Construction of a novel near-infrared fluorescent probe with multiple fluorescence emission and its application for SO2 derivative detection in cells and living zebrafish

Article abstract of DOI:10.1039/c8tb02030b

Sulfur dioxide (SO₂) in biological systems is an important gaseous signal molecule and plays important roles in physiological activities. It can be endogenously produced by enzymes in mitochondria during oxidation of sulphur-containing molecules. Thus, the development of probes for sulfur dioxide detection in biological environment is necessary. Here, a new near-infrared fluorescent probe (Rh-TPA) with multiple fluorescence emission was constructed and applied for SO₂ derivative detection. Rh-TPA was constructed via conjugation of a rhodamine analogue with a triphenylamine group. Rh-TPA exhibited a major emission peak at 740 nm and a shoulder peak at 810 nm. After interacting with SO₂ derivatives, the conjugated system dissociated into two smaller chromophores with two emission peaks (520 nm and 570 nm) in the visible region. The probe showed negligible cytotoxicity, as demonstrated by the MTT results. Biological imaging application experiments indicated that the probe can be used to image SO₂ derivatives in HeLa cells and living zebrafish.

Full text of DOI:10.1039/c8tb02030b

Journal of
Materials Chemistry B
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Construction of a novel near-infrared fluorescent
probe with multiple fluorescence emission and its
Cite this: J. Mater. Chem. B, 2018,
, 7060
application for SO derivative detection in cells
6
2
and living zebrafish†
ab
a
c
a
a
Keyin Liu,
*
Yunling Chen, Hui Sun, Shoujuan Wang and Fangong Kong*
2
Sulfur dioxide (SO ) in biological systems is an important gaseous signal molecule and plays important
roles in physiological activities. It can be endogenously produced by enzymes in mitochondria during
oxidation of sulphur-containing molecules. Thus, the development of probes for sulfur dioxide detection
in biological environment is necessary. Here, a new near-infrared fluorescent probe (Rh-TPA) with
multiple fluorescence emission was constructed and applied for SO
constructed via conjugation of a rhodamine analogue with a triphenylamine group. Rh-TPA exhibited a
major emission peak at 740 nm and a shoulder peak at 810 nm. After interacting with SO derivatives,
2
derivative detection. Rh-TPA was
Received 2nd August 2018,
Accepted 5th October 2018
2
the conjugated system dissociated into two smaller chromophores with two emission peaks (520 nm
and 570 nm) in the visible region. The probe showed negligible cytotoxicity, as demonstrated by the
MTT results. Biological imaging application experiments indicated that the probe can be used to image
DOI: 10.1039/c8tb02030b
rsc.li/materials-b
2
SO derivatives in HeLa cells and living zebrafish.
mitochondria during oxidation of sulphur-containing molecules
Introduction
8
such as H
) is widely known as a key pollutant existing in significance to develop a new method for SO
the atmosphere, which is predominantly generated by consump- in living organisms.
tion of fossil fuels, industry production and volcanic eruption for Many methods for SO
2
S, cysteine and homocysteine. Therefore, it is of great
Sulfur dioxide (SO
2
2
derivative detection
2
derivative detection have been developed
1
,2
many decades. Medical physiology studies have shown that in recent years, and they include electrochemical methods,
long time exposure to SO in the atmosphere or drinking water chromatography, colorimetry, titration, and gas chromatography–
2
2
À
9–15
containing high concentrations of sulfite (SO ) or bisulfite mass spectrometry (GC–MS).
Among them, the fluorescence
) may lead to several serious diseases such as respiratory imaging technique is one of the most powerful methods for SO
illness, lung disorders, cancer, neurological disorders, and cardio- detection in living organisms due to the advantages of noninvasive,
3
À
3,4
(
HSO
3
2
2
,5
16–19
vascular diseases. Moreover, sulfur dioxide (SO
2
) and its ionic real-time and in situ analysis.
) are very important chemical species that fluorescence imaging, recently, several highly sensitive fluorescent
are widely applied in fine chemical, food and pharmaceutical probes for SO detection have been reported based on modification
Due to these advantages of
2À
À
hydrates (SO
3
or HSO
3
2
3
,6
20
21–24
industries. However, excessive sulfite intake is harmful to human of traditional dyes such as fluorescence, coumarin,
and
Some of the probes are based on the
strict control. A recent study has demonstrated that SO can be fluorescence turn-on/turn-off response towards SO , which may
6,25–28
health, and the amount of sulfite as an additive must be under cyanine derivatives.
