An acylhydrazone coumarin as chemosensor for the detection of Ni2+ with excellent sensitivity and low LOD: Synthesis, DFT calculations and application in real water and living cells

Article abstract of DOI:10.1016/j.ica.2020.120144

A novel acylhydrazone coumarin fluorescent chemosensor C⁴ for detection of Ni²⁺ was designed and synthesized. The experimental results revealed a low detection limit of 2.1 × 10^?11 M with high selectivity and excellent sensitivity towards Ni²⁺. C⁴ showed a good linear relationship with the concentration of Ni²⁺ from 1.3 × 10^?6 to 1.6 μM. Moreover, a stable complex was formed between C⁴ and with Ni²⁺ and the binding ratio was proved to be 2: 1 by Job's plot and mass spectrum. The sensing ability of C⁴ towards Ni²⁺ was attributed to parity-forbidden transition according to fluorescence titrations and DFT calculations. The detection of Ni²⁺ in water samples illustrated C⁴ could be successfully applied for the detection of Ni²⁺ in real environmental samples. What's more, the fluorescence microscopy images of Hela cells demonstrated the high potential of the novel biosensor for the investigation of biological processes involving Ni²⁺, as well.

Full text of DOI:10.1016/j.ica.2020.120144

Journal Pre-proofs
Research paper
2
+
An acylhydrazone coumarin as chemosensor for the detection of Ni with ex‐
cellent sensitivity and low LOD: synthesis, DFT calculations and application
in real water and living cells
Wen Lu, Jichao Chen, Jiuzhou Shi, Zhong Li, Li Xu, Weina Jiang, Shilong
Yang, Buhong Gao
PII:
DOI:
Reference:
S0020-1693(20)31344-X
https://doi.org/10.1016/j.ica.2020.120144
ICA 120144
To appear in:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
17 September 2020
30 October 2020
17 November 2020
Please cite this article as: W. Lu, J. Chen, J. Shi, Z. Li, L. Xu, W. Jiang, S. Yang, B. Gao, An acylhydrazone
coumarin as chemosensor for the detection of Ni2+ with excellent sensitivity and low LOD: synthesis, DFT
calculations and application in real water and living cells, Inorganica Chimica Acta (2020), doi: https://doi.org/
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2
+
An acylhydrazone coumarin as chemosensor for the detection of Ni with
excellent sensitivity and low LOD: synthesis, DFT calculations and application in
real water and living cells
Wen Lu,a, *,† Jichao Chen,a, † Jiuzhou Shi, Zhong Li, Li Xu, Weina Jiang, Shilong Yang,
a
b
a, *
a
c
Buhong Gaoc
a
b
College of Science, Nanjing Forestry University, Nanjing 210037, China
National Engineering Research Center of Biomaterials, Nanjing Forestry University, Nanjing
10037, China.
2
c
Advanced Analysis and Testing Center, Nanjing Forestry University, Nanjing 210037, China
These authors contributed equally.
†
4
2+
Abstract: A novel acylhydrazone coumarin fluorescent chemosensor C for detection of Ni was
designed and synthesized. The experimental results revealed a low detection limit of 2.1 × 10-11
M
2
+
4
with high selectivity and excellent sensitivity towards Ni . C showed a good linear relationship
2
+
-6
with the concentration of Ni from 1.3 × 10 to 1.6 μM. Moreover, a stable complex was formed
4
2+
between C and with Ni and the binding ratio was proved to be 2: 1 by Job’s plot and mass
4
2+
spectrum. The sensing ability of C towards Ni was attributed to parity-forbidden transition
according to fluorescence titrations and DFT calculations. The detection of Ni2+ in water samples
4
2+
illustrated C could be successfully applied for the detection of Ni in real environmental
samples. What’s more, the fluorescence microscopy images of Hela cells demonstrated the high
2
+
potential of the novel biosensor for the investigation of biological processes involving Ni , as
well.
2
+
Keywords: Coumarin-based chemosensor, Ni , Low LOD, DFT, Bioimaging
1
. Introduction
Nickel is a malleable and ferromagnetic metallic element that can be highly polished. Due to the
resistance to corrosion, nickel is widely used in the manufacture of stainless steel and corrosion
resistant alloys, as well as for other applications, such as nickel plating, coinage, petroleum,
textile, printing, mold and medical treatments [1]. The increased demand for this heavy metal has
promoted the extraction of nickel from ores. However, the processes involve in the extraction and
utilization are not in accordance with the requirements of environmental protection. As a result, all
types of environmental pollution (i.e, air, water, and soil pollution) are already noticed and
associated with quantities of nickel above the tolerated limit, which have a negative impact on the
human, plants, and animal life [2-3]. Water and soil pollution by nickel harms the ecosystem by
reducing, for instance, the crop yield and aquatic products [4-5]. Nickel compounds from the
atmosphere can enter the body directly by the respiratory tract, and this excessive intake will
generate toxic effects [6-7], such as skin allergy, pulmonary fibrosis, cardiovascular system
toxicity, and carcinogenicity (particularly, in nose and kidney) [8-9]. Due to such toxic effects of
nickel, the environmental protection agency (EPA) has stipulated that children should not drink
water and other beverages containing a concentration of nickel ions higher than 0.04 mg/L [10].
Compared with the traditional analytical methods: Spectrophotometric Method [11-13],
Colorimetric Detection [14-15], Flow injection Spectrophotometry [16], Electrochemical Probe
[17], Atomic Absorption Spectrometry [18], Reversed-phase High Performance Liquid

