A new “ON-OFF” fluorescent and colorimetric chemosensor based on 1,3,4-oxadiazole derivative for the detection of Cu2+ ions

Article abstract of DOI:10.1016/j.jphotochem.2018.04.015

A symmetrical oxadiazole derivative (OXD) containing two pyrazole moieties was synthesized successfully and characterized by single crystal X-ray diffraction, nuclear magnetic resonance and mass spectrometry. The synthesized compound displayed “On-Off” fl

Full text of DOI:10.1016/j.jphotochem.2018.04.015

Accepted Manuscript
Title: A new “ON-OFF” fluorescent and colorimetric
chemosensor based on 1,3,4-oxadiazole derivative for the
detection of Cu2+ ions
Authors: Lin Wang, Qijing Bing, Jianxin li, Guang Wang
PII:
S1010-6030(18)30261-2
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.04.015
JPC 11230
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
25-2-2018
23-3-2018
7-4-2018
Please cite this article as: Lin Wang, Qijing Bing, Jianxin li, Guang Wang, A new “ON-
OFF” fluorescent and colorimetric chemosensor based on 1,3,4-oxadiazole derivative
for the detection of Cu2+ ions, Journal of Photochemistry and Photobiology A:
Chemistry https://doi.org/10.1016/j.jphotochem.2018.04.015
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A new “ON-OFF” fluorescent and colorimetric chemosensor based
on 1,3,4-oxadiazole derivative for the detection of Cu2+ ions
Lin Wang, Qijing Bing, Jianxin li, Guang Wang*
Faculty of Chemistry, Northeast Normal University, Changchun 130024, P. R. China
Corresponding author: Guang Wang
Tel: 86+431+85099544;
Fax: 86+431+85098768.
E-mail： wangg923@nenu.edu.cn (G. Wang)
Graphical Abstract
Highlights

A new “ON-OFF” fluorescent and colorimetric chemosensor based on 1,3,4-oxadiazole
derivative for the detection of Cu2+ ions

Lin Wang, Qijing Bing, Jianxin Li, Guang Wang*
Faculty of Chemistry, Northeast Normal University, Changchun 130024, P. R.
China

 ► Pyrazole-containing oxadiazole derivative as fluorescent chemosensor (sensor
1) was synthesized successfully.
 ► Sensor 1 displayed high selective and sensitive fluorescence sensing for Cu2+.
 ► Sensor 1 showed high anti-interference ability in the co-existence of other
metal ions.

► Sensor 1 also showed the ability of naked-eye detection for Cu2+ ions.
Abstract: A symmetrical oxadiazole derivative (OXD) containing two pyrazole
moieties was synthesized successfully and characterized by single crystal X-ray
diffraction, nuclear magnetic resonance and mass spectrometry. The synthesized
1

compound displayed “On-Off” fluorescent sensing towards Cu2+ with high selectivity
and sensitivity. Fluorescence and UV-vis absorbance spectra studies demonstrated
that the proposed sensor can detect Cu2+ directly in acetonitrile solution over a wide
range of metal ions including Ag+, Na+, K+, Hg2+, Mg2+, Cd2+, Co2+, Ni2+, Zn2+, Fe2+,
Pb2+, Ca2+, Mn2+, Ba2+, Fe3+, Al3+, and Cr3+. Moreover, the synthesized sensor
exhibits an instantaneous naked-eye color change from colorless to yellow with the
presence of Cu2+ ion. The fluorescence intensity of the sensor has shown a linear
response to Cu2+ in the concentration range of 0～15 μM with a detection limit of 2.14
μM. Fluorescence titration, 1H NMR spectrum, ESI-MS, and DFT calculation
confirmed that fluorescence quenching is caused by the complex formation between
the sensor and Cu2+ with 1:1 stoichiometry.
Keywords: Chemosensor; Fluorescence; Colorimetric; Oxadiazole; Copper ion.
1. Introduction
As the third most abundant transition metal ion in the human body, copper ions
plays pivotal roles as trace element in biological systems and process [1-2]. Cu2+ ion
is one kind of the essential trace metal ion to sustain normal human health and serves
as a cofactor for a wide variety of enzymes in all living organisms. Although Cu2+
ions are the essential trace elements after iron and zinc in the human body, they are
harmful to humans and organisms at high concentration [3]. Copper ions can
accumulate in the environment, which finally results in contaminated food and water.
According to the World Health Organisation (WHO), 1 mg·L-1 is the maximum
2

