A novel colorimetric fluorescent probe for SO2 and its application in living cells imaging

Article abstract of DOI:10.3390/molecules23040871

A novel chromenylium-based fluorescent probe was exploited for sulphur dioxide (SO₂) detecting. The probe displayed a remarkable fluorescence turn-on response towards SO₂ based on the nucleophilic addition reaction to the carbon-carbon double bond with 105 nm Stock shift. The probe was successfully applied for the quantification of SO₂.The linear detection range was from 0–160 μM with the detection limit as low as 99.27 nM. It also exhibited high selectivity for SO₂ than other reactive species and amino acids. Furthermore, cell staining experiments indicated that the probe was cell membrane permeable and could be used for high-performance imaging of SO₂ in living cells. The superior properties of the probe made it highly promising for use in chemical and biological applications.

Full text of DOI:10.3390/molecules23040871

molecules
Article
A Novel Colorimetric Fluorescent Probe for SO2 and
Its Application in Living Cells Imaging
Ming-Yu Wu 1, ID , Jing Wu 1, Yue Wang 1, Yan-Hong Liu 2 and Xiao-Qi Yu 2,
* *
1
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China;
w1249280681@sina.com (J.W.); chinakywy2009@163.com (Y.W.)
Key Laboratory of Green Chemistry and Technology, Ministry of Education, College of Chemistry,
Sichuan University, Chengdu 610064, China; yanhongliu@scu.edu.cn
2
*
Correspondence: wumy1050hx@swjtu.edu.cn (M.-Y.W.); xqyu@scu.edu.cn (X.-Q.Y.);
Tel.: +86-288-760-3202 (M.-Y.W.); +86-288-541-5886 (X.-Q.Y.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 March 2018; Accepted: 6 April 2018; Published: 10 April 2018
Abstract: A novel chromenylium-based fluorescent probe was exploited for sulphur dioxide (SO2)
detecting. The probe displayed a remarkable fluorescence turn-on response towards SO2 based
on the nucleophilic addition reaction to the carbon-carbon double bond with 105 nm Stock shift.
The probe was successfully applied for the quantification of SO2.The linear detection range was from
0–160 µM with the detection limit as low as 99.27 nM. It also exhibited high selectivity for SO2 than
other reactive species and amino acids. Furthermore, cell staining experiments indicated that the
probe was cell membrane permeable and could be used for high-performance imaging of SO2 in
living cells. The superior properties of the probe made it highly promising for use in chemical and
biological applications.
Keywords: fluorescent probe; SO2; colorimetric; living cell imaging
1. Introduction
As one of well-known air pollutants, sulphur dioxide (SO2) has been studied extensively in
toxicology [
sulphur-containing amino acids through biosynthetic pathways, such as transamination by aspartate
aminotransferase (AAT) [ ]. Moreover, SO2 play significant roles in physiological processes especially
in the regulation of cardiovascular function in synergy with NO and the lowering of blood pressure [4,5]
The abnormal endogenous levels of SO2 are believed to be linked with lung cancer, cardiovascular disease,
and a number of neurological disorders [ 10]. Toxicological studies further suggested that SO2 and its
1]. However, recent works show that SO2 is generated endogenously and mainly from
2,3
.
6–
derivatives could change the characteristics of voltage-gated sodium channels and potassium channels in
rat hippocampal neurons [11], affect thiol levels and, hence, affect redox balance in cells [12] and produce a
neuronal insult [13]. These observations have led to speculation that SO2 in the fourth gasotransmitter [14].
Thus, it is of great importance to develop novel analytical methods for the study of the biological and
pathological roles of SO2 in physiological systems [15,16].
In recent years, fluorescence probes attract a great deal of attention due to the superiorities of simple
operation, good selectivity, and high sensitivity as well as noninvasive imaging of biological molecules
and processes with high spatial and temporal resolution in real-time [17
mainly based on the nucleophilic addition reaction to the aldehydes, ketones, or carbon-carbon double
bonds [21 37]. Some of them had been applied for detection of SO2 in organelles or in tumors [24 26 36].
–20]. For SO2 detection, it was
–
– ,
However, some drawbacks of those probes are difficult to avoid, such as the small Stokes shift, high
detection limit, poor selectivity, remarkable interference from biothiols, or reactive oxygen species, and so
Molecules 2018, 23, 871; doi:10.3390/molecules23040871
www.mdpi.com/journal/molecules