Chromatography [19], the fluorescent chemosensors-based methods have attracted particular
attention due to its simple operation and convenience alongside with high sensitivity and
selectivity [20-28]. A great many efforts have been paid for designing and synthesizing sensors
with high selectivity, sensitivity, low detection limit and instantaneous response [29-31]. Many
fluorescent sensors for highly sensitive sensing Ni2+ have been widely reported with enhancement
of flurorescence [32-35]. Besides, a pyrazolopyrimidine-derived fluorescent chemosensor with
high sensitivity and selectivity towards Ni2+ has been developed based on intramolecular charge
transfer (ICT) which revealed fluorescence quenching [36]. Moreover, acridine
derived-fluorescent sensor was used to construct a novel chemosensor for Ni2+ detection at low
concentrations [37]. As we know, the concentration of nickel in natural freshwater is about 0.5
-9
2+
-8
ug/L (8.5× 10 M), while the lowest LOD for Ni in these reports is 2.16 × 10 M [29]. In this
work, we further reduce the LOD to 2.1 × 10-11 M, which will broaden the detection range for
nickel ion application in environmental protection, biology, and medicine.
Coumarin is a natural product found in black vanilla, wild vanilla, chamomile, and orchid.
Coumarin has high fluorescence quantum yield, large Stock's shift, adjustable photophysical and
photochemical properties, and good light stability [34]. As a result, coumarin has been developed
as a fluorescent chemosensor for the detection of various ions and molecules [38-47].
We synthesized
a
coumarin acylhydrazone-based fluorescent chemosensor, C4
(
)
(E)-N'-(2-chloro-6-hydroxybenzylidene)-7-(diethylamino)-2-oxo-2H-chromene-3-carbohydrazide
2
+
4
2+
, to detect Ni . The photo-physical properties of C chemosensor for Ni were studied in details
by fluorescence spectroscopy, absorption spectroscopy, and confocal fluorescence imaging. The
high sensitivity of this new sensor was proved by the low detection limit of 2.1 × 10-11 M. In
addition, it proved to be highly selective for Ni2+ based on the experiments performed in the
presence of other di- and trivalent cations.
2
. Experimental
2
.1. Characterization
UV-vis and fluorescent spectra were recorded on a LAMBDA950 spectrometer (Perkin Elmer
Precisely) and a LS 55 Fluorescence Spectrometer (Perkin Elmer), respectively, using a 1-cm
square quartz cell. The excitation wavelength of 426 nm (excitation/emission slit widths = 12 nm /
.5 nm) was used for all measurements. H and 13C NMR spectra were recorded on a Bruker
1
2
AVANCE III HD spectrometer operating at 600 MHz. Mass spectrometry was recorded on the
LTQ-Orbitrap (Thermo Fisher Scientific). The live-cell imaging of Hela cells was performed on a
Zeiss LSM710 Airyscan confocal laser scanning microscope.
2
.2. Synthesis of C4
Compound 2 (0.55 g, 2 mmol) and 2-chloro-6-hydroxybenzaldehyde (0.33 g, 2 mmol) were
dissolved in 20 mL anhydrous ethanol and refluxed for 6 h. After cooling to room temperature, the
4
1
yellow solid formed was washed with ethanol to obtain C (0.7954 g, 96%). H NMR (600 MHz,
DMSO-d ) δ (ppm) : 12.60 (s, 1H, NH), 12.07 (s, 1H, Ar-OH), 9.11 (s, 1H, CH=N), 8.74 (s, 1H,
6
C=CH), 7.73 (d, 1H, Ar-H, J=8.4 Hz), 7.32 (t, 1H, Ar-H, J=7.8 Hz), 7.04 (d, 1H, Ar-H, J=7.8 Hz),
6
4
.94 (d, 1H, Ar-H, J=8.4 Hz), 6.85 (d, 1H, Ar-H, J=9.0 Hz), 6.68 (s, 1H, Ar-H), 3.53~3.32 (m,
1
3
H, CH ), 1.16 (t, 6H, CH , J=7.2 Hz). C NMR (150 MHz, DMF-d ) δ (ppm): 161.70, 160.49,
2
3
7

