A new fluorescent probe based on imidazole[2,1-b]benzothiazole for sensitive and selective detection of Cu2+

Article abstract of DOI:10.1016/j.molstruc.2019.127384

A simple fluorescent chemosensor WS1 (N'-[(Z)-1H-indazol-3-ylmethylidene]imidazo [2,1-b] [1,3]benzothiazole-2-carbohydrazide) based on imidazole [2,1-b]benzothiazole was designed and synthesized. The probe showed specific recognition towards Cu²⁺ through colorimetric and “turn-off” fluorescence response in MeOH/H₂O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4) with detection limit of 4.32 × 10^?8 M. In addition, WS1 was a reversible fluorescence probe for the detection of Cu²⁺, the fluorescence intensity recovered to the similar state of free WS1 as PPi was added. And it showed a favorable potency for actual water monitoring. The proposed binding mode of WS1 with Cu²⁺ was verified by DFT calculation using Gaussian 09.

Full text of DOI:10.1016/j.molstruc.2019.127384

Journal Pre-proof
A new fluorescent probe based on imidazole[2,1-b]benzothiazole for sensitive and
2+
selective detection of Cu
Hanyu Wang, Songfang Zhao, Yuankang Xu, Linlin Li, Bing Li, Meishan Pei,
Guangyou Zhang
PII:
S0022-2860(19)31493-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127384
MOLSTR 127384
Reference:
To appear in: Journal of Molecular Structure
Received Date: 29 September 2019
Revised Date: 4 November 2019
Accepted Date: 7 November 2019
Please cite this article as: H. Wang, S. Zhao, Y. Xu, L. Li, B. Li, M. Pei, G. Zhang, A new fluorescent
2+
probe based on imidazole[2,1-b]benzothiazole for sensitive and selective detection of Cu , Journal of
Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.127384.
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A new fluorescent probe based on imidazole[2,1-b]benzothiazole for
sensitive and selective detection of Cu2+
Hanyu Wang,a Songfang Zhao,*b Yuankang Xu,a Linlin Li,a Bing Li,a
Meishan Pei,a Guangyou Zhang.*a
a. School of Chemistry and Chemical Engineering, University of Jinan,
Jinan 250022, China.
b. Henan Sanmenxia Aoke Chemical Industry Co. Ltd., Sanmenxia
472000, China.
Keywords: Fluorescent probe, PET, Benzothiazole derivative, Cu2+, DFT.
Abstract:
A
simple
fluorescent
chemosensor
WS1
(N'-[(Z)-1H-indazol-3-ylmethylidene]imidazo[2,1-b][1,3]benzothiazole-2
-carbohydrazide) based on imidazole[2,1-b]benzothiazole was designed
and synthesized. The probe showed specific recognition towards Cu2+
through colorimetric and “turn-off” fluorescence response in MeOH/H2O
(9:1 V/V) buffer solution (10 mM Tris, pH = 7.4) with detection limit of
4.32 × 10-8 M. In addition, WS1 was a reversible fluorescence probe for
the detection of Cu2+, the fluorescence intensity recovered to the similar
state of free WS1 as PPi was added. And it showed a favorable potency
1

for actual water monitoring. The proposed binding mode of WS1 with
Cu2+ was verified by DFT calculation using Gaussian 09.
1. Instruction
Recently, the research of fluorescent probes have been made
remarkable progress in fields of physiological and environmental
monitoring because of their specific recognition for target analytes [1-4].
Especially, fluorescence probes for transition metal ions have attracted
considerable attention due to their crucial applications in chemical,
biological and environmental processes [5-9]. Among the essential heavy
metals, copper is third most abundance element in human bodies and
plays a crucial role in various fundamental physiologic processes [10, 11].
It was reported that suitable concentration of copper in human body can
keep the normal physiological movement operating, whereas, disorders of
copper metabolism could lead to an increasing risk of various diseases
[12, 13]. Copper deficiency is likely to result in anemia-like symptoms as
well as hypopigmentation, irregularity in cholesterol metabolisms and
skeletal deformity, and excessive amounts of copper in the body could
lead to increase morbidity rate of various neurodegenerative diseases,
such as Alzheimer’s and Wilson’s disease [14-18]. Also, copper have
wide applications in lots of industrial fields such as electronics
manufacturing, petroleum industry, printing, medical equipment [19-22].
2

Numerous fluorescence probes for the detection of copper ion were
reported based on quinolone, coumarin, BODIPY, naphthoamide and
Rhodamine in last decade [23-27], but there was no report using
imidazo[2,1-b][1,3]benzothiazole derivative as copper sensitive
chemsensor. Imidazo[2,1-b]benzothiazole was often used as
pharmaceutical intermediate and play a significant role in drug discovery
[28-30]. In fact, the framework of imidazole[2,1-b]benzothiazole has
structural potential to be a fluorescent group because of its molecular
planarity
and
electrons
delocalization
[31,32].
Hence,
imidazo[2,1-b][1,3]benzothiazole is actual a potential platform for the
development of optical sensor for metal ion recognition. So, it is of great
meaning in scientific and applicable aspect if it is possible to develop new
fluorescence probes for detection of copper with satisfactory sensitivity
and selectivety with imidazo[2,1-b][1,3]benzothiazole as fluorophore.
Accordingly, in this report, a new Schiff base WS1 was designed
and
synthesized
based
on
imidazo[2,1-b][1,3]benzothiazole-2-carbohydrazide
and
1H-indazole-3-carbaldehyde. The molecule of WS1 possessed a large
electron delocalization might lead to a sufficient fluorescence, meanwhile,
the “N”, “O” atoms of the molecule could act as binding sites for
recognizing metal ions [33, 34]. Just as predicted, WS1 showed ideal
colorimetry and fluorescent response towards copper ion in MeOH/H2O
3