acceptable limit of copper for drinking water. High Cu2+ concentration in neuronal
cytoplasm can lead to Wilson’s disease, Menkes’s disease and Alzheimer’s disease
[4-5], overloading copper exhibits toxicity and causes irritation of nose and throat
resulting in nausea, vomiting, and diarrhea [6]. The traditional detection methods for
metal ions such as coupled plasma atomic emission spectrometry (ICP-AES) [7-8],
atomic absorption spectroscopy (AAS) [9], fluorescence techniques [10] and
electrochemical methods (EM) [11] require sophisticated equipment, tedious sample
preparation procedures. In addition, these analytical instruments are expensive and
need trained operators [12].
Compared to these traditional analytical methods, fluorescent chemosensors have
many advantages such as real-time monitoring with fast response times, intrinsic high
selectivity and sensitivity, ease of handling and low-cost [13-14]. Up to now, a lot of
fluorescent and colorimetric chemosensors of Cu2+ based on small organic molecules,
conjugated polymers, nanoparticles and biomolecules were reported [15-21].
Although the researchers have been made a lot of contribution to chemosensors,
development of new fluorescent chemosensors with high sensitivity and selectivity for
the detection of heavy metal ions in real samples is still urgent.
Most of oxadiazole derivatives have been used as fluorescent emitters for
OLEDs due to their excellent photophysical properties and chemical stabilities [22-
23]. Till now, to the best of our knowledge, only several reports are relative with the
oxadiazole derivatives as fluorophore in chemosensor and presented excellent sensing
3

properties for metal ions [24-25]. So, further exploring new fluorescence
chemosensors with high selectivity and sensitivity based on oxadiazole is significant.
On the basis of the above concept, we report herein a novel highly sensitive and
selective “on-off” fluorescent chemosensor towards Cu2+ based on a 1,3,4-oxadiazole
as the fluorophore and the pyrazole as the receptor (sensor 1 in Scheme 1). Sensor 1
contained two pyrazole units and presented symmetrical structure, which was hoped
to provide special steric configuration and selectively coordinate with metal ion. The
experimental results demonstrated that sensor 1 exhibited obvious fluorescence
change and a clearly visible color change from yellow to colorless upon the addition
of Cu2+ ions in acetonitrile medium.
Scheme 1. The synthetic route of sensor 1
2. Experimental
2.1. Materials and methods
All solvents were purchased from commercial sources and purified by standard
methods. Pyrazole was bought from Aladdin Industrial Corporation and used without
4

further purification. All other chemicals were obtained from Sinopharm Chemical
Reagent Co. Lid and were analytical grade reagent. Double-distilled water was used
for all experiments. The salts used in stock solutions of metal ions are CuCl2·2H2O,
NaCl, Ni(NO3)2·6H2O, Al(NO3)3·9H2O, MnCl2.4H2O, Zn(NO3)2·6H2O,
Cr(NO3)3·9H2O, Cd(NO3)2`4H2O, Fe(NO3)3·9H2O, Co(NO3)2·6H2O,
Mg(NO3)2·6H2O, KNO3, AgNO3, BaCl2·2H2O, HgCl2, PbCl2, CaCl2 and
FeSO4·7H2O.
1H NMR and 13C NMR spectra were obtained on Varian Unity Inova
Spectrometer 400 MHz. ESI-MS mass spectra were obtained on an agilent 1200
HPLC/Micro TOF Ⅱmass spectrometer. UV-vis absorption and fluorescent spectra
were recorded on Varian Cary 500 Spectrophotometer and Varian Cary Eclipse
Fluorescence Spectrophotometer, respectively.
2.2. Synthesis and characterization
2,5-(2-methylphenyl)-1,3,4-oxadiazole (Compound 3)
A mixture of o-toluic acid (6.8 g, 0.05 mol), hydrazine hydrochloride (2.6 g,
0.025 mol) and phosphoric acid (13.5 mL, 0.25 mol) in a 250 mL flask was stirred at
room temperature. Phosphorus oxychloride (4.6 mL, 0.05 mol) and phosphorus
pentoxide (21.3 g, 0.15 mol) were added dropwise into the above reaction mixture
successively. The mixture was heated to 140 ℃under stirring and nitrogen
atmosphere for 2 h. After cooling to room temperature, the mixture was poured into
ice water. The precipitate was obtained by filtration and washed with the solution of
5

NaHCO3 (85%) and deionized water. The crude product was recrystallized from
ethanol to give 2,5-(2-methylphenyl)-1,3,4-oxadiazole as a white crystal, 3.64 g,
yield, 58.2%. 1H NMR (600 MHz, CDCl3) δ 8.04 (d, J = 7.5 Hz, 2H), 7.48 – 7.33 (m,
6H), 2.77 (d, J = 19.8 Hz, 6H). 13C NMR (CDCl3, 600MHz, ppm): 164.43, 138.51,
131.84, 131.21, 129.00, 126.23, 123.07, 22.23. HR-ESI MS calcd for C16H14N2O
250.30 (3+, 100%), found 251.1185 m/z ([3+ H+]+).
2,5-bis [(2-bromomethyl)phenyl]-1,3,4-oxadiazole (Compound 2)
2,5-(2-methylphenyl)-1,3,4-oxadiazole (2.0 g, 8 mmol) was dissolved in purified
carbon tetrachloride (50 mL). After the solution was refluxed for 0.5 h under stirring,
N-bromosuccinimide (NBS, 4.27 g, 24.0 mmol) and diphenylperoxyanhydride (BPO,
0.24 g, 0.01 mol) were added. The mixture was further refluxed for another 5 h. After
the resulted mixture cooled down, the solvent was removed [25]. The residue was
washed with hot water three times, dried under vacuum and recrystallized with THF
and ethanol (THF:ethanol=1:1) to afford white solid of 2,5-bis [(2-
bromomethyl)phenyl]-1,3,4-oxadiazole, 2.17 g, yield, 66.4%. 1H NMR (400 MHz,
CDCl3, ppm) δ 8.14 (d, J = 7.6 Hz, 2H), 7.63 (d, J = 7.1 Hz, 2H), 7.53 (m, 4H), 5.20
(s, 4H). 13C NMR (CDCl3, 600MHz, ppm): 137.73, 132.15, 131.98, 129.65, 129.07,
100.00, 31.73. HR-ESI MS calcd for C16H12Br2N2O 408.09 (2+, 100%), found
408.9369 m/z ([2+ H+]+).
2,5-Bis[(2-pyrazolyl) methyl]-1,3,4-oxadiazole (sensor 1).
6

