A new highly selective and sensitive fluorescent probe for Zn2+ and its application in cell-imaging

Article abstract of DOI:10.1016/j.dyepig.2014.03.018

A new fluorescent probe, 3-((4-([2,2′:6′,2″-terpyridin]- 4′-yl)phenyl)ethynyl)-7-methoxy-2H-chromen-2-one (ZC-F7) composed of coumarin as the fluorophore and terpyridine as the receptor is designed and synthesized. Based on the intramolecular charge transfer (ICT) effect, the probe exhibits significant variation on emission wavelengths with shifts more than 100 nm after combined with Zn²⁺, in accordance with the conversion of emission colors from blue to green. Good selectivity and sensitivity of this probe towards Zn²⁺ can be found even on the ppb level in aqueous solution. The Job's plot test suggests a 1:1 stoichiometry between ZC-F7 and Zn²⁺. The application of the fluorescence probe in bio-imaging is also demonstrated, proving its potential usage in fields such as environment protection, water treatment and safety inspection.

Full text of DOI:10.1016/j.dyepig.2014.03.018

Dyes and Pigments 107 (2014) 45e50
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A new highly selective and sensitive fluorescent probe for Zn2þ and its
application in cell-imaging
,
Quan Hu a, Yiqun Tan b, Min Liu b, Jiancan Yu b, Yuanjing Cui b, Yu Yang b
*
a Department of Pharmacy, School of Medicine, Hangzhou Normal University, Hangzhou 310036, PR China
b State Key Laboratory of Silicon Materials, Cyrus Tang Center for Sensor Materials and Applications, Department of Materials Science & Engineering,
Zhejiang University, Hangzhou 310027, PR China
a r t i c l e i n f o
a b s t r a c t
A new fluorescent probe, 3-((4-([2,20:60,200-terpyridin]-40-yl)phenyl)ethynyl)-7-methoxy-2H-chromen-2-
one (ZC-F7) composed of coumarin as the fluorophore and terpyridine as the receptor is designed and
synthesized. Based on the intramolecular charge transfer (ICT) effect, the probe exhibits significant
variation on emission wavelengths with shifts more than 100 nm after combined with Zn2þ, in accor-
dance with the conversion of emission colors from blue to green. Good selectivity and sensitivity of this
probe towards Zn2þ can be found even on the ppb level in aqueous solution. The Job’s plot test suggests a
1:1 stoichiometry between ZC-F7 and Zn2þ. The application of the fluorescence probe in bio-imaging is
also demonstrated, proving its potential usage in fields such as environment protection, water treatment
and safety inspection.
Article history:
Received 5 January 2014
Received in revised form
17 February 2014
Accepted 11 March 2014
Available online 25 March 2014
Keywords:
Chemosensor
Fluorescent probe
Zinc
Ó 2014 Elsevier Ltd. All rights reserved.
Fluorescence
Intramolecular charge transfer
Cell-imaging
1. Introduction
Zn2þ because of their location at the same group. Recently, Jiang
et al. have reported an inspiring distinguishing method for Zn2þ
As the second most abundant transition metal ion in human
body, zinc plays an important role in the gene transcription, regu-
lation of metalloenzymes, neural signal transmission and apoptosis
[1e5]. Its deficiency causes acrodermatitis enteropathica [6], while
excess zinc may also cause serious neurological disorders such as
Alzheimer’s and Parkinson’s diseases [7e9]. It is found that the
imbalance in zinc may cause several health problem including su-
perficial skin diseases, prostate cancer, diabetes, and brain diseases
[10e12]. Thus, extensive research efforts have been devoted on
the quantitative measurement of trace Zn2þ in vivo [13e15].
Because of the lack of spectroscopic signature of Zn2þ, the method
of fluorescent probe has become one of the best choices for
detecting and tracking Zn2þ in cell-imaging and neurobiological
experiments [16].
and Cd2þ by introducing carbonyl group [28,29]. Ng et al. developed
two fluorescence sensors for differential detection Zn2þ and Cd2þ
based on BODIPY which can respond towards Zn2þ and Cd2þ
respectively [30]. Nevertheless, reports about the application of
these probes in cell-imaging have been barely mentioned.
