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An AIE + ESIPT ratiometric fluorescent probe for

monitoring sulfur dioxide with distinct ratiometric
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Cite this: J. Mater. Chem. B, 2018,

6, 1973 fluorescence signals in mammalian cells, mouse
embryonic fibroblast and zebrafish†
Yong Liu, ‡a Jing Nie, ‡b Jie Niu, a
Weishan Wang a
and Weiying Lin *a

Received 10th January 2018,
Accepted 27th February 2018
DOI: 10.1039/c8tb00075a
rsc.li/materials-b
Sulfur dioxide (SO2) is associated with serious diseases including lung cancer, cardiovascular diseases,
and many neurological disorders. However, discrimination of the physiological and pathological
functions of SO2 in different living systems is restricted by the lack of functional molecular tools. To
address this critical challenge, herein, we have developed a novel ratiometric probe, TPE-TE, for
monitoring SO2 with distinct ratiometric fluorescence signals in mammalian cells, mouse embryonic
fibroblasts, and zebrafish via a combination of an ESIPT mechanism and the aggregate fluorescence
method for the first time. The TPE-TE exhibits well-resolved emission peaks, high sensitivity, excellent
selectivity, and low cytotoxicity. Moreover, this probe possesses higher sensitivity in an aqueous solution
than the current probes. Taking advantage of these prominent features, we have achieved the detection
of endogenous and exogenous SO2 with distinct ratiometric fluorescence signals in mammalian cells
and mouse embryonic fibroblast. For the detection of endogenous SO2, probe-loaded HeLa cells exhibited
stronger ratiometric fluorescence signals than HepG2 cells. For the detection of exogenous SO2, it was found
that macrophage cells exhibited stronger ratiometric fluorescence signals than cancer cells for the first time.
Interestingly, mouse embryonic fibroblasts incubated with this probe showed unique ratiometric imaging.
Moreover, TPE-TE could be suitable for ratiometric SO2 imaging in living zebrafish.

Introduction with intramolecular hydrogen bonding.9–12 These probes with

In 2001, fluorescent dyes with aggregation-induced emission
(AIE) properties were first discovered by Tang’s group.1 These
fluorescent materials with AIE features exhibited weak fluores-
cence emission intensity in the solution state that became
intense in the aggregated state.2,3 The search for AIE materials
has become a novel technique to tackle the aggregation-caused
quenching (ACQ) of conventional fluorescent dyes.4–6 More-
over, fluorescent dyes with AIE property have shown significant
academic value in the biological imaging field.7,8 Excited-state
intramolecular proton transfer (ESIPT) is a photochemical
process occurring in the excited singlet state of a molecule
a
Institute of Fluorescent Probes for Biological Imaging, School of Materials Science
and Engineering, School of Chemistry and Chemical Engineering, University of
Jinan, Jinan, Shandong 250022, P. R. China. E-mail: weiyinglin2013@163.com
b
School of Chemical Engineering & Technology, China University of Mining and
Technology, Xuzhou, Jiangsu, 221116, P. R. China
† Electronic supplementary information (ESI) available: Experimental details
including synthesis, the characterization of the compounds, absorption and
fluorescence spectroscopic data, and living cells and in vivo imaging. See DOI:
10.1039/c8tb00075a
‡ Liu and Nie made equal contributions to this work.
the ESIPT mechanism have been studied intensively in recent
years because ESIPT usually alters the conjugation system of a
fluorophore and thus induces changes in the fluorescence
spectra.13–15 Herein, we have developed a unique ratiometric
fluorescent probe with AIE property for ratiometric imaging of
bioactive small molecules by a combination of the ESIPT
mechanism and the aggregate fluorescence method.
In 1986, it was first found that sulfur dioxide (SO2) was
produced endogenously from sulfur-containing amino acids,
such as L-cysteine, in mammals.16 L-Cysteine is catalyzed by
cysteine dioxygenase (CDO) to L-cysteine sulfinate, which is con-
verted into b-sulfinylpyruvate through transamination by aspartate
aminotransferase (AAT); then, b-sulfinylpyruvate spontaneously
decomposes into pyruvate and SO2.17,18 SO2 plays a key role in
the regulation of cardiovascular function in synergy with other
active small molecules (e.g. NO).19 Furthermore, epidemiological
studies have demonstrated that SO2 not only induces many
respiratory responses, but is also associated with serious diseases
including lung cancer, cardiovascular diseases, and many neuro-
logical disorders.20–22 Thus, to further understand the role of SO2
in biology, it is crucial to take these active small molecules into
account.

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Paper
Fig. 1 Strategies for the sensing of SO2.
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SO2 exhibits transient nature in live specimens.23,24 Thus,
the development of highly sensitive and selective fluorescent
probes, which can be used to determine the spatial and
temporal distributions of SO2 in live specimens, is very important.
To date, most of the fluorescent SO2 probes are intensity-based
and interfered by variations in the excitation intensity, probe
concentration, etc. To solve the abovementioned problems, a few
ratiometric fluorescent SO2 probes have been constructed.25–34
However, based on the research conclusions on SO2 probes drawn
from the dilute solution data, there have been no studies on an AIE
material for ratiometric sensing and imaging of SO2 in an aqueous
solution and in vivo (Table S1 in the ESI†). Since the AIE property
and ESIPT mechanism exhibit significant academic values in the
biological imaging field, development of ratiometric fluorescent
probes for ratiometric sensing of SO2 by the combination of the
ESIPT mechanism and the aggregate fluorescence method is impor-
tant. Thus, the goal of our study was to design an AIE + ESIPT
ratiometric fluorescent probe for the detection of SO2 in living cell
lines and zebrafish (Fig. 1).
In this regard, we report a new ratiometric fluorescent probe
(TPE-TE) that can ratiometrically monitor SO2 with distinct
ratiometric fluorescence signals by the combination of the
ESIPT mechanism and the aggregate fluorescence method.
