An AIE and PET fluorescent probe for effective Zn(ii) detection and imaging in living cells

Article abstract of DOI:10.1039/d0nj03667f

A sensitive fluorescent probe L for Zn2+ with aggregation-induced emission (AIE) properties has been synthesized. The probe L exhibits specific selectivity and high sensitivity towards Zn2+ in EtOH/H2O (4?:?1, v/v, HEPES = 20 mM, pH = 7.20) solvents, with the detection limit of 2.1 × 10-8 M. The potential sensing mechanism was analyzed via Job plot, MS and NMR titration, and the complexation ratio of the probe L and Zn2+ was 1?:?1. Furthermore, the as-prepared probe shows high sensitivity towards Zn2+ in a pH range of 4.8-12.0, combined with its wonderful cell permeability which ensures that it can work under physiological conditions and can image Zn2+ in living cells.

Full text of DOI:10.1039/d0nj03667f

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Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
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DOI: 10.1039/D0NJ03667F
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An AIE and PET fluorescent probe for effective Zn(Ⅱ) detection
and imaging in living cells
Received 00th January 20xx,
Accepted 00th January 20xx
Jin Wangac#, Lu Linxiab#, Chun Wangb, Minmin Wangac *, Jianfeng Jua, Jinli Zhua* and Tongming
Suna*
DOI: 10.1039/x0xx00000x
A sensitive fluorescent probe L for Zn2+ with aggregation-induced emission (AIE) properties has been synthesized. Probe L
exhibits specific selectivity and high sensitivity to Zn2+ in EtOH/H2O (4:1, v/v, HEPES = 20 mM, pH = 7.20) solvents, and the
detection limit is 2.1×10-8 M. The potential sensing mechanism was analyzed by Job’s plot, MS and NMR titration, the
complexation ratio of probe L and Zn2+ is 1:1. Furthermore, the as-prepared probe shows high sensitivity to Zn2+ in the pH
range of 4.8-12.0, combined with its wonderful cell permeability which ensures its work under physiological conditions and
can image Zn2+ in living cells.
highly hydrophilic substrates. To overcome this limitation, PET
molecules can be combined onto AIE fluorescent26.
1. Introduction
Herein, a new fluorescent probe L was designed and
Zn(II) is essential for biological processes ranging from gene
transcription, enzyme synthesis, cell growth and
division1,2,3,4,5,6. Malfunctioning Zn(II) homeostasis can result in
various diseases in the human body such as Alzheimer’s
disease, cancers and infantile diarrhea7,8,9,10,11,12. Therefore,
simple and accurate quantification and imaging of Zn2+ are
vital for early disease diagnosis13,14,15. In recent years, small
molecule fluorescent probe has been used to in sensitive
detection Zn2+ and imaging in living cells. However, previous
reported molecules are most often suitable only for organic
solvents or mixed aqueous in a narrow pH range, which limited
the utilization in practical samples16,17,18,19,20. Therefore, a
more sensitive probe with high response in water solution,
wide pH response, highly sensitive and selective is still
required to be developed in practical utilization.
Tetrastyrene show weak fluorescent in solutions, but emit
strongly in their aggregate states which has received much
attention. Such an abnormal property has been referred to as
aggregation-induced luminescence (AIE). AIE effect has been
exploited in highly selective and stable fluorescence sensors
for biological and chemical analytes, including metal
ions21,22,23,24,25. However, first generation AIE-probes require
high water solubility to ensure the low background signal,
which limits the AIE fluorescent to be only conjugated with
synthesized with tetrastyrene as the fluorescent group. As
expected, L has significant AIE phenomenon, and has good
sensitivity and selectivity for Zn2+. Through ESI-MS, Job, NMR
and IR mapping analysis, a potential light induced electron
transfer mechanism was proposed. More importantly, the L
has a high sensitivity to Zn2+ in the pH range of 4.8-12.0, and
the as-prepared probe with good biological compatibility could
be used to image Zn2+ in living cells.