The development of novel fluorescent probe for metal ions with
high sensitivity and selectivity has long been concerned by our
group [31e34]. We have developed a new fluorescent probe ZC-F1
based on intra-molecular transfer (ICT) effect for recognizing Zn2þ
from Cd2þ with the detection limit under ppb level [31]. However, it
cannot be used in bio-imaging because of two reasons: a) the poor
solubility of ZC-F1 in aqueous solution restricts its application
in vivo. b) The quantum yield of fluorophores baring ICT effect
usually reduces sharply in real practice after the electron with-
drawing group combined with metal ions, especially in solvents
with high polarity, for the decrease of the energy gap between
ground state and excited state [35e38]. To get over these obstacles,
a new fluorescent probe highly sensitive and selective towards
Zn2þ with better solubility and higher quantum yield is required.
Herein, we report a new fluorescent probe 3-((4-([2,20:60,200-
terpyridin]-40-yl)phenyl)ethynyl)-7-methoxy-2H-chromen-2-one
(ZC-F7). This probe contains 7-methoxycoumarin, which is known
In recent years, a lot of fluorescent probes for detecting Zn2þ
have been developed and most of them exhibit good performances
in the cellular use [17e27]. However, many fluorescence sensors
are still baring problems of selectivity, especially the interruption
from Cd2þ which possesses very similar chemical properties with
* Corresponding author. Tel.: þ86 571 87952334; fax: þ86 571 87951234.
E-mail address: yuyang@zju.edu.cn (Y. Yang).
http://dx.doi.org/10.1016/j.dyepig.2014.03.018
0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.

46
Q. Hu et al. / Dyes and Pigments 107 (2014) 45e50
Scheme 1. Synthesis of ZC-F7.
as a water soluble fluorophore and terpyridine as the receptor [39].
To overcome the fluorescence decrease resulted from metal ion
binding induced ICT enhancement, the methoxy group is also a
good choice as the electron donor, which possess less electron-
donating ability than other common donor such like amino
group, inducing weaker ICT effect and wider energy gap between
ground state and excited state. Thus, after combined with metal
ions, the energy gap may not be reduced so sharply and the fluo-
rescence enhancement in aqueous solution can be expected.
CDCl3):
d
¼ 8.78(t, 4H, J ¼ 12 Hz, ArH) 8.74(d, 2H, J ¼ 7 Hz, ArH)
7.97(s, 2H, ArH), 7.85(d, 2H, J ¼ 8 Hz, ArH), 7.67(d, 2H, J ¼ 2 Hz, ArH),
7.44(s, 2H, ArH); IR(KBr pallet, cmꢀ1): 1605s, 1584s, 1567s, 1541s,
1489s, 1468s, 1443s, 1409s, 1380s, 1074s, 1037s, 1007s, 990s, 888s,
821s, 786s, 733s, 703s, 660s, 627s, 615s, 575s, 497s, 476s, 449s,
420s; MS-ESI theoretical: m/z [M þ H]þ ¼ 388.04, found: 388.26;
Mp: 137.8 ꢁC; Anal. Calcd for C21H14BrN3: C, 64.96; N, 10.82; H, 3.63.
Found: C, 65.03; N, 10.79; H, 3.66.
2.2.2. 40-(4-ethynylphenyl)-2,20:60,200-terpyridine (4)
2. Experimental section
3 (1.94 g, 5 mmol), Pd(PPh3)2Cl2 (0.14 g, 0.2 mmol), 2-methyl-
3-butyn-2-ol (1.26 g, 15 mmol) and CuI (0.038 g, 0.2 mmol) were
added into a mixture of Et3N (4 mL) and toluene (16 mL) and
refluxed for 12 h. The solution was evaporated and purified by
chromatography using CH2Cl2 and EtOAC (20:1) as eluent,
resulting in white crystal, which is stirred into toluene with KOH
(0.56 g, 10 mmol) and refluxed for 2 h. The solution was then
extracted with CH2Cl2, and the organic phase was combined and
evaporated. The resulting black solid was purified by chroma-
tography, using EtOAC and petroleum (1:2) as eluent, afforded
2.1. Materials and measurements
Solvents and reagents were obtained from commercial source
and used as received without further purification. 1 and 2 are
synthesized according to literature [40].
1H NMR spectra were recorded in CDCl3 on a 500 MHz Bruker
Avance DMX500 spectrometer with tetramethysilane (TMS) as an
internal standard. Elemental analysis was performed using a
Thermo Finnigan Flash EA1112 microelemental analyzer. Differ-
ential scanning calorimetry (DSC) was performed on a Netzsch
Instruments 200 F3 at a heating rate of 10 K/min under nitrogen
atmosphere. Fluorescence emission spectra and excitation
spectra were obtained on a Hitachi F4600 fluorescence spectro-
photometer. UVevis absorption spectra were obtained using a
PerkineElmer Lambda spectrophotometer. The Fluorescence
decay curves were measured by an Edinburgh Instrument F900.