The TPE-TE exhibits excellent properties including well-resolved
emission peaks, high selectivity, and low cytotoxicity. Moreover,
this new probe exhibits higher sensitivity in an aqueous solution
than other probes. Considering the abovementioned properties, we
have achieved ratiometric imaging of endogenous SO2 with distinct
ratiometric fluorescence signals in mammalian cells, mouse
embryonic fibroblast, and zebrafish. For the detection of both
endogenous and exogenous SO2 by the proposed probe, different
cells lines have exhibited distinct results. Interestingly, compared to
the mammalian cancer cells, mouse embryonic fibroblast showed
unique ratiometric imaging results. Moreover, TPE-TE was capable
of ratiometrically imaging SO2 in living zebrafish.
Experimental
Materials
2-Aminothiophenol, DMAP, DCC, and levulinic acid were pur-
chased from J&K Chemical (Beijing, China). Phosphate buffered
saline (PBS), Dulbecco’s modified Eagle’s medium, high glucose,
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were obtained from Seikagaku Corporation (Japan). RAW
264.7 cells, HepG2 cells, HeLa cells, and calf serum were obtained
1974 | J. Mater. Chem. B, 2018, 6, 1973--1983
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from the College of Life Science, Nankai University (Tianjin,
China). The solvents used in the spectral measurement were of
chromatographic grade. All reagents were purchased from com-
mercial suppliers and used without further purification. Solvents
were purified by standard methods prior to use. Double-distilled
water was used throughout the experiments. TLC analysis was
performed on silica gel plates, and column chromatography was
conducted over silica gel (mesh 200–300); both the silica gel plates
and silica gel were purchased from the Qingdao Ocean Chemicals.
Apparatus
For all experiments, all the reagents used for synthesis
and analysis were obtained by commercial suppliers. These
reagents were used without further purification. Solvents were
purified by standard methods prior to use. For the synthesis
experiment, all the separation and purification procedures of
compounds were verified by TLC analysis. This method was
performed on silica gel plates; in addition, column chromato-
graphy was carried out by silica gel (mesh 200–300); silica gel
was obtained from the Qingdao Ocean Chemicals. For the
characterization of compounds, mass spectra were obtained
by an LCQ Advantage ion trap mass spectrometer, whose model
was Thermo Finnigan or Agilent 1100 HPLC/MSD spectro-
meter. The NMR spectra were obtained by the AVANCE III
400 MHz Digital NMR spectrometer. For the analysis experi-
ment, ultraviolet absorption spectra were obtained by the
Labtech UV Power PC spectrometer; fluorescence emission
spectra were obtained using the HITACHI F-4600 fluorescence
spectrophotometer. For biological imaging, fluorescence
imaging of the cells and tissue slices was conducted using
the Nikon A1MP confocal microscopy.
General procedure for the spectral measurement
The stock solution of the probe TPE-TE was prepared at
1 mM in DMF. The solutions of various testing species
were prepared from KBr, CoCl2, Cu2(SO4)2, NaF, NaNO3,
NaNO2, Na2SO3, o-Bu, ONOO, t-butylhydroperoxide (TBHP),
VC, ZnCl2, glutathione (GSH), H2O2, homocysteine (Hcy),
cysteine (Cys), NaClO, N2H4H2O, NaHS, and DEANONOate
in the double-distilled water. The test solution of the probe
TPE-TE (5 mM) in 2 mL PBS buffer was prepared by adding
10 mL of the probe TPE-TE stock solution to 2 mL PBS buffer.
The resulting solution was shaken before obtaining the
spectra. The titration and selectivity experiments of the probe
were carried out using the excitation wavelength of 380 nm,
and the excitation and emission slit widths in the spectral
measurement were 5 and 5 nm, respectively.
Quantum yields
The fluorescence quantum yields were calculated by the following
eqn (1):
  
Ar ðlr Þ ns2 Fs
Fs ¼ Fr (1)
As ðls Þ nr2 Fr
In the abovementioned equation, s and r stand for the
sample and the reference, respectively; F and F are the
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Journal of Materials Chemistry B
quantum yield and integrated emission intensity, respectively.
A and n are the absorbance and refractive index, respectively.
Preparation of solutions for TEM. Before conducting the
TEM experiment, some solutions were prepared; at first, we
prepared four water/DMF mixtures of TPE-TE (4 mM) with
different water fractions ( fw) including water/DMF ( fw = 0%)
and water/DMF ( fw = 100%) dispersions of TPE-TE; then, 10 mL
water/DMF solutions were added to copper wire mesh and
kept for 20 min. After this, residual liquid was absorbed with
a sanitary napkin. Finally, the TEM experiment was carried out.
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Cell viability evaluation by the MTT assays
Initially, HeLa cells were cultured in a 96-well plate in a 5% CO2
incubator at 37 1C. After 12 h, the cell culture medium was changed
to fresh medium containing TP-MIVC at different concentrations
(1, 5, 10, 20, and 30 mM). Then, after 24 h, the medium and the
excess probe were removed, and 10 mL 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg mL1 in PBS)
was added to the 96-well plate. Subsequently, the culture medium
was removed, and 100 mL DMSO was added to the dishes to dissolve
the formazan crystal product. Then, a 96-well plate was shaken for a
short period of time. Absorbance data at 490 nm was obtained by
Multiskan Spectrum. Finally, cell viability data was calculated by the
following equation: cell viability (%) = (OD490 sample  OD490 blank)/
(OD490 control  OD490 blank)  100%. OD490 sample stands for the cells
incubated with different concentrations of TP-MIVC, OD490 control
stands for the cells that are not treated with the probe, and
OD490 blank are the wells containing culture medium only.
Cell culture and imaging
Living RAW264.7 macrophages, HepG2 cells, and HeLa cells were
prepared in H-DMEM (Dulbecco’s modified Eagle’s medium, high
glucose) supplemented with 10% fetal bovine serum in a 5% CO2
incubator at 37 1C. Before the living cell imaging experiments,
living RAW264.7 cells were seeded in a confocal dish (density of
cells was 1  105 mL1). The cells were placed on glass coverslips
and allowed to adhere for 48 h. Cell imaging experiments could be
carried out as soon as the cells reached about 70% confluence.