1
1
59.90, 158.38, 153.76, 149.30, 147.34, 134.30, 132.38, 120.66, 116.58, 116.02, 110.87, 108.64,
4
+
08.58, 96.47, 45.04, 12.31. ESI-MS m/z: 436.1034 [C +Na] .
4
2
.3. Preparation of stock solution of C and metal ions
All reagents used in this work were purchased from professional commercial suppliers and were
used directly without further purification. The solutions of metal ions were prepared from salts of
AlCl ·6H O, Ca(CH COO) ·H O, Cd(CH COO) ·2H O, CoCl ·6H O, Cr(CH COO) ,
3
2
3
2
2
3
2
2
2
2
3
3
CuCl ·2H O,
FeCl ·4H O,
FeCl ·6H O,
CH COOK,
Mg(CH COO) ·4H O,
3 2 2
2
2
2
2
3
2
3
Mn(CH COO) ·4H O, Ni(CH COO) ·4H O, Pb(CH COO) ·3H O and Zn(CH COO) ·2H O in
3
2
2
3
2
2
3
2
2
3
2
2
-3
4
deionized water with a concentration of 10 M. The solution of C chemosensor (1.0 mM) was
dissolved in different solvents (THF, DMF and DMSO). For the fluorescence spectra
4
measurements, a concentration of 1.0 μM C (THF: PBS=99:1, v/v) was used while UV-vis
4
spectra measurements were performed with a C solution with a concentration of 10.0 μM (THF:
PBS=99:1, v/v).
2
.4. Limit of detection (LOD)
The limit of detection (LOD) and binding stoichiometry were evaluated based on fluorescence
titrations. A plot of the measured fluorescence intensity at the emission band 462.5 nm versus
concentration of Ni2+ was constructed to evaluate the limit of detection based on the equation
LOD = 3σ/k, where σ is the standard deviation of the emission of a blank solution and k is the
slope of the calibration curve. Each measurement was performed three times [48-49].
2
.5. Computational methodology
Density functional theory (DFT), model B3LYP [50-51], has been used to optimize the
4
4
geometric structures of C molecule and C -Ni (II) complex in the lowest singlet spin state S . The
0
4
geometries for the first excited singlet states S of C were optimized with TD-PBE0 method to
1
obtain their fluorescence emission energies and spectra. To analyze the nature of fluorescence
emission, natural transition orbital (NTO) method was applied based on the TDDFT calculations
[
52]. In all calculations, the solvation effect was evaluated based on the SMD solvation model
53], the energies were computed with the 6-31G* basis set for C, H, N, and O atoms and the SDD
[
effective core potential for Ni atom. All calculations were performed with Gaussian 09 program.
2
.6. Detection of Ni2+ in water samples
Water samples used in this work were ultrapure water and tap water from the Nanjing Forestry
University. Different concentrations of Ni2+ (0.1, 0.2 0.3 μM in ultrapure water and 0.2, 0.4, 0.6
4
μM in tap water) and C chemosensor (1 μM) were used for the measurements (pH=6.8-7.4). The
fluorescence intensity at 462.5 nm was then measured, and each sample was tested three times.
2
.7. Cell cytotoxicity study
4
The survival rate and toxicity of Hela for C was measured by MTT. Logarithmically grown
Hela cells (90% DMEM, 10% FBS, 5% Penicillin-Streptomycin Solution) were inoculated into
9
6-well plates at 10000 per well and incubated in incubator (37°C, 5% CO ) for 24 h. After then,
2
4
the medium was removed and washed with PBS for three times. 100 μL of C (The termination
concentration was 0.01-100 μM) was added, culturing the cell at 37°C, 5% CO for another 24 h.
2

