A highly selective fluorescent and colorimetric chemosensor for Zn II and its application in cell imaging

Article abstract of DOI:10.1002/ejic.201100804

A fluorescent, colorimetric chemosensor, L1, based on 8-aminoquinoline, has been developed as a sensor for Zn²⁺. L1 exhibited high selectivity and sensitivity towards Zn²⁺ over other common metal ions in a physiological pH window with a 1:1 binding mode. The ability of L1 to monitor intracellular Zn²⁺ in living cells has been examined by fluorescence microscopy and indicated that L1 is cell-permeable and biocompatible. We hope that such Zn²⁺-selective sensors will find application in biomedical and environmental detection. Copyright

Full text of DOI:10.1002/ejic.201100804

FULL PAPER
DOI: 10.1002/ejic.201100804
A Highly Selective Fluorescent and Colorimetric Chemosensor for ZnII and Its
Application in Cell Imaging
Guoqiang Xie,[a] Yanjun Shi,[a] Fengping Hou,[a] Hongyan Liu,[a] Liang Huang,[a]
Pinxian Xi,[a] Fengjuan Chen,[a] and Zhengzhi Zeng*[a]
Keywords: Fluorescence / Zinc / N ligands / Colorimetric chemosensor / 8-Aminoquinoline / Cell imaging
A fluorescent, colorimetric chemosensor, L1, based on 8-ami-
noquinoline, has been developed as a sensor for Zn2+. L1 ex-
hibited high selectivity and sensitivity towards Zn2+ over
other common metal ions in a physiological pH window with
a 1:1 binding mode. The ability of L1 to monitor intracellular
Zn2+ in living cells has been examined by fluorescence mi-
croscopy and indicated that L1 is cell-permeable and bio-
compatible. We hope that such Zn2+-selective sensors will
find application in biomedical and environmental detection.
small molecules and highly sensitive for real-time detection
in biological systems at physiological pH.
Introduction
The development of fluorescent chemosensors that have
the capability to selectively recognize and sense metal ions
is one of the most challenging fields in organic and supra-
molecular chemistry.[1] Sensors based on ion-induced
changes (e.g. intensity and/or emission wavelength) in fluo-
rescence have many advantages over other techniques,
which include their simplicity, high sensitivity, high selectiv-
ity, and instantaneous response.[2] So far, a number of excel-
lent metal-ion sensors based on metal–ligand coordination
or chemical reaction have been reported to detect transition
and heavy metal ions, such as zinc ions.[3] Zinc is the second
most abundant transition metal in human body after iron
and plays important roles in various pathological pro-
cesses.[4] Zn2+ does not give any spectroscopic or magnetic
signals due to its 3d104s0 electronic configuration. However,
fluorescence is an effective way to detect zinc in biological
systems.
We have synthesized a new 8-aminoquinoline derivative
L1 as a Zn2+ chemosensor, which shows high sensitivity and
selectivity for Zn2+ over other possible competitive cations
based on internal charge transfer (ICT) and chelation-en-
hanced fluorescence mechanisms. The mode of binding of
L1 with Zn2+ was investigated by UV/Vis spectrophoto-
metric titration and fluorescence titration. The ability of L1
to detect Zn2+ in living cells was examined by fluorescence
microscopy.
Results and Discussion
L1 was easily prepared from 8-aminoquinoline and
rhodamine 6G in high yield (Scheme 1), and its structure
was confirmed by 1H and 13C NMR spectroscopy, ESI-MS
(see Supporting Information), and X-ray diffraction (Fig-
ure 1).
Considerable efforts have been made to design and syn-
thesize fluorescent chemosensors for Zn2+ [5]
Among the
.
various fluorescent sensors, 8-aminoquinoline and its deriv-
atives are the first class of probes to have been developed
for Zn2+ [6]
.
They exhibit good photostability, high affinity
to metal ions, and satisfactory membrane permeability.[7]
Despite some commercial fluorescent probes for Zn2+ [8]
the
,
design of facile, easy to synthesize, nontoxic Zn2+-selective
sensors is still a challenging task, and there is a need for
the design and synthesis of such chemosensors, which are
[a] Key Laboratory of Nonferrous Metal Chemistry and Resources
Utilization of Gansu Province,
Lanzhou 730000, P. R. China
Fax: +86-931-8912582
E-mail: zengzhzh@yahoo.com.cn
zengzhzh@lzu.edu.cn
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201100804.
