A novel red-emitting fluorescent probe for the highly selective detection of Hg2+ ion with AIE mechanism

Article abstract of DOI:10.1016/j.chemphys.2020.110944

A novel red-emitting fluorescent probe TBTS for the highly selective detection of mercury (Hg²⁺) ion has been developed, which was constructed with triphenylamine-benzothiadiazole skeleton. Probe TBTS was barely emissive in HEPES buffer solution (pH = 7.4, 10 mM, with 3percent DMSO). As the presence of Hg²⁺ ion, the dithioacetal unit in probe TBTS was hydrolyzed to be aldehyde group, consequently resulting in the intense red emission via aggregation-induced emission (AIE) mechanism. This sensing behaviour was completed within 2.5 min, and the detection limit toward Hg²⁺ ion was calculated to be 90 nM. More importantly, probe TBTS exhibited the excellent biocompatibility, and it was capable of monitoring Hg²⁺ ion in living cells.

Full text of DOI:10.1016/j.chemphys.2020.110944

Journal Pre-proofs
A novel red-emitting fluorescent probe for the highly selective detection of
2
+
Hg ion with AIE mechanism
Bing Liu, Jia Liu, Jiaopu He, Jinsheng Zhang, Hongjun Zhou, Chao Gao
PII:
S0301-0104(20)30736-9
DOI:
Reference:
https://doi.org/10.1016/j.chemphys.2020.110944
CHEMPH 110944
To appear in:
Chemical Physics
Received Date:
Revised Date:
Accepted Date:
3 June 2020
27 July 2020
30 July 2020
Please cite this article as: B. Liu, J. Liu, J. He, J. Zhang, H. Zhou, C. Gao, A novel red-emitting fluorescent probe
2
+
for the highly selective detection of Hg ion with AIE mechanism, Chemical Physics (2020), doi: https://doi.org/
0.1016/j.chemphys.2020.110944
1
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Title: A novel red-emitting fluorescent probe for the highly selective
2
+
detection of Hg ion with AIE mechanism
Author names and affiliations:
Name
Country
Affiliations
E-mail
Wuhan Textile University, Wuhan
Bing Liu
China
450628456@qq.com
4
30073, Hubei, PR China
Wuhan Textile University, Wuhan
30073, Hubei, PR China
Jia Liu
China
China
China
China
378477751@qq.com
gc110312@126.com
wawj74@126.com
4
Wuhan Textile University, Wuhan
30073, Hubei, PR China
Jiaopu He
4
Wuhan Textile University, Wuhan
30073, Hubei, PR China
Jinsheng Zhang
Hongjun Zhou
4
Shanxi Zhendong Pharmaceutical
Co. Ltd, Changzhi 046000,
Shanxi, PR China
zhou73262811@126.
com
Wuhan Textile University, Wuhan
Chao Gao
China
chgao@wtu.edu.cn
4
30073, Hubei, PR China
Corresponding author:
Name: Chao Gao; Email: chgao@wtu.edu.cn
Permanent address of the corresponding author:
Hubei Key Laboratory of Biomass Fibers and Eco-dyeing ＆ Finishing,
department of Chemistry and Chemical Engineering, Wuhan Textile
University, Yangguang Avenue, Wuhan 430073, Hubei, PR China.
1

A novel red-emitting fluorescent probe for the highly selective
2
+
detection of Hg ion with AIE mechanism
a
a
a
a
b
Bing Liu , Jia Liu , Jiaopu He , Jinsheng Zhang* , Hongjun Zhou* , and
a
Chao Gao*
a
Hubei Key Laboratory of Biomass Fibers and Eco-dyeing & Finishing,
Wuhan Textile University, Wuhan 430073, Hubei Province, China.
Shanxi Zhendong Pharmaceutical Co. Ltd, Changzhi 046000, Shanxi
Province, China.
b
*
E-mail: wawj74@126.com; zhou73262811@126.com; chgao@wtu.edu.cn.
Abstract: A novel red-emitting fluorescent probe TBTS for the highly
2
+
selective detection of mercury (Hg ) ion has been developed, which was
constructed with triphenylamine-benzothiadiazole skeleton. Probe TBTS
was barely emissive in HEPES buffer solution (pH = 7.4, 10 mM, with 3%
2
+
DMSO). As the presence of Hg ion, the dithioacetal unit in probe TBTS
was hydrolyzed to be aldehyde group, consequently resulting in the intense
red emission via aggregation-induced emission (AIE) mechanism. This
sensing behaviour was completed within 2.5 min, and the detection limit
2
+
toward Hg ion was calculated to be 90 nM. More importantly, probe TBTS
exhibited the excellent biocompatibility, and it was capable of monitoring
2
+
Hg ion in living cells.
2
+
Keywords: Red-emitting fluorescent probe; Hg ion; Aggregation-induced
emission; Cell imaging.
1
. Introduction
2
+
Mercury (Hg ) was regarded as one of the most toxic heavy metal ions in
the environment, which could easily diffuse into human bodies via skin
2