Initially, control experiments were carried out. The culture
medium of the cells was added to a fresh medium containing
100 mM Na2SO3 and incubated for 1 h. Later, the medium was
removed and washed three times with PBS to remove excess Na2SO3.
After this, 1 mL of the medium containing 10 mM TPE-TE was added
followed by further incubation at 37 1C for 30 min. Finally, confocal
imaging was carried out in the blue (lex = 405 nm, lem =
425–475 nm) and red channels (lex = 405 nm, lem = 570–620 nm)
using Nikon A1MP confocal microscopy.
Fluorescence imaging in living zebrafish
Wild type zebrafish were obtained from Nanjing Eze-Rinka Biotech-
nology Co., Ltd. They were fed under optimal breeding conditions.
Before fluorescence imaging experiments, two groups of zebrafish
were fed for four days. They were transferred into a 30 mm glass
culture dishes using a disposable sterilized dropper. To these two
groups, TPE-TE (5 mM) was added followed by incubation for
30 min; the medium was removed followed by washing three times
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with a PBS buffer solution; after this, to one group, 1 mM SO2 was
added for another 30 min. Then, two groups were transferred into
new glass bottom dishes for imaging. Prior to imaging, we adopted
1% agarose gel for immobilization of zebrafish and put zebrafish
onto agarose with a little media to perform imaging. Fluorescence
images were obtained using the Nikon A1MP confocal microscope
with a 40 objective lens.
Synthesis
Synthesis of 1. The compound was synthesized according to
a literature procedure.35
Synthesis of TPE-TE-O. In a round-bottomed flask (25 mL)
equipped with a magnetic stirrer, a solution of the compound 1
(0.37 g, 1.0 mmol) and 2-aminothiophenol (0.15 g, 1.2 mmol) in
methanol (10 mL) was prepared. Then, 30% H2O2 (6.0 mmol)
and 37% HCl (3.0 mmol) were added, and the mixture was
stirred at room temperature for 2 h. The mixture was quenched
by adding H2O (10 mL), extracted with EtOAc (35 mL), and the
combined extracts were dried (Na2SO4). The corresponding
benzothiazoles were obtained after the removal of solvents
and purified by silica gel chromatography (eluent: n-hexane/
EtOAc = 4 : 1). 1H NMR (400 MHz, CDCl3) d 12.47 (s, 1H), 7.99–7.94
(m, 1H), 7.85 (dd, J = 8.0, 1.1 Hz, 1H), 7.50 (m, 1H), 7.42–7.35
(m, 2H), 7.20–7.02 (m, 16H), 6.88 (d, J = 8.6 Hz, 1H). 13C NMR
(101 MHz, CDCl3) d 169.45, 156.51, 151.81, 143.82, 143.67, 143.26,
140.87, 139.60, 135.92, 134.92, 132.68, 131.68, 131.49, 131.32,
131.24, 128.05, 127.84, 127.71, 126.92, 126.72, 126.61, 126.47,
125.42, 122.06, 121.49, 117.22, 115.99, 77.39, 77.27, 77.07, 76.75.
Synthesis of TPE-TE. A mixture of TPE-TE-O (50 mg,
0.1 mmol), levulinic acid (71.5 mg, 0.1 mmol), DCC (24 mg,
0.2 mmol), and DMAP (0.001 g, 0.01 mmol) in dichloromethane
(3 mL) at 25 1C for 8 h was dissolved in dichloromethane
(3 mL), and the reaction mixture were stirred at room temperature
for 8 h. The organic phase was washed with water and saturated
brine and dried over anhydrous magnesium sulfate overnight. After
CH2C12 was removed, the crude product was purified by column
chromatography with dichloromethane/methanol (20 : 1) as the
eluent, and finally, TPE-TE was obtained as a blue solid with a
yield of 68%. 1H NMR (400 MHz, DMSO-d6) d 8.12 (d, J = 7.9 Hz, 1H),
8.04 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 10.8 Hz, 1H), 7.54 (t, J = 7.5 Hz,
1H), 7.47 (t, J = 7.5 Hz, 1H), 7.22–7.12 (m, 10H), 7.11 (s, 1H), 7.10–
7.01 (m, 6H), 2.91 (dd, J = 11.3 Hz, 4.4 Hz, 4H), 2.14 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) d 207.10, 171.43, 162.05, 152.59, 146.82,
143.27, 143.15, 142.96, 142.34, 141.93, 139.36, 135.14, 134.57,
131.82, 131.18, 131.08, 131.04, 128.54, 128.54, 128.49, 128.34,
127.35, 127.22, 127.14, 126.15,125.20, 124.09, 123.40, 122.55,
40.42, 40.21, 40.00, 39.79, 39.58, 37.86, 30.00, 28.91. HRMS (ESI†)
(m/z): [M + H]+ calcd for C38H29NO3S: 580.1900, found, 580.1923.
Results and discussion
Preparation of the probe
In 2016, we first realized the highly sensitive detection of RNA
in a pure water system by an aggregation–disaggregation
method.36 Due to our interest in fluorescent dyes with AIE
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Fig. 2 The design strategy of the AIE + ESIPT fluorescent probe TPE-TE.
property, we herein introduced TPE-TE as the first SO2 ratio-
metric fluorescent probe with AIE property for sensing SO2 in
living cell lines and zebrafish. The key to this design is that this
unique probe should possess a SO2 site and the AIE property,
and more importantly, it should result in the emission of two
distinct fluorescence signals in the presence of SO2. As tetra-
phenylethene (TPE) is an archetypal AIE luminogen (Fig. S1A in
the ESI†),37 we have modified the compound TPE to exploit a
novel fluorescent dye. Moreover, we introduced a thiazole group into
the TPE-core to afford a novel AIE material, TPE-TE-O. TPE-TE-O
containing benzothiazole as ESIPT fluorophores could be used
to design new fluorescent probes with unique photophysical
properties.38,39
This probe is based on the novel AIE + ESIPT platform
TPE-TE-O with the sensing site levulinate moiety as the receptor
and fluorescence quencher.40 When the hydroxyl group in the
TPE-TE-O dye is protected, the excited-state proton transfer
from the hydroxyl group (proton donor) to nitrogen (proton acceptor)
will be blocked, and TPE-TE will show blue aggregation fluorescence.
