A colorimetric and fluorescent chemosensor for copper ions in aqueous media and its application in living cells

Article abstract of DOI:10.1039/c0nj00168f

Merocyanine dye 1 was prepared as an effective colorimetric and fluorescent chemosensor for copper ions in aqueous media, and displays a significant color change from reddish-purple to yellow with a large blue shift of 94 nm in its absorption spectrum in the presence of one equivalent of copper. As an on-off fluorescent sensor, it is feasible to detect copper ions in living cells due to its good cell permeability and high solubility under physiological conditions.

Full text of DOI:10.1039/c0nj00168f

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LETTER
www.rsc.org/njc | New Journal of Chemistry
A colorimetric and fluorescent chemosensor for copper ions in aqueous
media and its application in living cellsw
Huan-Huan Wang,ab Lin Xue,ab Zhang-Jian Fang,a Guo-Ping Liab and
Hua Jiang*a
Received (in Montpellier, France) 4th March 2010, Accepted 12th April 2010
First published as an Advance Article on the web 11th May 2010
DOI: 10.1039/c0nj00168f
Merocyanine dye 1 was prepared as an effective colorimetric and
fluorescent chemosensor for copper ions in aqueous media, and
displays a significant color change from reddish-purple to yellow
with a large blue shift of 94 nm in its absorption spectrum in the
presence of one equivalent of copper. As an on–off fluorescent
sensor, it is feasible to detect copper ions in living cells due to its
good cell permeability and high solubility under physiological
conditions.
To achieve good solubility in water and a suitable cellular
permeability, we employed indolium as a prochromophore,
due not only to its good water solubility but also to its wide
use in cyanine dyes.5 In addition, indolium styryl dyes were
prepared to probe various metal ions.6 On the other hand, the
DPA (di-2-picolylamine) moiety was chosen as a metal ion
receptor because it has been commonly employed to prepare a
host of chemosensors for transition metal ions such as zinc,
cadmium and copper, etc.1c,7 Herein, we have incorporated the
DPA moiety into indolium via a stryl group to achieve a novel
merocyanine dye, 1, which displays a significant color change
from reddish-purple to yellow with a large blue shift of 94 nm
in its absorption spectrum and a ‘‘turn-off’’ of its fluorescence
spectrum after exposure to copper ions in an aqueous buffer
(25 mM HEPES, 0.1 M NaClO4, pH 7.4, 25 1C). It turns out
that merocyanine dye 1 is a cellular permeable sensor that is
able to detect copper ions in living cells.
Copper, a co-factor in various enzymes and copper-based
pigments, is an essential element in the human body. Despite
its important roles in organisms, the accumulation of excess
amounts of copper ions or their misregulation can cause a
series of severe diseases. It is believed that the disruption of
copper homeostasis is implicated to some neurodegenerative
disorders like Alzheimer’s and Parkinson’s diseases.1 There is
therefore a need to develop chemosensors to monitor copper
ions in living cells or tissues. To this end, much effort has been
devoted to exploring chemosensors so as to improve our
understanding of the biological roles of copper ions in many
respects, for example, in neurophysiology and neuropathology.
Among the reported sensors for copper ions, fluorescent
sensors2 and colorimetric sensors3 have attracted much attention
because of their advantageous features of low cost, rapid
detection and analysis in situ using simple apparatus. In
particular, sensors with colorimetric and fluorescent features
for copper ions4 are appealing because they can monitor
copper ions both in solution by the naked eye and in living
cells by fluorescence microscopy. So far, only a few colorimetric
and fluorescent sensors based on the rhodamine chromophore
for copper ions have been reported.4a–f However, only few of
them4b,e exhibit good performance in pure aqueous media,
which is a very important factor for potential biological
applications. Therefore, developing new colorimetric and
fluorescent chemosensors for copper ions that behave well
under physiological conditions is highly desirable.
As shown in Scheme 1, merocyanine dye 1 was easily
synthesized from DPA derivative 3 and indolium salt 2 in
1
one step, and was characterized by H NMR, 13C NMR and
TOF-MS (see the ESIw).