Similarly, the medium was removed, washed with PBS for three times, and 100 μL of MTT
1mg/mL) was added to culture for 12 h. Removing the supernatant, adding 150 μL of DMSO to
(
each hole, and testing the absorbance at 590 nm with Filter Max F5 (MD, Amarican). Repeat the
experiment ten times and take the average.
2
.8. Cell imaging study
Hela cells were used for cell imaging. The medium contained 10% FBS and 5%
penicillin-streptomycin mixed solution. The samples were incubated at 37 °C in 5% CO . Hela
2
4
cells were inoculated in 6 well plates with coverslip and 1.5 × 10 cells per well. After 24 h, the
4
medium was removed and the cells washed three times with PBS (pH = 7.2). Then, the C ligand
-5
(
1 × 10 M) was added to the cells followed by incubation for 1 h. At the end of the process, the
medium was removed and the cells washed three times with PBS. Finally, 5 × 10-5 M Ni2+
solution was added and co-incubated for 1 h. The cover glass was washed three times with PBS
and double steamed water. The fluorescence of cells was observed by confocal laser scanning
fluorescence microscopy.
3
. Results and discussion
4
The C ligand was synthesized by a condensation reaction between a coumarin-based molecule
(compound 2) and 2-chloro-6-hydroxybenzaldehyde, as described in Scheme 1. The method of the
reference was used to synthesize compound 1 and compound 2 (Fig. S1-S7) [43].
Scheme 1. The reaction involved in the synthesis of C4.
.1. Sensing properties of C chemosensor for Ni2+
4
3
4
First, the role played by solvent on the spectral properties of C was evaluated. According to the
4
solubility of C , DMF, DMSO and THF were chosen for these tests. Hence, different solvents
4
(
THF, DMF, and DMSO) were selected to prepare C solution of 10 μM and 1 μM, and then the
UV-vis absorbance and fluorescence emission spectra were recorded respectively (Fig. S8). It can
be seen there is no obvious difference between the absorbance intensity for the molecule dissolved
in DMF, DMSO and THF. For the fluorescence properties, the effect of the solvent is more
4
obvious. The fluorescence emitted by C dissolved in THF was much stronger in comparison with
those emitted in DMF and DMSO. As the proportion of water in the solution increased, both
absorption and fluorescence intensity decreased. This may be caused by the decrease in solubility
4
of C with the addition of water, which favored the molecular aggregation. On the basis of UV-vis
absorption and fluorescence emission spectra, the best solvent was THF and it was used for further
investigations.

