Synthesis and application of a highly selective copper ions fluorescent probe based on the coumarin group

Article abstract of DOI:10.1016/j.saa.2017.09.028

A highly selective copper ions fluorescent probe based on the coumarin-type Schiff base derivative 1 (probe) was produced by condensation reaction between coumarin carbohydrazide and 1H-indazole-3-carbaldehyde. The UV–vis spectroscopy showed that the maximum absorption peak of compound 1 appeared at 439 nm. In the presence of Cu^{2 +} ions, the maximum peak decreased remarkably compared with other physiological important metal ions and a new absorption peak at 500 nm appeared. The job's plot experiments showed that complexes of 1:2 binding mode were formed in CH₃CN:HEPES (3:2, v/v) solution. Compound 1 exhibited a strong blue fluorescence. Upon addition of copper ions, the fluorescence gradually decreased and reached a plateau with the fluorescence quenching rate up to 98.73%. The detection limit for Cu^{2 +} ions was estimated to 0.384 ppm. Fluorescent microscopy experiments demonstrated that probe 1 had potential to be used to investigate biological processes involving Cu^{2 +} ions within living cells.

Full text of DOI:10.1016/j.saa.2017.09.028

Accepted Manuscript
Synthesis and application of a highly selective copper ions
fluorescent probe based on the coumarin group
Guangjie He, Xiangli Liu, Jinhe Xu, Liguo Ji, Linlin Yang, Aiying
Fan, Songjun Wang, Qingzhi Wang
PII:
DOI:
Reference:
S1386-1425(17)30747-3
doi: 10.1016/j.saa.2017.09.028
SAA 15462
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
12 July 2017
18 August 2017
12 September 2017
Please cite this article as: Guangjie He, Xiangli Liu, Jinhe Xu, Liguo Ji, Linlin Yang,
Aiying Fan, Songjun Wang, Qingzhi Wang , Synthesis and application of a highly selective
copper ions fluorescent probe based on the coumarin group, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.09.028
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ACCEPTED MANUSCRIPT
Synthesis and application of a highly selective copper ions
fluorescent probe based on the coumarin group
Guangjie Hea*, Xiangli Liua, Jinhe Xua, Liguo Jia*, Linlin Yanga, Aiying Fana, Songjun
Wangb, Qingzhi Wanga
a Department of Forensic Medicine, Xinxiang Medical University, Jinsui Road No.
601, Xinxiang, 453003, Henan Province, P. R. China
b
Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, East
Zhongshan Road No. 361, Shijiazhuang, 050017, Hebei Province, P. R. China
* Corresponding author: Tel./Fax: +86-373-3831183
E-mail: guangjiehe@163.com; liguoji_academy@126.com
Absract
A highly selective copper ions fluorescent probe based on the
coumarin-type Schiff base derivative 1 (probe) was produced by
condensation reaction between coumarin carbohydrazide and
1H-indazole-3-carbaldehyde. The UV-vis spectroscopy showed that the
maximum absorption peak of compound 1 appeared at 439 nm. In the
presence of Cu2+ ions, the maximum peak decreased remarkably
compared with other physiological important metal ions and a new
absorption peak at 500 nm appeared. The job’s plot experiments showed
that complexes of 1:2 binding mode were formed in CH3CN:HEPES (3:2,
v/v) solution. Compound 1 exhibited a strong blue fluorescence. Upon
addition of copper ions, the fluorescence gradually decreased and reached
a plateau with the fluorescence quenching rate up to 98.73%. The
detection limit for Cu2+ ions was estimated to 0.384 ppm. Fluorescent
1

