A new benzimidazole-based selective and sensitive ‘on–off’ fluorescence chemosensor for Cu2+ ions and application in cellular bioimaging

Article abstract of DOI:10.1002/bio.3586

Two new twinborn benzimidazole derivates (L and A), which bonded pyridine via the ester space on the opposite and adjacent positions of the benzene ring of benzimidazole respectively, were designed and synthesized. Compound L displayed fluorescence quench

Full text of DOI:10.1002/bio.3586

Received: 30 August 2018
DOI: 10.1002/bio.3586
Revised: 7 November 2018
Accepted: 29 November 2018
R E S E A R C H A R T I C L E
A new benzimidazole‐based selective and sensitive ‘on–off’
fluorescence chemosensor for Cu2+ ions and application in
cellular bioimaging
Yi He1
Qijing Bing2 Yingjuan Wei1 Heyang Zhang2 Guang Wang2
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1 College of Chemistry, Jilin University,
Changchun, P. R. China
Abstract
2 Faculty of Chemistry, Northeast Normal
University, Changchun, P. R. China
Two new twinborn benzimidazole derivates (L and A), which bonded pyridine via
the ester space on the opposite and adjacent positions of the benzene ring of
benzimidazole respectively, were designed and synthesized. Compound L dis-
played fluorescence quenching response only towards copper(II) ions (Cu2+) in
acetonitrile solution with high selectivity and sensitivity. However, compound A
presented ‘on–off’ fluorescence response towards a wide range of metal ions to
different degrees and did not have selectivity. Furthermore, compound L formed
a 1:1 complex with Cu2+ and the binding constant between sensor L and Cu2+
was high at 6.02 × 104 M−1. Job's plot, mass spectra, IR spectra, 1H‐NMR titra-
tion and density functional theory (DFT) calculations demonstrated the formation
of a 1:1 complex between L and Cu2+. Chemosensor L displayed a low limit of
detection (3.05 × 10−6 M) and fast response time (15 s) to Cu2+. The Stern–
Volmer analysis illustrated that the fluorescence quenching agreed with the static
quenching mode. In addition, the obvious difference of L within HepG2 cells in
the presence and absence of Cu2+ indicated L had the recognition capability for
Cu2+ in living cells.
Correspondence
Yi He, College of Chemistry, Jilin University,
Changchun 130025, P. R. China.
Email: heyi@jlu.edu.cn
Funding information
National Natural Science Foundation of China,
Grant/Award Number: 50873019
KEYWORDS
benzimidazole, cell imaging, copper ion, fluorescence chemosensor
method have been used to detect metal ions,[10–12] but it is difficult for
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1
INTRODUCTION
these techniques to execute on‐site detection. In recent years, the
Transition metal ions are required and play vital roles in diverse biolog-
ical processes.[1] Among them, the copper(II) ion (Cu2+) is an essential
trace element in biological and fundamental physiological processes,
such as a cofactor in a great many enzymes.[2–5] On the contrary,
overloading Cu2+ ions in the human body can induce neurodegenera-
tive diseases such as Menkes, Alzheimer and Parkinson.[6–9] Therefore,
the development of new methods to recognize and detect Cu2+ ions at
lower concentration and with higher sensitivity in the environment
and biological systems has become more important.
design and preparation of fluorescent chemosensors to recognize and
detect metal ions has attracted increasing interest due to their high
selectivity and sensitivity, on‐site monitoring, fast response and lower
cost.[13–17] So far, many Cu2+ fluorescent chemosensors based on dif-
ferent kinds of materials have been reported.[18–23]
The benzimidazole moiety as fluorescent material with high fluo-
rescence quantum yield has been used in optical devices[24–26] and
fluorescent chemosensors.[27,28] However, the reported fluorescence
chemosensors with benzimidazole as emitting moiety are not so abun-
dant, therefore development of new benzimidazole‐based fluorescent
chemosensors to detect Cu2+ is still noteworthy.
Up to now, many techniques such as coupled plasma atomic emis-
sion spectrometry, atomic absorption spectrometry and electrochemical
Luminescence. 2019;1–9.
wileyonlinelibrary.com/journal/bio
© 2019 John Wiley & Sons, Ltd.
1

2
HE ET AL.