The response of 1 to various metal ions was investigated in
aqueous buffer. It displays a very distinctive selectivity for
copper ions, as observed by the naked eye. When 1 was treated
with various metal ions, such as Cd2+, Pb2+, Zn2+, Ag+,
Hg2+, Cu2+, Mn2+, Fe2+, Cr3+, Ca2+, Mg2+, K+ and Co2+
in the buffer, only copper ions caused a distinct color change
from reddish-purple to yellow. In contrast, the other metal
ions didn’t give rise to any evident color change (Fig. 1a).
a Beijing National Laboratory for Molecular Sciences,
CAS Key Laboratory of Photochemistry, Institute of Chemistry,
Chinese Academy of Sciences, Beijing, 100190, P. R. China.
E-mail: hjiang@iccas.ac.cn; Fax: +86 10-62553316;
Tel: +86 10-62553316
b Graduate School of Chinese Academy of Sciences, Beijing,
P. R. China
w Electronic supplementary information (ESI) available: Synthetic
procedures, characterization of 1 and additional spectroscopic data.
See DOI: 10.1039/c0nj00168f
Scheme 1 Synthesis of merocyanine dye 1.
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Fig. 1 (a) The color change of 1 (12.5 mM) upon adding 1 equiv. of
various metal ions. (b) The color change of 1 (12.5 mM) upon adding
1 equiv. of copper ions in the presence of various other metal ions.
(c) The color change of 1 (12.5 mM) upon adding various amounts of
copper ions to the aqueous buffer.
Furthermore, the color change could be observed in copper/
other metal combined samples. All samples containing various
other metal ions exhibited a very distinguished color change
after being exposed to copper ions (Fig. 1b). It reveals that 1
can discriminate copper ions from other metal ions in a
colorimetric manner. In addition, to determine the threshold
of the color change by the naked eye, an increasing amount of
copper ions was added to the aqueous buffer of 1. As depicted
in Fig. 1c, the color of the buffer solution graded gradually
from reddish-purple to yellow in the presence of 0–4 equiv. of
copper ions. Accordingly, 1 equiv. of copper ions can be set as
the colorimetric threshold for determination by the naked eye.
Furthermore, we carried out UV-vis experiments to obtain
the detailed absorption properties and binding constant. As
shown in Fig. 2a, UV-vis experiments demonstrated that
chemosensor 1 (12.5 mM) exhibited very strong absorption
bands centered at 504 nm under identical conditions. As
expected, the absorption band displayed a blue shift from
504 to 410 nm in the presence of 4 equiv. of copper ions. In
addition, this spectral change could be reversed in the presence
of either EDTA or TPEN (Fig. S1, see the ESIw), whereas
Ag+, Fe2+, Co2+, Zn2+, Cd2+, Hg2+, Pb2+, Ca2+, Mg2+
and K+ didn’t have any effect on the absorption spectrum
of 1, which is consistent with the color change profile.
Meanwhile, when various amounts of copper ions were added,
an absorption band centered at 504 nm gradually decreased
and, concomitantly, a new band at 410 nm appeared with a
very clear isosbestic point at 444 nm, suggesting that the
binding of copper ions disturbs the ground state charge
transfer process in merocyanine (Fig. 2b).
Fig. 2 (a) UV-vis absorption spectra of 1 (12.5 mM) upon adding
4 equiv. of various metal ions. (b) The changes in the UV-vis absorption
spectrum of 1 (25 mM) upon titrating 0–4 equiv. of copper ions.
580 nm. Upon titration with various amounts of copper ions,
the emission intensity at 580 nm was gradually quenched. The
explanation for this phenomenon is partially due to the
coordination of copper ions, which decreases the electron-
donating ability of the nitrogen in the DPA moiety and
thereby inhibits the ICT process,8 and partially due to the
paramagnetic nature of copper ions. At the same time, we also
evaluated the emission properties of the sensor in the presence
of other metal ions. As expected, the fluorescence intensity of 1
remained unchanged upon adding 4 equiv. of other metal ions.