2. Experimental Section
2.1 Materials and apparatus
All chemicals were used as received and without any further
purification. All fluorescence performance tests were
performed on the F-7000 fluorescence spectrometer (Japan).
1
The H NMR and 13C NMR spectra were tested on a Bruker
400MHz NMR instrument with TMS as an internal standard.
FT-IR spectra testing was done on a Thermo Fischer Nicolet
IS50 FT-IR instrument using KBr plates. High-resolution mass
spectroscopy (HRMS) was measured on a Bruker Thermo
Scientific Q Exactive mass spectrometer. Fluorescence images
of cells were analyzed by inverted microscope (Leica
DMI6000B, Germany).
2.2 Synthesis
The synthesis of probe L could be achieved according to the
previous reported method17,27 with a slight modification
(Scheme 1). Through the condensation of 4-hydroxy-5-
aldehyde tetrastyrene and 2-hydrazine benzothiazole, a yellow
solid was obtained with a yield of 57.1%.
a. School of Chemistry and Chemical Engineering, Nantong University, Nantong
226019, P.R. China.
b. School of Textiles and Clothing, Nantong University, Nantong 226019, P. R. China.
c. Nantong Key Laboratory of Intelligent and New Energy Materials.
Corresponding authors, Email: mmwang0528@ntu.edu.cn; jinlizhu@ntu.edu.cn;
stm7314@ntu.edu.cn
2.2.1 Synthesis of compound 1:
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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The compound 1 was synthesis according to the previous 1H, J=1.84 Hz), 7.17-7.07 (m, 10H), 7.00-6.95 (m,V6ieHw)A,rt6ic.le7O4n(lidne,
reported work27. Typically, zinc powder (1.713 g, 0.026 mol) 1H, J=8.56 Hz). 13C NMR (100 MHz, DMSO-d6): 144.38, 131.18,
DOI: 10.1039/D0NJ03667F
and 20 mL anhydrous THF added to the reaction flask under 131.12, 131.07, 128.67, 128.50, 128.40, 128.29, 126.02.
nitrogen protection and cool temperature of the mixture to -5- (Figure S3, Figure S4).
2.2.3 Synthesis of compound 3:
0 °C. Then, titanium tetrachloride (1.3 mL, 0.012 mol) slowly
added in the above solution with stirring for 30 min. And then,
the reaction maintained 2.5 h at 85°C with string. The
temperature again cool to -5-0 °C, and pyridine (0.5 mL, 0.006
mol) dropwised with stirring for 10 min. Next, 5 mL mixture
THF solution of benzophenone (0.5 g, 2.75 mmol) and 4-
hydroxybenzophenone (0.6503 g, 3.27 mmol) slowly added to
the above established system. Then, the mixture solution heat
up to 85° C and refluxed for 12 h. The reaction was quenched
by the addition of 10% aqueous potassium carbonate. The
solvent was evaporated and the crude product was further
purified by chromatography on silica using petroleum ether
eluent to afford a white solid 1. Yield: 0.234 g (24.4%). IR (KBr,
v in cm-1): 3514, 3047, 1609, 1492, 1424, 1258, 1155, 749, 694.
1H NMR (400 MHz, DMSO-d6): 9.35 (S, 1H), 7.17-7.06 (m, 9H),
6.97-6.92 (m, 6H), 6,76 (d, 2H, J=8.56 Hz), 6.50 (d, 2H, J=8.56
Hz). 13C NMR (100 MHz, DMSO-d6): 156.42, 144.19, 144.05,
132.38, 131.20, 131.17, 131.13, 128,29, 128.22, 128.17,
127.86, 126.72, 115.13. (Figure S1, Figure S2).
