A novel OFF-ON-OFF fluorescence probe based on coumarin for Al3+ and F? detection and bioimaging in living cells

Article abstract of DOI:10.1016/j.saa.2018.12.022

A novel fluorescence probe L2 based on coumarin has been designed and synthesized. The probe L2 can be used for relay recognition of metal ions Al³⁺ and anion F^? in the aqueous HEPES buffer (0.05 M, pH = 7.4), and build a OFF-ON-OFF

Full text of DOI:10.1016/j.saa.2018.12.022

Accepted Manuscript
A novel OFF-ON-OFF fluorescence probe based on coumarin for
Al3+ and F− detection and bioimaging in living cells
Xu Tang, Zhi Zhu, Renjie Liu, Liang Ni, Yue Qiu, Juan Han, Yun
Wang
PII:
DOI:
Reference:
S1386-1425(18)31084-9
https://doi.org/10.1016/j.saa.2018.12.022
SAA 16651
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 21 August 2018
Revised date:
Accepted
date:
10 December 2018
11 December 2018
Please cite this article as: Xu Tang, Zhi Zhu, Renjie Liu, Liang Ni, Yue Qiu, Juan Han,
Yun Wang , A novel OFF-ON-OFF fluorescence probe based on coumarin for Al3+
and F− detection and bioimaging in living cells. Saa (2018), https://doi.org/10.1016/
j.saa.2018.12.022
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ACCEPTED MANUSCRIPT
A novel OFF-ON-OFF fluorescence probe based on coumarin for Al3+ and F-
detection and bioimaging in living cells
Xu Tanga*, Zhi Zhub, Renjie Liub, Liang Nib*, Yue Qiuc, Juan Hand, Yun Wangb
Institute for Advanced Materials, School of Materials Science and Engineering,
a
Jiangsu University, Zhenjiang, Jiangsu, 212013,China
b
School of Chemistry and Chemical Engineering, Jiangsu university, Zhenjiang,
Jiangsu, 212013,China.
c
Grimwade Centre for Cultural Materials Conservation, School of Historical and
Philosophical Studies, Faculty of Arts, University of Melbourne, Parkville, Victoria,
3052, Australia.
d School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013,
PR China
Corresponding author:
Xu Tang, E-mail address: tangxu1108@163.com
Liang Ni, E-mail address: niliang@ujs.edu.cn
Abstract
A novel fluorescence probe L2 based on coumarin has been designed and
synthesized. The probe L2 can be used for relay recognition of metal ions Al3+ and
anion F− in the aqueous HEPES buffer (0.05 M, pH=7.4), and build a OFF-ON-OFF
detection system. The probe showed high selectivity and sensitivity to target ions in
the process of relay recognition, and the corresponding detection limit could be as low

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as 0.014 µM (Al3+) and 0.03 µM (F−). Besides, the geometry optimizations of probe
L2 and [L2+Al3+] complex were carried out using the Gaussian 16 program based on
DFT, and the identification mechanism of the probe was also discussed by the mass
spectrometry and theoretical calculations. Moreover, the probe have also been
successfully applied to detection of target ions in living cells.
Keywords: Fluorescence probe, coumarin, Al3+, F−, cell imaging
1.Introduction
It is well known that the aluminum is the most abundant metal element in nature
and its reserves account for about 8% of the earth’s crust [1, 2]. At the same time, the
aluminum has the advantages of small density, good thermal conductivity and good
ductility. So it has also been widely used in various fields, such as industrial
production, aerospace materials, medical equipment, food additives, drinking-water
purification, and so on [3-6]. But the Al3+ is not a necessary trace element, and the
intake of Al3+ ions will remain in various organs of the body. First, Al3+ ions are very
easily deposited in brain tissue, which may cause brain damage and serious memory
loss. The existence of excessive Al3+ ions can also lead to a series of biochemical
reactions, which will affect the normal metabolism of human cells. Meanwhile, Al3+
ions can directly damage the activity of osteoblasts, thus inhibiting bone matrix
synthesis [7-9]. Therefore, the harm of Al3+ ions to human body is not to be
underestimated.
Similarly, the fluorine also play a very important role in many chemical and
biomedical industry [10,11]. The fluoride is the common ingredient of the anesthetic,