(9:1 V/V) buffer solution (10 mM Tris, pH = 7.4).
2. Experimental
2.1 Materials and reagents
All reagents and organic solvents were purchased from commercial
suppliers and used without further purification. Metal ions used were
stored in the salt solutions of NaCl, Li2SO4, KCl, MgCl2·6H2O, CaCl2,
ZnCl2, CdCl2·2.5H2O, HgCl2, AgNO3, CuCl2·2H2O, CoCl2·6H2O,
NiCl2·6H2O, MnCl2·4H2O, CrCl3·6H2O, AlCl3·6H2O, FeCl3, FeSO4. The
stock solutions of aforementioned were prepared in distilled water.
2.2 Experimental apparatus
1H MNR spectra were recorded on a Bruker AV III 400MHz NMR
spectrometer and 13C MNR spectra data were obtained by a Bruker AV III
100MHz NMR spectrometer with TMS as an internal standard and
DMSO-d6 as solvent. Fluorescence measurements were carried out using
a HITACHI Instruments F-4600 Fluorescence spectrophotometer (EX
WL: 385nm, EX slit: 10, EM slit: 10). UV-vis absorption spectra data
were taken on a SHimmadzu 3100 spectrometer. Infrared spectra were
determined on a Bruker Vertex 70 FT-IR spectrometer with KBr pellets.
Mass spectrum was performed under ESI mode on a Thermo Fisher
Scientific Exactive Plus mass spectrometer. All pH measurements were
made with a pH-10C digital pH meter. All reactions were monitored with
4

thin layer chromatography (TLC).
2.3 Experimental details for spectral studies
All tests described in this work were performed at room temperature
(25ꢀ) . WS1 was dissolved in MeOH/H2O (9:1 V/V) buffer solution (10
mM Tris, pH = 7.4) to afford the test solution (1 × 10-5 M). All the cations
solutions were prepared from NaCl, Li2SO4, KCl, MgCl2, CaCl2, ZnCl2,
CdCl2, HgCl2, AgNO3, CuCl2, CoCl2, NiCl2, MnCl2, CrCl3, AlCl3, FeCl3
and FeSO4 in distilled water with a concentration of 3 × 10-2 M,
respectively. The sensing for tap water sample was showed in MeOH/tap
water (9:1 V/V) solution. The Cu2+ solution were prepared from dissolved
CuCl2·H2O in tap water with a concentration of 3 × 10-2 M.
2.4 Theoretical calculation
DFT (density functional theory) structural was optimized with the
Gaussian 09 program. In all instances, the structures were optimized
using the B3LYP functional and the mixed basis set 6-31G/(d). Each
structure was subsequently subjected to TDDFT (time dependent density
functional theory) calculation using the B3LYP functional. For all
optimized structures, frequency calculations were carried out to confirm
the absence of imaginary frequencies. The molecular orbitals were
visualized and plotted with the Gauss View 5.0 program.
2.5. Calculation of quantum yield
The quantum yield was determined according to the following equation
5

[26b]:
ꢆ
ꢆ
ꢃ ꢄꢂꢅꢁ
ꢁ
ꢀꢁ = ꢀ
ꢂ ꢃ ꢄꢁꢅꢂ
ꢂ
Φ, F, A, n represents the quantum yield, the integrated area under the
corrected emission spectra, the absorbance intensity at the excitation
wavelength and the refractive index of solvent, respectively. In addition, s
refers to Rhodamine B as standard one, and u refers to the target one. The
quantum yield (Φ) of Rhodamine B dissolved in anhydrous ethanol is
0.97.
2.6. Calculation of the association constant
The association constant of complex WS1 with Cu2+ was calculated by
the Benesi–Hildebrand equation [35]:
1
1
1
=
+
ꢆꢑ
ꢊ
ꢎ
ꢃ − ꢃꢇ ꢈꢉ ꢃꢋꢌꢍ − ꢃꢇ [ꢏꢐ ] ꢃꢋꢌꢍ − ꢃꢇ
F is the fluorescence intensity of the solution of WS1 + Cu2+
complex in accordance with the concentration of Cu2+ at 465 nm. F0 is the
fluorescence intensity of free WS1. Fmin is the fluorescence intensity of
WS1 + Cu2+ complex in the presence of the maximum concentration of
Cu2+.
2.7 Syntheses
Synthesis
of
ethyl-imidazo[2,1-b][1,3]benzothiazole-2-carboxylate (1): 1 was
6