2-Aminobenzothiazole (0.35 g, 2.33 mmol) was dissolved in
15 mL of ethylene glycol, and 0.05 mL of concentrated sulfuric
acid was dropwised in the solution. Then, hydrazine hydrate
(0.88 g, 0.0176 mmol) added into the solution at 70 °C and
refluxed for 3 h. Then cooled the mixture solution to room
temperature and filtered. The filter cake recrystallized from
EtOH and finally obtained a white solid 3. Yield: 0.2031 g
(53.4%). IR (KBr, v in cm-1): 3319, 2872, 1650, 1557, 1449,
1281, 1128, 985, 757, 646. 1H NMR (400 MHz, DMSO-d6): 9.00
(s, 1H), 7.67 (dd, 1H, J1=7.80 Hz, J2 = 0.84 Hz), 7.31 (dd, 1H, J1=
7.94 Hz, J2= 0.48 Hz), 7.20 (td, 1H, J1= 7.26 Hz, J2 = 1.28 Hz),
6.98 (td, 1H, J1= 1.16 Hz, J2 = 7.58 Hz), 5.02 (s, 2H). 13C NMR
(100 MHz, DMSO-d6): 174.35, 153.93, 130.68, 125.59, 121.39,
120.41, 118.25. (Figure S5, Figure S6).
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2.2.4 Synthesis of probe L:
Compound 2 (0.17 g, 0.45 mmol) was first dissolved in EtOH
(15 mL), and compound 3 was dissolved in EtOH (10 mL) and
slowly dropwised in the above solution under nitrogen
atmosphere. The resulting mixture heated under reflux at 85°C
for 13 h. After cooling to room temperature, a white solid
precipitated. After suction filtration, the filter cake
recrystallized from EtOH to obtain a white solid L. Yield: 0.135
g (57.1%). IR (KBr, v in cm-1): 3431, 1616, 1475, 1424, 1272,
2.2.2 Synthesis of compound 2:
Typically, Compound 1 (0.1 g, 0.287 mmol) was added in a
reaction flask, and 0.8 mL of trifluoroacetic acid and 0.8 mL of
acetic acid were dropwised in the flask at 0 °C. Subsequently,
hexamethylenetetramine (0.12 g, 0.857 mmol) was added in
portions, then, the above mixture solution slowly heated up
and reflux for 3 h. The reaction was quenched by the addition
of water (20 mL) and extracted with DCM (3 × 50 mL). The
organic phase was dried by anhydrous sodium sulfate. The
solvent was evaporated and the crude product was further
purified by chromatography on silica using petroleum etheas
eluent to afford a yellow solid 2. Yield: 0.051 g (47.2%). IR (KBr,
1
1102, 752, 694. H NMR (400 MHz, DMSO-d6): 12.14 (d, 1H),
10.20 (d, 1H), 8.28 (d, 1H), 7.80 (d, 1H), 7.34 (t, 2H, J = 7.8),
7.26 - 7.14 (m, 11H), 7.09 - 7.02 (m, 6H), 6.88 (d, 1H, J = 8.6),
6.74 (d, 1H, J = 8.6). 13C NMR (100 MHz, DMSO-d6): 143.91,
142.81, 143.57, 140.43, 134.85, 133.79, 131.20, 131.13,
131.07, 128.44, 128.32, 128.29, 127.06, 127.92, 126.87,
122.03, 116.12. HRMS: Calced for [L - H]-, 522.17; Found
522.165. (Figure S7, Figure S8, Figure S9).
1
v in cm-1): 3424, 1665, 1334, 1190, 1109, 949, 831, 694. H
NMR (400 MHz, DMSO-d6): 10.69 (S, 1H), 10.10 (S,1H), 7.22 (d,
Scheme 1. Synthesis route of the probe L. a) zinc powder, titanium tetrachloride, pyridine, 85 °C; b) urotropine, acetic acid,
trifluoroacetate, 85 °C; c) ethylene glycol, H2SO4, hydrazine, reflux; d) EtOH, reflux.
2.3 General analytical procedures
fluorescence data were measured at excitation wavelength of
416 nm.