Interestingly, after the probe TPE-TE was treated with SO2, the
de-protection of levulinate of TPE-TE provided TPE-TLE-O with
AIE property41,42 that induced the ESIPT mechanism and showed a
yellow aggregation fluorescence signal (Fig. S1B in the ESI†).
Therefore, the AIE dye TPE-TE should be suitable for monitoring
SO2 by the combination of the ESIPT mechanism and the aggre-
gate fluorescence method (Fig. 2).
Optical properties
The synthetic route and the characterization spectra of the
compound TPE-TE are displayed in the ESI.† With TPE-TE in
hand, we set out to evaluate the absorption and fluorescence
emission spectra of the probes in various solutions. As shown
in Table S2 (ESI†) and Fig. S2 (ESI†), in pure aqueous systems,
the absorption and emission peak of the compound TPE-TE
emerged at 317 nm and 465 nm, respectively. However, the
absorption and emission peak emerged at 306 nm and 420 nm,
respectively, in organic solutions. Furthermore, the compound
TPE-TE possessed a larger Stokes shift in a pure aqueous
system than that in organic solvents. Moreover, the compound
TPE-TE exhibited stronger fluorescence intensity and higher
fluorescence quantum yield (F = 0.23) in the aqueous system than
those in organic solvents (F = 0.02 in DMF). As anticipated, the
1976 | J. Mater. Chem. B, 2018, 6, 1973--1983
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Journal of Materials Chemistry B
main reason causing the difference in the optical properties in
the pure aqueous phase and organic phase is the aggregation of
TPE-TE in a pure water system. The compound TPE-TE should be a
novel fluorescent dye with the AIE property.
Aggregation properties of TPE-TE in distinct polar
environments
To prove that the compound TPE-TE is a fluorescent material
with the AIE property, we have investigated optical properties of
TPE-TE in distinct polar environments. We have investigated
the fluorescence behavior of TPE-TE in water/DMF ( fw) mixtures.
We first prepared ten organic solutions of TPE-TE with different
water fractions ( fw); images of TPE-TE in water/DMF mixtures were
obtained under UV illumination (Fig. 3A). The result indicated that
the blue fluorescence signal of TPE-TE gradually became strong
with the increasing fw values. Then, we systematically changed the
fw value and further measured the emission spectra of TPE-TE in
water/DMF mixtures. The fluorescence intensity of TPE-TE at
470 nm gradually became strong with the increasing fw values
(Fig. 3B). Fluorescence characteristics of TPE-TE were consistent
with the images of TPE-TE in distinct polar environments (Fig. 3C).
The abovementioned results demonstrate that the compound TPE-
TE should be a novel fluorescent dye with the AIE property.
We envisioned that rational structural modifications on the
TPE-core could not change the AIE character of TPE. Conse-
quently, we further investigated the properties of TPE-TE-O in
distinct polar environments. The images and fluorescence
characteristics of TPE-TE-O in water/DMF mixtures are different
from those of TPE-TE. In the water/DMF ( fw = 0–40%) mixture
solution, TPE-TE-O showed weak blue fluorescence signals; in this
polar environment, fluorescence spectra (at 450 nm) of the probe
were consistent with the images of TPE-TE-O (Fig. 4A). However,
fluorescence intensity of the compound (at 565 nm) gradually
became strong with an increase in the fw values (Fig. 4B). Similar to
the case of TPE-TE, the fluorescence characteristics of TPE-TE-O
Fig. 3 (A) Images of TPE-TE in water/DMF mixtures obtained under
natural light and UV illumination; (B) fluorescence spectra of TPE-TE in
the water/DMF mixture; and (C) plots of the maximum emission intensity
of TPE-TE in water/DMF mixtures with different fw values. [TPE-TE] = 5.0 mM.
lex = 365 nm.
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Fig. 4 (A) Images of TPE-TE-O in water/DMF mixtures obtained under

natural light and UV illumination; (B) fluorescence spectra of TPE-TE-O in
water/DMF mixture; and (C) plots of maximum emission intensity of TPE-TE-O
at 450 nm in water/DMF mixtures with different fw values. [TPE-TE-O] =
5.0 mM. lex = 365 nm. Fig. 5 (A) Fluorescence spectra of TPE-TE and TPE-TE-O in a pure
organic solution (fw = 0%) and (B) water solution (fw = 100%). [TPE-TE] =
5.0 mM. lex = 365 nm.
were consistent with the images of TPE-TE-O in distinct polar
environments (Fig. 4C). The results demonstrate that TPE-TE-O is
the compound TPE-TE should form a red nanoparticle with
also a fluorescent material with the AIE property. Thus, TPE-TE
definite fw values. Then, we further investigated the aggregation
should be suitable for the ratiometric detection of biological
of TPE-TE in a pure aqueous solution by the dynamic light
analytes by an aggregate fluorescence method.