synthesized following previously reported method [36].
Synthesis of imidazo[2,1-b][1,3]benzothiazole-2-carbohydrazidꢁe 2ꢂꢃ
3 ml 80% hydrazine hydrate was added dropwise to 15 ml ethanol
solution of 142 mg (0.58 mmol) compound 1 and the mixture was stirred
for 6 hours at room temperature. Then the precipitate was filtered via
vacuum filtration and the filter cake was washed with ethanol (3 × 5 ml)
to obtain 109 mg (0.47 mmol) compound 2 as a white solid. Yield: 82%.
ESI-MS (Theoretical molecular weight: 232.04): m/z = 233.0456 [M +
H+]+. FTIR (KBr, cm-1): ν = 1369 (C–N), 1654 (C=O), 3112 (N–H)ꢄ3423
1
(N–H). H NMR (400 MHz, DMSO-d6, ppm): δ = 9.42 (s, 1H, –CH–),
8.85 (s, 1H, –NH–), 8.14 (d, J = 7.9 Hz, 1H, –CH–), 8.05 (d, J = 7.9 Hz,
1H, –CH–), 7.57 (t, J = 7.4 Hz, 1H, –CH–), 7.47 (t, J = 7.7 Hz, 1H,
–CH–), 4.45 (s, 2H, –NH, –NH).13C NMR (100 MHz, DMSO-d6, ppm): δ
= 161.34, 146.84, 141.33, 132.00, 129.90, 127.25, 126.27, 125.53, 115.68,
114.55.
Synthesis
N'-[(Z)-1H-indazol-3-ylmethylidene]imidazo[2,1-b][1,3]benzothiazole
-2-carbohydrazide (WS1): 95 mg (0.65 mmol)
of
1H-indazole-3-carbaldehyde was added to 15 ml ethanol suspension of
109mg (0.47 mmol) compound 2 and the mixture was stirred for 12 hours
at room temperature. After the reaction was completed which was
monitored with TLC, the mixture was filtered via vacuum filtration and
7

the filter cake was washed with ethanol (3 × 5 ml) to obtain 101 mg (0.28
mmol) compound WS1 as a yellow solid. Yield: 60%. ESI-MS
(Theoretical molecular weight: 360.07): m/z = 361.0785 [M + H+]+,
383.0596 [M + Na+]+. FTIR (KBr, cm-1): ν= 1617 (C=N), 1690 (C=O),
3115 (C–N)ꢄ3435 (N–H). 1H NMR (400 MHz, DMSO-d6, ppm): δ =
13.43 (s, 1H, –NH–), 12.02 (s, 1H, –NH–), 9.07 (s, 1H, –CH–), 8.88 (s,
1H, –CH–), 8.37 (d, J = 8.0 Hz, 1H, –CH–), 8.23 (d, J = 8.2 Hz, 1H,
–CH–), 8.09 (d, J = 8.0 Hz, 1H, –CH–), 7.63 – 7.59 (m, 1H, –CH–), 7.58
(d, J = 4.2 Hz, 1H, –CH–), 7.50 (t, J = 7.7 Hz, 1H, –CH–), 7.47 – 7.42 (m,
13
1H, –CH–), 7.28 (t, J = 7.4 Hz, 1H, –CH–). C NMR (100 MHz,
DMSO-d6, ppm): δ = 158.28, 147.08, 143.88, 141.64, 141.50, 141.10,
131.96, 130.04, 127.35, 127.33, 126.50, 125.58, 122.94, 122.14, 120.58,
117.31, 114.84, 110.87.
3. Results and discussion
3.1 Synthesis and structural characteristics of WS1
The synthesized route of WS1 was shown in scheme 1. WS1 was
synthesized by the dehydration condensation of compound 2 with
1H-indazole-3-carbaldehyde in ethanol at room temperature. The
1
chemical structure of compound 2 and probe WS1 was confirmed by H
NMR, 13C NMR, FTIR and ESI-MS spectrum. All the data in the spectra
were well accordance with the structures.
8

Scheme 1. The synthetic route of sensor WS1.
3.2 The absorbance response of WS1 toward metal ions
The colorimetric detectability of WS1 was measured with the
addition of various metal ions in MeOH/H2O (9:1 V/V) buffer solution
(10 mM Tris, pH = 7.4) of WS1 (1 × 10-5 M). As shown in Fig. 1a, the
solution of free WS1 (1 × 10-5 M) showed a higher energy absorption
band centered at 325nm which was attributed to π-π* transition. Whereas,
in the presence of 5 equiv. Cu2+, due to the complexion between WS1 and
Cu2+, the absorption intensity at 325nm decreased and a new absorption
band appeared which centered at 390 nm. However, the addition of equal
concentrations other metal ions (Ni2+, Co2+, Li+, Na+, K+, Mg2+, Ca2+,
Zn2+, Cd2+, Hg2+, Ag+, Mn2+, Fe3+, Fe2+, Cr3+ and Al3+) caused
inconspicuous change of absorption. And the color of test samples were
shown in Fig. 1c, it could be found that only the solution with 5 equiv.
Cu2+ changed from colorless to yellow which could be observed by naked
9

eyes. This consequence suggested that WS1 revealed colorimetric
specific recognition for Cu2+.
Fig. 1 (a) UV-vis absorption spectra of WS1 (1 × 10-5M) in MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris,
pH = 7.4) with various metal ions (5 × 10-5 M). (b) The absorption titration spectra of WS1 (1 × 10-5 M) in the
presence of different concentrations of Cu2+ (0 - 5 equiv.) in MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris,
pH = 7.4). (C) The image of visual colorimetric response with test metal ions.
In continuation of the above findings, UV-vis titration was carried
out in MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4) of
WS1 (1 × 10-5M) with gradual addition of Cu2+. As shown in Fig. 1b,
with the increasing of the concentrations of Cu2+, a new absorption band
at 390 nm appeared and the absorbance gradually increased, meanwhile,
the absorption at 325 nm exhibited slight decreased. Concomitantly there
were two isosbestic points at 292nm and 351nm, respectively. As shown
in Fig. S10, a spinodal appeared when the concentration of Cu2+ reached
10