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on. Therefore, it is necessary to exploit more efficient fluorescent probes for SO2 and its derivatives with
larger Stokes shifts and stronger intensities, in particular, with high sensitivity and selectivity.
In recently years, Yuan and Lin developed a series of chromenylium-based fluorescent probes with
larger Stokes shifts, substantial quantum yields, and photostability under physiological conditions which
are widely used to detection and imaging of bio-active species in vivo, such as H2O2, HClO, biothiols,
H2S, Sec, and so on [38–41]. Intrigued by their findings, we designed one novel fluorescent probe for SO2
by modifying chromenylium with an electron-withdrawing group (substituted pyridine) to synthesize
BPO-Py-Cl, which could be a selective probe to detect SO2 in HeLa cells based on the nucleophilic
addition reaction to the carbon-carbon double bond (Scheme 1).
Scheme 1. The detection process of BPO-Py-Cl with SO2.
2. Results
2.1. Synthesis and Characterization of BPO-Py-Cl
The synthesis route of the target molecule is illustrated in Scheme 2. It takes four steps to
complete its composition, as follows: BPOH was synthesized according to the literature [40
First, the Friedel-Crafts acylation reaction of 3-(diethylamino)phenol with phthalic anhydride can
obtain compound which could further react with cyclohexanone by a two-step cascade reaction to
acquire oxonium 5. Then, a condensation reaction between 4-hydroxybenzaldehyde with compound
,42].
3
5
will produce BPOH. Finally, BPO-Py-Cl could be obtained by a nucleophilic substitution reaction
1
of BPOH with the 2-chloropyridine derivative. The probe has been characterized by H-NMR,
13C-NMR, and HRMS. Detailed synthetic procedures and structure characterizations were given
in the experimental section and supporting information.
Scheme 2. Synthesis route of fluorescent probe BPO-Py-Cl.

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2.2. Optical Response of BPO-Py-Cl toward SO2
The optical property of BPO-Py-Cl (10
µM) was explored and the maxima absorption at 552 and
391 nm were observed in 10 mmol PBS (pH 7.4) buffer solution containing 20% DMSO as co-solvent
(Figure 1). After interacting with 500 M sulphite, the colour of the solution changed from purple
µ
to pale yellow under visible light and green fluorescence increased prominently under a 365 nm
ultraviolet lamp. The absorption peaks of BPO-Py-Cl were blue-shifted to 493 nm and 379 nm,
respectively. These results demonstrated that BPO-Py-Cl could detect SO2 efficiently.
Figure 1. The absorption spectra and colorimetric changes of BPO-Py-Cl (10
(500 µM) under visible and 365 nm ultraviolet lamps.
µM) interacting with SO2
2.3. pH-Dependent Fluorescence Response of the Probe toward SO2
The effect of pH on the fluorescent properties was examined. As shown in Figure 2, the fluorescent
intensity of the BPO-Py-Cl had almost no remarkable changes in 10 mmol PBS buffer solution
containing 20% DMSO when the pH increased from 4.0 to 10.0. However, upon addition of 500 µM
Na2SO3, the fluorescent intensity at 495 nm exhibited conspicuous enhancement when pH increased
from 4.0 to 6.0. When the pH continuously increased to 10.0, the fluorescent intensity decreased
dramatically. Therefore, pH 6.0 was chosen as the optimal condition for BPO-Py-Cl.
Figure 2. The pH dependent fluorescent changes of BPO-Py-Cl (10
(500 µM). The experiment was repeated three times (±S.D.).
µM) in PBS or after addition of SO2

