor sulfur dioxide in real time becomes an urgent requirement owing to the transient nature of sulfur dioxide in organisms. Here, a novel NIR ratiometric fluorescent probe for detection of SO₂ was developed based on a conjugation of coumarin and indol salt with excellent water solubility. The probe Mito-CI displayed highly sensitive (69 nM), fast response time (30 s), large Stokes shift (174 nm) and the NIR fluorescence emission wavelength (655 nm). In the reversibility process of the SO₃ ^?-probe Mito-CI system induced by HCHO in vitro was also detected. Besides, cell imaging showed that Mito-CI possesses mitochondria-targeted ability. Particularly, Mito-CI was proved to reversibly detect SO₂/HCHO in living cells.

Full text of DOI:10.1016/j.saa.2020.118253

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Mitochondria-targeted reversible ratiometric fluorescent probe for
monitoring SO2/HCHO in living cells
Tao Zhang, Caixia Yin, Yongbin Zhang, Jianbin Chao,
Guangming Wen, Fangjun Huo
PII:
S1386-1425(20)30231-6
https://doi.org/10.1016/j.saa.2020.118253
SAA 118253
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 3 January 2020
Revised date:
11 March 2020
1 March 2020
Accepted
date:
1
Please cite this article as: T. Zhang, C. Yin, Y. Zhang, et al., Mitochondria-targeted
reversible ratiometric fluorescent probe for monitoring SO2/HCHO in living cells,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2018),
https://doi.org/10.1016/j.saa.2020.118253
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2018 Published by Elsevier.

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Mitochondria-targeted reversible ratiometric fluorescent probe for monitoring
SO /HCHO in living cells
2
a
b
a
a
c,
Tao zhang , Caixia Yin, Yongbin Zhang , Jianbin Chao, Guangming Wen *
a,
Fangjun Huo *
a
Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006,
China.
b
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China.
c
Jinzhong University
Email: F.J. Huo, huofj@sxu.edu.cn
Abstract: Sulfur dioxide (SO ) maintains a certain steady state balance in the body,
2
high concentration SO will be harmful to human health. Seeking a suitable detection
2
method to monitor sulfur dioxide in real time becomes an urgent requirement owing
to the transient nature of sulfur dioxide in organisms. Here, a novel NIR ratiometric
fluorescent probe for detection of SO was developed based on a conjugation of
2
coumarin and indol salt with excellent water solubility. The probe Mito-CI displayed
highly sensitive (69 nM), fast response time (30 s), large Stokes shift (174 nm) and
the NIR fluorescence emission wavelength (655 nm). In the reversibility process of
−
the SO -probe Mito-CI system induced by HCHO in vitro was also detected.
3
Besides, cell imaging showed that Mito-CI posseses mitochondria-targeted ability.
Particularly, Mito-CI was proved to reversibly detect SO /HCHO in living cells.
2
Keywords: SO /HCHO; Near-infrared; Reversibly; Mitochondria; Cellular imaging.
2
1

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1
. Introduction
Formaldehyde and sulfur dioxide (SO ) are important components to maintain
2
the homeostasis of living organisms, the abnormal production of these components
lead to some critical diseases [1]. According to the latest research, formaldehyde
could deregulate the NO signaling pathway by mediating the nitrosylation of
S-protein. Formaldehyde assimilation and enhancement during protein S-nitrosation
may lead to extremely low local GSH concentrations. GSH is one of the main raw
materials for the production of endogenous SO [2-7], high concentrations of SO may
2
2
result in vasodilation by upregulating the NO/cGMP signaling pathway. These
conclusions indicated that formaldehyde and sulfur dioxide has a close relationship.
Therefore, accurate detection of sulfur dioxide in living organisms is crucial to the
overall health of biological systems.
At present, many research groups have developed a series of fluorescent probes
for detection of SO [8-20]. However, the real-time detection of SO in the body is
2
2
still a challenge. First, the limitations of most fluorescent probes are quite poor water
solubility [21-25]. Second, almost all reported probes caused high photodamage to
live samples [26-29]. Third, there are few reports about reversible detection of
fluorescent probes for SO /HCHO [30-34]. Thus, it is essential to exploit a fluorescent
2
probe with good water solubility, NIR emission wavelength and reversible detection
of fluorescent probes for SO /HCHO.
2
Herein, Mito-CI was found to be the perfect tool for solving above problems. It
showed highly sensitive (69 nM), fast response time (30 s), excellent water solubility,
2