2.4 Calculation of quantum yield
The probe L (1 mM) stock solution was prepared with pure
DMSO, and 10 mM metal ion stock solution was prepared with
deionized water. The final test solution is composed of 10 μM
The fluorescence quantum yields of compounds were
L, 20 mM HEPES buffer (pH = 7.40) and proper amount of Zn2+. measured using quinine sulfate as the standard (Φ=0.53, 0.1 M
The solution is mixed in EtOH/H2O (4:1, v/v) solution. All H2SO4) and calculated according to the following formula:
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media. As show in Figure 1a, with the increasViniegw AortficlewOantliener
fraction, the fluorescence emission intensity at 550 nm
I
unk
A
std
n
unk
nstd )2× ( std
) × (

unk
DOI: 10.1039/D0NJ03667F
I
std
Aunk
Φunk = Φstd × (
) × (
)
(1)
increased continuously. The enhanced emission were induced
by the aggregation of probe L in water and the fluorescence
intensity of probe L increased 50-fold from 0 % to 100 %. In
low water fractions, the smoothly photoinduced electron
transfer between tetrastyrene and N donor suppressed its
emission. However, with the increasing fraction of water, the
rotation of C = C double bond was limited to some extent,
Φunk, Iunk, Aunk, nunk and λunk are fluorescence quantum yield,
integrated emission intensity, absorbance of excitation
wavelength, refractive index of corresponding solution and
excitation wavelength of the substance to be tested,
respectively. Φstd, Istd, Astd, nstd and λstd are denote as
fluorescence quantum yield, integrated emission intensity,
absorbance of excitation wavelength, refractive index of
corresponding solution and excitation wavelength of
substances with as standards, respectively.
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which leads to the enhancement of fluorescence intensity28,29
.
2.5 Cell culture and fluorescence imaging
Hela cells were seeded on 35 mm glass-bottomed dishes
(NEST) and incubated in RPMI-1640 in an incubator (37 °C, 5%
CO2 and 20% O2) for 24 h. The cells were washed slightly 3
times with fresh RPMI-1640. Subsequently, the cells with
different concentrations of the L (0-10 μM) were incubated in
96 well-plates for 24 h. After washing with fresh RPMI-1640,
the cells pre-treated with sensor L were further incubated with
fresh RPMI-1640 containing 100 mM Zn(NO3)2•6H2O for 15
min. Cells were then analysed by inverted microscope (Leica
DMI6000B, Germany).
3. Results and Discussion
3.1 AIE characteristics of probe L
Figure 1. (a) Fluorescence spectra of the probe L (10 μM) in EtOH/H2O
solution with various water fraction; (b) the emission colour changes of
probe L in EtOH and increasing water fraction under UV light (365 nm).
3.2 Fluorescent sensing of Zn2+
To test the AIE characteristic of probe L, the effect of water
volume fraction on emission was evaluated in EtOH/H2O mixed
Figure 2. (a) Fluorescence spectra of probe L (10 μM) in the absence and presence of Zn2+, Mg2+, Ba2+, Na+, Ni+, Hg2+, Al3+, Mn2+, Co2+, Cu2+, K+, Li+, Fe2+, Ca2+
Fe3+. (b) Fluorescence intensity of L (10 μM) in the presence of selected metal ions (100 μM). (c) Fluorescence spectra of L (10 μM) in the presence of
,
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increasing concentrations of Zn2+ and (d) its corresponding limit of detection of L for Zn2+. All fluorescence analysis was studied in EtOH/H2O (4:1, v/v,
View Article Online
DOI: 10.1039/D0NJ03667F
HEPES = 20 mM, pH = 7.20).