contact and direct ingestion [1]. Moreover, Hg2+ ions was difficultly
biodegraded in bodies, but facile to be bio–accumulated through the food
2
+
chain [2]. The high level of Hg ion in organisms would inhibit enzymes
activity, disrupt the immune system, damage DNA, and even lead to death
[3-5]. Hence, it was greatly significant to develop an efficient pathway for
2
+
detecting Hg ion in ecological environment as well as biological systems.
2
+
At present, several analytical techniques for Hg ion have been utilized,
including inductively coupled plasma mass spectrum [6], surface-enhanced
Raman spectroscopy [7] and atomic absorption spectrometry [8]. However,
these conventional methods required the expensive instrument, complicated
procedure of sample preparation and professional operations, thus they were
2
+
unsuitable for the detection of Hg ion on site. By contrast, fluorescent
probes have attracted widespread attentions owing to the advantages of low
cost, easy operation, satisfactory selectivity and high sensitivity. So far, the
2
+
fluorescent probes with various recognition groups for Hg ion have been
developed [9-11]. In which, the dithioacetal unit was considered as the ideal
2
+
candidate, because its specific reaction with Hg ion would endow the
2
+
fluorescence probes with the high selectivity toward Hg ion. However,
most of these probes were constructed with the conventional fluorophores
that containing polycyclic aromatic skeleton, that they were generally used
3

in the aqueous media with a large amount of volatile and toxic organic
solvents for assisting their dissolution [12-18]. Moreover, they are facile to
suffer from the aggregation-caused quenching (ACQ) problem, because their
planar conformations readily generated intermolecular π-π stacking in
aggregate state, which would cause the emission quenching [19]. As well
known, these hydrophobic probes were prone to aggregate in hydrous
physiology system, thus the ACQ effect would weaken or even quench the
2
+
fluorescence signal toward Hg ion [20]. Obviously, the above drawbacks
severely limited these probes to be applied in organism. In comparison, the
aggregation-induced emission (AIE) active fluorophores successfully
overcame the ACQ shortcoming because of their intrinsic intense emission
in aggregation state. Recently, there were several AIE-active probes based
on dithioacetal moiety have been reported, whereas they commonly
2
+
exhibited the blue or green emission after the treatment with Hg ion
21-24]. In contrast, red-emitting probes were more suitable for bioimaging
[
application, because they could penetrate into deep tissues and reduce
background auto-fluorescence. Furthermore, red light possessed the low
energy, thus lessening the photo-damage to living cells, organisms and
tissues [25]. In table S1, the recent reported dithioacetal-based probes for
2
+
Hg ion have been summarized.
4

Red-emitting fluorophores possessed the small energy gap (Eg), and the
incorporation of electron-donor (D) and electron-acceptor (A) into
fluorophores was an efficient pathway to reduce the Eg value.
Benzothiadiazole (BT) unit was a typical electron-acceptor, which has been
widely used in the construction of fluorophores with long emission
wavelength. Moreover, triphenylamine (TPA) group exhibited the intense
electron-donating ability, and it was also a well-known AIE activator [19].
The TPA-BT based conjugates commonly displayed the AIE activity as well
as long wavelength emission [26,27], thus the introduction of dithioacetal
moiety into these conjugates was potential to construct the AIE-based probes
2+
for Hg ion with red emission.
In view of these considerations, a novel red-emitting probe TBTS based
2
+
on TPA-BT skeleton for Hg ion has been developed. Probe TBTS was
scarcely emissive in HEPES buffer (pH = 7.4, 10 mM, with 3% DMSO).
2
+
Upon the addition of Hg ion, the dithioacetal moiety in probe TBTS was
instantly hydrolyzed to be aldehyde group, consequently resulting in the
intense red emission via AIE mechanism. Probe TBTS exhibited the high
2
+
selectivity toward Hg ion, and the sensing behavior was accomplished
within 2.5 min. Prominently, probe TBTS displayed the low cytotoxicity to
2
+
HeLa cells, and it was capable for monitoring Hg ion in living cells.
5