In this article, two twinborn benzimidazole derivatives (L and A)
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2.2
Synthesis
with bound pyridine unit via ester space were designed and synthesized
(Scheme 1). The difference between these two molecules is the molec-
ular structures, one is a linear molecule (L) and the other is a bent‐type
molecule (A). It is hoped that the oxygen atom of the ester space and
nitrogen atoms of benzimidazole and pyridine units provide selective
coordination points towards metal ions. It was unexpected that the lin-
ear compound L displayed selective fluorescence quenching response
to Cu2+ but the bent‐type compound A indiscriminately responded to
most of the tested metal ions to different degrees. Compound L
displayed not only selective fluorescence response but also the recog-
nition ability to Cu2+ ions in living cells. In addition, the sensing mecha-
nism of L towards Cu2+ ions was also clearly studied.
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2.2.1
Synthesis of precursors 4‐(1H‐benzo[d]
imidazol‐2‐yl)phenol (compound 1) and 4‐(1H‐benzo[d]
imidazol‐2‐yl)phenol (compound 2)
Compounds 1 and 2 were synthesized using the following procedure:
Compound 1 – p‐hydroxy benzaldehyde (3.05 g, 25 mmol) and
sodium hydrogen sulfate (5.20 g, 50 mmol) were dissolved in 20 mL
anhydrous ethanol (C2H5OH) in a flask, the reaction mixture was
stirred at room temperature for
6 h. Then 20 mL N,N‐
dimethylformamide and o‐phenylenediamine (5.40 g, 50 mmol)
were added into the reaction mixture. After the mixture was heated
at reflux temperature for 4.5 h, it was cooled down to room tem-
perature and poured into 300 mL distilled water. The precipitate
was collected and recrystallized in anhydrous C2H5OH, faint yellow
solid powder was obtained, 8.34 g, yield 89.7%. 1H‐NMR
(600 MHz, DMSO) δ 12.72 (s, 1H), 8.10 (d, J 8.7 Hz, 2H), 7.61
(d, J 7.5 Hz, 1H), 7.49 (d, J 7.4 Hz, 1H), 7.19–7.09 (m, 4H), 4.91
(t, J 5.5 Hz, 1H).
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2
EXPERIMENTAL
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2.1
Materials and methods
All reagents were of analytical grade from Aladdin Chemical Com-
pany and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
The mass spectra were recorded on an Agilent mass spectrometer
1200 HPLC/Micro TOF II (Agilent, Santa Clara, USA). 1H‐NMR and
13C‐NMR spectra were measured on a Varian Unity Inova Spec-
trometer (500 MHz and 600 MHz), Varian, Walnut Creek, CA,
USA, using deuterated chloroform (CDCl3) and dimethyl sulfoxide
(DMSO‐d6) as solvents. Fluorescence spectra and absorbance spec-
tra were tested on a Varian Cary Eclipse fluorescence spectropho-
tometer (Varian, Walnut Creek, CA, USA) and Varian Cary 500 UV‐
visible spectrophotometer (Varian, Walnut Creek, CA, USA), respec-
tively. Fluorescence microscope images were taken on an Olympus
FV‐1000 Confocal Laser Scanning Microscope (Olympus, Tokyo,
Japan). A METTLER FE20 pH meter was used to measure pH
values of solutions (Mettler‐Toledo, Zurich, Suisse). The stock solu-
tions of all tested metal ions were prepared using the following
Compound 2 – yield, 80.8%. 1H‐NMR (600 MHz, CDCl3) δ 13.07
(s, 1H), 9.35 (s, 1H), 7.80–7.75 (m, 1H), 7.58 (dd, J 7.8, 1.5 Hz, 1H),
7.51 (dd, J 5.6, 3.1 Hz, 1H), 7.38 (ddd, J 8.5, 7.3, 1.6 Hz, 1H), 7.34–
7.30 (m, 2H), 7.14 (dd, J 8.3, 0.9 Hz, 1H), 6.99–6.95 (m, 1H).
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2.2.2
Synthesis of compounds L and A
Compound
compound
L
(4‐(1H‐benzo[d]imidazol‐2‐yl)phenyl picolinate) and
(2‐(1H‐benzo[d]imidazol‐2‐yl)phenyl picolinate) were
A
prepared also using the same procedure:
Compound L – picolinic acid (0.74 g, 6 mmol), compound 1
(1.89 g, 6 mmol) and 4‐dimethylaminopyridine (DMAP, 0.15 g,
1.25 mmol) were added into 40 mL purified dichloromethane in
100 mL flask, the mixture was stirred for 1 h at room temperature.