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2.4. Quantitative Determination of SO2
The fluorescent titrations were conducted in 10 mmol pH 6.0 PBS buffer containing 20% DMSO
as a co-solution. As expected, the fluorescence intensity of BPO-Py-Cl at 495 nm increased gradually
along with the increment of Na2SO3. To our delight, 400
as much as 11.7 times greater (Figure 3a and Figure S1). The quantum yields increased from 0.043
to 0.673 after interacting with 400 M Na2SO3. In the given concentration rage from 0 to 160.0 M,
µM Na2SO3 can lead to an intensity increase
µ
µ
the fluorescent signal intensity was linearly related to the concentration of Na2SO3 (Figure 3b), with
the detection limit of 99.27 nM.
2−
Figure 3.
(
a) Fluorescence titration of BPO-Py-Cl (10
µ
M) with SO3 (0, 20, 40, 60, 80, 100, 150, 200,
250, 300, 350, 400, 450, 500
µM) (
λ
= 390 nm, slit: 2.5 nm/5 nm) in a 10 mM PBS:DMSO = 8:2 pH
ex
6.0 buffer solution; and (
Slit: 2.5 nm/5 nm.
b
) linear fit of fluorescence intensity changes with Na2SO3.
λ
ex
= 390 nm.
2.5. Selectivity Experiments
Based on the excellent fluorescent properties observed with Na2SO3, the selectivity of the probe
for SO2 over other reactive species and amino acids were examined. As shown in Figure 4 and
Figure S2, 500
Meanwhile, 500
µ
M sulphite could lead to noteworthy fl−uorescent spectrum changes of BPO-Py-Cl
.
− − − 2− − 3− − 2−
M of representative anions (F , Cl , Br , I , CO3 , AcO , PO4 , SCN , S2O3 ),
µ
biologically-abundant metal ions (Li+, Na+, K+, Mg2+, Ca2+, Cu2+, Zn2+, Ni2+, Fe2+, Fe3+, Pb2+),
reactive oxygen species (NaClO, H2O2, TBHP), reactive nitrogen species (NO2 and NO3−), other
−
reactive sulphur species (SO42−, H2S, Cys, HCy, and GSH) and amino acids (Ala, Arg, Asp, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Val) hardly led to any change. Figure 5 shows the fluorescent
intensity and colour changes of the BPO-Py-Cl before and after the addition of sulphite or other
species under visible light and 365 nm UV lamp. Obviously, only sulphite caused a remarkable colour
change under the UV lamp.
Figure 4. Fluorescence spectrum changes of BPO-Py-Cl (10 µM) interacting with 500 µM amino acids
(Arg, Met, Ser, Asp, Gly, Ala, His, Val, Lys, Leu, Glu, Pro, Ile, Phe), reactive oxygen species (H2O2,
NaClO, TBHP), reactive nitrogen species (NO3−, NO2−), and reactive sulphur species (SO42−, Cys,
Hcy, GSH, H2S, SO2) in 10 mM pH 6.0 PBS buffer solution containing 20% DMSO,
2.5 nm/5 nm.
λ
ex
= 390 nm. Slit:

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Figure 5. Fluorescence intensity changes of BPO-Py-Cl with SO2 and other species. Inset: fluorescence
changes of BPO-Py-Cl upon addition of 500 M SO2 and other species under visible light and UV light
µ
(Ex = 365 nm). 1. BPO-Py-Cl, 2. Ala, 3. Arg. 4. Asp, 5. Glu, 6. Gly, 7. His, 8. Ile, 9. Leu, 10. Lys, 11. Met,
12. Phe, 13. Pro, 14, Ser, 15. Val, 16. H2O2, 17, NaClO, 18. TBHP, 19. NO3−, 20. NO2−, 21. SO42−, 22.
Cys, 23. Hcy, 24. GSH, 25. H2S, 26. SO2. The experiment was repeated three times (±S.D.).
2.6. Possible Mechanism
As reported by Lin, the carbon–carbon double bond linked with oxonium can provide the latent
nucleophilic addition site for SO2 [37]. From the absorption spectrum (Figure 1), it can be speculated
that Na2SO3 broke the conjugate system of BPO-Py-Cl. To verify the nucleophilic addition reaction of
sulphite to the carbon–carbon double bond, the spectrum of compound
PBS buffer solution containing 20% DMSO. As we can see in Figure 6 and Figure S3, compared with the
BPO-Py-Cl interacting with SO2, compound had a similar excitation and emission wavelength, which
5 was investigated in pH 6.0
5
demonstrated that both of them have similar conjugated systems. Therefore, nucleophilic addition
reactions of sulphite to the carbon–carbon double bond was considered as the possible mechanism.
Figure 6. The fluorescent spectrum of compound 5 and BPO-Py-Cl interacting with SO2.