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hypnotic, psychotropic drugs and military nerve gases. Meanwhile, fluorinated
compounds also can be used to prevent dental caries, and they are usually added to
some oral hygiene products. Fluorine is widely distributed in natural water, and
fluorine is also found in all tissues of human body, mainly accumulated in teeth and
bone tendons. A proper amount of F− ions is essential for the human body, but
excessive intake of fluoride is also harmful to people’s health. The fluorosis is a
typical symptom caused by chronic fluorosis [12, 13]. High concentration of fluoride
can also cause serious pulmonary edema, pulmonary hemorrhage, arrhythmia, nausea
and strong irritation in the eyes and upper respiratory tract. Even more, it will also
endanger human life [14, 15]. From the point of view of environment and human
health, it is very necessary to develop effective detection methods for fluoride.
In recent years, fluorescence sensing detection technology has attracted wide
attention. Compared with the traditional detection technology, the fluorescence
detection technology can show better detection results and have better application
value and potential. Therefore, the construction of fluorescent probes with specific
recognition performance has become one of the most popular research projects at
home and abroad [16, 17]. For the fluorescent probes that had been reported, most of
them were some metal ions fluorescent probes and anion fluorescent probes. And
many of them have some probes can be used for specific detection and identification
of Al3+ or F− [18-29]. However, many probes are usually limited by lengthy synthetic
routes or high detection limits, which cannot be applied to practical detection
applications. Most importantly, most of those probes are single response, although the

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selectivity of probes to the target ions is good, but the utilization ratio is not high
compared to the relay response or the multi response probe. Therefore, the design and
synthesis of fluorescent probes with high selectivity, high sensitivity and low cost,
which can break through a single response type, is still a challenging subject.
In this paper, coumarin was used as the fluorescent group, a novel fluorescent
probe (L2) with simple structure and good fluorescence response performance was
designed and synthesized. The probe L2 can be used for relay recognition of metal
ions Al3+ and anion F−, and build a OFF-ON-OFF detection system. The optical
properties and detection properties of probe L2 were systematically studied through a
series of UV-Vis spectra and fluorescence emission spectra. At the same time, the
influences of other common anion and cation on the detection effect of probe L2 were
also discussed. Finally, probe L2 was used for fluorescence imaging analysis of target
ions in living cells.
2. Experimental
2.1 Materials and Instruments
Unless otherwise noted, all the solvents and inorganic salts were purchased from
Sinopharm Chemical Reagent Ltd., The 4-hydroxy-4-biphenylcarbonitrile,
hexamethylenetetramine, Rhodamine B and hydrazine hydrate (85%) were purchased
1
from Aladdin Chemical Reagent Ltd., and used without further purification. The H
NMR spectra were recorded on an AVANCEⅡ 400 MHz spectrometer (Bruker
BioSpin) and 13C NMR spectra were recorded on 101 MHz spectrometers. Mass
Spectrometry (MS) were measured by a Liquid Chromatography-Ion Trap Mass

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Spectrometry (Thermo LXQ). Fluorescence spectra measurements were performed on
a Cary Eclipse fluorescence spectrometer (Variance, LTD, Australia) and the slit
widths were 5 nm for emission. Cell imaging experiments were applied on a Leica
DMI4000B inverted fluorescence microscope (Germany).
2.2 Synthesis of probe L2
Synthesis of compound 1: the methylumbelliferone (0.03 mol) and hexamine
(0.07 mol) in glacial acetic acid (50 mL) were refluxed for 8 h. Then added 75 mL 20%
of hydrochloric acid and continue heating for 45 min. After cooling to room
temperature, added 400 mL of ice water mixture and the product was extracted
several times with ethyl ether. All organic phase was mixed, and the solution was
dried over anhydrous sodium sulfate, filtered and the solvent was removed by vacuum
1
distillation, the yellow solid powder was obtained finally (1.06g, 17.3%). H NMR
(400 MHz, 25℃, CDCl3) δ 12.22 (s, 1H), 10.63 (s, 1H), 7.76 (s, 1H), 6.91 (s, 1H),
6.22 (s, 1H), 2.45 (s, 3H). 13C NMR (101 MHz, 25℃, CDCl3) δ 193.38 (s, 6H),
165.25 (s, 3H), 159.16 (s, 2H), 156.14 (s, 2H), 152.67 (s, 2H), 132.92 (s, 6H), 114.27
(s, 6H), 112.01 (d, J=8.4 Hz, 9H), 108.66 (s, 2H), 77.37 (s, 23H), 77.06 (s, 22H),
76.74 (s, 22H), 18.93 (s, 6H), 1.01 (s, 1H).
Synthesis of probe L2: 8-formyl-7-hydroxy-4-methylcoumarin (204 mg, 1.0
mmol) and benzoyl hydrazine (143 mg, 1.05 mmol) were dissolved in 30 mL ethyl
alcohol, and the mixture was refluxed for 6 h. After the reaction completed, removed
the solvent by vacuum distillation, and the crude products were washed with cold
ethanol for two times and dried by vacuum drying, the yellow powder products were