to 1 × 10-5 M, it was found that the complex ratio between WS1 and Cu2+
is 1:1. And based on the data of Fig. S11, the detection limit was
calculated to be 4.63 × 10-7 M according to the equation LOD = 3σ/s [37],
and the value was lower than the drinking water standard of WHO [38].
This consequence suggested that WS1 could be applied in colorimetric
detection of Cu2+ selectively and sensitively.
3.3 The fluorescence response of WS1 toward metal ions
The fluorescence detectability of WS1 was investigated by
examination the fluorescence intensity of WS1 with excitation at 385nm
in the presence of various metal ions. As shown in Fig. 2a, the
MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4) of free
WS1 (1 × 10-5 M) exhibited a strong fluorescence centered at 465nm with
a quantum yield (Φ) of 0.097, and a blue fluorescence color was observed
(in Fig. 2c). After the addition of 5 equiv. of Cu2+, WS1 showed a
significant fluorescence quenching. Whereas, equal concentrations of
other metal ions caused negligible changes in fluorescence intensity. As
shown in Fig. 2b, upon the addition of 5 equiv. Cu2+, the fluorescence
intensity at 465nm had declined around 80%. However, the addition of
other metal ions resulted unconspicuous change of fluorescence intensity.
Therefore, WS1 showed a highly selectivity towards Cu2+ over other
metal ions.
11

Fig. 2 (a) The emission spectra of WS1 (1 × 10-5 M) in MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH =
7.4) with various metal ions (5 × 10-5 M). (b) The emission intensity of WS1 (1 × 10-5 M) at 465nm in MeOH/H2O
(9:1 V/V) buffer solution (10 mM Tris, pH = 7.4) with various metal ions (5 × 10-5 M). (c) The image of
fluorescence response with the test metal ions. EX = 385nm.
Subsequently, the fluorescence titration was conducted in
MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4) of WS1 (1
× 10-5 M) to systematically measure the spectral activity for Cu2+. As
shown in Fig. 3, the fluorescence intensity at 465nm gradually decreased
and displayed a remarkable fluorescence quenching with the increasing of
the concentration of Cu2+ (0 - 5equiv.). Fig. S12 showed the changes of
fluorescence intensity as the concentration of Cu2+ increased from 0 to 5
× 10-5 M, and an excellent linear relationship was observed. As shown in
Fig. S13, the limit of detection (LOD) was calculated to be 4.32 × 10-8 M
according to the equation LOD = 3σ/s. In addition, Fig. S14 showed the
satisfactory linear relationship verifying the 1:1 binding ratio hypothesis
and the association constant as an estimated standard for the binding
12

ability was calculated to be 5.75 × 105 M-1 according to the formula (2).
These consequences suggested that WS1 could act as a fluorescence
probe for detection of Cu2+ with high selectivity and sensitivity by
fluorescence “trun off” response.
Fig. 3 The emission titration spectrum of WS1 (1 × 10-5 M) in the presence of different concentrations of Cu2+ (0 -
5equiv) in MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4). EX = 385nm.
3.4. The competitive experiments
The competitive experiment was executed in MeOH/H2O (9:1 V/V)
buffer solution (10 mM Tris, pH = 7.4) for investigating the
anti-interference ability of WS1 toward Cu2+. As shown in Fig. 4, no
obvious changes of fluorescence intensity were observed when 5 equiv.
competitive metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Zn2+, Cd2+, Hg2+,
Ag+, Mn2+, Fe3+, Fe2+, Co2+, Cr3+ and Al3+) were added into the WS1
solutions, respectively. Subsequently, when 5 equiv. Cu2+ were added to
the above solutions, the fluorescence intensity at 465 nm decreased
13

obviously. Fig. 4 showed that WS1 exhibited high selectivity for Cu2+
and the recognition toward Cu2+ was not interfered by other concomitant
metal ions in the test system. This result indicated that WS1 had a
possibility for specific recognition for Cu2+ in actual detection.
Fig. 4 Metal ions competition test of WS1 (1 × 10-5 M) in MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH
= 7.4). The red bars represent emission of WS1 and 5 equiv. of other metal ions. The black bars represent
fluorescence changes that occur upon addition of 5 equiv. of other metal ions to solution containing WS1 and 5
equiv. Cu2+. EX = 385 nm.
3.5. The pH effect of probe WS1
Fig. 5 showed the effect of pH on the fluorescence intensity of WS1
and WS1 + Cu2+ complex in MeOH/H2O (9:1 V/V) solution. WS1
exhibited a stationary fluorescence in the pH range from 2 to 11, and the
fluorescent quenching caused by Cu2+ was most conspicuous at pH = 7.4.
In addition, the sensor had an accepted fluorescence response toward
Cu2+ in the pH range of 5 - 8. When the pH less than 5 or more than 8, the
fluorescence quenching was gradually unconspicuous. The reasons for
these phenomenon were the protonation process of WS1 in acidic
14