Molecules 2018, 23, 871
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2.7. Living Cell Imaging
Encouraged by the aforementioned results, we further performed the capacity of probe for the
fluorescent imaging in living cells. First, a standard CCK-8 assay in HeLa cells was investigated at
different concentrations of BPO-Py-Cl. Figure 7 shows the changes of the cell survival rate with the
concentration of BPO-Py-Cl increasing, and the cell survival rate was about 85% after incubation with
2 µM BPO-Py-Cl for 24 h. With 10 µM BPO-Py-Cl, it decreased to 40%.
Figure 7. Cell viability by a standard CCK-8 assay. The experiment was repeated three times (±S.D.).
Finally, the utility of BPO-Py-Cl to image SO2 was next carried out for living cells (Figure◦8).
The HeLa cells were incubated with BPO-Py-Cl (10 µM) in culture medium for 30 min at 37 C,
and exhibited weak fluorescence in the green channel. In a control experiment, the cells were
pre-treated with BPO-Py-Cl for 30 min and further incubated with Na2SO3 (500 M) for another
µ
90 min. Obvious fluorescence increases in the green channel were observed compared with BPO-Py-Cl
alone. These results demonstrated that BPO-Py-Cl could be used for the detection and imaging of SO2
in living cells.
Figure 8. Cell image experiments of BPO-Py-Cl. HeLa cells were incubated with 10
µM BPO-Py-Cl
for 30 min, ( ) green channel, bright field, and the merged channel of green and bright field channels.
a–c
HeLa cells were incubated with 10
µ
M
BPO-Py-Cl for 30 min, then washed with PBS and incubated
) are the green channel, bright field, and the merged
2−
with SO3 (500
µM) for another 90 min; (
d–
f
channel of green and bright field channels.