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1
obtained (277 mg, 86.6%). H NMR (400 MHz, DMSO-d) δ 12.80 (s, 1H), 12.45 (s,
1H), 9.14 (s, 1H), 7.99 (d, J=7.4 Hz, 2H), 7.77–7.61 (m, 2H), 7.58 (t, J=7.4 Hz, 2H),
6.97 (d, J=8.8 Hz, 1H), 6.25 (s, 1H), 2.40 (s, 3H).13C NMR (101 MHz, DMSO) δ
163.11, 161.65,159.83, 129.12, 154.16, 153.01, 144.02, 132.91, 132.78, 129.14,
128.43, 128.12, 114.04, 112.18, 111.20, 106.06, 18.76 ppm. MS: calcd for
C18H14O4N2 [M+Na]+ (m/z): 345.33; found: 345.52. (Scheme 1)
Scheme 1. Synthesis process of the probe L2.
2.3 Spectral measurement
A series of metal ions stock solutions (10 mM) and anions stock solutions (10
mM) were prepared from corresponding chlorine salts (PbCl2, CdCl2, MgCl2, CoCl2,
CuCl2, NiCl2, FeCl2, MnCl2, HgCl2, KCl, LiCl, NaCl, AlCl3, FeCl3, CrCl3 and ZnCl2)
and corresponding sodium salts (NaCl, NaBr, NaI, NaF, NaNO2, NaNO3, NaClO4,
Na2CO3, NaHCO3, Na2SO4, NaHSO4 and CH3COONa). The 1.0 mM probe L2 stock
solution was prepared with dimethylsulfoxide and dilute the probe solution to 10 μM
with the HEPES (0.05 M, pH=7.4) buffer solution. The corresponding fluorescence
emission spectra of the probe system in the presence of different 10 equiv. metal ions
were measured. The fluorescence response of the probe was discussed based on the
measured results. And the emission spectra of the probe in the presence of different

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concentrations of Al3+ were recorded. Besides, the interference experiments of
common ions were also discussed. Meanwhile, the selectivity of [L2+Al3+] complex
system towards F− was further studied by the optical spectrogram determination. The
excitation wavelength was 334 nm.
2.4 Preparation of the real samples
The Black tea and toothpaste were purchase from the local supermarket. A series of
pretreatment processes for the sample were carried out before testing. Samples of
black tea were dried in an oven and ground into powder. Accurately weigh 0.2 g of the
powder sample and ashing it in a ceramic crucible at high temperature. After cooling,
the residue was dissolved with 0.5 mL hydrochloric acid, and then the volume was
fixed to 5.0 mL with the HEPES buffer solution (pH=7.4). Shake well for reserve.
Likewise, the 10 mg of toothpaste was dispersed in 5.0 mL of distilled water. The
mixture was sonicated, centrifuged and then filtered. The filtrate was used for
qualitative analysis of F−.
2.5 Cell imaging
Firstly, the RAMOS cells were seeded at a 24-well plate and incubated in
medium (supplementing with RPMI 1640, 10% fetal bovine serum) at 37 ◦C in a
thermostat for 24 h [30]. Then the RAMOS cells were treated with 10 μM probe L2
for 30 min. After washing the cells with PBS buffer for three times, and the cells were
further incubated with 50 μM Al3+ for 30 min. Then continue to incubated with 50 μM
F− for 30 min. The fluorescence imaging of cells in each stage were analyzed by the
inverted fluorescence microscope (Germany，DM2500).

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3. Results and discussion
3.1 The selectivity of probe L2 to Al3+
The fluorescence respond of the probe L2 were investigated by measuring the
fluorescence emission spectra in the presence of different metal ions including Pb2+,
Mg2+, Cd2+, Co2+, Cu2+, Ni2+, Fe2+, Al3+, Mn2+, Hg2+, K+, Li+, Na+, Fe3+, Cr3+ and Zn2+.
As shown in Fig.1, the single probe L2 presented an extremely weak fluorescence
emission at 461 nm (Φ=0.02). When 10 equiv Al3+ was added, the emission peak of
the probe system at 461 nm increased significantly (Φ=0.41). But under the same
conditions, the addition of other metal ions did not cause large fluctuations in the
fluorescence emission of the probe system. Similarly, the fluorescence emission of the
corresponding probe mixed solution after the addition of different metal ions under
the irradiation of 365 nm ultraviolet lamp showed that the addition of Al3+ ions
induced the appearance of the visible blue fluorescence of the naked eyes. The above
results showed that probe L2 has good selective fluorescence response to Al3+ ions.
The Job’s plot was used to predict the binding ratio between probe L2 and Al3+ and
the results were shown in Fig.S5. The fluorescence intensity of the system reached the
maximum when the molar ratio of probe L2 to Al3+ is 1:1. This conclusion indicated
that the stoichiometric ratio of probe L2 to Al3+ is 1:1.