After 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride
(EDC, 1.44 g, 7.5 mmol) was added, the resultant mixture was contin-
uously stirred under nitrogen gas for 24 h. The organic phase was
salts:
AgNO3,
Zn(NO3)2·6H2O,
KNO3,
Cd(NO3)2·4H2O,
Ni(NO3)2·6H2O, Cu(NO3)2·3H2O, Cr(NO3)3·9H2O, Al(NO3)3·9H2O,
Fe(NO3)3·9H2O, Mg(NO3)2·6H2O, BaCl2·2H2O, Co(NO3)2·6H2O,
MnCl2, SrSO4, CaCl2, PbCl2, HgCl2 and FeSO4·7H2O.
Compound L
Compound 1
Compound 2
Compound A
SCHEME 1 Synthesis of compounds L and A

HE ET AL.
3
washed with distilled water and then approximately 25 mL of dichlo-
romethane was evaporated under vacuum. The white product was
obtained by filtration, 1.37 g, yield 72,6%. 1H‐NMR (600 MHz,
DMSO) δ 12.99 (s, 1H), 8.85 (d, J 4.0 Hz, 1H), 8.32–8.23 (m, 3H),
8.12 (td, J 7.7, 1.7 Hz, 1H), 7.77 (ddd, J 7.7, 4.7, 1.0 Hz, 1H), 7.62
(s, 2H), 7.53 (d, J 8.7 Hz, 2H), 7.23 (dd, J 5.8, 3.0 Hz, 2H), 5.75 (s,
under the catalytic effect of DMAP and EDC. The structure of
compounds L and A were well characterized by high‐resolution
mass spectrometry (HRMS), 1H‐NMR, and 13C‐NMR analyses
(Figures S1–S8). It is hoped that the oxygen atom of the ester group
and the nitrogen atoms of benzimidazole and pyridine play a synergistic
effect to coordinate with special metal ions. The linear and non‐linear
molecular structures present different steric configurations and are
expected to display different coordination selectivity towards metal
ions.
1H). 13C‐NMR (151 MHz, DMSO)
δ 163.81, 152.27, 151.02,
150.56, 147.13, 138.28, 128.64, 128.56, 128.30, 126.29, 122.97,
55.38, 40.41, 40.28, 40.17, 40.14, 40.00, 39.86, 39.73, 39.72,
39.58. HRMS: m/z: found [M + Na+] 338.0913, molecular formula
C
19H13N3O2, requires [M + Na+] 338.3140.
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3.2
Absorption and fluorescence spectra of
Compound A – yield 68.5%. 1H‐NMR (600 MHz, CDCl3) δ 13.25
compounds L and A and their response towards metal
ions
(s, 1H), 9.12–9.01 (m, 1H), 8.80 (dd, J 7.9, 1.7 Hz, 1H), 8.36 (d, J
7.8 Hz, 1H), 8.06 (td, J 7.7, 1.7 Hz, 1H), 7.99 (dd, J 8.3, 1.0 Hz, 1H),
7.85 (dd, J 5.6, 2.9 Hz, 1H), 7.75 (ddd, J 7.6, 4.8, 1.1 Hz, 1H), 7.58
(dd, J 5.7, 3.0 Hz, 1H), 7.52 (ddd, J 8.3, 7.4, 1.8 Hz, 1H), 7.47–7.41
(m, 1H), 7.32–7.27 (m, 2H). 13C‐NMR (151 MHz, CDCl3) δ 161.40,
149.07, 148.39, 148.20, 147.15, 138.21, 130.31, 128.28, 126.29,
122.97, 122.33, 121.48, 119.55, 111.04, 77.25, 77.04, 76.83. HRMS:
m/z: found [M + H] 316.1089, molecular formula C19H14N3O2,
requires [M + H] 316.3320.
The fluorescence and absorbance spectra of compounds L and A (10 μM)
in CH3CN solutions are presented in Figure 1(a). The main absorbance
bands of L and A are located at 296 nm, which can be ascribed to π–π*
electron transition within their whole molecules.[17,18] In the same solu-
tions, compounds L and A displayed strong fluorescence emissions.
Compound L presented two emission peaks at 341 nm and 356 nm,
respectively. Compound A just presented one emission band at
461 nm and a large red‐shift compared with that of L, which may be
due to the larger π conjugation system in molecule A than L.