Scheme 1. Synthetic route to L1.
Eur. J. Inorg. Chem. 2012, 327–332
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G. Xie, Y. Shi, F. Hou, H. Liu, L. Huang, P. Xi, F. Chen, Z. Zeng
FULL PAPER
Figure 1. X-ray crystal structure of L1. All hydrogen atoms are
omitted for clarity (50% probability level for the thermal ellip-
soids).
The mode of coordination of L1 with Zn2+ was investi-
gated by spectrophotometric titration in acetonitrile solu-
tion. Figure 2a shows the spectral variation of L1 with the
gradual addition of Zn2+. The absorbance of L1 at 237 and
303 nm decreased linearly with increasing concentration of
Zn2+. In addition, two new absorption peaks appeared at
256 and 362 nm, and their intensity increased with the ad-
dition of Zn2+, which was accompanied by three isosbestic
points at 247, 282, and 340 nm, respectively. These phenom-
ena are expected to correspond to the coordination of L1
with Zn2+, which extended the conjugated system and re-
sulted in the appearance of the new absorption in the long-
wavelength region.[9] According to the linear Benesi–Hilde-
brand expression,[10] the measured absorbance [1/(A – A0)]
at 362 nm varied as a function of 1/[Zn2+] in a linear rela-
tionship (R2 = 0.9980), which indicates a 1:1 stoichiometry
between Zn2+ and L1 (Figure 2b). Moreover, there were no
absorption peaks between 450 and 700 nm, which indicates
that the spirolactam rhodamine was not ring-opened and
the N3 atom does not participate in the coordination to
Figure 2. (a) Absorption spectra of L1 (10 μm) in the presence of
Zn2+ (0–1 equiv.) in CH3CN. (b) Plot of 1/(A – A0) at 362 nm as a
function of 1/[Zn2+].
Zn2+
.
The spectrum of L1 (10 μm) showed a weak emission at
around 499 nm (λex = 365 nm, fluorescence intensity, F0 =
14.4, quantum yield, Φ0 = 0.005) in acetonitrile solution.
As shown in Figure 3, the addition of Zn2+ (0–15 μm) to
the solution produced a new emission band centered at
499 nm with an approximately 77-fold enhancement in in-
tensity (Φ = 0.537).
The fluorescence titration profile of L1 with Zn2+
demonstrates that the detection of Zn2+ is 1 μm in the range
of 0–15 μm of [Zn2+]. These results can be explained as fol-
lows. After binding with Zn2+, the intramolecular electron-
transfer process becomes forbidden, which enhances the
fluorescence emission.[11] Simultaneously, the electron
transfer from the nitrogen atom of the heterocycle to the
metal ion enhances the ICT process.[12] Another reason for
enhanced fluorescence could be that a more rigid structure
Figure 3. Fluorescence spectra of L1 (10 μm) in acetonitrile in the
presence of different concentrations of Zn2+ (0–2 equiv.) excited at
365 nm. Inset: Job plot to determine the stoichiometry of L1 and
Zn2+ in acetonitrile (the total concentration of L1 and Zn2+ is
25 μm).
forms when Zn2+ binds with L1. The association constant chiometry (Figure S1, Supporting Information). The Job
for Zn2+ was estimated to be 2.9ϫ104 m–1 on the basis of plot (Figure 3, inset) also confirmed that the binding mode
the linear fit of the titration curve that assumes a 1:1 stoi- had a 1:1 stoichiometry.
328
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A Highly Selective Fluorescent and Colorimetric Chemosensor for ZnII
When Zn2+ was added to the solution of L1, the colorless the same conditions, which reflects the lower affinity of L1
solution turned pink, and greenish fluorescence was ob- for Cd2+. The fluorescence of the L1/Zn2+ system was
served by the naked eye. However, the fluorescence did not quenched by Ni2+, Cu2+, and Hg2+, which may be due to
derive from the fluorophore of rhodamine 6G, because no nonradiative energy transitions or energy transfer between
emission peaks were observed between 530 and 650 nm the d orbital of the ions and the fluorophore.[14] These re-
when we excited the system between 480 and 520 nm. We sults demonstrate the high selectivity of L1 towards Zn2+
.