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large Stokes shift (174 nm) and the NIR fluorescence emission at 655 nm. Mito-CI
was successfully synthesized based on the coumarin and indol salt (Scheme 1). And
2
－
－
due to the nucleophilicity of SO3 /HSO3 , Mito-CI could detect SO with
2
ratiometric fluorescent emission. Moreover, cell imaging experiment showed that
Mito-CI could be used for reversible visualization of exogenous SO /HCHO and
2
mitochondria-targeted in living cells.
<
Inserted Scheme 1>
2
. Materials and methods
2
.1 Materials
Related materials are described in the support information.
.2. Synthesis of Mito-CI
2
2
.2.1 Compound 1
Diethylmalonate (6.08 g, 38 mmol), 4-diethylaminosalicylaldehyde (3.67 g, 19
mmol) and piperidine (2.0 mL) were mixed with ethanol (60 mL), then the mixture
was refluxed for 12 hours. After the reaction ended, solvent was evaporated under
reduced pressure. Next, a mixture of concentrated acetic acid (40 mL) and HCl (40
mL) was added, reactant was refluxed at 120 °C for 13 hours. The reaction mixture
was poured into 200 ml of 0°C water after cooling to 25 °C. Adding NaOH solution
(40 %) to improve pH up to 5, brown precipitate formed instantly. The mixture was
filtered and washed with water four times, drying it in vacuum, compound 1 was
1
obtained as a yellowish brown solid (3.92 g, 95 %); H NMR (600 MHz, DMSO-d ) δ
6
7
.82 (d, J = 9.3 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 6.68 (dd, J = 8.8, 2.5 Hz, 1H), 6.51
3

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(d, J = 2.3 Hz, 1H), 5.99 (d, J = 9.3 Hz, 1H), 3.42 (q, J = 7.0 Hz, 4H), 1.12 (t, J = 7.0
1
3
Hz, 6H). C NMR (151 MHz, DMSO-d ) δ 156.80, 144.96, 129.78, 109.22, 108.62,
6
9
7.04, 44.49, 12.77 (Fig. S1).
2
.2.2 Compound 2 and compound 3
Compound 2 and compound 3 was prepared according to the reported literature
[
35].
2
.2.3 Compound Mito-CI
Compound 2 (98.0 mg, 0.4 mmol) and compound 3 (92.9 mg, 0.4 mmol) were
mixed in ethanol (40 mL) and was refluxed for 12 hours. After the reaction finished,
evaporation of the solvent under reduced pressure. Next, the crude product was
purified by column chromatography on silica gel with CH Cl /MeOH (25:1) to get the
2
2
1
desired Mito-CI (102.87 mg, yield: 56 %). H NMR (600 MHz, DMSO-d ) δ 8.82 (s,
6
1
7
2
H), 8.21 (d, J = 13.4 Hz, 1H), 7.82 (d, J = 10.6 Hz, 2H), 7.80 (d, J = 8.0 Hz, 1H),
.61–7.54 (m, 2H), 7.52 (d, J = 7.3 Hz, 1H), 6.98–6.87 (m, 1H), 6.71 (s, 1H), 4.60 (s,
13
H), 3.56 (d, J = 6.6 Hz, 4H), 2.88 (s, 2H), 1.73 (s, 6H), 1.18 (t, J = 6.8 Hz, 6H). C
NMR (151 MHz, DMSO-d ) δ 171.76, 159.86, 158.06, 154.38, 141.50, 132.81,
6
1
1
29.33, 123.29, 112.84, 111.75, 109.88, 97.04, 51.85, 45.31, 32.14, 26.51, 21.55,
+
2.96. ESI-MS m/z: [Mito-CI + H ] Calcd. For 459.2278, Found 459.2283 (Fig. S2)
(Scheme 2).
<
Inserted Scheme 2>
2
.3 Fluorescence imaging
HepG2 cells were cultivated with DMEM contain 1 % penicillin-streptomyc and
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1
0 % fetal calf serum at 37°C. The cells were washed four times in absolute PBS
buffer solution before imaging. Fluorescence imaging of HepG2 cells was performed
at 460-510 nm (blue channel) and 630-680 nm (red channel). Then, colocation
imaging was proceed to study the mitochondrial distribution of Mito-CI in cells.
3
. Results and discussion
.1 Sensing Properties of Mito-CI toward SO3 /HSO3
Herein, we detected the fluorescence intensity of Mito-CI in the range of pH 3-9.
2
－
－
3
2
－
When SO3 (a standard source for SO ) was added, Mito-CI demonstrated the
2
optimal response in the pH scope of 6.0−9.0 (Fig. S3), which coincided with the
weakly alkaline environment of mitochondria [36]. This conclusion illustrated that
Mito-CI is suitable for monitoring SO of mitochondrial. Therefore, we chose pH 7.4
2
in the following spectral experiments. Then, the sensing properties of Mito-CI for
2
－
－
SO3 /HSO are tested. As shown in Fig. 1a, Mito-CI (10 µM) was added into 10.0
3
mM of PBS buffer, then the change of fluorescence spectrum was observed, the probe
2
－
showed a near-infrared fluorescence emission at 655 nm. Thereafter, when SO3
(0-100 µM) was gradually added, the fluorescence intensity at 655 nm was gradually
decreased, with a marked enhancement in fluorescence intensity at 481 nm. These
phenomena demonstrated that the π-conjugation of Mito-CI was interrupted. Then,
UV-Vis titration experiment was carried out (Fig. 1b), the probe exhibited a major
2
－
absorption band at 577 nm. Under the condition of gradually adding SO3 (0-150
µM), the absorption band at 577 nm gradually reduced, absorption peak at 423 nm
5