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microscopy experiments demonstrated that probe 1 had potential to be
used to investigate biological processes involving Cu2+ ions within living
cells.
Keywords: fluorescent probe, Cu2+ ions probe, coumarin, cell imaging
1. Introduction
Copper ions as an important existing form of copper element are
prevalent in lives; its content in the body is only less than iron and zinc1, 2.
It plays significant roles in pathophysiology metabolism and metabolic
activities of various enzymes in the organisms. For instance, copper ions
as a catalytic cofactor are involved in mitochondrial respiration, iron
absorption and redox process of a large number of enzymes such as SOD,
dopamine β-hydroxylase, lysyloxidase, tyrosinase, cytochrome oxidase,
etc3-5. The normal concentration of the copper ions in human body is
about 15.7 ~ 23.6 μM6. Thus abnormalities of the copper ions may lead to
some severe neurodegenerative diseases and disorders of growth and
metabolism7. Especially in recent years the environment pollution
exposed people for a long time in the circumstances of high concentration
of Cu2+ and induce to an emergence of series of neurodegenerative
diseases caused by copper metabolism disorders8-10, such as Alzheimer’s
diseases (AD), Menkes syndrome, Parkinson’s diseases (PD), Huntington
diseases (HD), familial amyotrophic lateral sclerosis (ALS) and many
others11-13. Therefore, design of a rapid, convenient, highly selective and
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sensitive detection method for copper ion is significant and always
attracts a great deal of attentions14-20
.
The fluorescent analysis method relative to the electrochemical
analysis method, inductively coupled plasma mass spectrometry21, flame
atomic absorption spectrometry22, flow injection analysis23,
spectrophotometry, etc., serving for determination of copper ions has the
advantages of high sensitivity, high selectivity, low cost, easy operation,
fast analysis, less sample size and real time in situ detection. In addition,
the detection of copper ions in the body could be realized by fluorescence
imaging technology24, 25. Thus a further research work is also needed
although some relate works have been reported.
Coumarin molecules possessed high fluorescence intensity, strong
light stability, good solubility, easy preparation, high molar absorption
coefficient and high fluorescence quantum yield, thus they were usually
served as chromophoric group to synthesis fluorescent probes26-31. Based
on this, a new fluorescent probe of coumarin-type schiff derivative
(compound 1) was synthesized by the condensation reaction between
coumarin carbohydrazide and 1H-indazole-3-carbaldehyde. This probe
was characterized by NMR and ESI-MS spectrums, and manifested
obviously selective to Cu2+ ions in the following experiments results.
Meanwhile, the interaction of this fluorescent compound with other metal
ions was also discussed by UV-vis absorption and fluorescent
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spectroscopy.
2. Experiment section
2.1 Instruments
The NMR spectra data were recorded with chemical shifts as ppm
with TMS as internal standard measured on a Bruker Ascend™ 400
spectrometer. Mass spectrometric data were measured on a Bruker
Microtof-QIII spectrometry. UV-vis absorption spectra data were
obtained by Shimadzu UV2600 spectrophotometer. Fluorescence spectra
were recorded with Edinburgh Instruments FS-5 fluorescence
spectrophotometer. Cell imaging was recorded with Leica DMI8 inverted
fluorescence microscope.
2.2 Reagents
All compounds were analytical grade and directly used as received.
4-(diethylamino)-2-hydroxybenzaldehyde, diethyl malonate, piperidine,
hydrazine hydrate (80%) and the other chemical reagents were purchased
from Energy Chemical Co. Ltd. Stock solutions (2.0×102 M) of the
perchlorate Mn2+, Fe3+, Fe2+, K+, Mg2+, Pb2+, Al3+, Zn2+, Ag+, Ni2+, Cd2+,
Ca2+, Na+, Cr3+, Co2+, Hg2+ and Cu2+ ions were prepared in aqueous
solutions. In the spectral measurements, the stock solutions of compound
1 were prepared in CH3CN:HEPES (3:2, v/v) solutions. A 3 mL solution
of compound 1 was filled in a quartz cell of 1 cm optical path length each
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time, by using a micro-syringe different stock solutions of metal ions
were added into the quartz cell gradually.
2.3 Synthetic procedures
COOEt
O O
CHO
OH
CH2(COOEt)2