condition, and the higher concentration of OH- in alkaline condition could
disturb the complex between WS1 and Cu2+. This consequence suggested
that WS1 had a potential for the detection of Cu2+ in actual samples.
Fig. 5 Effect of pH on fluorescence intensity at 465 nm for free WS1 (1 × 10-5 M) and WS1 + Cu2+ in
MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4). EX = 385 nm.
3.6 The reversibility of WS1 for recognizing toward Cu2+
In addition, the reversibility of WS1 was testified with the
fluorescence response of [WS1 + Cu2+] for various anions. As shown in
Fig. S15, the fluorescence intensity at 465nm decreased dramatically after
the addition of 5 equiv. Cu2+. Subsequently, the fluorescence intensity
recovered to the similar state of free WS1 as PPi was added. But with the
-
-
-
-
addition of other anions (BF4 , F-, Cl-, Br-, I-, NO3 , NO2 , S2-, HSO3 ,
2-
2-
3-
2-
-
-
S2O3 , SO4 , PO4 , CrO4 , HCO3 , H2PO4 ) to the solution of the WS1 +
Cu2+, the fluorescence was not recovered yet as that happened to PPi.
Moreover, Fig. 6 showed WS1 still displayed an acceptable fluorescence
15

response to Cu2+ after 4 cycles when Cu2+ and PPi was added in turn.
This consequence demonstrated that WS1 was a reversible fluorescence
probe for the detection of Cu2+.
Fig. 6. Fluorometric reversibility of WS1 upon sequential addition of Cu2+ and PPi in MeOH/H2O (9:1 V/V) buffer
solution (10 mM Tris, pH = 7.4). EX = 385nm.
3.7 The probable binding mechanism and the DFT calculation of WS1
and [WS1+Cu2+]
In order to discuss the possible binding mechanism, the Job’s plot
was obtained on the basis of fluorescence emission spectrum. As shown
in Fig. 7, the fluorescence emission intensity at 465nm reached the
maximum when the molar fraction of Cu2+ was 0.5. This result
demonstrated that the stoichiometry between WS1 and Cu2+ was 1:1. And
the ESI-MS spectrum date in Fig. S9 also verified the stoichiometry is 1:1
because of the peak at m/z = 423.0087 [WS1 + Cu2+]2+. Based on the
above data of emission and ESI-MS spectrum, a probable binding
mechanism between WS1 and Cu2+ was proposed as shown in scheme 2.
16

Upon the addition of Cu2+, the coordination between nitrogen atoms of
the imine (C=N), imidazole (C=N) and indazole (C=N) with
paramagnetic Cu2+ lead to the fluorescence quenching [39-42].
Fig. 7 Job’s plots of the complexation between WS1 with Cu2+.
In addition, the density functional theory (DFT) and
time-dependent density functional theory (TDDFT) calculations of WS1
and complex [WS1 + Cu2+] have been performed to acquire the optimized
structure of ground state and energy optimize structure of excited state
using the Gaussian 09 program at the B3LYP/6-31G basic sets. Fig. S16
showed the coplanarity of [WS1 + Cu2+] optimized structure decreased
compared with that of WS1. Then as shown in Fig. 8, the HOMO and
LUMO orbital of WS1 were spread over the indazole moiety, therefore,
electrons which excited to LUMO can transfer back to the HOMO orbital
which caused that WS1 exhibited a strong fluorescence. After the
addition of Cu2+, the frontier orbital of complex [WS1 + Cu2+] became
17

relatively different form free WS1 due to the paramagnetism of Cu2+. The
HOMO orbital was centered on the imidazole[2,1-b]benzothiazole moiety,
while the LUMO orbital was located at the indazole moiety. The HOMO
and LUMO orbital was distributed by the different moieties, this
corresponded to electron cloud distribution of PET mechanism process.
The occurrence of PET process lead to the conspicuous fluorescence
quenching. The energy gap between HOMO and LUMO orbitals of free
WS1 and complex [WS1 + Cu2+] were 4.182 eV and 3.341 eV,
respectively. The binding of WS1 with Cu2+ stabilized the system which
was certified from the lower energy gap of complex [WS1 + Cu2+]. These
calculation results support the probable binding mechanism in scheme 2.
Scheme. 2 The probable binding mode of WS1 for Cu2+.
18

Fig. 8 Frontier molecular orbitals (MOs) and energy diagrams of free WS1 and complex [WS1 + Cu2+].
3.8 The sensing test in actual water samples
To explore the application of WS1 in practical environmental
monitoring, the sensing test for Cu2+ in tap water was performed. As
shown in Table 1, WS1 exhibited satisfactory performance for Cu2+ in tap
water at low concentrations. Compared with the standard curve of Cu2+,
an acceptable recovery (96.75% – 107%) and RSD (1.86% – 4.24%) of
samples in tap water were obtained. Thus, this consequence demonstrated
that WS1 showed a favorable potency for the quantitative detection of
Cu2+ in actual water.
Table 1 the sensing test for Cu2+ in tap water samples.
Sample
Cu2+ added (mol/L)
Cu2+ recovered (mol/L)
Recovery (%)
RSD (%)
1
2
3
4
3 × 10-6
5 × 10-6
8 × 10-6
1 × 10-5
3.23 × 10-6
5.13 × 10-6
7.74 × 10-6
1.034 × 10-5
107
4.24
1.08
2.74
1.86
102.6
96.75
103.4
19