The fluorescence responses of the L towards various metal Figure 2b, other coexisting metal ions have little effect on the
ions were carried out in EtOH/H2O (4:1, v/v) at pH = 7.20. As detection results, but the addition of Cu2+, which may induced
shown in Figure 2a, under the excitation of 416 nm, probe L by the paramagnetic effect of Cu2+ 30. Therefore, all above
showed a weak fluorescence intensity, with the addition of results demonstrate that the prepared probe L has specific
Zn2+, the fluorescence intensity of the complex changed selectivity for Zn2+. Further we studied the change of the
significantly, and the fluorescence intensity at 484 nm fluorescence spectrum of probe L with the increase of Zn2+
increased by 30-fold. Meanwhile, the fluorescence quantum concentration. As shown in Figure 2c, the fluorescence
yield increased from 0.008 to 0.034. We further measured the intensity increases with the increase of Zn2+ concentration,
UV-vis spectra of the probe L and L-Zn2+. As shown in Figure which is caused by the chelation of probe L with Zn2+ 3,5,12. The
S10, the pure probe L show no absorption at 420 nm, while L- response of probe L to Zn2+ show a good linear relationship (R2
Zn2+ show stronger absorption intensity at 420nm. These = 0.98) in the range of 0-10 μM and the detection limit was
changes further indicate that probe L chelates with Zn2+. Upon calculated to be 2.1×10-8 M (Figure 2d). Therefore, the probe L
addition of various anions, only Zn2+ ions was able to trigger can detect Zn2+ in the environment and the biological system.
distinct fluorescence change. Meanwhile, the emission color The photostability properties of L-Zn2+ were evaluated by time-
change for probe L with Zn2+ and other ions were clearly course fluorescence measurements in EtOH/H2O (4:1, v/v,
shown in in Figure S11, under the excitation of a handheld HEPES = 20 mM, pH = 7.20). As shown in the Figure S12, the
ultraviolet lamp at 365 nm, only the L/Zn2+ complex has fluorescence intensity of L-Zn2+ shows slight changes in a
significant fluorescence. We further investigated the selectivity continues 20 min irradiation which proves that the L-Zn2+
of probe L to Zn2+ in the presence of other metals. As shown in complex have high photostability.
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3.3 Bonding mode studies
Figure 3. (a) A Job’s plot showing 1:1 ratio stoichiometry of the complex between L and Zn2+ in EtOH/H2O (4:1, v/v, HEPES = 20 mM, pH = 7.20). (b) ESI-
Mass spectrum of L + Zn2+ -H+ complex. (c) Association constant of L for Zn2+ through fluorescent intensity, assuming 1: 1 stoichiometry for association
between L and Zn2+ in methanol, λex = 416 nm. (d) The possible sensing mechanism of probe L to Zn2+
.
T
he Job's plot experiment is used to further verify the were kept constant (10 μM), and the molar fraction of L/L-Zn2+
combined stoichiometry between the analytical probe L and in the solution changed continuously. As shown in Figure 3a,
Zn2+. During the experiment, the concentrations of L and Zn2+ there is a turning point at a mole fraction of 0.5, indicating that
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the probe L forms a 1: 1 complex with Zn2+. In addition, a 416 nm, λem= 484 nm.
characteristic peak appeared at m/z = 588.0, which can be 3.5 Fluorescence Bio-imaging
attributed to [L+ Zn2+ -H+]+ in the ESI-MS spectrum of the L-Zn2+
View Article Online
DOI: 10.1039/D0NJ03667F
The cytotoxicity of as-prepared probe L was carefully
checked by the standard 3-(4,5-dimethyl-2-thiazolyl)-2,5
diphenyltetra-zolium bromide (MTT) assay 32,33,34,35,36. As
illustrate in Figure 5, the cell viability of all four cell lines tested
are not obviously affected the cell viability was greater than
80% after 12 h incubation with probe L at various
concentrations (0-100 μM), indicating that the probe L is safe
for cells (Figure 5a&b). Therefore, we applied the as-prepared
probe L for further fluoresces sensing Zn2+. As shown in Figure
5c, Hela cells containing only 10 μM of pure probe L were
incubated at 37 °C for 24 h, and there was no obvious
fluorescence. After adding RPMI-1640 solution containing 100