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2.3
Preparation of stock solutions
Selectivity is the first consideration for a chemosensor to be suit-
able in practical applications, so the absorbance and fluorescence
response towards different metal ions were the primary investigations
in the different solvent systems as follows, CH3CN, C2H5OH,
CH3CN–Tris (20 mM, pH 7.2, 1:1 v/v), C2H5OH–Tris (20 mM,
pH 7.2, 1:1 v/v). After the addition of a different metal ion (5 equiva-
lents) to L or A solution (10 μM) and mixing for 15 s, the UV‐visible
absorbance and fluorescence spectra were measured at room temper-
ature. For measurements of fluorescence spectra, the light of 305 and
330 nm was used as exaction wavelength for compounds L and A,
respectively. In the solvent systems of C2H5OH, CH3CN–Tris
(20 mM, pH 7.2, 1:1 v/v) and C2H5OH–Tris (20 mM, pH 7.2, 1:1 v/
v), all the absorbance and fluorescence of compounds L and A
displayed responses to most of the test metal ions but the responses
were to different degrees and there was no regularity. These results
indicated that most test metal ions affected the photophysical proper-
ties of compounds L and A. Therefore, in these three solvent systems,
The stock solutions of compounds L and A were prepared with the
concentration of 1 × 10−4 M in different solvents including acetonitrile
(CH3CN), C2H5OH, CH3CN–Tris (20 mM, pH 7.2, 1:1 v/v), C2H5OH–
Tris (20 mM, pH 7.2, 1:1 v/v).
The stock solutions of different metal ions (5 mM) in distilled
water were made of their inorganic salts: chloride salts (Cu2+, Co2+
Ni2+, Ba2+, Hg2+, Mn2+, Pb2+, Ca2+), nitrate salts (Al3+, Zn2+, Fe3+
Cr3+, K+, Mg2+, Cd2+ and Ag+), and sulfate salts (Sr2+ and Fe2+).
,
,
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3
RESULTS AND DISCUSSION
Synthesis and characterization
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3.1
The synthetic routes are shown in scheme 1. Compounds A and L
were synthesized by the esterification reaction of picolinic acid and
phenolic hydroxyl group of benzimidazole compounds with high yield
0.5
1.0
0.8
0.6
0.4
0.2
1600
1400
1200
1000
800
600
400
200
0
0.4
0.3
0.2
0.1
0.0
L
A
A
L
0.0
250 300 350 400 450 500 550
200 250 300 350 400 450 500
(nm)
wavelength
wavelength (nm)
FIGURE 1 (a) The absorbance and fluorescence spectra of compounds L and A in acetonitrile (CH3CN) solutions (10 μM) and fluorescence
spectrum of L in CH3CN solution (10 μM) upon the addition of Cu2+ ions (5 equivalents of L). λex = 305 nm for L and λex = 330 nm for A. (b)
The absorbance changes of compound L in CH3CN solutions (10 μM) upon the addition of 5 equivalents of different metal ions

4
HE ET AL.
compounds L and A were not suitable to be used as chemosensors of
metal ions.
spectra of L and largely quenched fluorescence intensity (Figure 2a).
The fluorescence intensity of L at 340 and 356 nm was quenched by
95.0% and 92.6% respectively upon the addition of 5.0 equivalents
of Cu2+, the fluorescence was changed from ‘on’ to ‘off’. The fluores-
cence photographs of L in CH3CN solution in the absence and pres-
ence of Cu2+ ions further illustrated the quenching effect of Cu2+
ion on the emission of L in Figure 2(b). This result indicated the excel-
lent fluorescence selectivity of L towards Cu2+ ion in CH3CN solution.
Although the additions of Cr3+, Al3+ and Fe3+ ions caused changes of
the emission band at 340 nm, they gave very little influence on the
fluorescence intensity at 356 nm. Therefore, monitoring fluorescence
intensity at 356 nm of L can recognize and quantify Cu2+ ions. Fur-
thermore, the reason for the fluorescence quenching was possibly
owing to formation of the complex L–Cu2+ and then the paramagnetic
characteristic of Cu2+ caused the fluorescence quenching. The elec-
trons of the excited states of benzimidazole can transfer to the
unpaired d‐orbital of copper ions and further quench the fluorescence
of L.[29–33] The results demonstrated that L can recognize Cu2+ with
high selectivity in pure CH3CN solution.
However, compound L displayed selective fluorescence response
to Cu2+ ions in pure CH3CN solution, only the addition of Cu2+ caused
significant fluorescence quenching of compound L (Figure 1a). The
absorbance spectra and colors of L in CH3CN did not change notice-
ably after the addition of the test metal ions (Figure 1b) indicating L
could not be used as a colorimetric chemosensor for metal ions. The
absorbance spectra changes of L induced by Hg2+ and Pb2+ were
ascribed to the interaction between Hg2+ and Pb2+ ions with CH3CN
molecules.[16,18] On the contrary, the absorbance and fluorescence
spectra of compound A in CH3CN solution did not show selective
responses towards metal ions (Figure S9), which indicated that com-
pound A could not be used as a chemosensor for metal ions.