A mixture of pyrazole (1.09 g, 16.0 mmol) and NaOH (0.64 g, 16.0 mmol) in
deionized water (5 mL) was stirred under heating in an oil bath at 40 oC for 5 min.
2,5-bis [(2-bromomethyl)phenyl]-1,3,4-oxadiazole (0.82 g, 2.0 mmol) in THF (10
mL) was added dropwise to the previous mixture in 10 min. The resulting solution
was then heated to 70 ℃for 24 h. The resulting mixture was cooled to room
temperature, poured into water, and filtered. The crude product was further purified
by column chromatography on silica gel (ethyl acetate: petroleum ether = 5:2) to give
white solid, 0.43 g, yield, 56.8%. 1H NMR (400 MHz, CDCl3, ppm) δ 8.08 (m, 2H),
7.60 (d, J = 2.5 Hz, 4H), 7.49 (t, J = 5.4 Hz, 4H), 7.01 (d, J = 6.7 Hz, 2H), 6.32 (t, J =
2.1 Hz, 2H), 5.99 (s, 4H); 13C NMR (CDCl3, 600MHz, ppm): 163.58, 139.96, 137.53,
132.11, 130.46, 129.18, 129.00 128.20, 121.17, 105.91, 54.11. HR-ESI MS calcd for
C22H18N6O 382.15 (1+, 100%), found 383.1622 m/z ([1+ H+]+), 405.1445 m/z ([1+
Na+]+) and found 315.1283 m/z ([1- pyrazol]+).
2.3. Preparation of sensor 1 and metal ion solutions
The stock solution of sensor 1 was prepared in acetonitrile with the concentration
of 1×10-4 M and further diluted according to the actual requirements.
The solutions of metal ions with a concentration of 2×10-2 M in water were
prepared using their inorganic salts, including nitrate salts (Fe3+, Al3+, Mg2+, K+, Cd2+,
Co2+, Cr3+, Zn2+ and Ag+), chloride salts (Cu2+, Cu+, Hg2+, Ni2+, Pb2+, Ca2+, Mn2+,
Ba2+, Na+) and FeSO4·7H2O.
2.4. Detection of sensing properties of sensor 1 for copper ions
7

2.4.1. Selective experiment
Both absorbance and fluorescence spectra were used to investigate the selective
response of sensor 1 towards metal ions. The different metal ion in water solutions
(20 equiv. of sensor 1) were added with pipette into sensor 1 solution (1×10-5 M),
respectively. After mixing them for 10 s, UV–vis absorbance and fluorescence spectra
were taken at room temperature.
2.4.2. Fluorescence titration
For each titration solution with different molar ratio of Cu2+ to sensor 1 was
prepared by adding different volumes [µL] of Cu2+ solution to sensor 1 solution
(1×10-5 M), the maximum added volume of the metal ion solution did not exceed 2%
volume of sensor 1 solution in order to avoid the effect of concentration on
fluorescence spectra. The concentration of sensor 1 in every solution was kept at
1×10-5 M. The fluorescence spectra were taken at room temperature after finishing the
solution preparation for 10 s.
2.4.3. Job’s plot measurement
The stoichiometry of complexes can be obtained using Job’s plot via the
measurement of fluorescence spectra. Job’s plot was drawn based on the measurement
of a series of solutions in which the molar concentrations of ligand and metal ion vary
but their sum remains constant. The fluorescence spectrum of each solution is
measured and then the maximum value of emission was plotted against the mole
8