μM Zn2+ and incubation for 15 min, a clear blue fluorescence
was observed under the microscope. The results show that
probe L has good membrane permeability and can be used for
imaging of Zn2+ in living cells (Figure 5d)
complex, further confirming the ratio is 1: 1 complexation
between L-Zn2+ (Figure 3b). Based on the 1: 1 complex, we
calculated the binding constant of L-Zn2+ according to the
Benesi-Hildebrand equation, and determined that the binding
constant of Zn2+ was 5.6×104 M-1 (Figure 3c). In order to better
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1
understand the binding sites of probe L and Zn2+, H NMR
titration experiments were performed in DMSO-d6. As shown
1
in Figure S13a (1), the H NMR spectrum of the probe L is
shown, the phenol-OH (Ha) in the probe L is observed at 12.09
ppm, and the Ha peak at 12.09 ppm disappears after adding 3
eq of Zn2+, indicating that Participated in the complexation
reaction between probe L and Zn2+. Meanwhile, the peak of
imine-NH (Hb) shifted from 12.24 ppm to 12.40 ppm. The
single-peak split of -CH-N- (Hc) was split into a double peak,
and the integrated area was basically unchanged. These results
confirm that probe L can chelate Zn2+ through the coordination
of oxygen atom of phenolic hydroxyl, nitrogen atom of imine
and nitrogen atom of thiazole ring. The hydrogen spectrum of
Zn2+ added with 2eq and Zn2+ added with 1eq remained almost
unchanged, which further proved that a 1: 1 complex was
formed between the probe L and Zn2+ (Figure S13b).
Therefore, a possible sensing mechanism was proposed as
show in Figure 3d, with the addition of Zn2+, the chelate
formed between the probe L and Zn2+, meanwhile, due to the
interruption of the PET phenomenon on pure probe L,
restoring the fluorescence emission enhancing of the probe L.
3.4 pH effect
In addition to specific selectivity and high-efficiency
sensitivity, exploring the sensing capabilities of probes under
different pH conditions is of great significance for the practical
application of probes. As shown in Figure 4, pure probes L
have only weak fluorescence intensity in the pH range of 2.0-
12.0. However, L+Zn2+ show high fluorescence responses in the
pH range of 4.8-12.0 which applies to typical mammalian cells
which intracellular pH value vary from 4.7 in lysosome to 8.0 in
mitochondria 31.
Figure 5. (a) Cell viability of Hela cells incubated with various concentration (0 -
100 μM) of the sensor L determined by MTT assay. (b) Optical transmission
images of HeLa cells incubated with probe L. (c) Fluorescent image of Hela cells
incubated with pure L (10 μM) and (d) L+Zn2+ (100 μM). Conclusions
4. Conclusions
In summary, a sensitive fluorescent probe L was synthesized a
single platform with AIE. The as-prepared probe L exhibits a
large pH response range towards Zn2+ and a remarkable
detection limit of 2.1×10-8 M. In the addition, probe L has good
membrane permeability and low cytotoxicity and can be
applied to the living cells imaging of the endogenous and
exogenous Zn2+ successfully. Overall, this new probe will also
show a foundation for subsequent research on disease
detection.
Figure 4. Effect of pH value on fluorescence intensity of the L (10 μM) and L
with 10 eq of Zn2+ in EtOH/H2O (4:1, v/v, HEPES = 20 mM, pH = 7.20). λex=
Conflicts of interest
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28.
29.
30.
M. Zhang, L. Gong, C. Sun, W. Li, Z. Chang, D. Qi, Spectrochim. Acta,
View Article Online
There are no conflicts to declare.
Part A, 2019, 214, 7-13.
DOI: 10.1039/D0NJ03667F
Y.Q. Ma, X.J. Sun, M.Q. Li, S. Zeng, Z.Y. Xing, J.L. Li, Chem. Pap., 2019,
73, 1469-1479.
Acknowledgements
L. Dong, G.R. Chen, X.P. He, S. Vidal, Org. Biomol. Chem., 2019, 17,
9251-9256.
This work was supported by National Natural Science
Foundation of China (No. 21776140, 21908114), and Natural
Science Foundation of Jiangsu Province (BK20190918) and
Natural Science Research Projects of Universities in Jiangsu
Province (19KJB430030). We are also very grateful to the
Nantong University Analytical Testing Center for its support for
testing.
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