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3. Materials and Methods
3.1. Materials and General Instruments
All chemicals and reagents were purchased from commercial suppliers Aladdin-Reagent
(Shanghai, China), Sigma-Aldrich (St. Louis, MO, USA), and TCI (Shanghai, China) and used
without any further purification if not declared. All the solvents for optical spectroscopic studies were
either HPLC or spectroscopic grade. TLC analyses were performed on GF 254silica gel. Silica gel
(HG/T2354-92, 200–300 mesh) used for column chromatographic purifications were obtained from
Qingdao Haiyang chemical Co., Ltd (Shandong, China). High-performance mass spectra were
performed on a Bruker Daltonics Bio TOF (Karlsruhe, Germany) mass spectrometer in electrospray
ionization mode. NMR spectra were recorded on a Bruker AMX-400 instrument using tetramethyl
1
silane as the internal reference for H-NMR (400 MHz) or CDCl3 as the internal standard for
13C-NMR (100 MHz). UV–VIS absorption spectra and fluorescent spectra were recorded by a UV-1900
UV–VIS spectrophotometer and F4600 spectrofluorimeter with a 10 mm quartz cuvette from Hitachi
PharmaSpec (Tokyo, Japan).
3.2. Synthesis and Characterization
2-(4-(Diethylamino)-2-hydroxybenzoyl)benzoic acid (
3): 16.5 g (100 mmol) 3-diethylamino phenol, 19.0 g
(128 mmol) phthalic anhydride, and 70 mL toluene were added to a 250-mL round round-bottom flask
◦
◦
◦
◦
which was heated to 80 C for 10 h, 90 C for 5 h, 100 C for 2 h, and 110 C for 1 h, successively under
N2. After cooling to RT, the precipitate was filtered off and washed with PhMe to obtain 28 g crude
purple solid (compound 3) (89.5% yield) which was used for the next reaction without purification.
9-(2-Carboxyphenyl)-6-(diethylamino)-1,2,3,4-tetrahydroxanthylium perchlorate (
(70 mL) was cooled to 0 C. Then, 6.6 mL of cyclohexanone (63.7 mmol) and 9.5 g of compound
4
): concentrated H2SO4
◦
3
(32 mmol) were added dropwise to the cooled H2SO4, respectively, with vigorous stirring. The mixture
was further stirred at 90 ◦C for 1.5 h, cooled down to room temperature, and poured onto ice (300
g). The resulting precipitate was filtered off and washed with cold water (100 mL) after 7 mL 70%
perchloric acid was added to the ice mixture. The precipitate was further purified by silica gel column
chromatography with DCM to DCM/MeOH (10/1, v/v). About 9.4 g of purple solid (compound 4)
was obtained (62% yield).
(E)-9-(2-Carboxyphenyl)-6-(diethylamino)-4-(4-hydroxybenzylidene)-1,2,3,4-tetrahydroxanthylium perchlorate
BPOH): 4-hydroxybenzaldehyde (0.53 g, 4.32 mmol) and 1.72 g (3.60 mmol) of compound were
(
5
mixed in 40 mL AcOH. Then the mixture was heated to 90 ◦C overnight under N2. Subsequently, the
solvent was removed under reduced pressure. After that, the crude product was dissolved in 50 mL
dichloromethane, washed with water (50 mL) three times, and dried by sodium sulphate. The crude
product was concentrated and purified with chromatography on a silica gel column with DCM to
DCM/MeOH (10/1, v/v) to get 1.35 g BPOH as dark purple solid (65% yield).
(E)-9-(2-Carboxyphenyl)-4-(4-((4-chloropyridin-2-yl)oxy)benzylidene)-6-(diethylamino)-1,2,3,4-
tetrahydroxanthylium (BPO-Py-Cl): About 174.0 mg (0.3 mmol) of BPOH was dissolved in
10 mL of DMF. Then, 441.0 mg (3.0 mmol) of 2,4-dichloropyridine and 207 mg (1.5 mmol) of K2CO3
were added into the above solution. After reacting for 3–5 h at 50 ◦C under supervision by TLC, the
solvent was removed under reduced pressure to obtain the crude product which as purified on a silica
gel column chromatography with DCM to DCM/MeOH (20/1, v/v) to obtain an aubergine solid of
1
70 mg BPO-Py-Cl (33.3% yield). H-NMR (400 MHz, CDCl3), 8.28 (d, 1H, J = 5.6 Hz), 7.99 (d, 1H,
J = 7.6 Hz), 7.68 (t, 1H, J = 7.6 Hz), 7.58 (t, 1H, J = 7.2 Hz), 7.49 (d, 2H, J = 8.4 Hz), 7.41 (s, 1H), 7.27
(d, 1H, J = 7.6 Hz), 7.13 (d, 2H, J = 8.4 Hz), 6.89–6.85 (m, 2H), 6.53 (d, 1H, J = 8.8 Hz), 6.45 (d, 1H,
J = 2.4 Hz), 6.39 (dd, 1H, J = 2.8 Hz, J = 8.8 Hz), 3.39 (q, 4H, J = 6.8 Hz), 2.86–2.82 (m, 1H), 2.69–2.65 (m,
1H), 2.11–2.07 (m, 1H), 1.75–1.63 (m, 3H), 1.20 (t, 4H, J = 6.8 Hz). 13C-NMR (100 MHz, CDCl3), 170.1,
166.4, 152.8,152.3, 152.2, 150.8, 149.4, 146.8, 134.5, 131.5, 131.2, 129.3, 128.6, 123.5, 120.6, 112.0, 111.5,