The results of the photophysical investigation on compounds L
and A indicated that only L was suitable for fluorescence sensing of
Cu2+ ions in CH3CN solution.
The reversible fluorescence response can further explain the for-
mation of the complex L–Cu2+. A solution of compound L (10 μM)
containing Cu2+ (2 equivalents of L) in CH3CN was added to an excess
of ethylenediamine tetraacetic acid (EDTA, 4 equivalents of L), the
fluorescence spectrum obtained was nearly restored to the original
spectrum of L solution (Figure S10). This result indicated that the fluo-
rescence quenching of L by Cu2+ was due to the reversible complex
formation process.
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3.3
Fluorescence sensing of compound L towards
Cu2+ ions
The earlier primary photophysical investigation of compound
L
towards metal ions showed that L can be used as a selective fluores-
cence chemosensor for Cu2+. In order to further confirm this conclu-
sion, the selectivity of L towards different metal ions was then
investigated in detail. After the additions of different metal ions (5
equivalents of L) into solutions of compound L (10 μM) in CH3CN
Job's plot is often used to evaluate complex ratio. In the series of
CH3CN solutions, the constant concentration of L and Cu2+ in each
solution was 50 μM and the molar ratios of L and Cu2+ ion were var-
iable. The emission intensity at 340 nm was plotted against the frac-
respectively, the metal ions such as K+, Ag+, Cd2+, Co2+, Mg2+, Hg2+
,
Ni2+,Mn2+, Ba2+, Pb2+, Ca2+, Na+ and Fe2+ did not impact any influence
on the fluorescence spectra. Furthermore, Cr3+, Al3+, Fe3+ ions did not
show any obvious influence on the fluorescence intensity of L at
356 nm but quenched the emission band at 340 nm and shifted emis-
sion to red. The Zn2+ was a special case, it made the fluorescence
spectrum of compound L increase and red‐shift, which could be
ascribed to the formation of a complex between L and Zn2+ ion to a
small extent. Only Cu2+ markedly impacted on the whole fluorescence
tion of L. The maximum appeared at 0.5 molar fraction of
L
indicating a 1:1 complex ratio between L and Cu2+. The 1:1 complex
ratio was further proved by electrospray ionization (ESI)‐HRMS spec-
tra. To prepare the mass spectrum sample, the excess solid
Cu(NO3)2·3H2O was added into the solution of L in DMSO‐d6. After
a sufficient reaction time, the liquid supernatant was used to measure
FIGURE 2 (a) Fluorescence response of compound L (10 μM) in acetonitrile (CH3CN) to different metal ions (5.0 equivalents of L). (b)
Fluorescence photographs of compound L in the absence and presence of 5 equivalents of Cu2+. (For an interpretation of the reference to the
color in this figure, the readers are referred to the website version of this article.)

HE ET AL.
5
the mass spectrum. As shown in Figure S11, a molecular‐ion peak at
m/z = 400.2713 belonged to [L + Cu2++H2O + 4H+]+ species (calcu-
lated 400.6945). Another molecular‐ion peak at m/z 458.2369
corresponded to the complex of [L + Cu2++CH3CN + Cl− + 3H+]+ (cal-
culated 458.1745). The results of the mass spectra also indicated that
both Cl− ion and H2O molecule took part in the coordination reaction
of L with Cu2+. To further confirm the 1:1 complex ratio and investi-
gate the role of the counter anion in the coordination process, the
complex reaction of L with Cu(NO3)2 was carried out. The mass
spectrum of the complex sample was also measured and shown in
Figure S12. As the previous mass spectrum, the molecular‐ion peak
at m/z = 400.2638 ascribed to [L + Cu2++H2O + 4H+]+ species also
appeared. Another different molecular‐ion peak at m/z 457.2557
corresponded to the complex of [L + Cu2++CH3CN + 2H2O + 2H+]+
(calculated 457.1037). The two peaks further demonstrated the 1:1
(Figure 4a), the Cu2+ solution in water was incrementally added into
L solution in CH3CN (10 μM). The added maximal volume of Cu2+
solution was smaller than 2% volume of L solution to avoid the large
change of the concentration of L. The fluorescence intensity of L at
356 nm was gradually quenched with the incremental Cu2+concentra-
tion (Figure S14). When the concentration of Cu2+ reached about 3.5
equivalents of L, the fluorescence of sensor 1 decreased to the equi-
librium value and all the fluorescence was almost quenched. The curve
of the fluorescence intensity at 356 nm as a function of Cu2+ concen-
tration in the range 2–17 μM was a straight line with a large correla-
tion coefficient of R2 = 0.99362. From this calibration curve, the
limit of detection (LOD) was calculated to be 3.05 × 10−6 mol/L
according to the equation of (3σ/K).[34] This LOD was lower than the
standard value of copper in drinking water (~20 μM) specified by the
US Environment Agency.[35] The LOD and linearity range provided
evidence that L was suitable for quantitative determination of Cu2+
concentration in water samples.