4. Conclusion
In summary, a fluorescence probe WS1 was designed and synthesized
based on imidazo[2,1-b][1,3]benzothiazole-2-carbohydrazide and
1H-indazole-3-carbaldehyde. The chemical structure of WS1 was
1
13
confirmed by H NMR, C NMR, FT-IR and ESI-MS. WS1 exhibited
high selectivity and sensitivity toward Cu2+ over other metal ions in
MeOH/H2O (9:1 V/V) buffer solution (10 mM Tris, pH = 7.4). Upon the
addition of Cu2+, a new absorption band appeared at 390nm with the
solution changed from colorless to yellow, and the colorimetric limit of
detection was 4.63 × 10-7 M. The fluorescence quenching occured
because WS1 complexed with Cu2+, the association constant of WS1 to
Cu2+ was calculated to be 5.57 × 105 M-1 and the limit of detection was
4.32 × 10-8 M which was sufficiently low to enable the detection of Cu2+
in practical applications. WS1 was a reversible fluorescence probe for the
detection of Cu2+ and the Job’s plot analysis supported the stoichiometry
between WS1 and Cu2+ as 1:1. In addition, due to its satisfactory
performance for Cu2+, WS1 was of the potential possibility to be applied
in the domains of biochemistry and biomedicine in the future.
Acknowledgement
The authors thank the Henan Sanmenxia Aoke Chemical Industry
Co., Ltd. w0920 for financial support. Financial support by the National
20

Natural Science Foundation of China (Grants 21708013) and the China
Postdoctoral Science Foundation (Grants 2017M620288).
References
[1] Q. Q. Wu, M. Mao, W. L. Liang, F. J. Stadler, Quinoline-derived
fluorescent probes for the discrimination of Cys from Hcys/GSH and
bioimaging in living cells, Talanta. 186 (2018) 110-118.
[2] X. Wang, Y. M. Zhou, C. G. Xu, H. H. Song, L. Li, J. L. Zhang, M.
Guo, A highly selective fluorescent probe for the detection of
hypochlorous acid in tap water and living cells, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy. 203 (2018)
415-420.
[3] H. H. Lv, Z. Q. Ren, H. Liu, G. B. Zhang, H. P. He, X. H. Zhang, S.
F. Wang, The turn-off fluorescent sensors based on thioether-linked
bisbenzamide for Fe3+ and Hg2+, Tetrahedron. 74 (2018) 1668-1680.
[4] H. Paritala, K. S. Carroll, A continuous spectrophotometric assay for
adenosine 5’-phosphosulfate reductase activity with sulfite-selective
probes, Analytical Biochemistry. 440 (2013) 32-39.
[5] S. Y. Qin, B. Chen, J. Huang, Y. F. Han, A thiocoumarin-based
colorimetric and ratiometric fluorescent probe for Hg2+ in aqueous
solution and its application in live-cell imaging, New J. Chem. 42
(2018) 12766-12772.
[6] J.L. Huang, B. Wang, J. G. Ye, B. Liu, H. Y. Qiu, S. C. Yin, A
21

Highly Copper-Selective Ratiometric Fluorescent Sensor Based on
BODIPY, Chin. J. Chem. 30 (2012) 1857-1861.
[7] K. Hanaoka, Y. Muramatsu, Y. Urano, T. Terai, T. Nagano, Design
and Synthesis of a Highly Sensitive Off-On Fluorescent
Chemosensor for Zinc Ions Utilizing Internal Charge Transfer,
Chem. Eur. J. 16 (2010) 568-572.
[8] S. Memon, A. A. Bhatti, A. A. Bhatti, Ü. Ocak, M. Ocak,
2−
Calix[4]arene Based Dual Fluorescent Sensor for Al3+ and S2O7 , J.
Fluoresc. 26 (2016) 1591–1599.
[9] T. Sun, Q. F. Niu, Z. R. Guo, T. D. Li, A simple highly sensitive and
selective turn-on fluorescent chemosensor for the recognition of Pb2+,
Tetrahedron Letters. 58 (2017) 252-256.
[10] L. S. Cui, J. U. Kim, H. Nomura, H. Nakanotani, C. Adachi,
Benzimidazobenzothiazole-based Bipolar Hosts to Harvest Nearly
All of the Excitons from Blue Delayed Fluorescence and
Phosphorescent Organic Light-Emitting Diodes, Angew. Chem. Int.
Ed. 55 (2016) 6864-6868.
[11] L. J. Tang, F. F. Li, M. H. Liu, R. J. Nandhakumar, Single sensor
for two metal ions: Colorimetric recognition of Cu2+ and fluorescent
recognition of Hg2+, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy. 78 (2011) 1168-1172.
[12] P. X. Xia, J. Y. Dou, L. Huang, M. Xua, F. J. Chen, Y. J. Wu, D. C
22

Bai, W. G. Li, Z. Z. Zeng, A selective turn-on fluorescent sensor for
Cu(II) and its application in imaging in living cells, Sensors and
Actuators B: Chemical. 148 (2010) 337-341.
[13] F. A. Abebe, E. Sinn, Fluorescein-based fluorescent and
colorimetric chemosensors for copper in aqueous media,
Tetrahedron Letters. 52 (2011) 5234-5237.
[14] Z. P. Dong, J. Jin, W. F. Zhao, H. M. Geng, P. Zhao, R. Li, J. T.
Ma, Quinoline group grafted carbon nanotube fluorescent sensor for
detection of Cu2+ ion, Applied Surface Science. 255 (2009)
9526-9530.
[15] H. Wang, D. L. Shi,1, J. Li, H. Y. Tang, J. Li, Y. Guo, A facile
fluorescent probe with a large Stokes shift for sequentially detecting
copper and sulfide in 100% aqueous solution and imaging them in
living cells, Sensors and Actuators B: Chemical. 256 (2018)
600-608.
[16] Q. M. Wang, S. S. Wu, Y. Z. Tan, Y. L. Yan, L. Guo, X. H. Tang, A
highly selective, fast-response and fluorescent turn on chemosensor
for the detection of Cu2+ ions and its potential applications, Journal
of Photochemistry and Photobiology A: Chemistry. 357 (2018)
149-155.
[17] T. Sun, Q. F. Niu, T. D. Li, Z. R. Guo, H. X. Liu, A simple,
reversible, colorimetric and water-soluble fluorescent chemosensor
23