fraction of ligand or metal ion. The maximum on the Job’s plot appear at the mole
ratio corresponding to the combining ratio of the complex.
3. Results and discussion
3.1. Crystal growth and X-ray crystallography
The molecular structure of sensor 1 was also confirmed by the single crystal
analysis. The colorless sensor 1 crystals with the size of 0.13 mm × 0.11 mm × 0.10
mm were obtained by slow evaporation method in DMSO after 6 days. Single-crystal
X-ray diffraction data for sensor 1 were recorded using a Bruker Apex CCD
diffractometer with graphite monochromated Mo Kα radiation (λ= 0.71073˚A) at 293
K. Absorption corrections were applied using a multi-scan technique. The structure
was solved by direct method of SHELXS-97 and refined by full-matrix least-squares
techniques using the SHELXL-97 program [26] within WINGX [27].
All non-hydrogen atoms were easily found from the Fourier difference maps and
refined with anisotropic temperature parameters. The hydrogen atoms were generated
geometrically. A summary of the crystal data, structure solution and refinement is
provided in Table S1.
Single-crystal X-ray structural analysis reveals that sensor 1 crystallized in the
orthorhombic space group Pca2(1). As shown in Fig. 1a, the crystal structure of
sensor 1 consists of two parts, 2,5-Diphenyl-1,3,4-Oxadiazole and 1-methyl-1H-
pyrazole. Adjacent molecules are packed together through π-π interactions (Fig. 1b)
between the 1,3,4-oxadiazole groups and the phenyl groups [the distances of centroid-
9

to-centroid and centroid-to-plane (based on the phenyl groups) are 3.62 and 3.54 Å,
respectively]. Then, the molecules are packed against each other through weak
interactions to afford fascinating supramolecular layers (Fig. 1c).
Crystallographic data for the structure reported in this paper has been deposited
with the Cambridge Crystallographic Data Centre, CCDC no. 1559079. Copies of this
information are obtained free of charge from the Director, CCDC, 12 UNION Road,
Cambridge 1EZ, UK (fax: 44 01223 336033, deposit@ccdc.cam.ac.uk or
http://www.ccdc.cam.ac.uk).
Fig.1. (a) View of the crystal structure of sensor 1 molecule. (b) View of the π-π interactions
between two sensor 1 molecules. (c) View of the packing structure of sensor 1 along the a
axis.
3.2. Photophysical properties (absorption and fluorescence spectra)
The absorbance and fluorescence spectra of sensor 1 in CH3CN solution are shown
in Fig. 2. The absorption band between 270~325 nm with the maximum absorption
peak at 279 nm and emission band between 300～470 nm with maximum emission
peak at 352 nm (λex = 270 nm) of sensor 1 are at similar wavelength range with its
10

parent compound 3 in scheme 1. This result indicated that the absorbance and
emission originated from oxadiazole group of sensor 1. The quantum yield (ΦF) of
sensor 1 was 0.33, determined at room temperature using quinine sulfate (ΦF=0.55 in
0.05 mol/L H2SO4) as standard [28]. The ΦF showed that the sensor 1 is a candidate
as fluorescence material.
Fig.2. The comparison of fluorescence and absorbance spectra of sensor 1 and compound 3 in
CH3CN solutions.
3.3. UV-vis absorbance spectra of sensor 1 with metal ions and colormetric sensing to
Cu2+
There are two pyrazoles in sensor 1 molecule and the nitrogen atoms on pyrazole
have the capability to coordinate with metal ions. According to our previous works
[24-25], it is anticipated that sensor 1 could selectively coordinate with metal ions via
the pyrazole units and further influence the absorbance and fluorescence spectra of
oxadiazole unit.
First of all, the recognition properties of sensor 1 towards different metal ions
including Fe3+, Al3+, Mg2+, K+, Cu+, Cd2+, Co2+, Cr3+, Zn2+, Ag+, Cu2+, Hg2+, Ni2+,
Pb2+, Ca2+, Mn2+, Ba2+, Na+ and Fe2+ were investigated by visual detection
11

experiments. After addition of different metal ions (20 equiv. of sensor 1) to the
sensor 1 solution in CH3CN (1×10-5 M), only the solution containing Cu2+ of sensor 1
turned from colorless to yellow straightway, shown in Fig. 3a. The result indicated
that the sensor 1 can recognize Cu2+ selectively.
The color changes of sensor 1 solutions upon the addition of Cu2+ were further
illustrated by the absorbance spectra, seen in Fig. 3b. The addition of Cu2+ enhanced
the absorbance intensity of sensor 1 to 2-fold with a slightly red-shift to 285 nm. On
the contrary, the other metal ions did not cause the obvious spectra changes, except that
Fe3+ induced the increase of absorbance of sensor 1 to some content. This result was
consistent with the color change of sensor 1 solution, upon the addition of Fe3+, the
color of sensor 1 solution displayed a slight yellow, shown in Fig. 3a. Another particular
case is the addition of Hg2+, a new absorbance peak at 240 nm appeared, but the
absorption intensity at 279 nm did not change and the color of solution did not present
obvious change. The blank experiment that the same amount of Hg2+ was added into
acetonitrile without sensor 1 demonstrated that the absorbance peak at 240 nm come
from the interaction of Hg2+ and acetonitrile (Fig. S4). So the addition of Hg2+ would
not affect the detection of Cu2+. Above consequence confirmed sensor 1 could be used
as a colorimetric sensor of Cu2+ with high selectivity and sensitivity. The obvious
change of the color and absorption intensity of sensor 1 upon the addition of Cu2+ was
attributed to the complexation between the electron-donating pyrazoles and acceptor
Cu2+. On the contrary, other competitive metal ions did not form stable complexes and
cause the negligible change of the absorption intensity, which may be due to the
unsuitable coordination geometry conformation of the two pyrazoles in sensor 1 and
the inappropriate ion radius and insufficient binding energy of these metal ions. The
12