complex ratio between
L . The molecular‐ion peak
and Cu2+
corresponded to NO3−‐containing complex was not found, which
−
indicated that the counter anion NO3 did not take part in the
The affinity of L with Cu2+ could be evaluated according to the
value of the binding constant (Ka) of the L–Cu2+ complex, which can
be calculated from the Benesi–Hildebrand (B‐H) plot based on the fol-
lowing equation.[36]
complex reaction.
Fast fluorescence response of a chemosensor is more suitable for
on‐site monitoring in practical applications. The fluorescence at
340 nm of L solution was monitored and rapidly decreased to equilib-
rium point within 15 s after addition of Cu2+ ions (Figure S13), which
indicated that L had the capability for on‐site monitoring.
High capacity of resisting interference is more beneficial to
chemosensors in practical applications and can be testified via the fol-
lowing experiment. The possible interference of other metal ions
1=ðF0– FÞ ¼ 1=fKaðF0–FminÞCg þ 1=ðF0–Fmin
Þ
where C is the concentration of Cu2+ solution, F 0 was the fluores-
cence intensity of L at 356 nm without Cu2+ and F was the fluores-
cence intensity upon the addition of different concentrations of
Cu2+. Thus, F min is the minimum value of the fluorescence intensity
upon the addition of excess Cu2+ ions. The experimental curve was
fitted according to the 1:1 complex ratio and the fitting curve was
almost a linear relationship with a high correlation coefficient of
0.99152 (Figure 4b), which also confirmed the 1:1 complex ratio
including Fe3+, Ag+, K+, Al3+, Cr3+, Mg2+, Cd2+, Co2+, Hg2+, Zn2+
,
Ni2+, Mn2+, Ca2+, Ba2+, Pb2+, Sr2+ and Fe2+ were taken into consider-
ation for a chemosensor in a practical application. After the addition of
5 equivalents of other metal ions to the solutions of L (10 μM) con-
taining 5 equivalents of Cu2+ in CH3CN respectively, their fluores-
cence intensities at 356 nm were measured and the results are
shown in Figure 3(b). In all the tested solutions, the addition of Cu2+
ions caused fluorescence quenching near to that of the L solution only
containing Cu2+ ions. The experiment proved that compound L can
recognize Cu2+ over a variety of coexisting metal ions.
between
6.02 × 104 M−1 from the intercept and slope of the linear fitting plot
L . The Ka value was calculated to be
with Cu2+
indicating strong affinity of L with Cu2+
.
|
3.4
Study on the fluorescence quenching
mechanism
The sensing property of L for Cu2+ ions was studied using fluores-
cence titration. The fluorescence emission titration experiments of L
(10 μM) with Cu2+ (0–5 equivalents) in CH3CN were carried out
Generally, there are two quenching mechanisms above fluorescence
quenching, static and dynamic quenching. Stern–Volmer curves are
1400
300
(a)
1200
1000
800
600
400
200
0
(b)
250
200
150
100
50
0
0.0
0.2
0.4
0.6
0.8
1.0
2+
[L]/[L]+
[
Cu
]
FIGURE 3 (a) Job's plot from the fluorescence intensity at 340 nm of L and Cu2+ in acetonitrile (CH3CN) solution with a total concentration of
50 μM (λex = 305 nm). (b) The fluorescence intensity changes at 356 nm of L (10 μM) in CH3CN solutions upon the addition of different metal ions.
The black bars represent the fluorescence intensity of L at 356 nm after the addition of different metal ions (5 equivalents of L), the red bars
represent the fluorescence intensity of the earlier solutions after a further addition of 5 equivalents of Cu2+, respectively

6
HE ET AL.