scattering (DLS) technique (Fig. 6). In the pure DMF solution,
AIE and ESIPT mechanism TPE-TE could not form aggregates as the compound TPE-TE
existed in its molecular form in a pure organic solvent; in an
Next, we investigated the AIE and ESIPT mechanism of TPE-TE
aqueous solution, the compound TPE-TE formed aggregates
and TPE-TE-O in a pure organic solution ( fw = 0%) and water
with an average size of 400 nm (Fig. 6A). Similar to the case of
solution ( fw = 100%). For the preparation of TPE-TE, introduc-
TPE-TE, DLS experiments of TPE-TE-O were carried out. TPE-TE-O
tion of levulinate moiety into the AIE + ESIPT platform
exhibited aggregate particles with an average size of 60 nm
TPE-TE-O resulted in a new probe, TPE-TE, for the detection
(Fig. 6B). The abovementioned results were in agreement with
of SO2. In the pure organic solution ( fw = 0%), as shown in
the optical properties of the compounds in distinct polar envir-
Fig. 5A, for TPE-TE, the hydroxyl group in the TPE-TE dye is
onments. The results further demonstrate that TPE-TE should be
protected, the excited-state proton transfer from the hydroxyl
capable of ratiometrically monitoring biological analytes by an
group to nitrogen will be blocked, and TPE-TE will show weak
aggregate fluorescence method.
fluorescence emission at 422 nm. For TPE-TE-O, de-protection
of levulinate of TPE-TE takes place; this induces the ESIPT Ratiometric responses of the probe to SO2
mechanism and a strong fluorescence signal is observed at
To examine whether the compound TPE-TE could ratiometri-
450 nm. Furthermore, TPE-TE-O will exhibit a new fluorescence
cally detect SO2 in an aqueous solution, we further studied a
emission peak at 565 nm. In the pure aqueous solution
fluorescence titration experiment of TPE-TE (5 mM) in the PBS
( fw = 100%), as shown in Fig. 5B, TPE-TE exhibits stronger
buffer solution. As shown in Fig. 7A, the fluorescence intensity
fluorescence intensity than that in the pure organic solution due to
of TPE-TE in the blue channel decreased; however, the degree
the generation of AIE; similarly, TPE-TE-O shows stronger fluores-
to which the fluorescence intensity was quenched was not
cence intensity than that in pure organic solution at 565 nm.
Moreover, compared to the case of the pure organic solution, the
fluorescence emission peak of the probe at 450 nm disappeared.
Thus, we envision that TPE-TE can ratiometrically monitor SO2 by
the combination of the ESIPT mechanism and the aggregate
fluorescence method. Thus, TPE-TE should be a novel AIE + ESIPT
fluorescent probe for the detection of SO2.

Investigation of the aggregation of TPE-TE by the DLS

technique
Through the study of the optical properties of TPE-TE in Fig. 6 (A) DLS measurements of TPE-TE and (B) TPE-TE-O dispersions in
distinct polar environments, we have preliminary inferred that water at 5 mM.

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Fig. 7 (A) Fluorescence spectra of TPE-TE (5.0 mM) in the (pH 7.4) PBS
buffer solution with the addition of SO32 (0–50 equiv.). Inset: The
fluorescence intensity changes at I567/I470 of TPE-TE with the addition of
SO32. (B) Normalized response of the fluorescence signal by changing the
concentration of SO32. lex = 380 nm. (C) Fluorescence spectra of TPE-TE
(5 mM) in the presence of various relevant analytes. (D) Fluorescence
responses of TPE-TE (5 mM) at 567 nm in the presence of various relevant
analytes. The concentrations of the representative analytes are as follows:
amino acids, 1 mM; GSH, 2 mM; cations and anions, 3 mM; and reactive
oxygen and nitrogen species, 0.2 mM. (E) Fluorescence emission spectra of
TPE-TE (5.0 mM) and TPE-TE-O (5.0 mM) in an aqueous solution. lex = 365 nm.
sufficient (Fig. 7A). With an increase in the concentrations of
SO32, a new red-shifted emission band centered at 567 nm
gradually emerged. Moreover, as can be seen in Fig. 7A (inset),
the fluorescence titration of the probe with SO32 lead to a moderate
increase in the ratios of emission intensities (I567/I470). Furthermore,
a definite emission point was observed at 567 nm that might
suggest that a new species was formed during the titration process.
The reaction product TPE-TE-O was confirmed using 1H NMR
spectroscopy (Fig. S3 in the ESI†). We have proved that TPE-TE
and TPE-TE-O are novel AIE materials. Thus, we concluded that
TPE-TE was capable of detecting SO32 in an aqueous solution by an
aggregate fluorescence method. Furthermore, as shown in Fig. 7B,
the detection limit of the probe was 0.9 mM, indicating that the
probe TPE-TE exhibited high sensitivity to SO32.
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Selectivity is an important criterion for developing fluores-
cent probes for the detection of bioactive small molecules.43 To
demonstrate the selectivity of TPE-TE, TPE-TE was treated with
various biological species, such as cations, anions, and oxidants,
and the fluorescence emission spectra were further studied. As
shown in Fig. 7C, the compound TPE-TE exhibited a significant
red-shift in the emission spectra in the presence of SO32. Other
representative species (Br, ClO, Co2+, Cu2+, Cys, F, GSH, H2O2,
Hcy, NO2, NO3, o-Bu, ONOO, SO42, TBHP, VC, Zn2+, H2S,
N2H4, SO32, and NO) had no obvious effect on the fluorescence
emission. When the abovementioned species were added, no
marked response was observed, and only SO32 responded with
an obvious increase in the ratios of the emission intensities
(I567/I470) (Fig. 7D). This clearly indicated that TPE-TE exhibited
excellent selectivity for SO32 in an aqueous solution.
In the presence of SO32, the de-protection of levulinate of
TPE-TE provides TPE-TE-O with AIE property. This induces the
ESIPT mechanism, which generates a yellow aggregate fluores-
cence signal of TPE-TE-O. This phenomenon was in agreement
with the fluorescence emission spectra of TPE-TE and TPE-TE-O in
the aqueous solution (Fig. 7E). Moreover, it is consistent with the
AIE and ESIPT mechanism of TPE-TE and TPE-TE-O in a pure
organic solution ( fw = 0%) and aqueous solution ( fw = 100%)
(Fig. 5). The abovementioned results demonstrate that TPE-TE is
capable of detecting SO32 in an aqueous solution by the combi-
nation of the ESIPT mechanism and the aggregate fluorescence
method. To the best of our knowledge, no studies have been
reported on a ratiometric fluorescent probe with the AIE property
for highly sensitive and selective detection of SO32 in an aqueous
solution by the combination of the ESIPT mechanism and the
aggregate fluorescence method.