for the naked-eye detection of Cu2+ in ~100% aqueous media and
application to real samples, Spectrochimica Acta Part A: Molecular
and Biomolecular Spectroscopy. 188 (2018) 411-417.
[18] J. Xu, Z. K. Wang, C. Y. Liu, Z. H. Xu, B. C. Zhu, N. Wang, K.
Wang, J. J. Wang, A colorimetric and fluorescent probe for the
detection of Cu2+ in a complete aqueous solution, Analytical
Sciences April. 34 (2018) 453-457.
[19] R. Kumar, H. Jain, P. Gahlyan, A. Joshi and C. N. Ramachandran,
A highly sensitive pyridine-dicarbohydrazide based chemosensor for
colorimetric recognition of Cu2+, AMP2-, F- and AcO- ions, New J.
Chem. 42 (2018) 8567-8576.
[20] N. Fredj, J. S. Kolar, D. M. Prichard, T. D. Burleigh, Study of
relative color stability and corrosion resistance of commercial
copper alloys exposed to hand contact and synthetic hand sweat,
Corrosion Science. 76 (2013) 415-423.
[21] A. T. Ozyılmaz, N. Colak, M. K. Sangun, M. Erbil, B. Yazıcı, The
electrochemical synthesis of poly(aniline-co-o-anisidine) on copper
and their corrosion performances, Progress in Organic Coatings. 54
(2005) 353-359.
[22] A. Taher, Corrosion Behavior of Copper – Nickel Alloy in Marine
Environment, Applied Mechanics and Materials. 799 (2015)
222-231.
24

[23] (a) Y. X. Chang, J. X. Fu, K. Yao, B. Li, K. X. Xu, X. B. Pang,
probes for sequential detection of Cu2+ and
Novel fluorescent
citrate anion and application in living cell imaging, Dyes and
Pigments. 161 (2019) 331-340.
(b) E. J. Song, G. J. Park, J. J. Lee, S. Lee, I. Noh, Y. Kim, S. J. Kim,
C. Kim, R. G. Harrison, A fluorescence sensor for Zn2+ that also acts
as a visible sensorfor Co2+ and Cu2+, Sensors and Actuators B.
Chemical. 213 (2015) 268-275.
(c) Y. P. Dai, P. Wang, J. X. Fu, K. Yao, K. X. Xu, X. B. Pang, A
quinoline-based Cu2+ ion complex fluorescence probe for selective
detection of inorganic phosphate anion in aqueous solution and its
application to living cells, Spectrochimica Acta Part A: Molecular
and Biomolecular Spectroscopy. 183 (2017) 30-36.
[24] (a) M. M. Guo, P. Y. Dong, Y. Feng, X. G. Xi, R. Shao, X. H. Tian,
B. C. Zhang, M. Z. Zhu, X. M. Meng, A two-photon fluorescent
probe for biological Cu (II) and PPi detection in aqueous solution
and in vivo, Biosensors and Bioelectronics. 90 (2017) 276-282.
(b) W. Lu, F. Huang, H. M. Hua, J. C. Chen, S. Qiu, F. Y. Zhao, J. Z.
Shi, L. Xu, S. L. Yang, X. W. Chi, Synthesis, crystal structure and
DFT calculations of a new coumarin-amide binuclear Cu (II)
complex, Journal of Molecular Structure. 1192 (2019) 115-121.
(c) X. H. Cheng, Z. C. Zhon, W N. Li, Coumarin-derived fluorescent
25

chemosensor for sulfide anion: displacement approach and
bioimaging application, Chinese Journal Of Luminescence. 38 (2017)
769-779.
(d) S. Feng, Q. M. Gao, X. Gao, J. Q. Yin, Y. Jiao, Fluorescent sensor
for copper(II) ions based on coumarin derivative and its application
in cell imaging, Inorganic Chemistry Communications. 102 (2019)
51-56.
[25] (a) B. C. Zhou, S. Y. Qin, B. Chen, Y. F. Han, Near-infrared
aza-BODIPY fluorescent probe for selective Cu2+ detection and its
potential in living cell imaging, Dalton Transactions. 46 (2017)
16251-16256.
(b) E. T. Ecik, E. Senkuytu, E. Okutan, G. Y. Çiftçi, Synthesis of
BODIPY-cyclotetraphosphazene triad systems and their sensing
behaviors toward Co(II) and Cu(II), Inorganica Chimica Acta. 495
(2019) 119009-119018.
(c) Y. T. Chen, H. H. Pan, F. Wang, Y. Y. Zhao, H. Y. Yin, Y. X. Chen,
J. L. Zhang, J. Z. Jiang, An ultrafast BODIPY single molecular
sensor for multi-analytes (acid/base/Cu2+/Bi3+) with different sensing
mechanism, Dyes and Pigments. 165 (2019) 279-286.
(d) Y. Huang, C. F. Li, W. J. Shi, H. Y. Tan, Z. Z. He, L. Y. Zheng, F.
G. Liu, J. W. Yan, A near-infrared BODIPY-based fluorescent probe
for ratiometric and discriminative detection of Hg2+ and Cu2+ ions in
26

