A highly sensitive and selective chemosensors for detection of Zn2+ and its application in live cell imaging

Article abstract of DOI:10.1016/j.ica.2020.119675

A novel salen derivative, named as 6,6′-((1E,1′E)-(1,2-phenylenebis(azaneylylidene))bis(methaneylylidene))bis(4-(4-iodobutyl)-2-methylphenol) (PA), was obtained for the detection of Zn²⁺. PA exhibited highly sensitive and selective via a chelation mechanism, which was promoted by Zn²⁺, accompanied by enhanced fluorescence. After adding various metal ions in the PA solution, only Zn²⁺ caused PA fluorescence enhancement. Other cations (such as Ag⁺, Al³⁺, Ba²⁺, Ca²⁺, Cd²⁺, Cr³⁺, Cu²⁺, Fe³⁺, Hg²⁺, K⁺, La³⁺, Li⁺, Mg²⁺, Na⁺, Ni²⁺ and Pb²⁺) cannot induce similar response. The binding stoichiometry and detection limit was 1:1 and 16.3 nM, respectively, which is superior to the reported Zn²⁺ fluorescent probes based on salen derivatives. The binding process was reasonably speculated by fluorescence measurements, infrared spectroscopy, ¹H NMR titrations, FT-IR, mass spectroscopy (MS) and density functional theory (DFT) calculation. In addition, PA can be used for Zn²⁺ fluorescence imaging in HeLa cells. This work indicated that this probe would be of great application prospect in medical diagnosis.

Full text of DOI:10.1016/j.ica.2020.119675

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A highly sensitive and selective chemosensors for detection of Zn2+ and its
application in live cell imaging
Yang Hu, LongChao Du, Jie Yang
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https://doi.org/10.1016/j.ica.2020.119675
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13 April 2020
Please cite this article as: Y. Hu, L. Du, J. Yang, A highly sensitive and selective chemosensors for detection of
Zn2+ and its application in live cell imaging, Inorganica Chimica Acta (2020), doi: https://doi.org/10.1016/j.ica.
2020.119675
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A highly sensitive and selective chemosensors for detection of Zn2+
and its application in live cell imaging
Yang Hu, LongChao Du*, Jie Yang
School of Chemistry and Chemical Engineering & the Key Laboratory of Environment-
friendly Polymer Materials of Anhui Province, Anhui University, Hefei, PRC
A
novel
salen
derivative,
named
as
6,6'-((1E,1'E)-(1,2-
phenylenebis(azaneylylidene))bis(methaneylylidene))bis(4-(4-iodobutyl)-2-
methylphenol) (PA), was obtained for the detection of Zn2+. PA exhibited highly
sensitive and selective via a chelation mechanism, which was promoted by Zn2+,
accompanied by enhanced fluorescence. After adding various metal ions in the PA
solution, only Zn2+ caused PA fluorescence enhancement. Other cations (such as Ag+,
Al3+, Ba2+, Ca2+, Cd2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, La3+, Li+, Mg2+, Na+, Ni2+ and Pb2+)
cannot induce similar response. The binding stoichiometry and detection limit was 1:1
and 16.3 nM, respectively, which is superior to the reported Zn2+ fluorescent probes
based on salen derivatives. The binding process was reasonably speculated by
1
fluorescence measurements, infrared spectroscopy, H-NMR titrations, FT-IR, mass
spectroscopy (MS) and density functional theory (DFT) calculation. In addition, PA
can be used for Zn2+ fluorescence imaging in HeLa cells. This work indicated that this
probe would be of great application prospect in medical diagnosis.
Keywords: Fluorescence enhancement; Salen base; Zinc ion; Cell imaging
1. INTRODUCTION
School of Chemistry and Chemical Engineering & the Key Laboratory of Environment-friendly Polymer Materials of Anhui Province,
Anhui University, Hefei, PRC
E-mail: dulongchao@sina.com