2
2
endogenously generated, and it plays a key role in physiological suffer from a comparatively low sensitivity or high fluorescence
7
environment. It can be endogenously produced by enzymes in background. Compared with fluorescence turn-on/turn-off-type
probes, ratiometric fluorescent probes can eliminate most of the
a
interference by measuring the ratio change of fluorescence inten-
sity at two different emission wavelengths. Hence, the develop-
ment of new fluorescent probes with multiple fluorescence
Key Laboratory of Pulp and Paper Science and Technology of Ministry of Education,
Qilu University of Technology (Shandong Academy of Sciences), Jinan,
Shandong 250353, P. R. China. E-mail: keyinliu@163.com, kfg@qlu.edu.cn;
Fax: +86-0531-89631168; Tel: +86-0531-89631168
22
emission is more beneficial for SO derivative detection in complex
2
b
c
Institute of Fluorescent Probes for Biological Imaging, University of Jinan, Jinan,
Shandong 250022, P. R. China
biological samples.
Herein, we have reported a new strategy for the construction
of a novel near-infrared fluorescent probe with multiple fluores-
Xuancheng product quality supervision and Inspection Institute, Xuan Cheng,
An Hui, 242000, P. R. China
†
2
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb02030b cence emission. The probe for SO derivatives was constructed
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Scheme 1 The synthesis route of probe Rh-TPA.
Fig. 1 (A) The general design strategy of a near-infrared fluorescent probe
with multiple fluorescence emission and (B) the design and detection
process of probe Rh-TPA toward SO
2
derivatives and potential application Bruker Avance 400 NMR spectrometer. Double-distilled water
in bioimaging.
was used in the solution preparation and measurement.
The fluorescence spectra were measured on a Hitachi F4600
spectrofluorimeter (Hitachi High-Tech Science), and the
through connection of two chromophores with an active conjugated
double bond (Fig. 1A). In the presence of SO , the conjugated double
bond was broken, and the near-infrared fluorescent probe was
separated into two distinct chromophores with emission at different
wavelengths.
As an example, we have developed a novel near-infrared
fluorescent dye (Rh-TPA) containing an active double bond in
the dye skeleton (Fig. 1B). Rh-TPA was constructed through
integration of a rhodamine-like structure (2) with 4-(diphenyl-
UV-visible absorption spectra were recorded on a Shimadzu
UV-2700 spectrophotometer (Shimadzu Suzhou instruments
Mfg. Co, Ltd). All animal procedures for this study were
approved by the Animal Ethical Experimentation Committee
of Shandong University according to the requirements of the
National Act on the use of experimental animals (China).
Images of cells and zebrafish were collected on a Leica SP8
microscope instrument.
2
amino)benzaldehyde (3) (Scheme 1). The triphenylamine group Synthesis procedures
in Rh-TPA is a notable organic building block extensively used
Synthesis of 4-(2-carboxyphenyl)-7-(diethylamino)-2-methyl-
chromenylium (2). Compound 1 and compound 2 were synthe-
sized and characterized according to a previously reported
2
9
in dye construction with excellent fluorescence performance,
which may endow Rh-TPA with novel fluorescence properties.
When interacting with SO derivatives, the conjugated system
2
30
method (Scheme 1). Briefly, 3-(diethylamino)phenol and
in Rh-TPA dissociated into two smaller chromophores with two
emission peaks in the visible region (Fig. 1). We envision that
the near-infrared fluorescent probe Rh-TPA can detect SO2
derivatives with clear fluorescence changes in both near infra-
red and visible-light regions.
phthalic anhydride were refluxed in toluene solution for 12 h
to acquire the crude product of compound 1, which was
purified by a silica gel column. Concentrated sulfuric acid
(
H SO ), 10 mL, was carefully added into a 100 mL round
2 4
bottom flask, and it was cooled to 0 1C by an ice bath. Then,
mL of acetone was added to the cooled H SO solution
3
2
4
dropwise, and compound 1 (0.6 g, 2.0 mmol) was added to
the mixture in sequence. The mixture was stirred at 0 1C for
Experimental
Reagents, materials and apparatus
30 min and then heated to 90 1C by an oil bath for 4 h. The
Unless otherwise stated, all reagents were purchased from mixture was cooled to room temperature and then poured into
commercial suppliers and used without further purification. 100 g crushed ice. Then, 2 mL of perchloric acid was added
All solvents used were purified by standard methods prior to dropwise to the mixture while stirring. The crude product was
use. Silica gel thin layer chromatography (TLC) plates and silica obtained by filtration and then purified by a silica gel column
gel (200–300 mesh) were purchased from Qingdao Ocean Ltd Co. by eluting with dichloromethane and methanol (v/v, 100/1–10/1).