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increased. The color of solution changed from dark purple to colorless, indicating that
2
－
－
Mito-CI can be used to detect SO3 /HSO3 with the “naked-eye”.
<
Inserted Figure 1>
2
－
.2 Sensing Properties of Mito-CI-SO3 toward HCHO
3
After the result of Mito-CI as a suitable fluorescent probe for the detection of
2
－
－
2－
SO3 /HSO , the reversibility of Mito-CI-SO3 were further studied. As shown in
3
Fig. 2a, under the condition of gradually adding HCHO (0-200 µM), the fluorescence
intensity at 655 nm slowly raised, with a marked decrease in the fluorescence
2
－
intensity at 481 nm. Then, UV-Vis titration experiment of Mito-CI-SO3 toward
HCHO were performed (Fig. 2b), when HCHO (0-300 µM) was gradually added, the
absorption band at 577 nm slowly enhanced, with a marked decrease in the absorption
band at 423 nm. The color of solution changed from colorless to dark purple
2
－
monitored by the “naked eye”. The absorption intensity of Mito-CI-SO3 ensemble
at 577 nm changed from the initial 0.59 to 0.49 (after adding HCHO). These
2
－
conclusions revealed that Mito-CI-SO3 can effectively reversible by addition of
HCHO, the level of recovery reached 83 %.
<
Inserted Figure 2>
.3 Time-dependent and Selectivity of Mito-CI
The time-dependent of Mito-CI toward SO3 (10 equiv.) showed that the
3
2
－
reaction was accomplished within 30 s (Fig. S4). The selectivity of the probe for
sulfur dioxide is also crucial in practical application. In the selective test, Mito-CI
2
－
was treated with SO3 and various analytes each (10 equiv.). Only upon addition of
6

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2
－
SO3 could make the fluorescence intensity at 481nm increase and decrease at 655
nm. Then, the competition tests were performed with the presence of corresponding
2
－
analytes. In Fig. S5, all the competing analytes did not interfere the detection of SO3
.
3
.4 LOD and Fluorescence Reversibility of Mito-CI
In order to obtain the detection limit of Mito-CI for SO3 /HSO , the relative
2
－
－
3
2
－
fluorescence emission intensities (I /I ) at different SO
3
concentrations (0-80
4
81 655
2