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Fig.1 Fluorescence spectra of probe L2 (10 µM) under the injections of 10 equiv.
different metal ions (Pd2+, Cd2+, Mg2+, Co2+, Cu2+, Ni2+, Fe2+, Al3+, Mn2+, Hg2+, K+,
Li2+, Na+, Fe3+, Cr3+ and Zn2+).
3.2 The anti-interference of the probe L2
The interference immunity of the probe to competing ions is an important
parameter to evaluate the practical application of a probe L2. So the anti-interference
performance of the probe system was tested when some common metal ions coexist.
As shown in Fig.2, probe L2 still has excellent single selectivity for the identification
of Al3+ in the presence of other interfering ions. The coexistence of other common
ions can not interfere with the recognition process of probe L2, and the fluorescence
intensity of system has not changed significantly. The above experimental phenomena
showed that the probe L2 has excellent anti-interference ability for Al3+ ion
recognition.

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L2+MI
L2+MI+Al3+
800
700
600
500
400
300
200
100
0
Pd2+ Cd2+ Cr3+ Fe3+ Cu2+Mg2+ Zn2+ Na+ Li+ Ni2+ K+ Co2+ Fe2+ Hg2+ Mn2+
Fig.2 Fluorescence selectivity of L2 (10 µM) toward Al3+ (10 equiv.) in the presence
of additional various metal ions (10 equiv.) in the same media.
3.3 Identification mechanism and binding mode
To verify that the probe L2 and Al3+ ions can form a stable complex with a ratio of
1:1, we analyzed the mass spectrum of the probe L2 system in the presence of Al3+.
As shown in Fig.3, there is a peak at m/z 345.55, which corresponded to the expected
mass of single probe L2 (calcd. for 345.33 [M+Na]+). The main peak located at m/z
456.61 (calcd. for 456.33 [L2+Al3++3Cl+H]+), which should corresponded to the peak
of the [L2+Al3+] complex. The results indicated that L2 and Al3+ formed a stable
complex with a ratio of 1:1, which is consistent with the result of Job’ plot.
Fig.3 The MS spectrum of probe L2 system in the presence of Al3+.

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Fig.4 DFT optimised structures of the probe L2 and [L2+Al3+] complex and the
corresponding HOMO and LUMO.
Besides, the geometry optimizations of probe L2, [L2+Al3+] complex were carried
out based on DFT using the Gaussian 16 program [31], in which the B3LYP function
and the def2-tzvp basis set were used. The optimized configurations and
corresponding the LUMO and HOMO [32] are shown in Fig.4, the results were
analyzed and the response mechanism of probe L2 to Al3+ was inferred as follows: the
single probe L2 molecule hardly showed any fluorescence due to the effect of the
internal charge transfer (ICT). The hydroxyl oxygen (-OH), acyl oxygen (C=O) and
ammonia nitrogen (C=N) atoms of the probe molecule can provide the sites for
binding of Al3+. The enhancement in fluorescence after the probe L2 combined with
Al3+ is probably due to the formation of a six member and a five member chelate ring
with the metal ion, which not only enhances the conjugation in the ligand framework

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but also increased the rigidity of the molecular assembly by restricting the free
rotations of the azomethine carbon. Besides, the chelation of L2 with Al3+ suppressed
the intramolecular charge transfer effect and the C=N double bond in the probe L2
structure also occurred the cis/trans isomeri-zation, which induced the chelation
enhanced fluorescence effect (CHEF). So the system presented a strong blue
fluorescence emission.
3.4 Relay identification
When a series of common anions were added to the [L2+Al3+] system, it is
surprising to found that the introduction of F− led to a large degree of quenching of
the fluorescence of the [L2+Al3+] system (Φ=0.02). This may be due to the
competitive combination of F− with Al3+, and to form the [AlF6]3− complexes. The
probe L2 returned to monomer form, so the fluorescence of the system disappeared.
−
−
However, the addition of other common anions such as Cl−, Br−, I−, NO2 , NO3 ,
−
2−
−
2−
−
ClO4 , CO3 , HCO3 , SO4 , HSO4 and CH3COO− did not cause the same optical
change under the same condition (Fig.5a). This phenomenon indicated that the
[L2+Al3+] complex can be used as a new probe system for the in situ identification of
F−.