4
Fig. 1. Fluorescence spectra of C in the presence of different metal ions (THF: PBS=99:1, v/v, λex = 426 nm, slit
width = 12, 2.5 nm).
4
2+
3+
2
2+
2+
3+
To evaluate the selectivity of C towards Ni , several metal ions (Al , Ca +, Cd , Co , Cr ,
2
+
2+
3+
+
2+
2+
+
2+
2+
2+
4
Cu , Fe , Fe , K , Mg , Mn , Na , Ni , Pb and Zn ) were added to the C solution and then
the fluorescence was recorded. For this test, 1 equiv. metal ions was used. As the experimental
results displayed in Fig. 1 show, only Ni2+ has an obvious fluorescence response. The color of
2
+
solution turned dark yellow from bright yellow with addition of Ni (Color change induced by the
addition of Ni2+ was seen in Fig. 2). The fluorescence intensity of the C solution with addition of
4
2
+
Ni quenched.
4
2+
4
To evaluate the binding ability of C to Ni , fluorescence titration profiles of C were carried
out by increasing the amounts of Ni2+ in the solution, as shown in Fig. 2. With increasing the
2
+
4
concentration of Ni , the intensity of the emitted fluorescence of C at 462.5 nm gradually
decreased. The lowest and stable fluorescence signal was obtained and a good linearity was
-6
obtained in the range in the fluorescence intensity in the concentration range of 1.3 × 10 – 1.6
2
+
2
μM Ni (R = 0.99238) according to LOD = 3σ/k (σ was shown in Table S1). To note, the limit of
detection was of 2.1 × 10-11 M, which is lower than that reported for other chemosensors. For
example, in Ghazali’s article, the LOD was 2.16 × 10-8 M [29] and 8.3 × 10-8 M [30] in
Velmurugan’s article. Many reported chemosensors’s LOD was higher than LOD in this paper
(Table 1).

Fig. 2. Standard curve for fluorescence emission intensity vs Ni2+ concentration (λex = 426 nm, slit width = 12, 2.5
nm). Insert: Color change induced by the addition of Ni2+ (1 equiv.) to the solution of C (C =1×10 mol/L, THF:
4
4
-6
PBS=99:1, v/v).
3
.2. Metal ion selectivity study
To investigate the practical utility of the synthesized C chemosensor, competitive titration was
4
3
+
2+
2+
2+
3+
2+
2+
3+
performed in the presence of various metal ions, i.e., Al , Ca , Cd , Co , Cr , Cu , Fe , Fe ,
+
2+
2+
2+
2+
K , Mg , Mn , Pb and Zn . For these experiments, the concentration of interfering ions was
4
four times higher than that of C . The results, expressed as fluorescence intensity versus the
composition of the sensing solution, are displayed in Fig. 3. It is obvious that irrespective of the
initial fluorescence intensity, it is quenched when Ni2+ is added to the “C + interfering ion”
solution. Interestingly, the level of quenching is similar in all cases, which indicates the high
4
2
+
affinity of the sensor for Ni . These results clearly show that the developed chemosensor is highly
2
+
selective for the detection of Ni .
4
4
4
Furthermore, to examine the stability of the C chemosensor, the solution of C and C -Ni (II)
was kept for a period of 48 hours and the fluorescence was measured at different intervals of time.
As shown in Fig. 4, the intensity of fluorescence keeps constant over the entire period which
means that the chemosensor was stable.
2
+
Table 1. Comparison of the reported sensors for Ni .
Target LOD (M)
Ref.
29
36
54
31
37
55
30
56
57
2
.16 × 10-8
.80 × 10-8
.30 × 10-8
.61 × 10-7
3
8
3
Ni2+
1.52 × 10-6
1
4
1
9
.91 × 10-6
.91 × 10-6
.00 × 10-5
.30 × 10-4

2
.10 × 10-11
This work
4
4
-6
Fig. 3. Fluorescence spectra of C in the presence of interfering metal ions (C =metal ions=1×10 mol/L, THF:
PBS=99:1, v/v,).
3
.3. Binding stoichiometry
4
2+
To assess the binding stoichiometry between the prepared C sensor and Ni , Job’s plot
4
2+
of fluorescence emission was recorded at different ratio of C and Ni . As shown in Fig. 5,
4
2+
the result demonstrated a ratio of 2:1 between C and Ni ion.
4
2+
The binding ability between C and Ni ion was measured by Stern-Volmer equation
[58] which is often used in quenched chemosensor.
Here, F is the fluorescence intensity of chemosensor in the absence of quenching agent,
0
F is the fluorescence intensity recorded in the presence of quenching agent, K is constant
sv
2+
of Stern-Volmer, [Q] is the concentration of quenching agent which is [Ni ]. The constant
K could be determined from the slop of the straight line of the plot of F /F against [Q].
sv
0
6
-1
The K was calculated to be 2 × 10 M . And the result confirmed the chemosensor had
sv
4
2+
an excellent binding ability between C and Ni ion.