presumed that the fluorescence of the system originated in
For practical applications, the sensing ability of L1
the fluorophore of 8-aminoqunoline when excited at towards Zn2+ at different pH values was investigated (Fig-
365 nm. Therefore, we can deduce a possible sensing ure 6). L1 showed no appreciable fluorescent response to
mechanism (Figure 4) in which the spirolactam rhodamine Zn2+ in acidic environments due to the more difficult pro-
is not ring-opened and three N atoms (labeled N4, N5, and tonation of the imino group (N5) of L1, which led to a
N6 in Figure 1) participate in the coordination to Zn2+ [13]
.
weak coordination of Zn2+ [15]
. However, L1 exhibited satis-
The species that forms in solution has a nearly planar struc- factory sensing abilities when the pH was increased from
–
ture. The fourth ligand (X) is NO3 by consideration of the 5.5 to 9.0. These data indicate that L1 could act as a fluo-
charge balance in solution.
rescent probe for Zn2+ under physiological pH conditions.
Figure 4. Proposed binding mode of L1 with Zn2+
.
An important property of chemosensors is their high
selectivity towards the analyte over the other competitive
metal ions. Therefore, we determined the fluorescence in-
tensity of L1 at 499 nm in the presence of various metal
ions. As shown in Figure 5, the addition of Zn2+ gave rise
to a prominent fluorescence enhancement in an acetonitrile
solution of L1, whereas the addition of other metal ions,
such as K+, Na+, Ca2+, and Mg2+, which exist at high con-
centrations in human cells, did not show any significant
color or spectral changes. Fluorescence enhancement of L1
was observed in the presence of Cd2+ because the chemical
properties of Cd2+ are similar to those of Zn2+. However,
the fluorescence intensity of L1 in the presence of Cd2+ is
far below that in the presence of Zn2+ (FZn/FCd = 2.8) under
Figure 6. Fluorescence intensities of L1 (10 μm) in the absence and
presence of Zn2+ (20 μm) at various pH values in CH3CN/H2O
(95:5, v/v). (λex = 365 nm).
We also investigated the time evolution of the response
of L1 to 2 equiv. of Zn2+ in CH3CN (Figure 7). We found
that the interaction of L1 with Zn2+ was completed in less
than 2 min. Therefore, this system could be used to track
Zn2+ in cells and organisms in real-time.
Figure 5. Fluorescence intensities of L1 (10 μm) in the presence of
various metal ions and ion mixtures in acetonitrile. [K+], [Na+],
[Ca2+], and [Mg2+] = 5 mm, [Zn2+], [Mn2+], [Cr3+], [Pb2+], [Cd2+],
[Co2+], [Fe2+], [Ni2+], [Ag+], [Hg2+], [Cu2+] = 20 μm (λex = 365 nm,
λem = 499 nm).
Figure 7. Time evolution of the response of L1 (10 μm) to 2 equiv.
of Zn2+ in CH3CN.
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G. Xie, Y. Shi, F. Hou, H. Liu, L. Huang, P. Xi, F. Chen, Z. Zeng
FULL PAPER
As we developed L1 to be used with cells, we quantified treated cells in a culture medium at 37 °C for 0.5 h, a signifi-
its effects on the viability of L929 fibroblast cells in vitro. cant increase in fluorescence from the intracellular area was
Cell viability was evaluated by using a modified 3-(4,5-di- observed (Figure 9d). These results demonstrate that L1
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) could be used as a sensor to detect intracellular Zn2+ in
assay. Figure 8 shows the viability of cells treated with L1 living cells.
over a range of concentrations for 24, 48, and 72 h. L1 does
not negatively affect cell viability over the full range of con-
centrations measured, which indicates that they exhibit no Conclusions
cytotoxicity and could be used for intracellular detection.
We have developed a fluorescent, colorimetric chemo-
sensor based on 8-aminoquinoline for the detection of Zn2+
in living cells. L1 displayed high selectivity, sensitivity, and
a fast time response to Zn2+ in a physiological pH window
with a 1:1 binding mode. The ability of L1 to monitor intra-
cellular Zn2+ in living cells was examined by fluorescence
microscopy, which indicated that L1 is cell-permeable and
biocompatible. We hope that such Zn2+-selective sensors
will find application in biomedical and environmental de-
tection.