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(a)
800
[L2+Al3+]+other anions
600
400
200
[L2+Al3+]+F-
0
400
450
500
550
600
Wavelength (nm)
Fig.5 Fluorescence spectra of [L2+Al3+] (10 µM) upon the addition of different anions.
λem=461 nm.
Similarly, the anti-interference performance of the [L2+Al3+] complex system as a
probe for the detection of F− was also discussed. As shown in Fig.6, the [L2+Al3+]
complex could still achieved a single identification of F− in the presence of other
−
−
−
2−
−
−
equivalent anions (Cl−, Br−, I−, NO2 , NO3 , ClO4 , CO3 , HCO3 , SO42−, HSO4 and
CH3COO−). As a result, the [L2+Al3+] complex has excellent single selectivity and
anti-interference ability for F−. In other words, the probe L2 could be used to build a
OFF-ON-OFF relay recognition detection system.
[L2+Al3+]+anions
[L2+Al3+]+F−+other anions
800
700
600
500
400
300
200
100
0
F− HCO3− Cl− ClO4− Br− CO32− Ac− HSO4− I− NO3− SO42−NO2
−
Fig.6 Fluorescence response of probe [L2+Al3+] (10 µM) toward F− (100 µM) in the
presence of various anions.
3.5 Optimization of determination condition (pH and response time)
The fluorescence response of probe L2 (10 μM) toward Al3+ and the [L2+Al3+]

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toward F− under different pH conditions was further studied, so as to determined the
suitable pH condition for the detection of the Al3+ and F− ions by the probe L2 and
probe [L2+Al3+] complex. As shown in Fig.7, the fluorescence emission intensity of
probe L2 in pH=3~12 is not significantly changed without adding Al3+ ions. It can
also be seen from the results that strong acid or strong base conditions had great
influence on the detection of Al3+ by probe L2. And in the range of pH 5.0~9.0, the
probe L2 system has the best effect on the detection of Al3+ and the fluorescence
emission intensity of the system tends to be stable. Meanwhile, the experimental
results that the detection effect of probe [L2+Al3+] is not ideal for F− under the
condition of over acid (pH<5), This is due to the low rate of coordination reaction
between Al3+ and F− under acidic conditions. Under the condition of over alkali, some
of Al3+ ions will form the [Al(OH)₄]− with hydroxyl groups, which seriously affected
the accuracy of F− content detection. Therefore, all the experiments were carried out
in HEPES buffer solution (0.05M, pH=7.4).
800
[L2+Al3+]
L2
[L2+Al3+]+F-
600
400
200
0
3
4
5
6
7
8
9
10 11 12 13
pH
Fig. 7 Fluorescence intensity of L2 (10 µM) at various pH values from 3 to 12, under
the absence and presence of Al3+ and F− (λex=334 nm, λem=461 nm).
The response time of probe L2 to Al3+ and [L2+Al3+] complex system to F− were

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further studied. The intensity of fluorescence emission of the corresponding test
solution were recorded every 10 s in the experiment. The results as shown in Fig.8,
the fluorescence emission intensity of probe L2 did not change with time. With the
addition of 10 µM Al3+, the fluorescence emission intensity of the probe system at 461
nm increased instantaneously, and the fluorescence intensity of the system could
reached the maximum after about 1 min, then the fluorescence intensity tended to be
stable. When the 10 equiv. F− was added to the [L2+Al3+] complex system, the
fluorescence intensity of the system decreased sharply, and the fluorescence
quenching process of the system required only 2 min. The above experimental results
showed that the probe L2 can realize fast relay recognition and detection for Al3+ and
F−. This study also provided a basis for the optimization of our detection process.
900
(b)
800
600
400
200
0
(a)
600
300
0
L2+Al3+
L2
0
30
60
90
120 150 180 210
0
30
60
90
120
150
180
210
Time(s)
Time(s)
Fig.8 (a) Time course of compound L2 (10 µM) responding to Al3+ (100 µM); (b)
Time course of ensemble [L2+Al3+] (10 µM) responding to F− (100 µM).
3.6 Fluorescent titration experiments (Al3+ and F-)
To future study the fluorescence response sensitivity of probe L2 to Al3+, the
fluorescence titrations of probe L2 (10 µM) in the presence of different concentration
of Al3+ (0~10 equiv.) were carried out. The results were shown in Fig.9(a), the

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fluorescence emission intensity at 461 nm of the probe L2 system increased gradually
with the increase of the addition of Al3+ concentration. And the fluorescence intensity
of 461 nm was enhanced to the maximum when the amount of Al3+ ions is up to 1.0
equiv. With continuously increasing the amount of Al3+, the fluorescence intensity of
the system tended to be stable and no longer enhanced. Moreover, there are a good
linear relationship (Fig.S6, R2=0.998) between the fluorescence emission intensity
and the concentration of Al3+ (0~1.0 equiv.). And the detection limit (L=3σ/slope,
slope=1.671×107, σ=0.08) of probe L2 to Al3+ can be as low as 0.014 µM [33, 34].
Based on the linear relationship between 1/[Al3+] and 1/[F-F0] in Fig.S7 and
1
1
1