Metal ions are widely present in tissue cells and body fluids, affecting human
physiological and pathological health all the time.[1, 2] In general, proper levels of
metal ions can effectively maintain biological processes. Once the ion is excessive or
missing, it will cause homeostasis imbalance, leading to a series of serious diseases.[3-5]
Among metal ions, zinc ions are abundantly present in living organisms due to the
existence of various coordination characteristics.[6] The content of zinc in the human
body is second only to iron, and it is the second most transition metal element. It is
widely present in human cells and plays an important role in cell metabolism, gene
expression, body immunity, and nerve transmission.[7-11] Although the metal proteins
of Zn2+ ions are tightly bound, a few Zn2+ are still free in the cell. 5-20% of Zn2+ is
accumulated in presynaptic cells in the brain, and many free Zn2+ are also found in cells
of the hippocampus.[12] Zn2+ controls the excitability of the brain and displays a pivotal
role in synaptic plasticity.[13] Meanwhile, Changes in zinc concentration often indicate
the occurrence of certain diseases. For example, too high or too low zinc concentration
may lead to physiological disorders such as enzyme activity disorders, gene expression
errors, apoptosis, and blocked neurotransmission.[14, 15] Therefore, designing a
chemical sensor that can sensitively and efficiently detect Zn2+ is a very crucial subject
in the field of neurobiology.[16, 17]
Because they show a pivotal role in diverse biological systems, the detection of
metal ions is particularly important in the fields of biomedicine and analysis.[18-21]
Over the past few decades, several analytical tools have been used to analyze Zn2+, such
as ICP-AES, AAS, and instrumental neutron activation analysis.[22-25] However, the
cost of the instrument is high and sample preparation is cumbersome.[26] The
fluorescent probe technology, which has the advantages of low cost, fast response time,
simple operation, and low detection limit, has become a research method for
2

quantitative and qualitative analysis of ions and molecules in biological cells. More
importantly, Zn2+ can be detected while maintaining cell viability. These advantages
have prompted researchers to develop new chemical sensors and apply them to the
fields of chemistry, biochemistry, and cell imaging.[27-32] Therefore, chemical sensors
such as acetaminoquinoline[33], iminocoumarin[34] and tetrazole[35] have been
developed to detect Zn2+ ions. However, research on Salen-based chemical sensors has
not received sufficient attention. Salen ligands have excellent photophysical properties.
Moreover, these salen ligand not only comprises N2O2 coordination pocket, which can
easily accommodate various metal ions, but also can easily introduce various
substituents into the aromatic ring to adjust the selectivity and sensitivity of the
fluorescent chemical sensor.
Here, we designed a novel Salen-based zinc ion sensor PA (Scheme 1). Compound
PA could detect Zn2+ by a significant fluorescence enhancement and their biological
imaging in Hela cells was studied. They were expected to be applied to the qualitative
analysis and quantitative detection of metal ions in the environment or in biomedicine.
3

Scheme 1. Synthsis of PA.
2. EXPERIMENTAL
2.1. Materials and instrumentation
Reactions were carried out under inert atmosphere in Schlenk glassware or in a
purified nitrogen-filled drybox. Except ethanol was distilled using sodium, solvents
(THF, CH2Cl2, CH3CN,) were dried using a purification system composed of calcium
hydride. N, N-dimethylformamide (DMF) was HPLC grade and had no fluorescent
impurities. The solutions of various metal ions were prepared from nitrate salts.
Deionized water was used in the experiment. General chemical reagent were purchased
from Shanghai Macklin Biochemical Co, Aladdin Industrial Co, or Shanghai Richjoint
Chemical Reagents Co., and used with as received.
The samples were made into KBr tablets, and the Fourier transform infrared (FTIR)
1
spectra were collected on a Vertex80. H-NMR spectra were recorded on a NMR
spectrometer (AVANCEII, 400MHz) at room temperature. Samples were dissolved in
CDCl3 or CD3SOCD3 with tetramethylsilane as an internal standard. All spectral
characterizations were performed in HPLC-grade solvents in a 10 mm quartz cell at a
constant temperature of 20°C. UV-vis spectra were carried out in a Shimadzu UV-3600
spectrophotometer. The fluorescence emission spectra were obtained on a Perkin Elmer
LS-55 FL spectrophotometer. The mass spectra were obtained on a Bruker Autoflex III
time of flight mass spectrometer.
2.2. Synthesis
4