4
Fig. 4. Fluorescence spectra of C -Ni (II) complex at different intervals of time.
4
2+
Fig. 5 Job’s plot of C -Ni complex in THF: PBS=99:1, v/v.
3
.4. Mass spectrometry studies
4
2+
The binding mode of C with Ni was analyzed by mass spectrometry, as well (Fig. 6a). The
4
mass fraction at m/z = 413.1142 belongs to free C ligand, whereas the mass fraction at m/z =
4
+
8
83.1544 is associated with the complex formed between ligand and metal ion, [Ni(C ) +H] , in
2
4
+
the stoichiometric ratio of 2 : 1 m/z=436.1027 was the ion peak of [C +Na] . The chemical
formula of the complex is C H O N Cl Ni. In addition, the experimental spectrum is in line with
4
2
39
8
6
2
the simulated one, as shown in Fig. 6b.

4
Fig. 6. Experimental (a) and simulated mass spectrum (b) of C -Ni (II).
3
.5. Theoretical calculation studies
According to the Job plot, fluorescence titration, and ESI-mass analysis, we proposed the
4
possible structure of 2: 1 C -Ni(II) complex. The computational study was performed by using the
density functional theory (DFT) and time dependent density functional theory (TDDFT) methods
in parallel with the natural transition orbital (NTO) method to gain further insight into the ‘turn
4
off’ mechanism of C sensor.
The theoretical results confirmed the energy and the charge transfer model proposed to explain
the mechanism of the fluorescence quenching. According to Kasha's rule, the fluorescence is
generated when the electrons are transferred from S to S . We calculated the vertical electronic S
1
0
1
4
4
to S transitions of C and C -Ni (II) by TDDFT method. Table S2 display the orbital pairs
0
involved in the electronic transition and accountable for the intense fluorescence emission of C4
4
4
and C -Ni (II) complex. Clearly, the electronic transition of C molecule is a Local Excitation
4
(
LE), C having a S →S excitation energy of 2.7624 eV (448.83 nm) and an oscillator strength of
0
1

1
.7219, which could be attributed to the intense absorption. In addition, the theoretical absorption
4
at 448.8 nm (S →S excitation) of C in is well agreement with the experimental absorption of
0
1
4
4
62.5 nm while the dominant MO transition is MO108 → MO109. C -Ni (II) has a S →S
0 1
excitation energy of 0.8253 eV and an oscillator strength of 0.0001 which inferred to
4
parity-forbidden. Yet, no dominant MO transition was observed for C -Ni(II), the largest
contribution of a single MO pair is only of 26.71%, which makes it impossible to identify the
nature of this excitation based on MO pair only. Therefore, the natural transition orbital (NTO)
4
distribution of the complex C -Ni (II) was calculated. As shown in Fig. 7, the optical emissions
are mainly attributed to the d-d orbital transitions in Ni atom from NTO224 to NTO225.
To summarize, the computational results along with the experimental data were demonstrated
4
2+
the complex formation between C and Ni . In addition, it is confirmed that the fluorescence
quenching in the presence of Ni2+ may due to the parity-forbidden transition according to the
Laporte selection rule.
4
Fig. 7 (a)The orbitals involved in the principal electronic transition S
0
- > S
1
of C ; (b) Dominant natural transition
4
0 1
orbital (NTO) pairs of the principal electronic transition S →S in C -Ni(II).