piperidine
NH2NH2
Et2N
Et2N
3
H
N
NH
N
N
O
O
O
N
N
H
O
NHNH2
Et2N
O
O
Et2N
O
2
)
2
1
O 4
l
C
(
u
C
-
ClO4
Cu2+
NH
N
O
O
N
HClO4
N
Cu2+
-
Et2N
O
ClO4
-
ClO4
complex 1-Cu2+
Scheme.1 synthetic route of compound 1 and the presumable structure of complex 1-Cu2+
The synthetic procedures were shown in Scheme 1. The coumarin
hydrazine (compound 2) and ethyl-7-(diethylamino)-2-oxo-2H-chromene
-3-carboxylate (compound 3) were synthesized according to the previous
work32, 33. Compound 1 (probe) was synthesized conveniently from the
reaction of compound 2 with 1H-indazole-3-carbaldehyde and
1
13
characterized by H NMR, C NMR, and ESI-MS (Fig.S1, S2, S3), and
the complex 1-Cu2+ was prepared by compound 1 and Cu(ClO4)2·6H2O
under reflux condition for 3 h. The experimental details could be found in
Supplementary Information.
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3. Results and Discussions
3.1 The experiment of water solubility
The application of the probe in vivo was obviously influenced by its
water solubility, so a fluorescent stability test of the probe was proceeded
in varied solutions in which the ratio of CH3CN and HEPES buffer were
changed (Fig.S4). The results indicated that the fluorescent intensity was
stable until the proportion of the HEPES in the solution was up to 40%. A
drastically decreasing was observed when more HEPES solution was
added. So the buffer solution (CH3CN:HEPES = 3:2, v/v) was used to
proceed the following tests.
3.2 The pH dependence of the probe
For reducing the system error caused by the pH, the pH dependent
experiment of the compound 1 and compound 1-Cu2+ were proceeded in
the solutions (CH3CN:HEPES = 3:2, v/v) (Fig.S5). The results
demonstrated that the fluorescent intensity of both compounds was no
obviously changed in the pH 5.5-7.5. Thus the pH range of the solutions
in the following tests was controlled in 7.2-7.4 and which also indicated
that the probe could be applied in physiological condition.
3.3 The UV-vis spectra responses of the probe
Metal ions selectivity of the probe was investigated by UV-vis
spectrometry. Various equivalent metal ions such as Mn2+, Fe3+, Fe2+, K+,
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Mg2+, Pb2+, Al3+, Zn2+, Ag+, Ni2+, Cd2+, Ca2+, Na+, Cr3+, Co2+, Hg2+, Cu2+
-
(as ClO4 salts) were added into solutions (10 μmol/L, CH3CN:HEPES =
3:2, v/v) of compound 1. The intensity of maximum absorption peak (439
nm) weaken and slightly red shift was induced by addition of Cu2+ and
Hg2+ ions while other metal ions were not observed obviously (Fig.S6).
These results indicated that the probe could selectively recognized Cu2+
ions. Then the UV-vis spectra titration experiment of the probe 1 with
Cu2+ ions was also proceeded (Fig.1). With the increasing concentration
of the Cu2+ ions, obviously, the sharp decreasing and red shift of the
maximum absorption peak at 439 nm were observed in the titration
experiments, approximately 6 equivalents Cu2+ ions could induce
equilibrium. Meanwhile, a new absorption peak at 500 nm appeared
which indicated that a new species was formed in the solution.
Fig.1 The UV-vis spectra titration experiments of the probe 1 (10 μmol/L) with Cu2+ ions in
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solution (CH3CN:HEPES = 3:2, v/v). Insert: UV-vis titration profile of compound 1 upon addition
of Cu2+, the absorption spectra were recorded at 439 nm.
For further understanding the binding mode of the compound 1 with
the copper ions, a job’s plot experiment was carried on (Fig.S7) in a
solution (CH3CN:HEPES = 3:2, v/v). The results indicated that the
binding mode of the compound 1 relative to Cu2+ was 1:2. The binding
constant was also calculated from the UV-vis spectra titration dates by
employing the Benesi–Hildebrand (B-H) relation34-36. The constant value
was calculated as K = 1.18 109 M-2 (R=0.994) (Fig.S8).
3.4 The fluorescent spectra responses of the probe
The fluorescence experiments of the probe were also investigated.
The results demonstrated that the maximum emission peak of the
compound 1 appeared at 483 nm (excited at 439 nm). Only the copper
ions could lead the fluorescence of the compound 1 (10 μmol/L) to
quench dramatically, which was similar to the results that obtained in the
UV-vis spectra experiments. The fluorescent quenching rate in the
solution (CH3CN:HEPES = 3:2, v/v) was evaluated up to 98.73%. Other
metal ions such as Mn2+, Fe3+, Fe2+, K+, Mg2+, Pb2+, Al3+, Zn2+, Ag+, Ni2+,