The synthetic route to PA was illustrated in Scheme 1. Compound 1-4 were
prepared according to literatures reported previously.[36] Detailed synthetic procedures
were described below.
Synthesis of PA: Compound 4 (1.04g, 3.27mmol) was dissolved in the ethanol in
a three-neck round flask under a N2 atmosphere. The reaction mixture was stirred at 80℃
for 8 hours. After completion of reaction, the solution was placed in the ice bath for 2h.
The product was a yellow solid 0.43g 43%). IR (KBr) (Fig. S5): 3180cm-1(Ar-OH),
1618cm-1(C=N). 1H-NMR (400MHz, DMSO) (Fig. S6): δ=13.07(s, 2H, Ar-OH), 8.86
(s, 2H, C=N), 7.48-7.37(m, 4H, Ar-H), 7.29(s, 2H, Ar-H), 7.17(s, 2H, Ar-H), 3.29-
3.25(m, 4H, CH2I), 2.58(m, 4H, Ar-CH2), 2.17(s, 6H, Ar-CH3), 1.85-1.57 (m, 8H,
CH2CH2). 13C-NMR (400MHz, CDCl3) (Fig. S7): 164.41, 156.01, 144.17, 136.10,
131.46, 127.99, 126.56, 121.92, 121.05, 117.03, 34.99, 32.10, 29.22, 15.34, 6.96. ESI-
MS m/z (Fig. S8): [PA+H]+, 709.87.
2.3. Preparation of working solutions and determination of UV–Vis and fluorescence spectroscopy
The sensor PA (3.54 mg, 5.0×10-3 mmol) was dissolved in 5mL of DMF to prepare
a 1.0×10-3 mol/L stock solution. Take 1 mL from the stock solution and add to a 100
mL volumetric flask. Use a pipette to take 1 mL of the stock solution into a 100 mL
volumetric flask and prepare a measurement solution with a final concentration of 1×10-
5
mol/L. The stock solutions (1.0×10-3 mol/L) of various metal ions (Ag+, Al3+, Ba2+,
Ca2+, Cd2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, La3+, Li+, Mg2+, Na+, Ni2+, Pb2+ and Zn2+) were
prepared in deionized water with corresponding nitrates. In the spectrum measurement
experiment, 3 mL (1×10-5 mol/L) of the working solution was added to a quartz optical
5

cell with an optical path of 1cm, and then an equal amount of a metal ion solution (3
μL) was added. The fluorescence spectra of the samples were recorded at an excitation
wavelength of 328 nm. Both the excitation and emission slit widths were 20nm.
3. RESULTS AND DISSCUSSION
3.1. Synthesis
The synthesis of salen bases probe (PA) is shown in Scheme 1. First, using Al2O3
as a catalyst, N2 was introduced under low temperature conditions, and acylation of o-
cresol and 4-chlorobutyryl chloride occurs in CH2Cl2 to form compound 1 (Fig. S1).
Subsequently, compound 1 and triethylsilane reacted in trifluoroacetic acid at room
temperature. Si-H bond breaks, and H attacks C=O in compound 1 to form compound
2 (Fig. S2). In the next step, compound 2 was introduced into the aldehyde group by
using acetonitrile as a solvent and reacting with paraformaldehyde and triethylamine
under a N2 atmosphere to obtain compound 3 (Fig. S3). The chloro group of compound
3 was substituted with I- in a refluxing solvent of CH3CN to get compound 4 (Fig. S4).
The condensation reaction of compound 4 with amine compounds was a crucial step to
synthesize the desired product. The intermediates compound 1-4 and final product PA
1
were characterized with H-NMR, FT-IR and ESI-MS spectra. The formation of PA
was confirmed by the appearance of a characteristic N=CH band at about 1618 cm-1 in
its FTIR spectrum. The 1H-NMR spectrum of PA exhibited imino proton (-CH=N-) at
8.86 ppm, which confirmed the formation of PA.
3.2. Spectral studies
The selectivity to various metal cation of PA was investigated by UV-vis and
6