2
. Experimental section
2
.1. Materials and Instruments
Unless additional notification, all the chemicals and solvents were
purchased from reagent companies. Double-distilled water was used in the
experiments, and the chromatogram grade of DMSO was used in
fluorescence testing. Melting points were determined with SGW X-4
1
instrument. The H NMR (400 MHz) chemical shifts were measured that
1
3
using CDCl as the internal reference (CDCl : δ = 7.26 ppm). The C NMR
3
3
(
100 MHz) chemical shifts were given using CDCl -d as the internal
3
6
standard (CDCl : δ = 77.16 ppm). High-resolution mass spectra (HRMS)
3
were obtained with a Bruker-Q-TOF mass spectrometer. The fluorescence
spectra were tested in a quartz cells with the HITACHI F-2500 fluorescence
spectrometer. The pH measurements were performed by Sartorius PB-10
meter. Dynamic light scattering (DLS) experiments were investigated with
ZEN3600 Malvern particle sizer. Fluorescence imaging was studied with the
OLYMPUS IX71 fluorescence microscope.
2
.2 The synthesis of compound 1
Compound 1 was synthesized according to our previous method [28],
which was described as follows:
a
mixture of compound
6

4
4
-formylphenylboronic acid pinacol cyclic ester (278 mg, 1.2 mmol),
-bromo-7-[4-(diphenylamino)phenyl]-2,1,3-benzothiadiazole (457 mg, 1
mmol), Pd(dppf)Cl (74 mg, 0.1 mmol) and K PO (636 mg, 3 mmol) in 1,
2
3
4
4
-dioxane/H O (4:1, v/v, 5 mL) was refluxed overnight under N
2
2
atmosphere. Then, the organic phase was separated and the aqueous phase
was extracted with CH Cl . The combined organic phase was concentrated
2
2
using rotavapor, and the residue was purified by a silica gel column
(PE/EtOAc = 10/1, v/v) to provide compound 1 as a red solid (289 mg,
1
yield: 60%). m.p. 197.6-199.4 °C. H NMR (400 MHz, CDCl ): δ (ppm)
3
1
0.11 (s, 1H, aldehyde H), 8.17 (d, J = 8.0 Hz, 2H, -ArH), 8.05 (d, J = 8.4
Hz, 2H, -ArH), 7.90 (d, J = 8.8 Hz, 2H, -ArH), 7.78-7.86 (m, 2H, -ArH),
.29-7.33 (m, 4H, -ArH), 7.19-7.23 (m, 6H, -ArH), 7.09 (t, J = 7.4 Hz, 2H,
7
1
3
-
ArH); C NMR (100 MHz, CDCl ): δ (ppm) 191.94, 154.19, 154.03,
3
1
1
48.56, 147.51, 143.56, 135.89, 134.27, 130.47, 130.17, 130.06, 129.54,
29.15, 127.11, 125.19, 123.65, 122.75.
2
.3 The synthesis of probe TBTS
A mixture of compound 1 (241 mg, 0.5 mmol) and methyl thaioglycolate
(113 μL, 1.2 mmol) was dissolved in dry DCM (5 mL), then p-TsOH (42
mg, 0.22 mmol) as catalyst was added. The reaction mixture was stirred at
o
5
0 C under N atmosphere for 6 h. After that, the solvent of reaction
2
7