All the reactions were monitored by silica thin layer chromato- Compound 2 (0.21 g, 0.63 mmol) was obtained. Yield: 35%.
1
graphy (TLC), and the intermediates were purified by a silica gel
4
H NMR (400 MHz, MeOD-d ) d 8.30 (dd, J = 7.7, 1.1 Hz, 1H), 7.83
1
13
column. H NMR and C NMR spectra were recorded on a (dd, J = 7.5, 1.4 Hz, 1H), 7.78 (dd, J = 7.7, 1.4 Hz, 1H), 7.49–7.41
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(
4
m, 1H), 7.41–7.34 (m, 2H), 7.23–7.17 (m, 2H), 3.75 (q, J = 7.1 Hz, with 20 mM Rh-TPA for 30 min. In the control group, the larvae
H), 2.81 (s, 3H), 1.34 (t, J = 7.1 Hz, 6H). were only exposed to 20 mM of Rh-TPA for 30 min before
Synthesis of SO2 probe Rh-TPA. Compound 2 (0.1 g, fluorescence imaging.
.3 mmol) and 4-(diphenylamino)benzaldehyde (0.09 g, 0.33 mmol)
0
were added to a 50 mL round bottom flask; then, 10 mL of AcOH
was added. The mixture was reacted at 90 1C under nitrogen
protection for 6 hours, and the reaction was monitored by TLC.
After compound 2 was consumed, the reaction mixture was
cooled to room temperature and the solvent was removed using
an evaporator. The crude product was further purified by a
silica gel column by eluting with dichloromethane and methanol
Result and discussion
The spectral response of probe Rh-TPA towards SO derivatives
2
The photophysical properties of the intermediates 2 and 3
and the SO derivative probe Rh-TPA in solution were explored
2
(Fig. S1–S4, ESI†). Interestingly, in the absorption spectra,
Rh-TPA showed a maximum absorption peak at around 640 nm
and a shoulder peak at 495 nm. In the fluorescence spectra, the
probe showed an emission band at 740 nm and a shoulder peak at
(
v/v, 100/1–10/1). Rh-TPA (0.035 g, 0.06 mmol) was obtained.
1
Yield 20%, H NMR (400 MHz, MeOD-d ) d 8.10 (d, J = 15.8 Hz,
4
1H), 7.96 (d, J = 6.6 Hz, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.59
810 nm (Fig. S3, ESI†). The fluorescence emission is probably due
(
(
dd, J = 6.1, 3.7 Hz, 2H), 7.47 (d, J = 9.5 Hz, 1H), 7.37 (m, 5H), 7.17
m, 10H), 6.98 (d, J = 8.7 Hz, 2H), 3.69 (q, J = 7.0 Hz, 4H), 1.31
31
to the p* - p electron transition in Rh-TPA. Then, we investi-
gated the spectral changes in Rh-TPA towards SO derivatives. As
shown in Fig. 2, in the absorption spectra, the absorption at
13
2
(
1
1
1
t, J = 7.1 Hz, 6H). C NMR (100 MHz, MeOD-d
58.87, 155.40, 151.38, 146.32, 143.27, 134.55, 130.40, 130.27,
29.75, 129.46, 129.29, 129.22, 128.47, 127.44, 126.65, 125.87,
24.78, 120.05, 116.34, 116.23, 114.98, 113.52, 95.75, 45.38, 38.81,
3.35, 30.20, 28.69, 22.63, 11.36. HRMS (ESI) m/z calcd for
4
) d 165.07, 162.63,
2
À
650 nm decreased sharply with the addition of SO3 ; meanwhile,
a new absorption band emerged at 420 nm.
In accordance with the change in absorption spectra, a
considerable fluorescence change was also observed in the
emission spectra (Fig. 3). As shown in Fig. 3(a and b), with
3
+
C
40
H
34
N
2
O
3
Na [MÀH + Na] : 613.2462, found: 613.2426.