fluorescence spectroscopy. As shown in Figure S9, absorption spectra were measured
upon addition of various metal cations (Ag+, Al3+, Ba2+, Ca2+, Cd2+, Cr3+, Cu2+, Fe3+,
Hg2+, K+, La3+, Li+, Mg2+, Na+, Ni2+, Pb2+ and Zn2+). PA shows weaker absorption peaks
at 278nm and 328nm. It can be attributed to the intermolecular charge transfer (ICT) of
long conjugated molecules and the C=N group issued transition of π-π*, respectively.
Upon the addition of Zn2+ to the solution of PA(10μmol), the absorption spectrum of
the mixed solution changed greatly, and the maximum absorption wavelength was red-
shifted from 328nm to 419nm. The cause of this phenomenon may occur on the ligand-
to-metal charge transfer (LMCT) between PA and Zn2+ ions. The fluorescence
spectrum of PA (10.0 µM) and PA upon addition of various metal ions were recorded
at the excitation wavelength of 328 nm in DMF. As shown in Figure 1, the addition of
Zn2+ greatly stimulated the fluorescence of PA, and the fluorescence intensity was
increased 14 times compared to the original. Accompanying the blue shift phenomenon,
the emission peak shifted from 484 nm to 440 nm. At the same time, this phenomenon
was detected with the naked eye under a 365 nm UV lamp. We assumed that a
fluorescent complex was formed between PA and Zn2+. Since the complex PA+Zn2+
could inhibit both the photoinduced electron transfer (PET) processes and the C=N
isomerization (rotation) process in the salen structure, the fluorescence of the system
was enhanced significantly. Under identical conditions, no obvious response could be
observed upon the addition of other ions including Ag+, Al3+, Ba2+, Ca2+, Cd2+, Cr3+,
Cu2+, Fe3+, Hg2+, K+, La3+, Li+, Mg2+, Na+, Ni2+ and Pb2+. It was hinted that PA can be
used as a fluorescence sensor with good selectivity for Zn2+.
7

None
Ag+
Al3+
Ba2+
Ca2+
Cd2+
Cr3+
Cu2+
Fe3+
Hg2+
K+
6000
4000
2000
0
Zn2+
Zn2+
Others
La3+
Li+
Mg2+
Na+
Ni2+
Pb2+
Zn2+
400
500
600
Wavelength (nm)
Figure 1. Fluorescence responses of PA (10 µM) in DMF upon the addition of 1.0 equiv various
metal ions (λex=328nm); The insert shows the fluorescence color of PA with Zn2+ and other metal
ions under 365 nm UV lamp.
In order to understand the sensing process and binding form of PA to Zn2+,
fluorescence titration were conducted that a gradually increasing amounts of Zn2+ was
added to a solution of PA in DMF, and a significant fluorescence enhancement was
observed. As shown in Figure 2, during the titration of Zn2+ with PA, a new emission
band was formed at 440 nm. This was because the addition of Zn2+ inhibited the
occurrence of the PET process of PA and C=N isomerization. When Zn2+ was added to
1 equivalent, it could be observed that the fluorescence emission intensity reaches a
maximum. The absolute quantum yield of PA was 0.03 and PA+Zn2+ was 0.48 in DMF.
To support the binding stoichiometry of PA+Zn2+ complex, Job plot method was
performed. From the results shown in Figure 3, it could be seen that the highest point
appeared at mole fraction of 0.50 for Zn2+. The results had indicated that the binding
modes of PA+Zn2+ was stoichiometry of 1: 1.
8

8000
6000
4000
2000
0
1.5equiv
0
400
500
600
Wavelength (nm)
Figure 2. Fluorescence titration of PA (10µM) upon the addition of Zn2+ (0-1.5equiv) in
DMF (λex=328nm).
9

4000
3000
2000
1000
0
0
3
6
9
[Zn2+]/[Zn2++PA]
Figure 3. Job’s plots for PA and Zn2+. The total concentration PA and Zn2+ kept at 10μmol/L
with λex=360nm.
According to the modified Benesi-Hildebrand equation, the binding constant of
PA+Zn2+ could be determined from the fluorescence titration curve of the
corresponding complex. Fluorescence spectral data was analyzed using the following
equation (1):
1
1
1
(1)
=
+
Ka(Fmax - Fmin)[Zn2 +
]
F - F0
Fmax - F0
Ka was the binding constant of Benesi-Hildebrand equation. F0, F, and Fmax were
the emission intensities at 440 nm of PA, at tested zinc concentration, and at a
concentration of complete interaction, respectively. As shown in Figure 4a, there was
an excellent linear relationship between 1/(F-F0) and the reciprocal of the concentration
of Zn2+ (R2=0.9904) in the range of 0 to 1.4 equivalents of Zn2+ ion concentration. The
association constant (Ka) was 2.94×104 M-1.
10