mixture was moved by rotary evaporators, and the obtained residue was
purified by silica gel chromatographic column (PE:EtOAc = 5:1, v/v), which
afforded pure intermediate product as a red solid (204 mg). Subsequently,
the intermediate product was dissolved in CH CN (5 mL), and 3N NaOH
3
(
1.5 mL) was added, the mixture was stirred at 35 ºC for 9 h. Then, the
mixture was neutralized by 1N HCl and the pH value was maintained around
.0. The mixed solution was extracted with DCM for three times, and the
6
combined organic phase was washed with saturated sodium chloride
aqueous solution for two times. After that, the organic layer was dried by
anhydrous Na SO , and then concentrated to get the probe TBTS as an
2
4
1
orange-red solid (165 mg, 49%). M.p.: 105.7-107.4 ºC. H NMR (400 MHz,
CDCl ): δ (ppm) 7.98 (d, J = 8.0 Hz, 2H, -ArH), 7.89 (d, J = 8.4 Hz, 2H,
3
-
(
(
ArH), 7.76-7.77 (m, 2H, -ArH), 7.64 (d, J = 8.0 Hz, 2H, -ArH), 7.28-7.32
m, 4H, -ArH), 7.19-7.23 (m, 6H, -ArH), 7.08 (t, J = 7.4 Hz, 2H, -ArH), 5.43
1
3
s, 1H), 3.52 (s, 1H), 3.49 (s, 1H), 3.29 (s, 1H), 3.26 (s, 1H); C NMR (100
MHz, CDCl ): δ (ppm) 170.44, 154.13, 148.25, 147.51, 138.38, 137.75,
3
1
1
33.25, 131.81, 130.77, 130.02, 129.54, 129.42, 128.28, 127.26, 125.03,
+
23.44, 122.85, 53.44, 33.84. HRMS (ESI ): calcd for C H N O S
3
5
28
3
4 3
+
[
M+H] 650.1236, found 650.1232.
2
.4 Photophysical measurements
8

2
+
2+
2+
2+
2+
2+
2+
3+
+
The chloride salts of Hg , Ca , Cd , Zn , Mg , Co , Ni , Cr , K ,
+
2+
2+
3+
3+
+
Na , and nitrite salts of Cu , Pb , Fe , Al and Ag were used. The stock
solutions (3 mM) of these metallic ions were prepared in HEPES buffer (10
mM, pH =7.4) for the experiments. The stock solution of probe TBTS (3
mM) was prepared in DMSO, and then diluted to get the DMSO-HEPES
mixed solutions for fluorescence measurements.
2
.5 The calculation of detection limit
The detection limit (DL) of probe TBTS toward Hg ion was calculated
2
+
according to the equation “DL = 3σ/k”. In which, the value of
signal-to-noise ratio (S/N) was at “3”. Moreover, “σ” was the standard
deviation of blank measurements, and “k” represented the slope between
2
+
fluorescence intensity and the concentrations of Hg ion.
2
.6 Cell culture and fluorescence imaging
The HeLa cells were incubated in Dulbecco's Modified Eagle's Medium
o
(
DMEM) supplemented with 10% fetal bovine serum at 37 C (under 5%
CO atmosphere) for 24 h, which were then seeded in a 96-well plate.
2
Subsequently, the HeLa cells and probe TBTS (20 μM) were cultured in
DMEM with 3% DMSO as the co-solvent for 1 h, and washed twice with
PBS buffer (pH = 7.2) to remove the extracellular TBTS. After that, these
9

cells were divided into the control group and experimental group, and Hg2+
ion (60 μM) was added into the experimental group. The cells were then
o
cultured for 40 min at 37 C, which were washed with PBS for three times
before imaging experiments.
3
. Results and discussion
3
.1. Synthesis and characterization
Probe TBTS was synthesized according to scheme 1. In brief, compound
reacted with methyl thioglycolate to generate the intermediate product,
1
which was then hydrolyzed in 3N NaOH (aq) and finally acidized in 1N
HCl. After the purification, probe TBTS was obtained as an orange-red
1
13
solid. The chemical structure of TBTS was confirmed by H NMR, C
NMR and HRMS (ESI †).
O
S
S
N
N
O
N
N
OH
OH
HS
S
S
1
)
OCH3
N
CHO
N
2
) 3N NaOH,
1
N HCl
1
TBTS
O
Scheme 1. The synthetic route of probe TBTS
2
+
3
.2. Sensing behaviour of probe TBTS to Hg ion
The fluorescence response of probe TBTS toward Hg2+ ion was
investigated by fluorescence titration experiments. As shown in figure 1,
1
0