2
the concentration increase of an SO derivative, a remarkable
Cell culture and fluorescence imaging
fluorescence turn-on was observed at both 520 nm and 570 nm.
Synchronously, the emission peaks at 740 nm and 810 nm
HeLa cells were obtained from the Institute of Biochemistry
and Cell Biology, Shanghai Institute of Biological Sciences
decreased sharply with the addition of SO derivative with the
2
(SIBS), Chinese Academy of Science. HeLa cells were cultured
fluorescence intensity change of nearly 10-fold (Fig. 3c and d).
When evaluated by using the change in the ratiometric fluores-
cence intensity of the two emission peaks (F520/F740), up to
in Dulbecco’s modified Eagle’s medium (DMEM) containing
1
0% fetal bovine serum and antibiotics (100 units per mL
À1
penicillin and 100 mg mL streptomycin) and maintained at
2
60-fold fluorescence enhancement was observed with the SO to
2
37 1C in an incubator atmosphere of 5% CO and 95% air. A
probe ratio change (Fig. 3e). The detection limit of Rh-TPA
fresh stock solution of HeLa cells was seeded into glass bottom
À6
toward SO
2
derivatives was about 3.2 Â 10 M, as calculated
À5
dishes with a density of about 1 Â 10 cells per dish. The cells
from the linear fit analysis (Fig. 3f). The time-dependent
were then incubated at 37 1C in an incubator for 24 h before
fluorescence change of Rh-TPA toward SO derivatives was also
2
fluorescence imaging. In the experimental group, the cells were
measured. As shown by Fig. 4, the fluorescence at 520 nm
increased steadily, whereas the fluorescence at 740 nm
decreased over 80 min. The detection limit was lower than
2
À
pre-treated with 50 mM of SO3 for 20 min and then exposed to
0 mM of Rh-TPA for another 20 min at room temperature.
1
In the control group, the cells were only exposed to 10 mM of
Rh-TPA for 20 min before fluorescence imaging. The flow
cytometry experiments were performed with a Moflo XDP
the allowed limit of additives in food industry; thus, the probe
5
2
is suitable for SO derivative detection in living animals.
The fluorescence change mechanism was also proved by
H NMR and LC-MS analyses. As shown in H NMR spectra
(Beckman Coulter) instrument, red channel: lex = 640 nm, filter:
1
1
750 Æ 20 nm; green channel: lex = 488 nm, filter: 550 Æ 20 nm.
(Fig. S5, ESI†), after the reaction with sulphite, the proton shifts
Zebrafish maintenance and imaging
The wild-type zebrafish were purchased from Nanjing Eze-Rinka
Ltd Co. China, and all zebrafish experiments were performed
fully according to the international ethics guidelines. Zebrafish
were cultured at 28 1C and maintained at optimal breeding
conditions on a 12 h light/12 h dark cycle. The embryos of
zebrafish were transferred into glass dishes filled with E3 media
(
15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO , 1.0 mM CaCl ,
4 2
0
.15 mM KH PO , 0.05 mM Na HPO , 0.7 mM NaHCO ; pH 7.5).
2 4 2 4 3
2
À
Fig. 2 The absorption spectra change of Rh-TPA titrated with SO
3
.
For the fluorescence imaging experiments, 4-day-old larvae were
transferred into a 96-well plate by a disposable transfer pipette in
1
(
a) The absorption spectra change of 10 mM of Rh-TPA titrated with
2À
increasing concentrations of SO
3
in PBS (pH 7.4, 10 mM). (b) The
2À
mL of culture medium. In the experimental group, the larvae
absorption changes at 650 nm with the SO
for 20 min and then treated Error bars are ÆSD, n = 3.
3
to probe ratio increase.
2À
were pretreated with 50 mM of SO
3
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to Rh-TPA via 1,2 conjugated addition, which has been demon-
2
5,26
strated in previous reports.