Experimental Section
Materials and General Methods: 1H and 13C NMR spectra were
recorded with a Varian Mercury-400 spectrometer with Me4Si as
the internal standard and CDCl3 as the solvent. Mass spectra were
performed with an HP-5988A spectrometer (EI at 70 eV). Absorp-
Figure 8. MTT assay of L929 cells cultured for 24, 48, and 72 h in
media that contain different amounts of L1.
tion spectra were determined with a Varian UV-Cary 100 spectro-
Bright field microscopy images of cells grown in the pres-
ence and absence of L1 confirmed the biocompatibility of
L1 (Figure S2).
photometer. Fluorescence measurements were performed with a
Hitachi F-4500 spectrofluorimeter. Both excitation and emission
slits were 5 nm. Quantum yields were determined by an absolute
method by using an integrating sphere with an Edinburgh Instru-
ment FLS920. The pH values were adjusted by the addition of HCl
(0.1 m) or NaOH (0.1 m). The volume of the added solutions was
less than 20 μL to leave the concentration of L1 unchanged. All
pH measurements were carried with a pH-10C digital pH meter.
All titration experiments were run twice for reliable data. All im-
aging experiments were performed with a fluorescence-inverted
microscope (DMI 4000 B, Leica Microsystem) with excitation
between 340 and 380 nm. The total magnification was 400ϫ.
The cationic solutions were prepared from NaClO4, K2CO3,
Mg(ClO4)2, Ca(NO3)2, FeSO4, Pb(NO3)2,Co(NO3)2, NiCl2,
Zn(NO3)2, Zn(ClO4)2 AgNO3, CdSO4, HgCl2, and CuSO4 in dis-
tilled water with a concentration of 0.01 m or 0.001 m. The volume
of the cationic solutions added was less than 100 μL to leave the
concentration of L1 unchanged. All synthetic materials were pur-
chased from commercial suppliers and used without further purifi-
cation. CH3CN for spectroscopy was of HPLC reagent grade with-
out fluorescent impurities.
The ability of L1 to detect Zn2+ in SCABER (human
bladder cancer) cells was also examined. The cells were sup-
plemented with L1 (10 μm) in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine se-
rum (FBS) at 37 °C for 3 h and showed very weak intracel-
lular fluorescence as determined by fluorescence micro-
scopy (Figure 9b). With the addition of Zn2+ (40 μm) to the
Crystal Structure Determination: Single-crystal X-ray diffraction
measurements were carried out with a Bruker SMART 1000 CCD
diffractometer operating at 50 kV and 30 mA by using Mo-Kα radi-
ation (λ = 0.71073 Å). The selected crystal was mounted inside a
Lindemann glass capillary for data collection, which used the
SMART and SAINT software.[16a,16b] An empirical absorption cor-
rection was applied by using the SADABS program.[16c] The struc-
ture was solved by direct methods and refined by full-matrix least
squares on F2 by using the SHELXTL-97 program package.[16d,16e]
All non-hydrogen atoms were subjected to aniostropic refinement,
and all hydrogen atoms were added in idealized positions and re-
fined isotropically. Crystal data for L1: Mr = 640.77, triclinic, space
Figure 9. (a) Bright field image and (b) fluorescence microscopy
image of SCABER cells loaded with L1 (10 μm) incubated for 3 h.
(c) Bright field image and (d) fluorescence microscope image of
SCABER cells loaded with L1 (10 μm) incubated for 3 h with the
addition of Zn2+ (2 equiv.). Scale bar = 50 μm.
¯
group P1, a = 10.4814(6) Å, b = 13.0286(7) Å, c = 13.1038(7) Å, α
330
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2012, 327–332

A Highly Selective Fluorescent and Colorimetric Chemosensor for ZnII
= 79.464(3)°, β = 80.073(3)°, γ = 81.045(3), V = 1718.62(16) Å3, Z
the absorbance of L1-treated cells to the absorbance of control
= 2, ρcalcd. = 1.238 gcm–3, R(reflections) = 0.0591(3051), wR2(re- cells.
flections) = 0.0974(6351). CCDC-825020 contains the supplemen-
Supporting Information (see footnote on the first page of this arti-
cle): Materials and general methods; schematic molecular struc-
tures; experimental details; additional NMR and ESI-MS data.