probe TBTS (20 μM) was barely emissive in HEPES buffer (10 mM, pH
2
+
=
7.4, with 3% DMSO). Upon the continuous addition of Hg ion, the
fluorescence intensity of solution was clearly increased. Moreover, the
2
+
emission peak displayed a slight red-shift, because the present Hg ion
promoted dithioacetal unit of probe TBTS to turn into aldehyde group,
which enhanced the electron withdrawing ability and benefited the
intramolecular charge transfer (ICT) effect, consequently leading to the
red-shift of emission peak. While 70 μM of Hg2+ ion was added, the
fluorescence intensity of the solution reached to maximum, and this solution
displayed an intense emission at 613 nm. Moreover, the dynamic light
scattering (DLS) data indicated the aggregation of this solution, that the
average size of generated nano-aggregates was around 227.9 nm (figure
S1a). In comparison, the average size of nanoparticles in aqueous solution of
2
+
probe TBTS without Hg ion was only around 8.2 nm, indicating that probe
TBTS was well dissolved in this aqueous mixture (figure S1b). As known
2
+
from the above results, the addition of Hg ion caused the aggregation of
probe TBTS in aqueous solution, consequently resulting in the intense red
emission.
1
1

Figure 1. The fluorescence spectra of probe TBTS (20 μM) in HEPES buffer
10 mM, pH =7.4, containing 3% DMSO) with different amounts of Hg2+
ion, excitation at 443 nm. Insert: the photos of the solutions without and
(
2
+
with Hg ion (70 μM) that irradiated at 365 nm light.
2
+
3
.3. Sensing sensitivity of probe TBTS to Hg ion
2
+
In order to assess the sensing sensitivity of probe TBTS toward Hg ion
in aqueous solution, the detection limit (DL) was calculated according to the
acquainted equation “DL = 3σ/k” (S/N=3). Gratifyingly, the fluorescence
intensity at 613 nm showed a great linear correlation with the concentrations
2+
of Hg ion from 0 μM to 60 μM (figure S2). The detection limit (DL) of
2
+
probe TBTS toward Hg ion was calculated to be 90 nM, which was much
lower than many other dithioacetal-based probes [22-24,29-31], further
2
+
illustrating the high sensitivity of probe TBTS toward Hg ion.
1
2

3
.4. Sensing selectivity and competitive experiments
Subsequently, the sensing selectivity of probe TBTS toward Hg ion in
HEPES buffer (10 mM, pH =7.4, with 3% DMSO) was studied. As known
2
+
2
+
from the fluorescence spectra, the addition of Hg ion (60 μM) into aqueous
solution of probe TBTS remarkably enhanced the fluorescence intensity,
2
+
2+
2+
2+
2+
2+
whereas other metallic ions (such as Ca , Cu , Pb , Cd , Zn , Mg ,
2
+
2+
3+
3+
3+
+
+
+
Co , Ni , Al , Cr , Fe , Ag , K and Na ions) caused the negligible or
even no enhancement (figure 2). Furthermore, the fluorescence difference of
these solutions could be clearly distinguished by naked-eyes.
Figure 2. The fluorescence spectra of probe TBTS (20 μM) in HEPES buffer
(10 mM, pH =7.4, containing 3% DMSO) with different metallic ions (60
μM), excitation at 443 nm. Insert: the photos of these solutions in spot plate
that irradiated under 365 nm light.
1
3

For further estimating the sensing selectivity, the fluorescence response of
2
+
probe TBTS toward Hg ion in competitive environments were studied. As
shown in figure 3, the addition of other competitive metallic ions (60 μM)
into the aqueous solution of probe TBTS (20 μM) caused the negligible
improvement of fluorescence intensity. Upon the sequential addition of Hg2+
ion (60 μM), the fluorescence intensity of these solutions was significantly
improved. The above results implied that probe TBTS was able to detect
2
+
Hg ion even in competitive environments.
Figure 3. The fluorescence intensity (I613 nm) of probe TBTS (20 μM) in
HEPES buffer (10 mM, pH =7.4, with 3% DMSO) upon the addition of
2
+
various cations (60 μM, blue bars) and sequential addition of Hg ion (60
μM, red bars), excitation at 443 nm.
3
.5. Time-dependent and pH-dependent experiments
The response time was an important index for evaluating the fluorescence
1
4