Cd2+, Ca2+, Na+, Cr3+, Co2+, Hg2+, Cu2+ (as ClO4 salts) induced the
fluorescence almost no responses (Fig.2). As to the mechanism of
fluorescence of probe 1 was quenched by Cu2+ ions, it might be attributed
to the photoinduced electron transfer (PET) mechanism or d-d electron
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paramagnetic quenching mechanism of Cu2+ ions37-40
.
Fig.2 The fluorescence intensity variation (at 483 nm) of the compound 1 (10 μmol/L) in solutions

(CH3CN:HEPES = 3:2, v/v) after addition of various metal ions (as ClO4 salts)
For further proving the selectivity of probe to copper ions, the
competitive fluorescent experiments of compound 1 with copper ions
under the existences of other different metal ions were carried on (Fig.S9).
The fluorescence intensity (at 483 nm ) of the probe (10 μmol/L,
CH3CN:HEPES = 3:2, v/v) was not changed distinctly under equivalent
various metal ions such as Mn2+, Fe3+, Fe2+, K+, Mg2+, Pb2+, Al3+, Zn2+,

Ag+, Ni2+, Cd2+, Ca2+, Na+, Cr3+, Co2+ and Hg2+ (as ClO4 salts). An
obviously decreasing of the fluorescent intensity (at 483 nm) relative to
compound 1 was observed when the copper ions were added subsequently.
This was coincident with the results observed in UV-vis experiments that
the probe had a good selectivity to copper ions relative to other metal
ions.
The fluorescent titration experiments were further investigated in
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solution (CH3CN:HEPES = 3:2,v/v) (Fig.3). It was indicative that an
obviously decreasing of the fluorescent intensity was observed along with
the copper ions were added. Approximately 4 equivalents copper ions
added lead the fluorescence quenching completely, instead of 2
equivalents that observed in the job’s plot experiments, which might be
due to the moderate strength binding ability between compound 1 and
copper ions. For further interpreting this point, the binding constant (K)
for the complex formed between compound 1 and Cu2+ ions was
determined by employing Benesi–Hildebrand (B-H) relation34-36, it was
calculated as K = 1.91 109 M-2 (R=0.994) (Fig.S10), which was similar
to the result obtained from the UV-vis spectra titration dates.
Fig.3 The fluorescence spectra titration experiments of the compound 1 (10 μmol/L) with Cu2+

ions (as ClO4 salts) in the solution (CH3CN:HEPES = 3:2, v/v). Insert: the fluorescece titration
profile of compounds 1 upon addition of Cu2+ (excited at 439 nm, recorded at 483 nm).
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In order to investigate the sensitivity of the probe to copper ions, the
minimum limit test was performed in the solution (CH3CN:HEPES = 3:2,
v/v). The minimum limit was calculated to 6 μM (0.384 ppm, decreased
up to 11.67%) when the concentration of probe was 1 μM. A good fitting
curve demonstrated that there was a good linear relationship between the
fluorescent intensity of probe and the concentration of copper ions
(Fig.4).
Fig.4 The fitting curve of the fluorescent intensity (at 483 nm) of the probe (1 µM) relative to
copper ions in the solution (CH3CN:HEPES = 3:2, v/v).
3.5 The Fluorescent images of probe in living cells
The fluorescent methods to monitor the biological species are
significant. Fluorescence images of HepG-2 cells were observed under a
Leica DMI8 inverted fluorescence microscopy. Firstly, HepG-2 cells were
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incubated for 24 hours in RPMI-1640 culture medium containing 10%
fetal bovine serum. Then compound 1 (2 μM) was added and continually
incubated for 3 hours. It showed a clear green-blue intracellular
fluorescence (Fig 5a), which demonstrated that compound 1 was cell
permeable. After stained with the probe for 3 hours and rinsed with PBS
buffer three times, HepG-2 cells were supplemented with 5 equivalents of
Cu2+ for another 5 minutes; a remarkable decreasing of the fluorescence
intensity was observed (Fig 5b). The cells were still alive and the probe
showed no obviously toxic and side effects during the imaging
experiments (approximately 2 hours). These results were indicative that
compound 1 could be used to detect intracellular Cu2+ ions. Therefore,
this probe had potential application value to investigate biological
processes involving Cu2+ ions within living cells.
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Fig.5 Fluorescence images of HepG-2 cells incubated with the probe (2 μM) (a), further incubated
with addition of Cu(ClO4)2 (5 equivalents) (b) , and their corresponding bright field images (c, d).
4. Conclusions
In summary, based on coumarin carbohydrazide and
1H-indazole-3-carbaldehyde, a new fluorescent probe 1 was synthesized
for detection of Cu2+ ion. The UV-vis and fluorescent spectral
experiments indicated that the probe has excellent selectivity and
sensitivity toward Cu2+ ions. The 1:2 binding ratio of the compound 1
with Cu2+ ions was confirmed by the jobs’ plot experiments. The results
of competitive experiments further confirmed its outstanding selectivity
in the presence of other metal ions. The detection limit of the probe to
copper ion was 0.384 ppm. The Fluorescent microscopy experiments
demonstrated that probe 1 could be used to investigate biological
processes involving Cu2+ ions within living cells. Its other applications in
life science are still underway.
Acknowledgments:
This work was financially supported by the National Natural Science
Foundation of China (No.21371148), the Doctoral Scientific Research
initiation
Foundation
of
Xinxiang
Medical
University
(No.XYBSKYZZ201649), the Hebei Key Laboratory of Forensic
Medicine (No.KF201601), and the Disciplinary group of Psychology and
Neuroscience, Xinxiang Medical University.
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Graphical abstract
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Highlights:
a) Based on the coumarin-type Schiff base derivative a novel fluorescent probe for
copper ions detection was produced.
b) The probe displayed high selective and low detection limit for Cu2+ ions.
c) The probe could be used to investigate biological processes involving Cu2+ ions
within HepG-2 cells.
17