Benesi-Hildebrand formula (
) [35], the binding
F  F k F  F c Fmax  F
0 

0
max
0
constants (k) between probe L2 and Al3+ ion were calculated to be 5.453×104 M−1.
Compared with Al3+ fluorescent probe that has been reported (Table 1), the probe L2
showed lower detection limit and higher sensitivity to Al3+. In contrast, the probe L2
seems to be more valuable.
Subsequently, the sensitivity of [L2+Al3+] complex system to F− was also
determined by the fluorescent titration experiment. As shown in Fig.9(b), the
fluorescence emission peak of [L2+Al3+] system at 461 nm decreased linearly with
the increase of F− ions content. When the concentration of F− ions reached 100 µM,
the fluorescence emission of [L2+Al3+] system is completely quenched. Through the
linear curve between the concentration of F− and fluorescence emission intensity in
Fig.S8, the detection limit of [L2+Al3+] to F− can be calculated to be 0.03 µM. Its
value is much lower than the maximum acceptable value of F− (4.0 mg L-1, 211 µM）

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in drinking water provided by the US Environmental Protection Agency. [42]
800
800
800
800
(a)
(b)
600
400
200
0
600
400
200
0
600
400
200
0
600
400
200
0
F-
Al3+
0
2
4
6
8
10
12
14
16
0
20
40
60
80
100
Al3+/10-5
M
[F-](M)
400
450
500
550
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Fig.9 (a)Fluorescence titration of L2 (10 µM) in the presence of different
concentration of Al3+ (0~10 equiv.), λex=334 nm. Inset: fluorescence emission
intensity at 461 nm changes with increasing concentration of Al3+; (b) Fluorescence
titration of [L2+Al3+] (10 µM) in the presence of different concentration of F− (0~10
equiv.), λex=334 nm. Inset: fluorescence emission intensity at 461 nm changes with
increasing concentration of F−.
Table 1 Comparison of different fluorescent sensors for Al3+ detection.
Fluorescent probes
Detection limi（t µM）
Reference
1.37
Sens. Actuators B, 2014, [36]
0.82
Spectrochim. Acta A, 2014, [37]
Anal. Chim. Acta, 2015, [38]
0.0195

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21.7
8
Inorg. Chim. Acta, 2015, [39]
RSC Adv, 2014, [40]
5.2
Synthetic. Met, 2015, [41]
0.014
This work
3.7. Analysis of real samples
In order to verify the practical application value of the probe L2, the detection of
trace target ions in the real samples (Black tea and Tooth paste) was studied.
Preparation of the samples to be analyzed was performed as described above in
Section 2.4. The detection data of standard addition and recovery experiments were
shown in Table 2. The content of Al3+ ions detected in Black tea samples is 0.07 μM,
the recovery rate of standard addition can reach as high as 99.6%. Alike, The content
of F− ions detected in Tooth paste samples is 0.04 μM and the recovery rate can reach
as 100.2%. This result showed that the probe can be applied to the detection of trace
target ions in real samples.
Table 2 The measured data of target ions from the real samples.
Sample
Target ions
Added (μM)
Found (μM)
Recovery (%)

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0
0.07±0.01
5.05±0.02
0.04±0.01
5.05±0.03
Black tea
Al3+
5.0
0
99.6%
Tooth paste
F−
5.0
100.2%
3.8 Cell imaging
In order to verify the practical application value of the probe L2, the fluorescence
imaging application of probe L2 in living cells was preliminarily explored. The cells
used in the imaging experiments were human lymphocytic tumor cells (RAMOS).
First, the cells were cultured by probe L2 and the cells imaging under the bright field
and fluorescence field was observed under inverted fluorescence microscope. The
cells were then treated with Al3+ and F− ions in turn. The corresponding cells imaging
were shown in Fig.10, the Fig.10a and Fig.10b are the imaging of cells after probe L2
treating under bright field and fluorescence field. At this time, the cells showed no
obvious fluorescence phenomenon. After Al3+ treatment, the cells showed strong blue
fluorescence emission (Fig.10c). But the fluorescence was quenched due to the
subsequent addition of F− (Fig.10d). This series of phenomena indicated that the
probe L2 has good cell permeability and can be used for the identification and
detection of target ions in biological systems. This is of great help in studying the
physiological functions of Al3+ and F− in organism.