probe. In view of this, the time-dependent experiments of probe TBTS
toward Hg2+ ion was executed (figure S3a). As time elapsing, the
fluorescence intensity of probe TBTS (20 μM) in HEPES buffer (10 mM,
pH =7.4, with 3% DMSO) with Hg2+ ion (60 μM) was dramatically
enhanced, which reached to the maximum within 150 s, suggesting the faster
response toward Hg2+ ion than many other dithioacetal-based probes
[13,17,18,21,31]. Furthermore, the pH influence on the sensing behavior of
2
+
probe TBTS toward Hg was studied. As shown in figure S3b, the alkaline
conditions (pH ≥ 9) significantly quenched the fluorescence of probe TBTS
in aqueous solution with Hg2+ ion, which might be attributed to the
－
2+
generation of mercuric oxide from the reaction of OH ion with Hg ion,
2
+
thus preventing Hg ion to trigger the hydrolysis of dithioacetal group in
probe TBTS. Moreover, the fluorescence intensity of this aqueous solution
was also weakened in acid condition (pH ≤ 5), because the acid condition
was harmful for the emission of compound 1 in aqueous solution (figure S4).
+
The acid hydrogen ion (H ) might attach with nitrogen atom of
benzothiadiazole in compound 1, which enhanced its hydrophilicity and
hampered the aggregation in aqueous solution, consequently weakening the
fluorescence intensity. Even so, the fluorescence intensity of probe TBTS in
2
+
aqueous solution with Hg ion exhibited an obvious improvement within
1
5

pH range from 5 to 8, indicating that probe TBTS was able to detect Hg2+
ion in biological environment.
3
.6 Sensing mechanism
After that, the sensing mechanism of probe TBTS toward Hg ion was
2
+
studied. In order to expediently understand this sensing mechanism, the
product from the reaction mixture of probe TBTS with HgCl in
2
CH CN-H O was obtained, whose structure was analyzed by TLC plate and
3
2
1H NMR. In TLC plate, this product exhibited the similar polarity and
1
fluorescence property with compound 1 (figure S5). Moreover, its H NMR
spectra were essentially identical to compound 1, that the peaks of
dithioacetal signal at 5.43 ppm (1H, a single peak) and 3.26-3.52 ppm (4H, 4
single peaks) in probe TBTS were disappeared, while a new signal of
aldehyde proton over 10.00 ppm (1H, a single peak) in compound 1 was
found (figure 4). Obviously, the generated product from the reaction mixture
2
+
of probe TBTS with Hg ion was confirmed to be compound 1.
1
6

1
Figure 4. Partial H NMR spectra of compound 1, isolated product from
reaction of probe TBTS with HgCl , and probe TBTS in CDCl .
2
3
Additionally, the fluorescence properties of probe TBTS and compound 1
were studied in DMSO-HEPES buffer mixed solutions with different
volume fractions of HEPES buffer (f ). As known from figures S6, probe
H
TBTS and compound 1 showed the weak emission in pure DMSO. Upon the
increase of f , their fluorescence intensity was both slightly decrease.
H
Because the present HEPES buffer improved the solvent polarity, which
compelled these D-A type compounds to form the twisted charge-separated
conformation by intramolecular rotation, consequently weakening the
fluorescence intensity [32]. While f was up to 40%, the fluorescence
H
1
7