-0.0007
-0.0008
-0.0009
-0.0010
-0.0011
-0.0012
-0.0013
1400
1200
1000
800
600
400
200
0
-4
intercept=-4.79819x10
(a)
-4
slope=-7.96442x10
(b)
2
R
=0.99152
4
-1
Ka=6.02x10
M
325
350
375
400
425
0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
1/[Cu2+](105 mol/L)
Wavelength / nm
FIGURE 4 (a) The changes of fluorescence spectra of L (10 μM) in acetonitrile (CH3CN) solution with incremental addition of Cu2+ (0–5
equivalents of L). (b) The Benesi–Hildebrand plot of the titration experiment of L with Cu2+ ions in CH3CN solution
often used to investigate the nature of the quenching mechanism in
the complexing process. Two different forms of fluorescence intensi-
ties (ln F 0/ F or F 0/ F ) were plotted as a function of Cu2+ concentra-
tion, in which, the linear curve belongs to the true quenching mode.[37]
1:1 L–Cu2+ complex formation, the density functional theory (DFT)
calculations, 1H‐NMR titration and IR spectra were carried out.
The DFT calculation was completed using the B3LYP/6‐31G(d)
level of Gaussian 09 package to further study the interaction mecha-
nism between L and Cu2+ [38]
The optimized ground‐state geometries
.
of the complex showed a 1:1 binding stoichiometry in Figure 6(a).
Compound L provided two coordination sites, the nitrogen atom of
pyridine and the oxygen atom of the carbonyl via lone‐pair electrons.
The frontier orbital energies and shapes were presented in
Figure 6(b), the main highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) of L were cen-
tered on the benzimidazole moiety and pyridine ring. On the contrary,
all the HOMO and LUMO of the L–Cu2+ complex were localized in the
pyridine part indicating the existence of a charge transfer from benz-
imidazole to Cu2+ and further causing the fluorescence quenching.
The energy gap between the LUMO and HOMO of L–Cu2+ was calcu-
lated to be 0.33 eV and was much smaller than that of L (3.82 eV),
which indicated the formation of a stable complex. The HOMO energy
(−12.69 eV) of L–Cu2+ complex was very much smaller than that of L
(−6.06 eV) indicating electrons on the HOMO of the complex was
more stable and had lower reactive activity.[39,40] In addition, the
absorbance spectrum of L was also optimized by DFT calculations
under the consideration of the solvent effect, Figure S15. The opti-
mized maximum absorbance band was at 298 nm, which was very
close to the experimental value (296 nm) and also indicated that the
DFT calculation results were in very good agreement with the exper-
imental results. The earlier results confirmed the 1:1 stoichiometry
of the L–Cu2+ complex and indicated that the nitrogen atom of pyri-
F0
½Mꢀ
ln ¼ eK
Static quenching
SV
F
F0
¼ 1 þ KSV½Mꢀ Dynamic quenching
F
where F and F 0 are the fluorescence intensities of L at 356 nm in the
presence and absence of Cu2+, respectively, [M] is the concentration
of Cu2+ and KSV is the Stern–Volmer quenching constant. As shown in
Figure 5, F 0/F (black line) did not show a linear relationship with Cu2+
concentration, which suggested the fluorescence quenching did not
belong to dynamic quenching. On the contrary, the plot of ln F 0/F
values against Cu2+ concentrations was a linear curve; the fitting curve
with a high correlation coefficient (R2 = 0.99521) was superimposed
together with the experimental plot. This result illustrated that the
fluorescence quenching process was concurrent with the Perrin model
of static quenching (blue line) and proves that fluorescence quenching
occurs intramolecularly in the L–Cu2+ complex.
|
3.5
Possible sensing mechanism
Aforementioned results of photophysical studies and mass spectra
suggested that the sensing response of L towards Cu2+ was ascribed
to the formation of 1:1 L–Cu2+ complexes. To further prove the
dine and oxygen atom of carbonyl coordinated with Cu2+
.
1H‐NMR titration in DMSO‐d6 also provided obvious proof of
coordination interaction between L and Cu2+, the results are shown
in Figure S16. The changes of the integral area and position of hydro-
30
3.5
25
20
15
10
5
Y=1.09483X-0.34148
3.0
2.5
2.0
1.5
1.0
0.5
0.0
2
R =0.99521
gen peaks illustrated the coordination interaction between L and Cu2+
.
static
The peak at 8.80 was ascribed to the hydrogen adjacent to the nitro-
gen atom of the pyridine unit and vanished completely after the addi-
tion of 0.5 equivalents Cu2+ of L, which indicated the participation of
the nitrogen atom of pyridine in the coordination of L with Cu2+. On
the contrary, the peak at 5.75 corresponding to the hydrogen of imine
of benzimidazole decreased gradually with Cu2+ increasing but did not
shift or vanish, which showed that the nitrogen atom of amine did not
take part in the coordination process. The other proton peaks
dynamic
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5
[Cu2+](10-5 mol/L)
FIGURE 5 Stern–Volmer quenching analysis plots of L upon the
addition Cu2+

HE ET AL.