3
.6. The proposed binding mechanism
Based on the results of mass spectrometry, and DFT studies, the possible binding mechanism in
4
2+
C -Ni (II) complex is shown in Scheme 2. Ni coordinated to the phenolic hydroxyl of
salicylaldehyde and N atom in C=N Schiff base. N atom in C=N is a perfect coordination atom
and often be used to coordinate with metal ions [69-63]. As a result, the fluorescence is quenched
[64-65] and the coordination mode is similar to reported work [60].
4
2+
3
.7. Application of C for detection of Ni in water samples
4
2+
To prove the practical application of C for Ni detection, experiments were carried out with
ultrapure and real water samples. In the linear range of Ni2+ concentration, water sample was
spiked with standard Ni2+ solution at different concentrations. The results showed good values for
the recovery and relative standard deviation (R.S.D.), which proved the ability of the chemosensor
2
+
to successfully detect Ni in environmental samples (Table 2).
Cl
Cl
O
O
N
Ni2+
N
Cl
O
HN
NH
Ni2+
O
O
O
O
N
N
H
OH
O
O
N
O
O
N
N
Fluorescence "ON"
Fluorescence "OFF"
Scheme 2. Proposed mechanism for detection of Ni2+ by C4.
Table 2. Determination of Ni2+ in water samples from different sources.
Sample
Added(μM)
Found (μM)
0.10
Recovery (%)
101.11±0.29
98.10±0.34
RSD (%)
2.91
Ultrapure water
0.10
0
0
.20
.30
0.19
1.75
0.31
100.74±0.27
104.34±0.61
109.68±0.56
103.14±0.90
0.91
Tap water
0.20
0.21
2.95
0
0
.40
.60
0.44
1.28
0.62
1.46

Fig. 8. Fluorescence images of Hela cells incubated with C4 (10 μM) (a), further incubated with Ni2+ (5
equivalents) (c), and their corresponding bright field images (b, d).
3
.8. Cell cytotoxicity study and cell image experiment
4
We further investigated the fluorescence imaging in Hela cells treated with C (10 μM)
4
for 12 h. Before carrying out the cell image experiment, the cell cytotoxicity of C was
4
performed by MTT. The IC of C with Hela cells is 21.83 after incubating for 12 hours,
50
4
meaning C is low toxicity to Hela cells and could be used for bioimaging. As shown in
4
Fig. 8, a strong blue fluorescence was observed with addition of C into Hela cells (Fig.
2+
8
a). Then, Ni (50 μM) was added and incubated for 1 h, and a fluorescence emission can
2+
be observed, but of much lower intensity in comparison with the sample without Ni (Fig.
8c). This indicates that the cell membrane excellent permeability for C4, which once
2+
internalized in the cell can detect exogenous Ni .
4
. Conclusion
In summary, a new coumarin-based selective and sensitive chemosensor for Ni2+ ion
detection was developed and characterized by NMR, and ESI-MS techniques. This
2+
chemosensor exhibited a turn-off fluorescence response towards Ni , whose intensity was
quenched in the presence of this ion. The detection limit was as low as 2.1 × 10-11 M. The
sensor also selectively recognized Ni2+ in the presence of various competing ions.
According to fluorescence titrations, and DFT calculations, the sensing ability of C4

towards Ni2+ was attributed to parity-forbidden transition. The binding stoichiometry
4
2+
between C and Ni was of 2: 1. The new developed ligand proved to be a good
chemosensor for detection of Ni2+ in real water samples. In addition, the bio-imaging of
4
Hela cells demonstrated the permeability of the cell membrane for C followed by the
2+
2+
detection of Ni , demonstrating the possible utility in sensing of Ni in living cells.
Acknowledgements
This work was supported by the National Key Research and Development Program of
China (2017YFD060070602), Innovation Fund for Young Scholars of Nanjing Forestry
University (CX2017017, CX2017004), and the priority academic program development of
Jiangsu higher education institutions.
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4
2+
A novel acylhydrazone coumarin fluorescent chemosensor C for determination of Ni was
designed and synthesized with lower detection limit （2.1 × 10-11 M）. Water samples and
4
cell imaging experiments illustrated that C could be applied to the detection of practical
samples and in living cells.
Highlights
1
2
3
. A novel acylhydrazone coumarin fluorescent chemosensor for determination of
2
+
Ni was synthesized.
. The experimental results revealed a low detection limit of 2.1 × 10-11 M with high
selectivity and excellent sensitivity.
4
. Water samples and cell imaging experiments illustrated that C could be applied to
the detection of practical samples and in living cells.