The selectivity of probe Rh-TPA towards SO
2
derivative
Among all the factors of a probe for small molecule detection,
the selectivity of a probe is one of the key fundamental para-
meters because of the complexity of biological samples. Thus,
the fluorescence responses of Rh-TPA towards other reactive
À
À
species including reactive oxygen species (H
2
O
2
À
, ClO , TBHP,
ꢀ
OH), reactive nitrogen species (NO, HNO, NO , NO3 ), reac-
2
2
À
2À
tive sulfur species (H S, S O , SO4 , Cys, Hcy, GSH) and
2
2 3
other traditional small molecule species in the physiological
environment were also measured. Fig. 5(a–c) demonstrate that
2
À
Rh-TPA exhibited excellent selectivity toward SO
3
, and other
traditional small molecules almost caused negligible inter-
ference to the detection process. Moreover, detection could be
achieved with multiple fluorescence changes at both 520 nm
and 740 nm, and the ratio change of the fluorescence signals
(
F520/F740) greatly enhanced the detection sensitivity. Peroxy
À
nitrite (ONOO ) is an important species with strong oxidizing
ability. The fluorescence change of Rh-TPA toward ONOO
3
2
À
2
À
.
was also measured (Fig. S7, ESI†); the result indicated that it
exhibited no fluorescence interference to the detection of
Fig. 3 The fluorescence spectra change of Rh-TPA titrated with SO
3
2À
(a and c) 10 mM of Rh-TPA titrated with increasing amounts of SO
3
in PBS
(pH 7.4, 10 mM, with 10% DMSO as co-solvent), (a) excitation by 440 nm and sulphite derivatives.
(
c) excitation by 640 nm. (b and d) The fluorescence intensity changes at
2À
5
20 nm and 640 nm with SO
3
to probe ratio change (b) monitored at
2
Detection of SO in living cells
5
20 nm and (d) monitored at 740 nm. (e) The ratiometric fluorescence intensity
2À
change (F520/F740) with SO
3
to probe ratio. Error bars are ÆSD, n = 3.
With the above measured properties for SO derivative detection
2
in solution, the potential application of Rh-TPA for fluorescence
2
imaging of SO derivatives in biological environment was
explored. First, we performed an MTT assay to evaluate the
cytotoxicity of Rh-TPA toward HeLa cells. The results showed
that more than 90% of cells survived even after 20 mM of Rh-TPA
Fig. 4 The time-dependent fluorescence change of Rh-TPA in the
2À
presence of SO
of Rh-TPA in the presence of 30 equivalents of SO
40 nm and 640 nm, respectively. (b and d) The fluorescence intensity
changes at 520 nm and 640 nm with time, respectively.
3
. (a and c) The fluorescence spectra change of 10 mM
2À
3
, (a) and (c) excited at
4
Fig. 5 The selectivity experiment of Rh-TPA in the presence of various
of the double bond in Rh-TPA at 8.04 ppm and 7.65 ppm kinds of species. (a and b) The bar graph of fluorescence intensity changes
of 10 mM of Rh-TPA in the presence of 30 equivalents of various kinds of
species; numbers 1–19 represent blank, Ca , Mg , Zn , Br , Cl , Ac ,
shifted upfield to 4.89 ppm and 5.76 ppm, respectively. The
2+
2+
2+
À
À
À
result was further confirmed by LC-MS analysis (Fig. S6b, ESI†);
the prominent peak at m/z of 673.2362 (retention time, 1.80 min)
corresponding to the addition product was identified in the
À
ꢀ
À
À
2À
2À
2À
H
2
O
2
, ClO , OH, GSH, Hcy, Cys, NO
The fluorescence intensity was monitored at 520 nm (a) and 740 nm (b).
c) The ratio changes of fluorescence at 520 nm and 740 nm (F520/F740).
2
, NO
3
, NO, S
2
O
3
, S , SO
3
.
(
HR-MS spectra. The results suggested that sulphite was added Error bars are ÆSD, n = 3.
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Fig. 6 The confocal fluorescence images of SO
a–d) HeLa cells were incubated with 10 mM of Rh-TPA for 20 min; (a) bright
field, (b) green channel, (c) red channel, (d) merge. (e–h) HeLa cells were
preincubated with SO derivatives for 20 min and then incubated with Rh-TPA
for 20 min before fluorescence imaging; (e) bright field, (f) green channel,
2
derivatives in HeLa cells.
Fig. 7 The confocal fluorescence images of SO
2
derivatives in zebrafish.
(
(
a–d) Zebrafish were incubated with 20 mM of Rh-TPA for 30 min; (a) bright
field, (b) green channel, (c) red channel, (d) merge. (e–h) Zebrafish were
preincubated with SO derivatives for 20 min and then incubated with
0 mM of Rh-TPA for 30 min before fluorescence imaging; (e) bright field,
f) green channel, (g) red channel, (h) merge. Scale bar: 500 mm.