The limit of detection (LOD) was also calculated based on fluorescence titration.
First, the emission intensity of PA in DMF (10 μM) needed to be measured separately
10 times to obtain the standard deviation of the blank measurement. Second, 0-42 μM
Zn2+ was added to the PA (10 μM) solution, and a good linear relationship was found
between the fluorescence intensity at 440 nm and the Zn2+ concentration between 0 and
42 μM. Finally, the detection limit was calculated according to the following formula:
detection limit=3σ/K, where K was the slope of the fluorescence intensity and the Zn2+
concentration, and σ was the standard deviation of the blank measurement. As shown
in Figure 4b, the detection limit was determined as 1.635×10-8 M pointing to the high
detection sensitivity. Compared with the previously reported values of Zn2+-
fluorescence sensors, the LOD of PA presented here was lower.[37, 38]
a
y=1.39E-5x-0.3854
R2=0.9904
0.6
0.4
0.2
0.0
20000
40000
60000
1/[Zn2+] M-1
11

b
6000
4500
3000
1500
y=2.86106E8x+269.97
R2=0.9919
0.000005
0.000010
0.000015
0.000020
[Zn2+]
Figure 4. (a) A certain linear relationship between reciprocal of fluorescence intensity [1/F-
F0] and reciprocal of concentration of Zn2+ solution (λex=328nm); (b) A linear relationship
between F/F0 and [Zn2+].
High selectivity was also a valid criterion for the success of the probe design. In
order to evaluate the affinity of the probes for different metal ions, the competition
experiments of PA (10 μM) were performed with various metal ions (10 μM) in DMF
solution. The corresponding fluorescence intensity was shown in Figure 5. Obviously,
in addition to the slight fluorescence change caused by Cu2+ and Fe3+, Zn2+ can cause
significant fluorescence enhancement in different metal ion solutions. When other
common metal ions are present, such as Ag+, Al3+, Ba2+, Ca2+, Cd2+, Cr3+, Hg2+, K+,
La3+, Li+, Mg2+, Na+, Ni2+, and Pb2+, they did not interfere with the detection of Zn2+.
These results indicated that PA can be used for the selective and sensitive detection of
Zn2+ in DMF.
12

PA+Zn2+
PA+ions+Zn2+
8000
6000
4000
2000
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
+i
3l
a
2i
2
2
3
2
2
3
2
3
2
g
r
K
L
a
a
d
u
e
g
a
g
b
A
A
N
N
C
L
F
B
P
C
C
C
H
M
Figure 5. Fluorescence spectra of PA (10μM) containing Zn2+ (1.0equiv) and other
competitive metal ions (1.0equiv) in DMF solution.
3.3 Binding mechanism of PA to Zn2+
To confirm the complexation mechanism between PA and Zn2+ ions, 1H-NMR of
PA in the absence and presence of Zn2+ was measured using a NMR spectrometer. As
shown in Figure 6, the chemical shifts at 13.07 and 8.86 ppm may be assigned to the
phenolic hydroxyl group and N=CH band of the probe PA, respectively. Once Zn2+ was
added, the phenolic hydroxyl groups of the probe PA completely disappeared at δ13.07,
indicating that Zn2+ and the phenolic hydroxyl groups of the probe PA may have been
combined. In addition, the combination of Zn2+ changed the chemical shift of N=CH
band. Compared with the free probe, the peak of N=CH band in PA+Zn2+ had moved
from the position of δ 8.86 to the position of δ 9.85, indicating that Zn2+ may react with
N=CH band. Similarly, FT-IR studies clearly show that the phenolic hydroxyl bands of
PA appearing at 3180 cm-1 in the presence of Zn2+ ions disappear. At the same time, the
13