Ratiometric imaging of endogenous SO2 in different cells lines
SO2 widely exists in different cells lines and plays a key role in
apoptosis and oxidative stress.44 Thiosulfate sulphurtransferase
(TST) is a widespread enzyme in nature, which commonly localizes
in different cells lines.45 Biological bisulfite can be produced
endogenously from thiosulfate via TST. We selected different cell
lines to further study the ratiometric imaging of SO2. Cell viability
of TPE-TE was determined by the standard 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays; the results
indicated that the probe TPE-TE had no cytotoxicity for living cells
(Table S3 in the ESI†). Via a further biological study, we verified if
the probe could be used for the detection of endogenously
produced bisulfite inside the cells.
At first, we chose HepG2 cells and further investigated the
ratiometric imaging of endogenous SO2. HepG2 cells were
incubated with the probe TPE-TE (5 mM) for 30 min and then
washed with the PBS buffer solution before the images were
obtained. The imaging results exhibited weak blue and red
fluorescence signals in the blue channel and red channel,
respectively (Fig. 8A(II and III)). Furthermore, ratiometric
images were obtained (Fig. 8A(IV)). Another group of HepG2
cells was incubated with the probe TPE-TE (5 mM) for 30 min
followed by washing with PBS buffer and further incubation
with 500 mM GSH for 0.5 h before the images were obtained.
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Fig. 8 The first line (parallel): (AI–AIII) HepG2 cells were incubated with
TPE-TE (5 M) for 30 min; (AIV) the ratiometric images of AII and AIII. The
second line: (BI–BIII) HepG2 cells were incubated with TPE-TE (5 mM) for
30 min and then with 500 mM GSH for 0.5 h; (BIV) the ratiometric images
of BII and BIII. The third line: (CI–CIII) HepG2 cells were incubated with
TPE-TE (5 mM) for 30 min and then with 500 mM GSH and 250 mM Na2S2O3
for 0.5 h; (CIV) the ratiometric images of CII and CIII. The fourth line:
(DI–DIII) HepG2 cells were incubated with TPE-TE (5 mM) for 30 min, then
with 1 mM SNAP for 0.5 h, followed by 500 mM GSH and 250 mM Na2S2O3
for another 0.5 h. Images were acquired from 425 to 475 nm for blue
fluorescence and from 570 to 620 nm for red fluorescence. lex = 405 nm.
Scale bar = 20 mm.
Compared to the case of Fig. 8A(I–IV), no significant fluores-
cence changes were observed in Fig. 8B(I–IV). In addition,
HepG2 cells were incubated with 500 mM GSH and 250 mM
Na2S2O3 for 0.5 h, and a clear fluorescence change was observed
in the HepG2 cells (Fig. 8C(I–III)). Moreover, ratiometric fluores-
cence images of HepG2 cells were obtained (Fig. 8C(IV)). The
probe-loaded HepG2 cells were pre-treated with 1 mM TST
inhibitor SNAP and then incubated with 500 mM GSH and
250 mM Na2S2O3 for 0.5 h; no significant fluorescence change
was observed (Fig. 8D(I–III)). In the presence of GSH and Na2S2O3,
the mean fluorescence intensities of the probe in red and blue
channels displayed a slight increase (Fig. S4 in the ESI†). The
abovementioned results indicated that TPE-TE was capable of
ratiometrically imaging endogenous SO2 in HepG2 cells and was
comparable to the probes reported earlier.46
In addition, the ratiometric images of endogenous SO2 in
HeLa cells were investigated. HeLa cells were incubated with
the probe TPE-TE (5 mM) for 30 min, and images were obtained
via a confocal microscope. The imaging results indicate no
fluorescence signal in the blue and red channels (Fig. 9A–C).
Probe-loaded HeLa cells were pre-treated with GSH (Fig. 9D–F)
or GSH, Na2S2O3, and TST inhibitor SNAP (Fig. 9G–I), and no
significant fluorescence signals were observed clearly in both
channels. However, probe-loaded HeLa cells were pre-treated
with GSH and Na2S2O3, and strong blue and red fluorescence
signals were clearly observed (Fig. 9K and L). Moreover, a clear
ratiometric image was obtained (Fig. 9M). These results demon-
strated that the probe TPE-TE was capable of ratiometrically
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Fig. 9 The first line (parallel): (A–C) HeLa cells were incubated with TPE-TE
(5 mM) for 30 min; the second line (D–F) HeLa cells were incubated with
TPE-TE (5 mM) for 30 min and then with 500 mM GSH for 0.5 h; the fourth
line: (G–I) HepG2 cells were incubated with TPE-TE (5 mM) for 30 min, then
with 1 mM SNAP for 0.5 h, followed by 500 mM GSH and 250 mM Na2S2O3
for another 0.5 h. The third line: (J–L) HeLa cells were incubated with
TPE-TE (5 mM) for 30 min and then with 500 mM GSH and 250 mM Na2S2O3
for 0.5 h. The fourth line: (M) the ratiomertic images of K and L. Images
were acquired from 425 to 475 nm for blue fluorescence and from 570 to
620 nm for red fluorescence. lex = 405 nm. Scale bar = 20 mm.
imaging endogenous SO2 in living HeLa cells. Compared to
HepG2 cells, HeLa cells treated with GSH and Na2S2O3 exhibited
stronger fluorescence emission signals. Furthermore, the TPE-TE
exhibited stronger emission intensities in the HeLa cells treated
with GSH and Na2S2O3 than those in the HeLa cells untreated with
GSH and Na2S2O3 (Fig. S5 in the ESI†).