colors change of the solutions from shallow to deep yellow was illustrated by the
absorbance titration experiment of sensor 1 (1×10-5 M) with Cu2+ (0~40 equiv) in
CH3CN solution, as shown in Fig. S5. Upon gradual increasement of Cu2+, the
absorption intensity of the solution at 290 nm increased gradually.
Fig.3. (a) The photographs of sensor 1 solutions containing different metal ions. (b) The
absorbance spectra of sensor 1 in CH3CN solution and different metal ions. In every sample,
the concentration of sensor 1 is 1×10-5 M and the metal ion is 20 equiv. of sensor 1. (For
interpretation of the reference to color in this figure legend, the reader is referred to the web
version of this article.)
The colorimetric sensing ability of sensor 1 to Cu2+ can be realized via the
following manner. The series of standard solutions with constant concentration of
sensor 1 (1×10-5 M) and different concentration of Cu2+ in CH3CN were prepared,
their yellow color gradually deepened with the concentration of Cu2+ (Fig. 4a). When
the water sample was added into sensor 1 solution and the color would be compared
with the above series of standard solutions, the concentration of Cu2+ in water sample
could be evaluated roughly.
The competitive experiments could further confirmed the selectivity of sensor 1
to Cu2+. Therefore, the competitive experiments were carried out by measurement of
the absorbance spectra of the series of sensor 1 in CH3CN solutions (1×10-5 M)
13

containing 20 equiv. of Cu2+ and 20 equiv. of the other metal ions. As shown in Fig.
4b, the ordinate values are the absorbance at 279 nm. This column diagram obviously
showed that other coexistent competition cations had no influence on the colorimetric
sensing of the sensor 1 to Cu2+, which also confirmed the high colorimetric selectivity
of sensor 1 to the Cu2+.
Fig.4. (a) Naked eye detection of sensor 1 to Cu2+ (concentration of sensor 1 is 1×10-5 M). (b)
Selectivity of sensor 1 (1×10-5 M) in CH3CN solution over other competitive metal ions (20
equiv. of sensor 1). (For interpretation of the reference to color in this figure legend, the
reader is referred to the web version of this article.)
3.4. Fluorescence sensing of sensor 1 to Cu2+
Based on the absorbance properties of sensor 1 with metal ions, it is predicted
that sensor 1 could selectively fluorescence sensing towards to Cu2+ over other
competitive metal ions.
High selectivity toward target metal ion over the other potentially competitive
ions is a very important parameter to evaluate the nature of a chemosensor. The
selectivity of sensor 1 towards metal ions was investigated in different solvent
systems including DMSO, CH3CN, DMSO:H2O (1:1, V:V), THF:H2O (1:1, V:V) and
CH3CN:H2O (1:1, V:V). Sensor 1 displayed satisfied result only in pure CH3CN,
14

which may be ascribed to the weak solubility in aqueous solution. Although the metal
ions detection was in CH3CN solutions, the metal ions were in pure water solutions,
so this method can be applied in the detection of metal ions in water. Therefore, the
fluorescence response of sensor 1 towards various metal ions (20.0 equiv.) was tested
in CH3CN solution (1×10-5 M). Sensor 1 emitted blue fluorescence emission and
presented strong fluorescence emission peak at 352 nm when it was excited at 270
nm. Most metal ions such as Fe3+, Al3+, Mg2+, K+, Cd2+, Co2+, Cr3+, Zn2+, Ag+, Hg2+,
Ni2+, Pb2+, Ca2+, Mn2+, Ba2+, Na+ and Fe2+ only induced negligible decrease in
fluorescence spectra of sensor 1. Only Cu2+ intensively impacted the fluorescence
spectra of sensor 1 and quenched the fluorescence at a significant extent (Fig. 5a), the
fluorescence intensity at 352 nm was quenched by 71% after addition of 20.0 equiv.
Cu2+, the fluorescence changed from “on” to “off”. The fluorescence images in Fig.
5b also proved the quenching phenomenon of Cu2+ on the fluorescence of sensor 1,
the fluorescence of sensor 1 was obviously quenched. These observations indicated
that sensor 1 had excellent fluorescence selectivity to Cu2+ ion in CH3CN solutions.
The fluorescence of sensor 1 was quenched due to the forming of sensor 1-Cu2+
complexes, the molecule of sensor 1 and Cu2+ ion was close-connected in complex
structure and the paramagnetic properties of Cu2+ quenched the fluorescence of sensor
1. This is ascribed to the unpaired d (9) electrons of Cu2+ ions, the electron transfer
between the excited states of oxadiazole and the d-orbitals of copper ions took place.
This process can effectively quench the excited states of the oxadiazole and further
15