M) was measured. We could get the linear equation I /I = 0.10c + 9.63 (R =
4
81 655
2
－
.9980), where c is the SO3 concentration. The calculated result is 69 nM according
0
to the formula (LOD=3Sb/m) [37-41], which can cover the endogenous SO2
concentration of the organism (Fig. 3A). Next, after adding HCHO, we performed the
2
－
reversible detection of Mito-CI toward SO3 . As shown in Fig. 2b, the reversibility
could be repeated 3 times (Fig. 3B).
<
Inserted Figure 3>
3
.5 Proposed mechanism
The sensing mechanism of Mito-CI for SO3 /HSO3 was examined by mass
spectrometry. Mito-CI displayed a characteristic peak at m/z = 459.2283 (Fig. S2). A
2
－
－
2
－
mixture solution of Mito-CI with SO3 was injected into ESI-MS analysis (Fig. S6).
It was found that the peak at m/z = 539.1869 displayed a characteristic peak. A
2
－
mixture of Mito-CI+SO3 with HCHO was added in ESI-MS analysis, the new
characteristic peak appeared at m / z = 459.2280 (Fig.S7)., which accorded with the
+
value of the theoretical calculation [M + H] (calcd = 459.2278). Therefore, the
7

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2
－
－
sensing mechanism of Mito-CI for SO3 /HSO3 was based on nucleophilic addition
Scheme 1). And the free bisulfite group is effectively captured by HCHO to drive the
dissociation of the adduct Mito-CI -SO to the formation of the probe Mito-CI [1].
(
3
3
.6 Cellular Imaging
In vivo imaging experiments, we used the MTT method to test the toxicity of
Mito-CI in cells, and the result indicated that Mito-CI was appropriate for living cells
under 10.0 µM (Fig. S8). Next, HepG2 cells were cultured in PBS buffer with
Mito-CI (10 µM) for 20 minutes at 37 °C (Fig. 4). Then, the cells were observed to
have a distinct red fluorescence, and a weak blue fluorescence, which was consistent
with the fluorescence test in vitro. The red fluorescence was obviously weakened and
2
－
blue fluorescence was obviously enhanced after the addition of SO3 (40 µM).
These results showed that Mito-CI can successfully detect exogenous SO in living
2
cells. Moreover, when HCHO (40 µM) was mixed with the just stained HepG2 cells
for another 20 min, it was observed that the red fluorescence enhanced and the blue
fluorescence reduced, indicating that Mito-CI could be reversible in living cells by
HCHO indeed. In a co-localization test, the combined images obtained with the
2
－
mitochondrial dye Mito-Tracker Green and Mito-CI-SO3 adduct displayed that the
2
－
fluorescence of Mito-Tracker Green and Mito-CI-SO3 co-localized good with a
high Pearson coefficient of 0.81 (Fig. 5), which indicated the capability of the
2
－
Mito-CI in detecting SO3 in mitochondria.
<
<
Inserted Figure 4>
Inserted Figure 5>
8

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4
. Conclusions
In summary, a mitochondria-targeted ratiometric reversible detection of
SO /HCHO fluorescent probe Mito-CI was firstly obtained in our work. Mito-CI
2
displayed highly sensitivity (69 nM), fast response time (30 s), large Stokes shift (174
nm) and the NIR fluorescence emission. Besides, we carried out reversible detection
of SO /HCHO in absolute PBS buffer solution, it could be reversible at least 3 times.
2
Last but not least, we also successfully reversibly detected the exogenous SO /HCHO
2
in HepG2 cells. These results will lead to a deeper understanding of SO2
concentration-dependent biological effects.
Acknowledgments
We thank the National Natural Science Foundation of China (No. 21775096),
One hundred people plan of Shanxi Province, Shanxi Province "1331 project" key
innovation team construction plan cultivation team (2018-CT-1), 2018 Xiangyuan
County Solid Waste Comprehensive Utilization Science and Technology Project
(2018XYSDJS-05), Shanxi Province Foundation for Returness (2017-026), Shanxi
Collaborative Innovation Center of High Value-added Utilization of Coal-related
Wastes (2015-10-B3), Shanxi Province 2019 annual science and technology activities
for overseas students selected funding projects, Innovative Talents of Higher
Education Institutions of Shanxi, Scientific and Technological Innovation Programs
of Higher Education Institutions in Shanxi (2019L0031), Key R&D Program of
Shanxi Province (201903D421069), China Institute for Radiation Production and
Scientific Instrument Center of Shanxi University (201512).
9