C-O band at 1648 cm-1 was significantly enhanced in the FT-IR spectrum (Figure S10).
Formation of PA+Zn2+ was also evidenced by ESI-mass test. The MS spectrum of the
PA+Zn2+ complex in DMF solution was shown in Figure S11. The MS spectrum
exhibited that the peak of m/z: 772.43 was suggestive to [PA+Zn2++H+]+. These results
supported the coordination of phenol-OH and imine nitrogen with Zn2+ in PA after
adding Zn2+. Based on the analysis of 1H-NMR, FT-IR, ESI-mass test and Job plot, the
1:1 binding of PA with Zn2+ was suggested, as displayed in Scheme 2.
Figure 6. 1H-NMR titration spectral of PA upon addition of Zn2+ in CD3SOCD3-D2O
(v/v=1:1).
14

Scheme 2. The proposed binding mode of Zn2+ with PA.
In order to understand the possible mechanism of the interaction between PA and
Zn2+ from a theoretical perspective, the geometric structure of the probe PA and
PA+Zn2+ was optimized by density functional theory (DFT) based on the previously
estimated binding model. The Gaussian 09 program was used to obtain the singlet
excited states of the probe PA and PA+Zn2+ at the B3LYP/6-31G level. From the
calculation results (Figure 7), The energy gap between the LUMO and HOMO levels
in the probe PA was 3.99 eV, while the energy gap between the LUMO and HOMO
levels in PA+Zn2+ was 3.74 eV. It could be found that the band gap energy (3.74 eV)
between HOMO and LUMO of PA+Zn2+ was lower than the band gap energy (3.99
eV) of the probe PA, which indicated that the probe PA formed a more stable complex
after combining with Zn2+. As shown in the Figure 7, the LUMO electron density of the
probe PA was mainly distributed in the phenolic hydroxy-benzene moiety. The HOMO
electron density of the phenolic hydroxy-benzene moiety of the probe PA decreased
relative to the LUMO electron density, and the HOMO electron density of the o-
phenylenediamine moiety increased. These results indicated that the PET pathway of
the phenolic hydroxy-benzene moiety to the o-phenylenediamine moiety of the probe
PA was open. Once PA was coordinated with Zn2+, the LUMO and HOMO electron
density of PA+Zn2+ were evenly distributed in the phenolic hydroxy-benzene moiety
and o-phenylenediamine moiety. And after PA coordinated with Zn2+, the LUMO and
HOMO electron density decreased. These results indicated that the PET pathway of the
phenolic hydroxy-benzene moiety to the o-phenylenediamine moiety of the probe PA
was blocked. As the previous experimental results was shown, PA and PA+Zn2+ had
similar band gap energies between LUMO and HOMO, suggesting their similar
absorption peaks. Not surprisingly, the experimental results were confirmed by the
15

conclusions of theoretical calculations.
Figure 7. (a) Optimized structures of PA and PA+Zn2+; (b) HOMO/LUMO energy levels and the
contour diagrams of PA and PA+Zn2+ by DFT calculation.
3.4 Cyclic Voltammetry Experiments
Cyclic voltammetry (CV) was used to conduct electrochemical studies to further
understand the sensing behavior of PA and Zn2+ ions. Initially, a 0.1M TBAP
(tetrabutylammonium perchlorate)/CH3CN solution was used as the electrolyte, and the
scanning rate of 0.1 V/s was used to study the electrochemical behavior of PA. It can
be seen from Figure 8 that the sensor PA had two oxidation peaks (Epa1=0.985V, Epa2=-
0.585V) and one reduction peak (Epc=-1.041V) in the potential range of -1.6V to 1.6V.
When Zn2+ was added to the DMF solution of PA, it caused obvious behavior in the
CV curve. It can be seen from the figure that after the interaction between PA and Zn2+,
the redox peak current slightly decreased, and a negative shift occurred. Compared with
the previous CV curve of PA, a new reduction peak appeared at the position of Epc=-
0.718V. At the position of Epa2=-0.585 V, the oxidation peak of PA+Zn2+ decreased
16