Fluorescence quantum yield is measured with integrating sphere
on Edinburgh Instrument F900. Fluorescence images were ob-
tained on confocal laser scanning microscopes (CLSM, fluoview
FV1000, Olympus).
pale solid 4 (0.94 g, 48%). 1H NMR (500 MHz, CDCl3):
d
¼ 8.68(t,
4H, J ¼ 7 Hz, ArH), 8.62(d, 2H, J ¼ 8 Hz, ArH), 7.84(m, 4H, J ¼ 8 Hz,
ArH), 7.71(d, 2H, J ¼ 8.5 Hz, ArH), 7.32(t, 2H, J ¼ 6 Hz, ArH), 3.11(s,
1H, CH); IR(KBr pallet, cmꢀ1): 3202s, 3063m, 1653w, 1605s,
1585s, 1566s, 1540m, 1510m, 1466s, 1441m, 1412s, 1388m, 1110m,
1076m, 1038m, 990s, 847m, 839s, 825w, 788s, 744m, 734s, 678m,
659m, 623m, 578s, 534m, 516w; MS-ESI theoretical: m/z
[M þ H]þ ¼ 334.13, found: 334.21; Mp: 142.1 ꢁC; Anal. Calcd
for C23H15N3: C, 82.86; N, 12.60; H, 4.54. Found: C, 82.83; N,
12.73; H, 4.64.
Table 1
Linear photophysical properties of ZC-F7 and ZC-F7-Zn.a
2.2. Synthesis
b
3
ꢂ 104L molꢀ1 cmꢀ1
sc, ns
F
d, %
labs, nm
2.2.1. 40-(p-Bromophenyl)-2,20:60,200-terpyridine (3)
ZC-F7
ZC-F7-Zn
6.25
6.42
2.85
3.30
6.2
13.0
385
395
4-bromobenzaldehyde (1 g, 5.4 mmol) and 2-acetylpyridine
(1.3 g, 10.8 mmol) were stirred into methanol (120 mL) followed
by addition of NaOH (0.22 g, 5.4 mmol) and NH4OH 30 mL. The
mixture was refluxed for 36 h, and then cooled down to room
temperature. The precipitate was filtered and washed by methanol
and water to obtain white powder (0.8 g, 40%). 1H NMR (500 MHz,
a
All the compounds were dissolved in solvents (water:DMSO ¼ 99:1) at the
concentration of 1 ꢂ 10ꢀ6 M.
b
The molar absorption coefficient of ZC-F7 and ZC-F7-Zn.
The fluorescence lifetime of ZC-F7 and ZC-F7-Zn.
Fluorescence quantum yield of ZC-F7 and ZC-F7-Zn excited at the respective
c
d
maximum absorption wavelengths.

Q. Hu et al. / Dyes and Pigments 107 (2014) 45e50
47
Fig. 3. Fluorescence intensities of ZC-F7 at 444 and 530 nm in the solution with
different concentration of Zn2þ and their linear fits.
Fig. 1. Absorption spectra of ZC-F7 in DMSO/water (99:1) at 100 nM upon titration of
Zn2þ
.
2.2.3. 3-((4-([2,20:60,200-terpyridin]-40-yl)phenyl)ethynyl)-7-
methoxy-2H-chromen-2-one (ZC-F7)
imaging, the cells were passed and platedon glass-bottomed dishes.
For labeling, the growth medium was removed and replaced with
DMEM without FBS. The cells were treated and incubated with
2 (1.01 g, 4 mmol) and 4 (1.32 g, 4 mmol) were solved into a
mixture of Et3N (4 mL) and toluene (16 mL). CuI (0.038 g, 0.2 mmol)
and Pd(PPh3)2Cl2 (0.14 g, 0.2 mmol) were stirred into the solution
and heated at 80 ꢁC for 12 h under argon atmosphere. The resulting
solution was evaporated and purified by chromatography using
CH2Cl2 and petroleum as eluent to yield yellow solid ZC-F7 (1.02 g,
10
mL of 1 mM ZC-F7 in DMSO stock solution (10
m
M ZC-F7) at 37 ꢁC
under 5% CO2 for 15 min, and provided with 5
mL of fresh media that
contained either 1 mM or 0 mM ZnCl2. Then, the cells were incu-
bated for another 15 min at the conditions mentioned above. Prior
to imaging, cells were rinsed three times with phosphate buffered
saline (PBS).