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pyrene-hemicyanine fluorescent probe for colorimetric and ratiometric detection
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Figure captions
2
－
Scheme 1 Diagram of Mito-CI for SO3 /HCHO detection.
Scheme 2 Synthesis of the probe Mito-CI.
Fig. 1 (A) The fluorescence intensity of Mito-CI (10 µM) with various concentrations
2
－
SO3 (0−100 µM) in absolute PBS buffer solution (10.0 mM) after 30 s equilibrium,
λex = 413 nm, slit: 5 nm/10 nm. (B) UV-vis spectrum of Mito-CI (10.0 µM) in the
2
－
presence of different amounts of SO3 (0−150 µM) in absolute PBS buffer solution
after 30 s equilibrium, (10.0 mM, pH = 7.4).
Fig. 2 (A) Fluorescent spectral changes with HCHO (0−200 M) in absolute PBS
buffer solution (10.0 mM, pH = 7.4) after 3 min equilibrium, λ = 413 nm, slit: 5
ex
－
nm/10 nm. (B) UV-vis spectrum of Mito-CI-SO3 in the existence HCHO (0−300
M) in absolute PBS buffer solution after 3 min equilibrium, (10.0 mM, pH = 7.4).
2
－
Fig. 3 (A) Reversibility of Mito-CI (10 µM) toward SO3 (100 μM) with HCHO
200 µM). (B) The linear curve obtained from relative fluorescence emission
intensities (I /I ) of Mito-CI (10 µM) with Na SO from 0–80 µM.
(
4
81 655
2
3
2
－
Fig. 4 Cellular images of SO3 and HCHO with dual-channel: (a1−d1): HepG2 cells
were cultivated with 10 µM of Mito-CI for 20 min; (a2−d2): HepG2 cells were
2
－
cultivated with 10 µM of Mito-CI for 20 min, next cultivated with 40 µM of SO3
2
－
for 20 min; (a3−d3): HepG2 cells containing Mito-CI were cultivated with SO3 (40
µM) for 20 min, next cultivated with HCHO (40 µM) for another 20 min. Red
channel (λ = 630-680 nm. λ = 560 nm), Blue channel (λ = 460-510 nm. λ = 458
em
ex
em
ex
nm).
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Fig. 5 Co-localization imaging of HepG2 cells. Cells were cultivated with Mito-CI
(10 µM) and 0.2 µM Mito-Tracker Green at 37 °C for 20 min. (a) Red channel (λem =
6
38-747 nm. λ = 633 nm), (b) Green channel (λ = 500-540 nm. λ = 488 nm). (c)
ex
em
ex
Combined image of (a) and (b). (d) Intensity scatter plot of Mito-Tracker Green and
Mito-CI.
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Scheme 1
Scheme 2
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Figure 1
(a)
(b)
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Figure 2
(a)
(b)
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Figure 3
(a)
(b)
Figure 4
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Figure 5
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Author contributions
Tao zhang carried out synthesis and spectrometry Caixia yin and Fangjun huo’s idea and
writing
Jianbin Chao’s NMR
Yongbin Zhang’s sample detection and cell imaging
Guangming Wen support for experiments.
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Declaration of interest statement
We have no any interest conflict.
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Graphical abstract
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Highlights
a) A near-infrared and ratiometric fluorescent probe for SO was reported.
2
b) Probe Mito-CI showed highly sensitivity (68 nM), excellent water solubility.
c) Probe Mito-CI was successfully applied to mitochondria-targeted and reversible for
2
－
monitoring SO /HCHO in living cells.
3
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