Compared to the mammalian cancer cells (HepG2 and HeLa
cells), the 3T3 cells are mouse embryonic fibroblasts.47 As is known,
the detection of SO2 mainly focuses on mammalian cancer cells.48
To find the difference of SO2 in mammalian cancer cells and 3T3
cells, we investigated the ratiometric images of endogenous SO2 in
3T3 cells. We have proved that TPE-TE is capable of ratiometrically
imaging endogenous SO2 in 3T3 cells under similar experimental
conditions and methods as in the case of mammalian cancer cells.
However, compared to mammalian cancer cells, as shown in
Fig. 10, the probe-loaded 3T3 cells exhibited a stronger blue
fluorescence signal under the same imaging conditions; the
probe-loaded 3T3 cells showed stronger ratiometric fluorescence
signals than the mammalian cancer cells. In addition, the TPE-TE
still presented stronger emission intensities in 3T3 cells treated
with GSH and Na2S2O3 than those in the 3T3 cells untreated with
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Fig. 10 The first row (vertically): 3T3 cells were incubated with TPE-TE (5 mM)
for 30 min, (A) bright field, (E) fluorescent image of probe in blue channel,
(I) fluorescent image of probe in red channel, (M) the ratio images of E and I.
The second row: 3T3 cells were incubated with TPE-TE (5 mM) for 30 min, and
then with 500 mM GSH for 0.5 h, (B) bright field, (F) fluorescent image of probe
in blue channel, (J) fluorescent image of probe in red channel, (N) the ratio
images of F and J. The third row: 3T3 cells were incubated with TPE-TE (5 mM)
for 30 min, and then with 500 mM GSH and 250 mM Na2S2O3 for 0.5 h;
(C) bright field, (G) fluorescent image of probe in blue channel, (K) fluorescent
image of probe in red channel, (O) the ratio images of G and K. The fourth row:
3T3 cells were incubated with TPE-TE (5 mM) for 30 min, then with 1 mM SNAP
for 0.5 h, followed by 500 mM GSH and 250 mM Na2S2O3 for another 0.5 h.
(D) bright field, (H) fluorescent image of probe in blue channel, (L) fluorescent
image of probe in red channel, (P) the ratio images of H and L. Images were
acquired from 425 to 475 nm for blue fluorescence, and from 570 to 620 nm
for red fluorescence. lex = 405 nm. Scale bar = 20 mm.
GSH and Na2S2O3 (Fig. S6 in the ESI†). This result was similar to
that obtained with mammalian cancer cells.
Fig. 11 Cell images of SO2 in (A) RAW 264.7 cells and (B) HepG2 cells untreated and treated with Na2SO3; (AI–AIII) Images of RAW 264.7 cells and
(BI–BIII) HepG2 cells untreated with Na2SO3; (AIV–AVI) images of RAW 264.7 cells and (BIV–BVI) HepG2 cells treated with Na2SO3 in the blue emission
channels (lex = 405 nm, lem = 425–475 nm); (AVII) ratiometric images of RAW 264.7 cells and (BVII) HepG2 cells in the blue and red emission channels
(lex = 405 nm, lem = 570–620 nm); (AVIII) the ratio of emission intensities profiles in RAW 264.7 cells and (BVIII) HepG2 cells untreated and treated with
Na2SO3. Scale bar = 20 mm. Statistical analyses were performed with the Student’s t-test (n = 4). The error bars represent standard deviation (S.D.).
1980 | J. Mater. Chem. B, 2018, 6, 1973--1983
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Journal of Materials Chemistry B
For in situ imaging of mammalian cells (HepG2 cells), the
in situ spectrum exhibits a strong red fluorescence signal at
590 nm. However, upon in situ imaging of the mouse embryonic
fibroblast, a weaker red fluorescence signal was observed in the
red channel than that in the HepG2 cells under identical experi-
mental conditions (Fig. S7 in the ESI†).
Photostability is also a key criterion for developing excellent
fluorescent probes.49 Fluorescence images of both channel of
cells incubated with TPE-TE (5 mM), 500 mM GSH, and 250 mM
Na2S2O3 were acquired at different times under successive
excitation (Fig. S8A in the ESI†). The mean intensities of both
channels at different times were obtained (Fig. S8B in the ESI†).
The abovementioned results demonstrated that TPE-TE showed
high photostability at different times under successive excita-
tion. We had envisioned that the probe was capable of real-time
imaging endogenous SO2 in different cell lines.
Ratiometric imaging of exogenous SO2 in different cell lines
At first, we prepared two groups of cell lines untreated and
treated with Na2SO3; three groups of cell lines (RAW264.7
macrophages, HepG2 cells, and HeLa cells) were incubated
with TPE-TE (8 mM) for 30 min and then washed with PBS
buffer solution to remove the free probes; finally, the fluores-
cence imaging for three groups of cells lines was carried out.
As shown in Fig. 11A(I–III), RAW264.7 macrophages untreated
with Na2SO3 emitted a weak blue fluorescence signal and red
fluorescence signal in the blue (Fig. 11A(II)) and red channels
(Fig. 11A(III)), respectively. However, compared to the cells
untreated with SO2, RAW264.7 macrophages treated with
Na2SO3 displayed a stronger blue emission (Fig. 11A(V)) and
red emission (Fig. 11(VI)). Moreover, we further obtained the
ratiometric fluorescence image of RAW264.7 macrophages
(Fig. 11A(VII)), in good agreement with the SO2-induced ratiometric
fluorescence response in an aqueous solution. The ratios of the
mean fluorescence intensities in red and blue channels displayed a
large increase from 0.5 to 3.5 (Fig. 11A(VIII)). The abovementioned
This journal is © The Royal Society of Chemistry 2018

Journal of Materials Chemistry B
results indicated that the probe TPE-TE was capable of ratiome-
trically imaging exogenous SO2 in RAW264.7 cells.