55%). 1H NMR (500 MHz, CDCl3):
d
¼ 8.74(m, 4H, ArH) 8.68(d, 2H,
J ¼ 8.0 Hz, ArH) 7.90(m, 5H, ArH), 7.71(d, 2H, J ¼ 8.5 Hz, ArH), 7.41(d,
1H, J ¼ 8.5 Hz, ArH), 7.36(m, 2H, ArH), 6.89(m, 1H, ArH), 6.85(d, 1H,
J ¼ 2.5 Hz, ArH), 3.90(s, 3H, CH3); IR(KBr pallet, cmꢀ1): 3049w,
2925w, 1727s, 1620s, 1600, 1584s, 1565s, 1506s, 1465s, 1440s,
1414m, 1388s, 1364s, 1317m, 1278s, 1226s, 1194w, 1143s, 1121m,
1073w, 1040m, 1016m, 989m, 968m, 917w, 831s, 790s, 767s, 733m,
684m, 660m, 626w, 622w, 575m, 529m; MS-ESI theoretical: m/z
[M þ H]þ ¼ 508.16, found: 508.23; Mp: 185.3 ꢁC; Anal. Calcd for
3. Results and discussion
3.1. Design and synthesis of ZC-F7
In the newly designed fluorescent probe ZC-F7, 7-
methoxycoumarin, which is selected as the reporter for its good
solubility in water is linked with Zn2þ selective receptor terpyridine
via ethynyl group as the conjugated bridge. Thus, ICT effect can be
expected in this probe. As a promising strategy, intramolecular
charge transfer (ICT) was widely used in the design of fluorescent
probes [41e44]. Probes based on ICT generally have structures
containing an electron withdrawing group conjugated to an elec-
tron donating group, which permit ICT from the donor to the
acceptor upon excitation, accompany with large Stokes’ shift
associated with ICT efficiency [45e47]. After combined with metal
C
33H21N3O3: C, 78.09; N, 8.28; H, 4.17. Found: C, 78.49; N, 8.32; H,
4.13.
2.3. Cell culture
HeLa human cervical carcinoma cells were cultured in Dulbec-
co’s Modified Eagle’s Medium (DMEM, Neuronbc) supplemented
with 10% fetal bovine serum (FBS, sijiqing) penicillin (100 units/ml,
Boster), and streptomycin (100
mg/mL, Boster). Two days before
Fig. 2. Fluorescence spectra of ZC-F7 in DMSO/water (99:1) at 100 nM excited at
395 nm upon titration of Zn2þ
Fig. 4. Job’s plot analysis of the stoichiometry of ZC-F7 and Zn2þ (excited at 395 nm
and monitored at 530 nm).
.

48
Q. Hu et al. / Dyes and Pigments 107 (2014) 45e50
Zn2þ and the probe. As shown in Fig. 2, a broadband emission
0.8
0.6
0.4
0.2
0.0
3000
2500
2000
1500
1000
500
em
Zn2+
abs
spectrum with split peaks located at 425 and 444 nm can be
observed. While with the titration of Zn2þ, the initial fluorescence
bands gradually disappear and a new emission peak at 530 nm
owing to the combination of probe and Zn2þ is observed. It is worth
noting that the fluorescence intensity of ZC-F7 at both 444 nm and
530 nm has well fitted linear relationships with [Zn2þ], as we can
see in Fig. 3, and significant enhancement of fluorescence intensity
can be observed even when [Zn2þ] are as low as 10 nM, which equal
to 0.65 ppb, indicating that ZC-F7 is highly sensitive for Zn2þ even
at ppb level. Job’s plot is also performed to confirm the possible
binding modes of ZC-F1 with Zn2þ and certify a 1:1 stoichiometry
(Fig. 4).
other ions
0
300
400
500
600
700
Wavelength (nm)
3.3. Selectivity
Fig. 5. Absorption and fluorescence emission spectra of ZC-F7 at 100 nM with Ni2þ
,
Cd2þ,Cr3þ, Fe3þ, Naþ, Ca2þ, Pb2þ, Al3þ, Co2þ, Cu2þ, Mg2þ, Kþ, Fꢀ, Clꢀ, NOꢀ3 , PO4ꢀ, CO23ꢀ
and SCNꢀ upon excitation of 395 nm. The competing ions are at concentration of
To verify the feasibility of ZC-F7 in complicated environment,
representative interferences of biological and environmental in-
100 equiv. (10 m
M) and Zn2þ is 12 equiv.