7
FIGURE 6 (a) Optimized ground‐state geometries and orbital energy level of L and L–Cu2+. Color code: C (gray), O (red), N (blue), H (white), Cu2+
(orange). (b) LUMO and HOMO orbital shapes of L–Cu2+ complex and L. (For interpretation of the reference to color in this figure legend, the
readers are referred to the website version of this article.)
gradually decreased with the increment of Cu2+ ions accompanied
with the high‐field shift. These results clearly revealed the occurrence
of a complex reaction between L with Cu2+ and the nitrogen atoms on
of the imino group did not take part in the coordination reaction. In
addition, most of the peaks in the fingerprint region vanished upon
the coordination effect. These results indicated that the nitrogen atom
of pyridine and the oxygen atom of the carbonyl groups of L were coor-
pyridine were the coordination sites for Cu2+
.
Infrared (IR) spectra results also provided the evidence of the coor-
dination interaction of L with Cu2+. The complex L–Cu2+ sample for IR
spectrum was prepared as for the samples for HRMS spectra. The
absorption peak of carbonyl of ester of L appeared at 1754 cm−1, the
broad absorption peak at 3424 cm−1 was caused by the formation of
hydrogen‐bond due to the existence of oxygen and nitrogen atoms
(Figure S17). After the formation of the complex, the carbonyl absorp-
tion peak disappeared indicating the participation of the carbonyl group
in the coordination process with Cu2+. Meanwhile, the peak of the
stretching vibration of benzene skeleton was extruded and presented
a very strong peak at 1600 cm−1. On the contrary, the amine peak at
3424 cm−1 did not vanish, which demonstrated that the nitrogen atom
dination points for Cu2+
.
Based on the earlier evidence, the proposed binding mode of L
with Cu2+ and the fluorescence quenching mechanism are shown in
Scheme 2.
|
4
ANALYTICAL APPLICATIONS
From the earlier experimental results, L can recognize and detect Cu2+
in water samples. To prove this result, the recovery experiment of
water samples and fluorescent images in living cells were executed.
|
4.1
Recovery experiments in water samples
The drinking and tap water samples were used in Cu2+ recovery exper-
iments, each sample was measured with four replicates and the results
are shown in Table 1. The recovery and relative standard deviation
(RSD) values of four samples were satisfactory. The recovery results
proved that sensor L could be useful in the quantitative detection of
Cu2+ in environmental water samples.
SCHEME 2 The proposed binding mode of L with Cu2+ and its
fluorescence quenching mechanism
|
4.2
Fluorescence bioimaging for living cells
TABLE 1 The analytical results of copper(II) ion (Cu2+) in water
samples (n = 4)a
The high selective and sensitive fluorescence response of L towards
Cu2+ ions encouraged us to study the possibility of Cu2+ recognition
in living cells via the cell imaging experiments. The liver cancer HepG2
cells were chosen for the fluorescence imaging experiments. First, we
prepared a solution (20 μM) of L in DMSO and a solution of Cu2+
(40 μM) in empty medium. Then well 1 and 2 were loaded with
500 μL cell suspension, respectively. Then, well 1 was added with
10 μL Cu2+ solution and incubated at 37°C for 30 min and then
washed twice with phosphate‐buffered saline (PBS) solution. After
Samples
Added (μM) Foundb (μM) Recovery (%) RSDc (%)
Tap water
4.0
8.0
4.08
8.08
3.96
7.99
102.0
100.75
99.0
0.21
0.43
0.31
0.29
Drinking water 4.0
8.0
99.88
aConditions: [Sensor L] = 10 μM in acetonitrile solution. bMean values of
four determinations. cRelative standard deviation of four determinations.

8
HE ET AL.
FIGURE 7 Confocal fluorescence images of living HepG2 cells. (a) Bright field image of the HepG2 cells only pre‐incubated with L. (b)
Fluorescence image of (a). (c) Overlay image of (a) and (b). (d) Bright field image of HepG2 cells pre‐incubated with Cu2+ for 30 min and then
incubated with L. (e) Fluorescence image of (d). (f) Overlay image of (d) and (e)
that, wells 1 and 2 were added with 10 μL L solution respectively and
incubated for 90 min. After being washed twice with PBS solution, the
two kinds of treated cells were placed on glass slides for fluorescence
imaging.
ORCID
Yi He https://orcid.org/0000-0002-2616-0497
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As shown in Figure 7, the cells only incubated with L showed a
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in living cells. This result confirmed that L can coordinate with Cu2+
ions and recognize Cu2+ ions in living cells.
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