significantly. These changes may be due to the formation of a complex between the
probe PA and Zn2+ through the O atom and the N atom of the C=N group. The CV
curve results indicated that PA and Zn2+ ions formed a complex, as displayed in Scheme
2
0.00008
PA
PA+Zn2+
0.00004
0.00000
-0.00004
-1
0
1
Potential (V)
Figure 8. CV profiles of PA and PA+Zn2+.
3.5 In vitro cytotoxicity and imaging Zn2+ in Living Cells
Based on the good selectivity and sensitivity of probe PA, the cytotoxicity of probe
PA was measured by MTT assay before imaging Zn2+ in living cells. HeLa cells were
incubated with different concentrations of probe PA (1, 2.5, 5, 10, 15, 20, 25, 30 μM)
for 24 hours. As shown in Figure 9, even when the probe PA was at a concentration of
30 μM, the cell viability of HeLa cells was still higher than 80% for an incubation time
up to 24 hours. These results indicated that probe PA had low cytotoxicity and good
17

biocompatibility to live HeLa cells
Subsequently, the practical utility of this probe was explored. In order to explore
the application prospects of PA in biological systems, we applied the probe PA to live
Hela cells and performed biological imaging experiments to study the sensing
performance of the probe PA on Zn2+ in the cell matrix. The probe PA was dissolved
in DMSO (10-3 M) for future use. HeLa cells were incubated with PA solution (10 μM)
for 30 minutes, and the change in fluorescence intensity was observed by a confocal
fluorescence microscope (Figure 10). As expected, we did not observe fluorescence
emission after incubating HeLa cells with probe PA (10 μM) in DMEM for 30 minutes
at 37°C. However, in another experimental group, when HeLa cells were pretreated
with 10 μL of probe PA (10 μM) for 30 minutes, and then pretreated with Zn2+ (10 μM)
for 30 minutes, we could detect significant blue fluorescence. The merged image under
the bright field image on the fluorescence image shows that the cells pretreated with
the probe retained a good morphology, and PA could successfully enter the cells and
accumulate in the cytoplasm. This strong fluorescent emission confirmed that the probe
was permeable to the cell membrane. These results indicated that probe PA could detect
intracellular Zn2+ in living cells, and probe PA had good biocompatibility and cell
permeability. Therefore, it may be a satisfactory chemical sensor for Zn2+ ion imaging
in living cells.
18

100
50
0
1
2.5
5
10 15 20 25 30
Concentration of PA (M)
Figure 9. Viability of HeLa cells in the presence of PA as measured by using MTT assay.
Figure 10. Confocal images of Hela cells: (a1) incubated with PA (10 μM); (a2) further incubated
with Zn2+ (10 μM); (b1-b2) bright-field images; (c1-c2) merged images.
4. CONCLUSION
19

In summary, we had successfully prepared a Zn2+ fluorescent probe PA with high
selectivity and sensitivity. The probe exhibited a remarkable fluorescence enhancement
upon addition of Zn2+ in DMF solution. The binding stoichiometry and detection limit
were 1:1 and 16.3 nM, respectively, which is superior to the reported Zn2+ fluorescent
1
probes. The theoretical calculations of Zn2+ probe, H-NMR, FT-IR, ESI-mass, and
cyclic voltammetry tests can agree well with the identification mechanism. In addition,
the probe had good biocompatibility and low cytotoxicity, and could be used to detect
Zn2+ in living HeLa cells.
ACKNOWLEDGEMENTS
The
authors
are
thankful
for
financial
support
from
the Anhui Provincial Natural Science Foundation (No.1908085MB51), and Anhui
Province Key Laboratory of Enviorment-friendly Polymer Materials.
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Author Contribution Statement
LongChao Du: Conceptualization, Methodology, Funding acquisition.
Yang Hu: Investigation, Methodology, Data curation, Software, Writing-Original draft
preparation.
Jie Yang: Software.
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Graphical Abstract
25

Highlights
 A novel salen derivative was obtained for the detection of Zn2+.
 The probe exhibited a remarkable fluorescence enhancement upon addition of
Zn2+ in DMF solution.
 The probe had good biocompatibility and low cytotoxicity, and could be used
to detect Zn2+ in living HeLa cells.
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