quenched the fluorescence. This quenching mechanism is same as the many previous
reports [29-33].
Fig.5. (a) Fluorescence response of sensor 1 (1×10-5 M) in CH3CN towards to various metal
ions (20.0 equiv.). (b) Fluorescence images of sensor 1 after addition of 20 equiv. Cu2+. (For
interpretation of the reference to color in this figure legend, the reader is referred to the web
version of this article.)
Fast response of sensor 1 to Cu2+ is more suitable for real-time detection in
practical samples. The experiment found that the fluorescence was instantly quenched
to equilibrium point after addition of Cu2+ into sensor 1 solution within 10 seconds, so
the synthesized sensor 1 could meet the requirement of real-time detection.
The reversibility of fluorescence response process of sensor 1 toward Cu2+ was also
investigated with ethylenediaminetetraacetic acid disodium salt (EDTANa2). After
addition of excess EDTA (2 equiv. of Cu2+) to sensor 1 (1×10-5 M) and Cu2+ (20
equiv. of sensor 1) solution, the fluorescence spectrum of the resulted solution
restored to the original spectrum of sensor 1 (Fig. S6), which indicates that the Cu2+
recognition is a complexing and reversible process.
16

The anti-jamming ability is also important for one fluorescence sensor to be used
in practical applicability, the possible interference ions including Fe3+, Al3+, Mg2+, K+,
Cd2+, Co2+, Cr3+, Zn2+, Ag+, Hg2+, Ni2+, Pb2+, Ca2+, Mn2+, Ba2+, Na+ and Fe2+ were
measured through competitive experiments. The competition experiment was carried
out by monitoring the change of fluorescence intensity at 352 nm upon addition of 20
equiv. Cu2+ and 20 equiv. other metal ions to a solution of sensor 1, the results are
shown in Fig. 6a. The solutions of sensor 1 both containing addition of Cu2+ and
different coexistent cations displayed significant fluorescence quenching and the
quenching efficiencies (F0-F/F0) are the basically same as that caused by Cu2+ only.
The fluorescence selectivity of sensor 1 was similar with the result of competitive
experiment of UV-vis spectroscopy. These results indicated that sensor 1 had the
capability of fluorescence recognition of Cu2+ ion with high selectivity and was not
influenced by the coexisting metal ion.
The stoichiometry of sensor 1-Cu2+ complex was determined by Job’s plot
method. As shown in Fig. 6b, the emission intensity at 352 nm was plotted against the
fraction of Cu2+ under a constant total concentration (10 μM) of sensor 1 and Cu2+.
The Job’s plot with a maximum at 0.5 molar fraction indicated 1:1 binding
stoichiometry of sensor 1 and Cu2+ [34].
17

Fig.6. (a) The fluorescence intensity changes of sensor 1 (1×10-5 M) to various metal ions.
The black bars represent the fluorescence intensity of sensor 1 in the presence of
miscellaneous metal ions (20 equiv.), the red bars represent the fluorescence intensity of the
above solution upon further addition of 20 equiv. of Cu2+ (λem = 352 nm). (b) Job’s plot from
the fluorescence emission spectra of sensor 1 and Cu2+ in CH3CN solution with the total
concentration of 10 μM. λex = 270 nm, λem = 352 nm.
In order to evaluate the sensing properties of sensor 1 towards Cu2+, fluorescence
titration experiment of sensor 1 (1×10-5 M) with Cu2+ (0~50 equiv) in CH3CN
solution was carried out. The fluorescence intensity of sensor 1 at 352 nm was
gradually quenched with the continuous growing concentration of Cu2+ from 0 to 0.5
mM (Fig. 7a). When the concentration of Cu2+ reached about 50.0 equiv of sensor 1,
the fluorescence intensity of sensor 1 decreased from 855 to 70 and the fluorescence
was almost quenched.
The binding constant for the interaction of sensor 1 with Cu2+ ion was estimated
using the data obtained in fluorescence titration studies. The association constant (Ka)
for the sensor 1-Cu2+ complex was evaluated using the Benesi-Hildebrand (B-H) plot
according to the following equation [35].
1/(F0-F) = 1/{Ka(F0 – Fmin)C}+1/(F0 –Fmin)
18

Where F0 and F denoted the fluorescence intensities of sensor 1 only and in the
presence of Cu2+, C is the concentration of Cu2+ solution. Fmin is the minimum
fluorescent intensity at 352 nm in the presence of Cu2+. Linear fitting of the
experiment plot based on the 1:1 binding stoichiometry of sensor 1 and Cu2+ was
examined and the fitted curve was almost superimposed over the experimental plot
with a correlation coefficient over 0.98003 (Fig. 7b), which confirmed the 1:1 binding
stoichiometry of sensor 1 and Cu2+. Ka was obtained from the slope and intercept of
the linear plot. In this way, the association constant was calculated to be Ka =
4.38×104 M-1.
Fig. 7. (a) Fluorescence response of sensor 1 in CH3CN solution (1×10-5 M) upon addition of
Cu2+ (0~50 equiv.). λex = 270 nm, λem = 352 nm. (b) Benesi-Hildebrand plot of sensor 1 (1×10-
5) in CH3CN solution in the presence of Cu2+ (0~15 μM)) (R2=0.98003).
Additionally, the fluorescent detection limit (LOD) of sensor 1 for Cu2+ was also
obtained from the calibration curve of titration experiment (Fig. S7). The calibration
curve of the fluorescence intensity at 352 nm versus [Cu2+] displayed a linear graph
and the regression coefficient was 0.99342. The detection limit was calculated to be
2.14 μM from the equation (3σ/K) [36], which is much lower than the limit of copper
in drinking water (~20 µM) permitted by US Environment Agency (EPA) [37].
19