living cells, Talanta. 198 (2019) 390-397.
[26] (a) Y. Wang, P. D. Mao, W. N. Wu, X. J. Mao, X. L. Zhao, Z. Q.
Xu, Y. C. Fan, Z. H. Xu, A novel colorimetric and ratiometric
fluorescent Cu2+ sensor based on hydrazone bearing
1,8-naphthalimide and pyrrole moieties, Sensors and Actuators B.
251 (2017) 813-820.
(b) Z. Liu, W. He, M. S. Pei, G. Y. Zhang, A fluorescent sensor with
a detection level of pM for Cd2+ and nM for Cu2+ based on different
mechanisms, Chem. Commun. 51 (2015) 14227-14230.
(c) S. Y. Park, W. Kim, S. H. Park, J. Han, J. Lee, C. Kang, M. H.
Lee, An endoplasmic reticulum-selective ratiometric fluorescent
probe for imaging a copper pool, Chem. Commun. 53 (2017)
4457-4460.
(d) G. J. He, C. X. Liu, X. B. Liu, Q. Z. Wang, A. Y. Fan, S. J. Wang,
X. L. Qian, Design and synthesis of a fluorescent probe based on
naphthalene anhydride and its detection of copper ions, PlOS. ONE.
12 (2017) e0186994.
[27] (a) Y. Q. Ge, X. L. Zheng, R. X. Ji, S. L. Shen, X. Q. Cao, A new
pyrido[1,2-a]benzimidazole-rhodamine FRET system as an efficient
ratiometric fluorescent probe for Cu2+ in living cells, Analytica
Chimica Acta. 965 (2017) 103-110.
(b) Y. C. Zhao, C. Chen, B. Chen, P. Y. Wang, Z. B. Wu, H. Li, B. A.
27

Song, S. Yang, Tunable fluorescent probes for selective detection of
copper(II) andsulphide with completely different modes, Sensors
and Actuators B. 241 (2017) 168-173.
(c) Q. P. Diao, P. Y. Ma, L. L. Lv, T. C. Li, X. H.Wang, D. Q. Song,
Phenanthroline-fluorescein molecular hybrid as a ratiometric and
selective fluorescent chemosensor for Cu2+ via FRET strategy:
synthesis, computational studies and in vitro applications,
Supramolecular Chemistry. 29 (2017) 616-626.
[28] S. K. Maddili, Z. Z. Li, V. K. Kannekanti, R. R. Y.
Bheemanaboina, B. Tuniki, V. K. R. Tangadanchu, C. H. Zhou,
Azoalkyl ether imidazo[2,1-b]benzothiazoles as potentially
antimicrobial agents with novel structural skeleton, Bioorganic &
Medicinal Chemistry Letters. 28 (2018) 2426-2431.
[29] J. R. Wu, H. Luo, T. Wang, H. M. Sun, Q. Zhang, Y. H. Chai,
Diverse synthesis
of pyrimido[1,2-a]benzimidazoles
and
imidazo[2,1-b]benzothiazoles via CuI-catalyzed decarboxylic
multicomponent reactions of heterocyclic azoles, aldehydes and
alkynecarboxylic acids, Tetrahedron. 75 (2019) 1052-1063.
[30] I. V. Rassokhina, T. A. Tikhonova, S. G. Kobylskoy, I. Y. Babkin,
V. Z. Shirinian, V. Gevorgyan, I. V. Zavarzin, Y. A. Volkova,
Synthesis of Imidazo[2,1-b]thiazoles via Copper-Catalyzed A3 ꢀ
Coupling in Batch and Continuous Flow, J. Org. Chem. 82 (2017)
28

9682-9692.
[31] J. Gao, J. Zhu, L. Chen, Y. Shao, J. Zhu, Y. Huang, X. Wang, X.
Lv, The benzimidazo[2,1-b]benzothiazole backbone was synthesized
though cyclization of 2-mercaptobenzimidazole with onitrophenyl
bromide, Tetrahedron Letter. 55 (2014) 3367-3373.
[32] C. F. Li, G. P. Yong, Y. Z. Li, Phosphorescent iridium (III)
2-phenylpyridine complexes: Efficient color tuning by novel
ancillary ligands, Inorganic Chemistry Communications. 13 (2010)
179–182.
[33] Y. Ren, D. Fan, H. Z. Ying, X. Li, Rational design of the
benzothiazole-based fluorescent scaffold for tunable emission.
Tetrahedron Letters. 60 (2019) 1060-1065.
[34] I.
Kaur,
P.
Kaur,
K.
Singh,
based
2-(4-Amino-2-hydroxyphenyl)benzothiazole
Schiff-base:Complexation/decomplexation driven photo physical
3−
tuning of fluorescence leading to Cu2+ and PO4 detection. Sensors
and Actuators B: Chemical. 257 (2018) 1083-1092.
[35] X. F. Bao, X. W. Cao, X. M. Nie, Y. Xu, W. H. Guo, B. J. Zhou, L.
Y. Zhang, H. Liao, T. Pang, A new selective fluorescent chemical
sensor
for
Fe3+based
onrhodamine
B
and
a
1,4,7,10-tetraoxa-13-azacyclopentadecaneconjugate and its imaging
in living cells, Sensors and Actuators B: Chemical. 208 (2015)
29