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Fig.10 Images of RAMOS cells: (a) bright field and (b) fluorescence field images of
cells incubated with probe L2 (10 μM); (c) fluorescence field image of cells further
incubated with Al3+ (100 μM) (d) fluorescence field image of cells further incubated
with F− (100 μM).
4. Conclusions
A novel schiff base fluorescent probe (L2) based on coumarin fluorescence was
designed and synthesized. The probe L2 can be successfully applied to relay
recognition detection of Al3+ and F−. First, the probe L2 has high selectivity and
anti-interference ability for Al3+. The main phenomenon is that the system changed
from no fluorescence to strong blue fluorescence emission. And the titration
experiments also showed that the probe L2 has excellent sensitivity to Al3+ and the
minimum detection limit can be as low as 0.014 µM. In addition, the [L2+Al3+]
complex can also be used as a new probe for in-situ relay identification of F− ions.
The addition of F− will cause the fluorescence emission quenching of [L2+Al3+]
complex system. The detection limit of probe [L2+Al3+] for F− can be as low as 0.03
µM. Meanwhile, the identification mechanism of the probe was also discussed by the
mass spectrometry and DFT calculations. Moreover, the probe L2 can also be used as
a target ions detector to successfully detect the Al3+ and F− in living cells.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
31470434, 21576124, 21507047 and 21676124), the China Postdoctoral Science

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Foundation funded project (No. 2017M610308), and the project supported by the
Science Foundation of Jiangsu Entry-exit Inspection Quarantine Bureau
(No.2017KJ47). Meanwhile, thanks to Spartan HPC computing service of University
of Melbourne.
Referents
[1] R. Ali, S.S. Razi, P. Srivastava, M. Shahid, A. Misra, RSC Adv. 5 (2015)
61513-61520.
[2] J. Kumar, M.J. Sarma, P. Phukan, D.K. Das, Dalton Trans. 44 (2015) 4576-4581.
[3] Y.J. Chang, P.J. Hung, C.F. Wan, A.T. Wu, Inorg. Chem. Commun. 39 (2014)
122-125.
[4] R. Azadbakht, T. Almasi, H. Keypour, M. Rezaeivala, Inorg. Chem. Commun. 33
(2013) 63-67.
[5] A. Banerjee, A. Sahana, S. Das, S. Lohar, S. Guha, B. Sarkar, S.K. Mukhopadhyay,
A.K. Mukherjeea, D. Das, Analyst.137 (2012) 2166-2175.
[6] K. Boonkitpatarakul, J. Wang, N. Niamnont, B. Liu, L. Mcdonald, Y. Pang, M.
Sukwattanasinitt, ACS Sensors. 1 (2015) 144-150.
[7] T.P. Flaten, Brain. Res. Bull. 55 (2001) 187-196.
[8] A. Campbell, A. Becaria, D.K. Lahiri, K. Sharman, S.C. Bondy, J. Neurosci. Res.
75 (2004) 565-572.
[9] T. Han, X. Feng, B. Tong, J. Shi, L. Chen, J. Zhi, Y. Dong, Chem. Commun. 48
(2012) 416-418.
[10] S. Purser, P.R. Moore, S. Swallow, V. Gouverneur, Chem. Soc. Rev. 37 (2008)

ACCEPTED MANUSCRIPT
320-330.
[11] R. Martínezmáñez, F. Sancenón, Chem. Rev. 103 (2003) 4419-4476.
[12] L. Wang, G. Fang, D. Cao, J. Fluoresc. 24 (2014) 1757.
[13] S. Madhu, M. Ravikanth, Inorg. Chem. 53 (2014) 1646-1653.
[14] J. Xiong, L. Sun, Y. Liao, G.N. Li, J.L. Zuo, X.Z. You, Tetrahedron. Lett. 52
(2011) 6157-6161.
[15] S. Rivadehi, E.F. Reid, C.F. Hogan, S.V. Bhosale, S.J. Langford, Org. Biomol.
Chem. 10 (2012) 705.
[16] M. Bates, B. Huang, G.T. Dempsey, X. Zhuang, Science., 317 (2007) 1749-1753.
[17] C.R. Li, J.C. Qin, B.D. Wang, L. Fan, J. Yan, Z.Y. Yang, J. Fluoresc. 26 (2016)
345-353.
[18] Y.S. Mi, D.M. Liang, Y.T. Chen, X.B. Luo, J.N. Xiang, RSC Adv. 4 (2014)
42337-42345.
[19] M. Mukherjee, B. Sen, S. Pal, S. Banerjee, S. Lohar, P. Chattopadhyay, RSC Adv.
4 (2014) 64014-64020.
[20] L. Yan, Z. Kong, W. Shen, W. Du, Y. Zhou, Z. Qi, Anal. Biochem. 500 (2016)
1-5.
[21] X. Yong, M. Su, W. Wang, Y. Yan, J. Qu, R. Liu, Org. Biomol. Chem. 11 (2013)
2254-2257.
[22] R. Liu, Y. Gao, Q. Zhang, X. Yang, X. Lu, Z. Ke, W. Zhou, J. Qu, New. J. Chem.
38 (2014) 2941-2945.
[23] P. Ashokkumar, H. Weißhoff, W. Kraus, K. Rurack, Test-Strip-Based