3.5. Stern-Volmer analysis
Stern-Volmer analysis was used to study the nature of the fluorescence
quenching or enhancement process in the complexation of metal ions. The plots
obtained emission intensities (F0/F or lnF0/F) against Cu2+ ion concentration and
showed a linear graph [38].
F
0
= 1 + 퐾푆푉[M] dynamic quenching
F
ln F = 푒퐾
static quenching
0
[M]
푆푉
F
F0 and F are the fluorescence intensity of sensor 1 at 352 nm under the absence and
presence of Cu2+, respectively. Ksv is the Stern-Volmer quenching constant and [M] is
the concentration of Cu2+. As seen in Fig. 8, under the low concentration, F0/F (black
line) was not linear with quencher (Cu2+) concentration, and thus failed to obey the
Stern-Volmer model of dynamic quenching. However, the lnF0/F values were linear
with quencher concentration, the fitting linear relationship R2 was 0.99699. This result
was in agreement with the Perrin model of static quenching (blue line), suggesting
that all the added quencher (Cu2+) were bound to sensor 1, and the quenched sensor 1
molecules lied within the quencher’s active sphere. This result met the Perrin model
which further supports that fluorescence quenching in this systems occurs
intramolecularly.
20

Fig.8. Stern-Volmer quenching constant analysis plots for dynamic quenching and static
quenching of sensor 1 by Cu2+.
3.6. Proposed mechanism
To further clarify the coordination behavior of sensor 1 with Cu2+, density
functional theory (DFT) calculations at the B3LYP/6-31G(d) level of Gaussian 09
package [39] were carried out to provide a theoretical basis on the mechanism of
interaction between sensor 1 and Cu2+. The optimized ground-state geometries in Fig.
9a revealed a 1:1 binding stoichiometry of sensor 1 and Cu2+ ion. There were two
coordination bonds between Cu2+ and nitrogen atoms in pyrazoles via the lone pairs
of nitrogen atoms.
The frontier orbital energies and shapes can help us to get more insight into the
interaction of sensor 1 with Cu2+. As can be seen from Fig. 9b, both HOMO and
LUMO of sensor 1 were mainly centered on the oxadiazole moiety and benzene ring,
while the HOMO of sensor 1-Cu2+ complex was localized in whole molecular
backbone. On the other hand, the energy gap between the HOMO and LUMO of
21

sensor 1-Cu2+ was calculated to be 1.40 eV, which was much smaller than that of
sensor 1 (4.83 eV). A molecule with high HOMO energy will be more unstable and is
easy to react. These results suggested the frontier molecular orbitals of sensor 1-Cu2+
complex were better stabilized than those of sensor 1. The above explanation based on
DFT had also confirmed 1:1 binding mode of sensor 1-Cu2+ complex.
Fig.9. (a) Optimized ground-state geometries of sensor 1 and senor 1- Cu2+. Color code: C
(black), N (blue), H (white), O (red), Cu2+ (orange). (b) Frontier orbital shapes of sensor 1 and
sensor 1-Cu2+ complex.(For interpretation of the reference to color in this figure legend, the
reader is referred to the web version of this article.)
Above results of spectral studies suggested that the sensing behavior of sensor 1
towards Cu2+ ion involves the formation of a 1:1 complex. The fluorescence of sensor
1 was quenched because of the paramagnetic properties of Cu2+ via the forming of
sensor 1-Cu2+ complexes. The excess solid Cu2+ salt was added into sensor 1 solution
in DMSO-d6, after enough mixing and complexes formation, the upper clear solution
was used in ESI-mass spectrometry analysis. As shown in Fig. S8, ESI-mass spectra
gave a molecular-ion peak at m/z = 445.1486 belonged to [1+Cu2+-H+]+ species.
Additionally, the other molecular-ion peak at m/z 479.1141 appeared in mass spectra,
which matched with the complex corresponded to [1+Cu2++Cl--2H+] and indicated Cl-
22

ion participated in the coordination reaction with Cu2+. These results demonstrated the
formation of complexes between sensor 1 and Cu2+ with 1:1 stoichiometry.
To further demonstrate the complex effect of sensor 1 and Cu2+, 1H NMR
titration of sensor 1 and Cu2+ in DMSO-d6 were conducted. As shown in Fig. 10, the
integration and peak shifts of spectra afforded the obvious evidence of the proposed
binding mode. The peaks at 6.34, 7.54 and 7.88 ppm were corresponded to the proton
Ha and Hb of pyrazole rings and Hc of benzene ring separately. With the addition of
Cu2+ to the solution of sensor 1, sensor 1-Cu2+ complex was gradually formed.
Consequently, the proton signal of Ha, Hb and Hc displayed significant downfield shift
with a gradual broadening. At the existence of 2.0 equiv. Cu2+ ion, these proton
signals almost merged with the base line and disappeared. These observations clearly
indicated that the nitrogen atoms on pyrazole were the binding sites for Cu2+.
Fig.10. 1H NMR titration experiment of sensor 1 in the presence of different amounts of Cu2+
in DMSO-d6.
23