tary crystallographic data for this paper. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
Preparation of 2-Chloro-N-(quinol-8-yl)acetamide: To a cooled,
stirred solution of 8-aminoquinoline (288 mg, 2.0 mmol) and pyr-
idine (0.23 mL, 2.8 mmol) was added a chloroform (10 mL) solu-
tion of 2-chloroacetyl chloride (0.15 mL, 2.0 mmol) dropwise over
1 h. After 2 h at room temperature, a brown-yellow solid was ob-
tained by removing the solvent under reduced pressure. The crude
product was purified by silica gel column chromatography using
dichloromethane as eluent to afford 2-chloro-N-(quinol-8-yl)acet-
amide (362 mg, 80%).
Acknowledgments
This study was supported by the Fundamental Research Funds for
the Central University (Lzujbky-2011-18), the Foundation of Key
Laboratory of Nonferrous Metals Chemistry and Resources Utili-
zation of Gansu Province, and the State Key Laboratory of Applied
Organic Chemistry.
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Preparation of L1: 2-Chloro-N-(quinol-8-yl)acetamide (110 mg,
0.5 mmol), N-(rhodamine-6G)lactam-ethylenediamine (228 mg,
0.5 mmol), and potassium iodide (8 mg) were dissolved in acetoni-
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was removed under reduced pressure to obtain a pale-yellow solid,
which was purified by silica gel column chromatography using
dichloromethane/petroleum ether (5:1, v/v) as eluent to afford L1.
Yield: 83.8%. 1H NMR (400 MHz): δ = 11.234 (s, 1 H), 8.743–
8.738 (d, 1 H), 8.726–8.462 (m, 1 H), 8.101–8.076 (m, 1 H), 7.903–
7.882 (m, 1 H), 7.505–7.424 (m, 4 H), 7.357–7.326 (m, 1 H), 7.075–
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3.372 (t, 2 H), 3.201 (s, 2 H), 3.126–3.110 (m, 4 H), 2.562–2.532 (t,
2 H), 1.805 (s, 6 H), 1.278–1.242 (t, 6 H) ppm. 13C NMR
(100 MHz): δ = 170.40, 168.69, 153.67, 148.39, 147.36, 138.84,
135.80. 134.33, 132.38, 131.13, 128.29, 127.93, 127.84, 127.03,
123.80, 122.75, 121.37, 121.28, 117.90, 116.20, 105.82, 96.42, 65.14,
53.19, 48.23, 40.03, 38.18, 16.41, 14.57 ppm. ESI-MS: 641.1
[M + 1]+.
Cell Incubation and Imaging: The SCABER cells were provided by
Cells Bank of the Chinese Academy of Science (Shanghai, China).
Cells were grown in H-DMEM (high glucose) supplemented with
10% FBS in an atmosphere of 5% CO2/95% air at 37 °C. Cells
(5ϫ108/L) were plated on 18 mm glass coverslips and allowed to
adhere for 24 h. Uptake experiments of Zn2+ were performed in
the same medium supplemented with 20 μm Zn(NO3)2 for 0.5 h.
Before the experiments, cells were washed with phosphate-buffered
saline (PBS) and incubated with 10 μm L1 at 37 °C for 3 h. Cell
imaging was carried out after washing the cells with PBS.
Assessment of Biocompatibility: The biocompatibility was deter-
mined in L929 mouse fibroblast cell lines. The cell viability was
evaluated by using the modified MTT assay. The cells were plated
at a density of 1ϫ105 in 96-well plates 24 h prior to exposure to
the materials. Different concentrations of L1 with saturated sur-
faces [by interaction with DMEM/F12 (1:1) for 24 h before use]
were added to the wells, and the cells were incubated for 24, 48,
and 72 h. After treatment, 10 μL of MTT (5 mg mL–1 in PBS) was
added into each well. After 4 h of incubation, culture supernatants
were aspirated, and purple insoluble MTT product was redissolved
in dimethyl sulfoxide (150 μL) over 10 min. The concentration of
the reduced MTT in each well was determined spectrophotometri-
cally by subtraction of the absorbance reading at 630 nm from that
measured at 570 nm using a microplate reader. All MTT experi-
ments were performed five times, and the maximum and minimum
were discounted. The results were expressed as the mean Ϯ stan-
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