Then, similar to the case of RAW264.7 macrophages, images
of cancer cells (HepG2 cells and HeLa cells) were obtained
(Fig. 11B and Fig. S9 in the ESI†). The ratios of mean fluores-
cence intensities (HepG2 cells) in red and blue channels also
displayed a large increase from 0.5 to 1.6 (Fig. 11B(VIII)). The
results demonstrated that the probe TPE-TE could ratiometri-
cally image exogenous SO2 in living cancer cells. To date, no
studies have been reported on the development of ratiometric
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SO2 fluorescent probes with AIE property for ratiometric
imaging of SO2 in different cell lines (Table S1 in the ESI†).
Compared with cancer cells, RAW 264.7 macrophages
treated with Na2SO3 exhibited stronger fluorescence emission
signals. Furthermore, the TPE-TE exhibited a higher ratio of
emission intensities in RAW 264.7 macrophages treated with
Na2SO3 than that in the HepG2 cells. The abovementioned
results demonstrated that the macrophages exhibited stronger
ratiometric fluorescence signals than the cancer cells. To the best
of our knowledge, this was the first time that an AIE + ESIPT
ratiometric probe was used to study different ratiometric fluores-
cence signals of SO2 in different cell lines.
Although fluorescence intensity of TPE-TE in blue channel is
decreased in vitro, the degree to which it has diminished is not
enough (Fig. 7A). For the imaging of endogenous and exogenous
SO2 in HepG2, 3T3, and RAW263.7 cells, the mean fluorescence
intensities of probe in the blue channels displayed very little
increase. However, fluorescence intensity of the blue channel in
the absence and presence of SO2 had no obvious change.
In contrast, in HeLa cell imaging, the mean fluorescence
intensities of the probe in the blue channels exhibited an
obvious change. It has been universally observed in vitro.
Changes of the mean fluorescent intensities in red channel
were consistence with in vitro test. We speculate that the
difference between the mean fluorescence intensities of the
probe in vitro and in vivo should be permissible since the cell is
an extremely complex environment. Thus, the results obtained
in vitro could not be completely equivalent to the results
obtained in vivo.32
Ratiometric imaging studies in vivo
Significantly, simultaneous one-photon imaging of SO2 in vivo
was demonstrated using zebrafish as the vertebrate model.
As shown in Fig. 12, probe-loaded zebrafish exhibited strong
blue fluorescence and weak red fluorescence with Ired/Iblue of
0.09 at the chest (Fig. 12B, C and J). When incubated with
5.0  106 M Na2SO3 for 1 h, it was found that the blue
fluorescence decreased obviously and the red fluorescence
increased obviously with an Ired/Iblue of 0.40 at 405 nm excita-
tion at the chest of the zebrafish (Fig. 12F, G and J). Moreover,
ratiometric fluorescence imaging of SO2 is conducted (Fig. 12I);
it clearly demonstrates that the chest of the zebrafish has an
interestingly small amount of SO2.
Quantitative distribution characteristics of SO2. Ratiometric
fluorescence imaging results demonstrate that SO2 is mainly dis-
tributed in the head and eye region of the zebrafish. These results
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Fig. 12 Images of zebrafish treated with 5.0  106 M TPE-TE in the
absence (A–D) or presence (E–H) of 1  103 M Na2SO3. (I) Ratiometric
fluorescence imaging of TPE-TE to SO2; (J) ratios of Iblue/Ired obtained
from (B and C) and (F and G). Blue channel: lex = 405 nm, lem =
425–475 nm; red emission channels: lex = 405 nm, lem = 570–620 nm.
Statistical analyses were performed with Students t-test (n = 4). The error
bars represent standard deviation (S.D.).
indicate that TPE-TE can be suitable for the simultaneous one-
photon imaging of SO2 in vivo. To the best of our knowledge, this is
the first report on the fluorescence imaging of SO2 in vivo using
one-photon modes.
In living zebrafish treated with Na2SO3, although fluores-
cence signal of TPE-TE in blue channel is decreased, the degree
to which it has diminished is not enough in the chest region of
the zebrafish. This is quite consistent with the basic findings
in vitro.
Conclusion
In summary, we introduced a thiazole group and a levulinate
group into the TPE-core to develop a novel AIE + ESIPT ratio-
metric SO2 probe, TPE-TE, for the first time. Ratiometric
sensing of TPE-TE to SO2 has been mainly conducted on the
basis of an ESIPT mechanism and the aggregate fluorescence
method. The TPE-TE exhibits excellent properties such as the
well-resolved emission peaks, high sensitivity, high selectivity,
and low cytotoxicity. The abovementioned mechanism and
excellent properties enable us to demonstrate that the
AIE + ESIPT fluorescent probe TPE-TE can ratiometrically
monitor endogenous SO2 with distinct ratiometric fluorescence
signals in different cell lines. For the detection of endogenous
SO2, HeLa cells treated with GSH and Na2S2O3 exhibited
stronger fluorescence emission signals and higher emission
intensities than the HepG2 cells; compared to the mammalian
cancer cells, mouse embryonic fibroblast showed unique ratio-
metric imaging results. For the detection of exogenous SO2, it
was first found that macrophage cells exhibited stronger ratio-
metric fluorescence signals than cancer cells. Moreover, TPE-TE
could be suitable for ratiometric fluorescence imaging of SO2 in
living zebrafish. We expect that this unique ratiometric probe
J. Mater. Chem. B, 2018, 6, 1973--1983 | 1981

Paper
will be useful for dissecting the complicated roles of endogen-
ous SO2 in the complex signal transduction and anti-oxidative
processes. In addition, the modular design strategy may be
extended to construct powerful AIE + ESIPT fluorescent probes
for imaging a variety of biomolecules in the field of biological
applications.
Conflicts of interest
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There are no conflicts of interest to declare.
Acknowledgements
This work was financially supported by the NSFC (21472067,
51503077, 21672083), the Taishan Scholar Foundation
(TS 201511041), and the startup fund of the University of
Jinan (309-10004). Doctor start up fund of University of
Jinnan (160082102) and NSFSP (ZR2015PE001) are also
acknowledged.
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