Based on the above results, the binding mode of sensor 1 with Cu2+ and
fluorescence quenching mechanism were proposed in Scheme 2.
Scheme 2. The proposed mechanism for Cu2+ detection by sensor 1.
4. Conclusions
Herein we report a highly selective and sensitive “on-off” fluorescent
chemosensor based on oxadiazole and pyrazole for Cu2+ in presence of coexisting
metal ions. The fluorescence quenching was caused by the complex formation
between sensor 1 and Cu2+, the fluorescence and 1H NMR titrations, ESI-mass and
DFT calculation evidenced the sensor 1-Cu2+ complex with 1:1 stoichiometry. Sensor
1 also displayed the naked-eye detection capability for Cu2+ via the color change from
colorless to yellow. Both fluorescent and colorimetric sensing of sensor 1 towards
Cu2+ displayed high sensitivity and selectivity, and also was not interfered by the co-
existence of other metal ions. Sensor 1 displayed great potential for detection for
Cu2+, so we hope these results will promote the rational design for a more excellent
chemosensor for Cu2+ in the future.
24

Acknowledgement
This work was supported by the National Natural Science Foundation of China (no.
50873019) and Research Fund for the Doctoral Program of Higher Education of China
(20120043110007).
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Figure Caption:
Scheme 1. The synthetic route of sensor 1
Scheme 2. The proposed mechanism for Cu2+ detection by sensor 1.
Fig.1. (a) View of the crystal structure of sensor 1 molecule. (b) View of the π-π interactions
between two sensor 1 molecules. (c) View of the packing structure of sensor 1 along the a
axis.
Fig.2. The comparison of fluorescence and absorbance spectra of sensor 1 and compound 3 in
CH3CN solutions.
Fig.3. (a) The photographs of sensor 1 solutions containing different metal ions. (b) The
absorbance spectra of sensor 1 in CH3CN solution and different metal ions. In every sample,
the concentration of sensor 1 is 1×10-5 M and the metal ion is 20 equiv. of sensor 1. (For
interpretation of the reference to color in this figure legend, the reader is referred to the web
version of this article.)
Fig.4. (a) Naked eye detection of sensor 1 to Cu2+ (concentration of sensor 1 is 1×10-5 M). (b)
Selectivity of sensor 1 (1×10-5 M) in CH3CN solution over other competitive metal ions (20
equiv. of sensor 1). (For interpretation of the reference to color in this figure legend, the
reader is referred to the web version of this article.)
Fig.5. (a) Fluorescence response of sensor 1 (1×10-5 M) in CH3CN towards to various metal
ions (20.0 equiv.). (b) Fluorescence images of sensor 1 after addition of 20 equiv. Cu2+. (For
interpretation of the reference to color in this figure legend, the reader is referred to the web
version of this article.)
Fig.6. (a) The fluorescence intensity changes of sensor 1 (1×10-5 M) to various metal ions.
The black bars represent the fluorescence intensity of sensor 1 in the presence of
miscellaneous metal ions (20 equiv.), the red bars represent the fluorescence intensity of the
above solution upon further addition of 20 equiv. of Cu2+ (λem = 352 nm). (b) Job’s plot from
the fluorescence emission spectra of sensor 1 and Cu2+ in CH3CN solution with the total
concentration of 10 μM. λex = 270 nm, λem = 352 nm.
Fig. 7. (a) Fluorescence response of sensor 1 in CH3CN solution (1×10-5 M) upon addition of
Cu2+ (0~50 equiv.). λex = 270 nm, λem = 352 nm. (b) Benesi-Hildebrand plot of sensor 1 (1×10-
5) in CH3CN solution in the presence of Cu2+ (0~15 μM)) (R2=0.98003).
Fig.8. Stern-Volmer quenching constant analysis plots for dynamic quenching and static
quenching of sensor 1 by Cu2+.
28

Fig.9. (a) Optimized ground-state geometries of sensor 1 and senor 1- Cu2+. Color code: C
(black), N (blue), H (white), O (red), Cu2+ (orange). (b) Frontier orbital shapes of sensor 1 and
sensor 1-Cu2+ complex.(For interpretation of the reference to color in this figure legend, the
reader is referred to the web version of this article.)
Fig.10. 1H NMR titration experiment of sensor 1 in the presence of different amounts of Cu2+
in DMSO-d6.
29