intensity of compound 1 in mixed solution was clearly improved. But for
probe TBTS, its fluorescence intensity was dramatically enhanced until f =
H
9
9%. The DLS results revealed the aggregation of these solutions, that the
average size of generated nano-particles in these solutions was at 335 nm
and 247 nm, respectively (figure S7). Comparing with compound 1, probe
TBTS was more hydrophilic due to the carboxyl groups, thus it only could
be aggregated in the aqueous solution with less DMSO. These results
implied that probe TBTS and compound 1 both possessed the AIE activities,
and the restricted intramolecular rotation in aggregate state was responsible
for the improvement of fluorescence intensity. Moreover, as known from the
fluorescence spectra of compound 1 in its AIE studies (figure S8a),
compound 1 almost displayed the same emission peak as that of probe
2
+
TBTS with Hg ion in DMSO-HEPES buffer (v/v, 3:97) mixture (figure
2
+
S8b). Which further certified that the mixture of probe TBTS with Hg ion
produced compound 1, whose aggregation resulted in the intense red
emission.
As known from the above results, the sensing mechanism of probe TBTS
2
+
toward Hg ion was illustrated in scheme 2. Probe TBTS was dissolved in
HEPES buffer (10 mM, pH =7.4, with 3% DMSO), that the intramolecular
2
+
rotation quenched the emission. As the treatment with Hg ion, probe
1
8

TBTS was hydrolyzed to be compound 1 with the serious hydrophobicity,
thus its aggregation brought the intense red emission via AIE mechanism.
2
+
Scheme 2. The sensing mechanism of probe TBTS toward Hg ion.
3
.7 Cytotoxicity assay and fluorescence imaging
Stimulating by the excellent fluorescence response of probe TBTS toward
2
+
Hg ion, the cell imaging experiments were further performed. At first, the
cytotoxicity of probe TBTS towards HeLa cells at different concentrations
(0-40 μM) were studied by the standard MTT assays (figure S9). The result
showed that the cellular viability maintained at nearly 90%, illustrating the
low cytotoxicity of probe TBTS to living HeLa cells. Afterwards, the cell
imaging experiments were carried out. In the control group, HeLa cells were
only incubated with probe TBTS (20 μM) for 1 h at 37 ºC, that no
fluorescence was observed in HeLa cells (figure 5b). Subsequently, the cells
2
+
were continuously incubated with Hg ion (60 μM) for another 40 min, the
red fluorescence could be distinctly observed in the intracellular region
(figure 5d). Moreover, the bright-field images (figure 5a and 5c) showed
1
9

that the cells were alive during the incubating period. Therefore, probe
TBTS displayed good cell membrane permeability, and it was promising to
2
+
monitor Hg ion in living cells.
Figure 5. Fluorescence microscopy images of HeLa cells: (a) and (b) were
bright field images and fluorescence images of HeLa cells incubated with
probe TBTS alone for 1 h, respectively; (c) and (d) were bright field image
and fluorescence image of HeLa cells with probe TBTS that further
2
+
incubated with Hg ion for another 40 min, respectively.
Conclusion
In conclusion, a novel AIE-based fluorescence probe TBTS toward Hg2+
ion with red emission has been explored. Probe TBTS was barely emissive
2
+
in HEPES buffer (with 3% DMSO). As the presence of Hg ion, the
dithioacetal moiety in probe TBTS was hydrolyzed to be aldehyde group,
2
0

consequently bringing about the intense red emission via AIE mechanism.
2
+
Probe TBTS displayed the high selectivity toward Hg ion, and its detection
limit (90 nM) was much lower than many other dithioacetal-based
fluorescence probes. Moreover, probe TBTS showed the low cytotoxicity to
2
+
HeLa cells, and it was successfully applied in cell imaging of Hg ion.
Acknowledgments. We sincerely acknowledge the school foundation of
Wuhan Textile University (183010) and the open foundation of Hubei Key
Laboratory of Biomass Fibers and Eco-dyeing＆Finishing (STRZ2017020)
for financial supports.
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A novel red-emitting fluorescent probe for the highly selective
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detection of Hg ion with AIE mechanism
a
a
a
a
b
Bing Liu , Jia Liu , Jiaopu He , Jinsheng Zhang* , Hongjun Zhou* , and
a
Chao Gao*
a
Hubei Key Laboratory of Biomass Fibers and Eco-dyeing & Finishing,
Wuhan Textile University, Wuhan 430073, Hubei Province, China.
Shanxi Zhendong Pharmaceutical Co. Ltd, Changzhi 046000, Shanxi
Province, China.
b
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*
E-mail: wawj74@126.com; zhou73262811@126.com; chgao@wtu.edu.cn.
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Graphic Abstract:
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A novel AIE-based fluorescence probe TBTS for the detection of Hg ion
in aqueous solution and living cells has been reported.
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