2
2
2
(g) red channel, (h) merge. Red channel: lex = 640 nm, filter: 750 Æ 20 nm;
(
green channel: lex = 488 nm, filter: 550 Æ 20 nm. Scale bar: 20 mm.
3
3
model widely used in pharmaceutical and biological research.
Herein, we investigated the fluorescence detection of SO in
was internalized for 24 h (Fig. S8, ESI†), suggesting that it has
relatively low cytotoxicity and can be applied to image SO2
derivatives in living cells. We then investigated cell imaging
2
zebrafish by Rh-TPA. Four-day-old zebrafish were used in the
experiments (Fig. 7). In the experimental group, zebrafish were
preincubated with an SO derivative for 30 min and then further
2
properties toward SO
experimental group, HeLa cells were pre-incubated with 50 mM
of SO derivatives for 20 min and then incubated with 10 mM of
2
derivatives in HeLa cells (Fig. 6). In the
incubated with 20 mM of Rh-TPA for another 30 min. In the control
group, zebrafish were incubated with 20 mM of Rh-TPA only before
fluorescence imaging. As shown in Fig. 7(a–d), a fluorescence signal
was observed in the red channel in the zebrafish incubated with the
probe only. However, in the experimental group, bright fluores-
cence increase was observed in the green channel and the fluores-
cence signal in the red channel diminished sharply (Fig. 7e–h). The
experimental results indicated that Rh-TPA can potentially be used
2
Rh-TPA for 20 min. In the control group, HeLa cells were only
incubated with 10 mM of Rh-TPA for 20 min at 37 1C in an
incubator before fluorescence imaging measurements. As
expected, in the control group, fluorescence signal was observed
only in the red channel (Fig. 6a–d). However, in the experimental
group, the fluorescence in the red channel decreased dramatically,
and considerable fluorescence enhancement was observed in the
green channel synchronously (Fig. 6e–h). Therefore, Rh-TPA can
as an efficient probe for SO derivative detection in living animals.
2
2
detect SO successfully with clear fluorescence signal change in
both red and green channels. The fluorescence change was also
observed by flow cytometry studies (Fig. S9, ESI†). Compared with
the observations for cells incubated with the probe only (Fig. S9c
and d, ESI†), the fluorescence in the red channel in the cells
Conclusions
In summary, we have constructed a novel near-infrared fluores-
cent probe for SO2 derivatives with multiple fluorescence
emission by incorporating triphenylamine group in Rh-TPA.
The probe showed near infrared fluorescence emission peaks at
2À
incubated with the probe decreased when SO
3
was added,
whereas the fluorescence increased in the green channel under
the same conditions (Fig. S9e and f, ESI†).
7
40 nm and 810 nm; after interacting with SO
emission peaks emerged in the visible light region. The probe
could detect SO derivatives with both colorimetric and ratio-
metric characteristics and excellent selectivity in solution. As
indicated by biological experiments, Rh-TPA could detect SO
2
derivatives, two
Furthermore, Rh-TPA was applied for detecting endogenous
2
SO in living cells. N-Benzyl-2,4-dinitrobenzenesulfonamide
2
was synthesized (Scheme S1, ESI†) and used as an intracellular
7
,25
SO generator.
As shown in Fig. S10 (ESI†), when the cells
2
2
were treated with Rh-TPA, a bright fluorescence signal was
observed in the red channel (Fig. S10a–d, ESI†). However,
in the cells pretreated with an SO donor and then incubated
2
with Rh-TPA, the fluorescence in the red channel diminished;
meanwhile, the fluorescence in the green channel increased
sharply (Fig. S10e–h, ESI†). The results indicated that the probe
derivatives efficiently in HeLa cells and zebrafish with remark-
able fluorescence changes in both red and green channels. As
indicated by the photophysical and biological experiments, the
probe exhibited excellent near infrared emission properties and
good biocompatibility; thus, it can potentially be applied as a
novel unit in many fields such as organic photovoltaic materials,
optical materials and drug delivery areas in the future.
can be used for detecting endogenously generated SO in cells
2
effectively.
Detection of SO
2
derivatives in vivo
Conflicts of interest
With the probe in hand, we then explored the ability for SO
2
detection in living animals. Zebrafish is a typical vertebrate animal There are no conflicts to declare.
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