terests such as Ni2þ, Cd2þ,Cr3þ, Fe3þ, Naþ, Ca2þ, Pb2þ, Al3þ, Co2þ
,
Cu2þ, Mg2þ, Kþ, Fꢀ, Clꢀ, NO3ꢀ, PO4ꢀ, CO32ꢀ and SCNꢀ are introduced to
investigate their impact on the selectivity of the probe towards
Zn2þ. As shown in Fig. 5, addition of main group metal ions,
including Kþ, Ca2þ, Naþ, Mg2þ and Al3þ exert no influence on the
ions, the energy level of ICT state is supposed to be reduced, leading
to both spectra shift and fluorescence intensity variation [47e50].
As shown in Scheme 1, the compound ZC-F7 can be synthesized
in several steps from commercially available chemicals in high
yield. Details for synthesis are described in the experiment section.
Photophysical parameters of ZC-F7 and ZC-F7-Zn such as quantum
yield, fluorescence lifetime, absorption peaks and absorption co-
efficients are listed in Table 1.
fluorescence intensity before and after treating ZC-F7 with Zn2þ
,
whereas Fe3þ, Co2þ, Cu2þ, Ni2þ, Cr3þ and Pb2þ, quench the fluo-
rescence slightly. Anions such as Fꢀ, Clꢀ, NOꢀ3 , POꢀ4 , CO32ꢀ and SCNꢀ
cause no interruption on the fluorescence spectra of the probe. In
addition, Cd2þ, as an important disruptor, exhibits little disturbance
even at 100 equiv. level, indicating that the probe can tell Zn2þ from
Cd2þ effectively. Moreover, all the interferences mentioned above
exhibit almost no influence on the absorption spectra.
3.2. Optical response of probe ZC-F7
To test the usefulness of ZC-F7 for Zn2þ detection, the absorption
and fluorescent spectra and its response after titrated with Zn2þ are
first studied in aqueous solution (water:DMSO ¼ 99:1). With the
titration of Zn2þ, the absorption peak at 385 nm decreases gradu-
ally and a new absorption maximum at 395 nm appears as we can
see in Fig. 1. The isosbestic point at 391 nm suggests the complex of
3.4. Cell imaging
Taking advantage of the excellent sensing properties for Zn2þ
in vitro, the assessment whether ZC-F7 can detect Zn2þ in live cells
Fig. 6. Images of HeLa cells incubated with ZC-F7 (10
contrast (DIC) images, while Panels (b), (c), (e) and (f) show the corresponding confocal fluorescence images collected at 450e530 nm (b and e) and 550e650 nm (c and f). The
wavelength for excitation is 405 nm. The scale bar is 50 m.
mM) and ZnCl2 at the concentration of 5 mM (aec) and 0 mM (def) for 30 min. Panels (a) and (d) show differential interference
m

Q. Hu et al. / Dyes and Pigments 107 (2014) 45e50
49
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dium at 37 ꢁC, which showed strong intracellular fluorescence in
the window of 450e530 nm, while almost no fluorescence can be
observed in the detection range of 550e650 nm. Other cells were
firstly treated and incubated with 10
stock solution (10
M ZC-F7) at 37 ꢁC under 5% CO2 for 15 min, and
then incubated with DMEM containing ZnCl2 (5 M) for another
mL of 1 mM ZC-F7 in DMSO
m
m
15 min at 37 ꢁC. After incubation, the cells were washed with PBS to
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gested that the cells were viable throughout the imaging experi-
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brane permeable and could response to the presence of Zn2þ in live
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4. Conclusion
A new fluorescent probe for Zn2þ with high sensitivity and
selectivity was designed and synthesized and its response was
studied. The results showed that the probe can detect Zn2þ by two
signal channels and minor interruption can be observed from other
representative interferences of biological and environmental in-
terests, including Cd2þ, which usually plays an important role as a
disruptor. This probe exhibited apparent signal change on the ppb
level indicating its potential use in the environmental detection of
trace Zn2þ. In addition, the cell-imaging experiment suggested that
ZC-F7 was membrane permeable and could reveal the distribution
of Zn2þ in live cells. Moreover, these cells were all viable during the
experiments, showing a potential usage of ZC-F7 as an indicator for
Zn2þ in vivo.
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Acknowledgment
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The authors gratefully acknowledge the financial support for
this work from the National Natural Science Foundation of China,
China (Nos. 51229201 and 51372221), and Natural Science Foun-
dation of Zhejiang Province (No. LY12E02004).
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