Molecules 2018, 23, 871
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108.9, 108.5, 104.8, 97.3, 44.5, 27.2, 23.1, 22.4, 12.6. HRMS (ESI): m/z [M]+ calcd. for C36H32ClN2O4:
591.2045; found 591.2043.
3.3. Preparation of the Test Solutions
Na2SO3 was used as the source of SO2. A probe stock solution of the BPO-Py-Cl (2.0 mM) was
prepared in −DMSO. Other stock solutions of representative anions (F−, Cl−, Br−, I−, CO32−, AcO−,
3−
2−
PO4 , SCN , S2O3 ), biologically-abundant metal ions (Li+, Na+, K+, Mg2+, Ca2+, Cu2+, Zn2+, Ni2+
,
Fe2+, Fe3+, Pb2+), reactive oxygen species (NaClO, H2O2, TBHP), reactive nitrogen species (NO2
and NO3−), other reactive sulphur species (SO42−, H2S, Cys, HCy, and GSH), and amino acids (Lys,
−
Glu, Leu, Met, Arg, His, Phe, Ser, Ile, Val, Ala, Asp, Pro,) were prepared in deionized water at a
concentration of 50 mM. All the test solutions were prepared by mixing 75
µL of each species stock
solution and 15 L of the probe stock solution in 3.0 mL with PBS buffer/DMSO (8:2, v/v) solution.
µ
The absorption and emission spectra were recorded after the test solution was incubated at room
temperature for 2 h.
3.4. Quantum Yields
The fluorescence quantum yield (Φ) was calculated using the following formula:
2
Φx = Φs × (As/Ax) × (Dx/Ds) × (nx2/ns )
where the subscripts s and x refer to the standard material and test sample, respectively.
Φs and Φx
represent the fluorescence quantum yield of the standard and test sample; ns and nx represent the
refractive index of the solvents used; As and Ax represent the absorption intensity at the excitation
wavelength of the standard and test sample; Ds and Dx represent the integral of the fluorescence
intensity of the standard and test sample. Fluorescein in 0.1 M NaOH (Φs = 0.85) was used as the
standard [43].
3.5. CCK-8 Assay for the Cell Cytotoxicity
The cytotoxicity of the probe was determined by CCK-8 assays. HeLa cells were first seeded in
96-well plates (about 7000 cells per well) and cultured overnight for 70–80% cell confluence. After the
cells were incubated with BPO-Py-Cl at different concentrations (2, 4, 6, 8, 10
µM in DMSO/cell culture
medium = 1:49) for twenty-four hours, 10 L of CCK-8 mixed in 90
µ
µL of PBS was added to each well
of the 96-well assay plate for additional 1 h incubation at 37 ◦C. The absorbance was measured at a
wavelength of 450 nm using an ELISA plate reader (Model 550, BioRad, Hercules, CA, USA). All the
samples were repeated three times.
3.6. Determination of the Detection Limit
The detection limit was calculated based on the fluorescence titration [44] with the following equation:
detection limit = 3σbi/m
where σbi is the standard deviation of blank measurements, m is the slope between intensity versus
sample concentration (signal-to-noise ratio of 3:1). The standard deviation of blank measurements was
determined when the emission intensity of probe (10 µM) without Na2SO3 was measured 10 times.
Then, fluorescent titrations were conducted under the present conditions. A good linear relationship
between the fluorescence intensity and the concentration of Na2SO3 was obtained, the slope between
intensity versus Na2SO3 concentration was m in the above-mentioned formula.

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3.7. Fluorescence Imaging of SO2 in Living Cells
HeLa cells were incubated in DMEM medium (which contained 10% foetal bovine serum and
◦
1% antibiotic-antimycotic) at 37 C in an incubator (5% CO2/95% air). Then cells were seeded in
confocal dishes (4
times with physiological PBS and incubated with BPO-Py-Cl (10
washed with physiological PBS three times to remove free probes, cell imaging was carried out after
the cells were further incubated with Na2SO3 (500 M) for another 90 min. Meanwhile, the control
experiment was performed. The cells were incubated with BPO-Py-Cl (10 M) for 30 min and then
×
103 per well) and incubated for 24 h. Before staining, the cells were washed three
µ
M) for 30 min at 37 ◦C. After being
µ
µ
washed with PBS three times. Then, cell imaging was performed. The confocal fluorescent images
were recorded for the green channel, corresponding to the wavelength range 460–530 nm, which was
monitored for excitation at 405 nm.
4. Conclusions
In summary, a novel chromenylium derivative bearing the electron-withdrawing group
BPO-Py-Cl was synthesized, and the probe exhibited a remarkable turn-on fluorescence response
toward SO2 with a 105 nm Stock shift. It exhibited high sensitivity and selectivity with a low detection
limit (99.27 nM) for SO2. In addition, the probe was cell membrane permeable and could be successfully
applied to the imaging of SO2 in living cells.
Supplementary Materials: The supplementary materials are available online.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (no. 21708030)
and the Fundamental Research Funds for the Central University (no. 2682016CX102). We also thank the
Comprehensive Training Platform of Specialized Laboratory, College of Chemistry, Sichuan University for
laser scanning confocal imaging.
Author Contributions: M.-Y.W. and X.-Q.Y. conceived and designed the work. M.-Y.W. carried out the synthetic
work and wrote the paper. J.W., Y.W., and Y.-H.L. performed the fluorescence properties assay. All authors read
and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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