ACCEPTED MANUSCRIPT
Fluorometric Detection of Fluoride in Aqueous Media with a BODIPY-Linked
Hydrogen-Bonding Receptor. Angew. Chem.Int. Edi. 53 (2014) 2225-2229.
[24] X. Zhu, J. Wang, J. Zhang, Z. Chen, H. Zhang, X. Zhang, Sensors. 15 (2015)
1611-1622.
[25] A. Guo, R. Zhu, Y. Ren, J. Dong, L. Feng, Spectrochim Acta A. 153 (2016)
530-534.
[26] Q.M. Wang, W. Gao, X.H. Tang, Y. Liu, Y. Li, J. Mol. Struct. 1109 (2016)
127-130.
[27] B.K. Datta, C. Kar, A. Basu, G. Das, Tetrahedron. Lett. 54 (2013), 771-774.
[28] H.Y. Jeong, S.Y. Lee, J. Han, M.H. Lim, C. Kim, Tetrahedron. 73 (2017)
2690-2697.
[29] E. Yıldız, Ş. Saçmacı, M. Saçmacı, A. Ülgen, Food. Chem. 237 (2017) 942-947.
[30] X. Tang, Z. Zhu, Y. Wang, J. Han, L. Ni, H. Zhang, J. Li, M. Yanli, Sens.
Actuators, B: Chem. 2018, 262: 57-63.
[31] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R.
Cheeseman, G. Scalmani, V. Barone, G.A. Petersson, H. Nakatsuji, X. Li, M.
Caricato, A.V. Marenich, J. Bloino, B.G. Janesko, R. Gomperts, B. Mennucci,
H.P. Hratchian, J.V. Ortiz, A.F. Izmaylov, J.L. Sonnenberg, D. Williams-Young, F.
Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D.
Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda,
O. Kitao, H. Nakai, T. Vreven, K. Throssell, J. A. Montgomery, Jr., J.E. Peralta, F.

ACCEPTED MANUSCRIPT
Ogliaro, M.J. Bearpark, J.J. Heyd, E.N. Brothers, K.N. Kudin, V.N. Staroverov,
T.A. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A.P. Rendell, J.C.
Burant, S.S. Iyengar, J. Tomasi, M. Cossi, J.M. Millam, M. Klene, C. Adamo, R.
Cammi, J.W. Ochterski, R.L. Martin, K. Morokuma, O. Farkas, J.B. Foresman,
and D.J. Fox, Gaussian, Inc., Wallingford CT, 2016.
[32] J.F. Zhang, C.S. Lim, S. Bhuniya, B.R. Cho, J.S. Kim, Org. Lett. 13 (2011)
1190-1193.
[33] J. Chao, Z. Li, Y. Zhang, F. Huo, C. Yin, H. Tong, Y. Liu, Sens. Actuators, B:
Chem. 228 (2016) 192–199.
[34] X. Tang, Y. Wang, J. Han, L. Ni, H. Zhang, C. Li, J. Ji, Y. Qiu, Dalton. Trans. 47
(2018) 3378-3387.
[35] H.A. Benesi, J.H. Hilderbrand, J. Am. Chem. Soc. 71 (1949) 2703–2707.
[36] O. Alici, S. Erdemir, Sens. Actuators, B: Chem. 208 (2015) 159-163.
[37] J.C. Qin, J.C. Qin, T.R. Li, B.D. Wang, Z.Y. Yang, L. Fan, Spectrochim Acta A.
133 (2014) 38-43.
[38] B.K. Datta, D. Thiyagarajan, C. Kar, A. Ramesh, G. Das, Anal. Chim. Acta. 882
(2015) 76-82.
[39] J. Zhao, Y. Zhao, S. Xu, N. Luo, R. Tang, Inorg. Chim. Acta. 438 (2015) 105-111.
[40] Y.S. Mi, D.M. Liang, Y.T. Chen, X.B. Luo, J.N. Xiang, RSC Adv. 4 (2014)
42337-42345.
[41] J.C. Qin, Z.Y. Yang, Synthetic. Met. 209 ( 2015) 570-576.
[42] B. Zhu, F. Yuan, R. Li, Y. Li, Q. Wei, Z. Ma, B. Du, X. Zhang, Chem. Commun.

ACCEPTED MANUSCRIPT
47 (2011) 7098-7100

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Graphical Abstract

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Highlights
• A novel fluorescence probe L2 based on coumarin has been designed and
synthesized.
• The probe L2 can be used for relay recognition of metal ions Al3+ and anion F−
and build a OFF-ON-OFF detection system
•
•
The corresponding detection limit could be as low as 0.014 µM (Al3+) and 0.03
µM (F−)
The probe have also been successfully applied to detection of target ions in living
cells.