With these findings at hand, we decided to rate the performance
of 1 in living cells. Fibroblast NIH 3T3 cells (mouse embryonic
fibroblast cell line) were incubated with 10 mM 1 in Dulbecco’s
modified Eagle’s medium (DMEM, Hyclone) supplemented
with 10% fetal calf serum (FCS, Hyclone), 1% penicillin
(Hyclone) and 1% streptomycin (Hyclone) at 37 1C in a
5 : 95 CO2–air incubator. The cells were then washed with
PBS to remove excess 1. A strong fluorescence emission was
observed in the optical window at 560–610 nm, as shown in
Fig. 4b. After it had been treated with 10 equiv. of copper ions
for 20 min, intracellular fluorescence was almost completely
suppressed, as shown in Fig. 4c. Subsequently, upon treating
the cells with an excess amount of TPEN (N,N,N0,N0-tetrakis-
(2-pyridylmethyl)ethylenediamine), the fluorescence intensity
could be readily reversed. Thus, 1 is cell-permeable and can
respond to copper ions within living cells.
According to Job’s plot, 1 and copper ions form a 1 : 1
stoichiometry complex (Fig. S2, see the ESIw). On the basis of
a 1 : 1 stoichiometry and a linear Benesi–Hildebrand expression,
the measured 1/(A ꢁ A0) at 410 nm was plotted against
1/[Cu2+] to yield a binding constant of 15052 Mꢁ1 by a linear
fit (R = 0.992) (Fig. S3, see the ESIw).
We next performed fluorescence titration experiments
(Fig. 3). When a solution of 1 (2.5 mM) was excited at
444 nm, sensor 1 showed a strong fluorescence emission at
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the indolium moiety with various receptors may generate
many new colorimetric and fluorescent chemosensors for other
specific metal ions in aqueous media.
We thank the Chinese Academy of Sciences ‘‘Hundred
talents program’’, the National Natural Science Foundation
of China (90813002) and the State Key Laboratory of Fine
Chemicals, Department of Chemical Engineering, Dalian
University of Technology (KF0708) for financial support.
Experimental section
Synthesis of 1
2 (29 mg, 0.16 mmol) and 3 (50 mg, 0.165 mmol) were
dissolved in ethanol with piperidine as the catalyst and the
mixture refluxed for 2 h. The solvent was then evaporated
and the residue purified by chromatography (silica gel,
DCM/0–5% methanol) to give a dark red solid (37.51 mg,
yield 40%). 1H NMR (400 MHz, CD3OD, ppm) d 8.59 (2H, d,
J = 4.0 Hz), 8.04 (3H, m), 7.66 (2H, m), 7.50 (5H, m), 7.21
(4H, m), 6.91 (2H, d, J = 8.0 Hz), 4.96 (4H, s), 4.25 (3H, s),
1.76 (6H, s). 13C NMR (100 MHz, CD3OD, ppm): d 180.9,
157.1, 154.8, 153.8, 149.1, 142.7, 142.0, 137.6, 133.3, 128.8,
128.2, 123.7, 122.7, 122.3, 121.5, 113.3, 105.8, 56.5, 51.4, 25.5.
Mass (MALDI-TOF): m/z 459.2 [M ꢁ I]+. HRMS (ESI) calc.
for C31H31N4: 459.2543; found: 459.2537. mp: 135–137 1C.
Cell culture
Fig. 3 (a) Fluorescence profiles of 1 (2.5 mM) upon titration with
0–4 equiv. of copper ions in an aqueous buffer (lex = 444 nm). (b) The
fluorescence intensity enhanced index (F/F0) in the presence of 4 equiv.
of various metal ions.
NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Hyclone) supplemented with 10% fetal calf
serum (FCS, Hyclone), 1% penicillin (Hyclone) and 1%
streptomycin (Hyclone) at 37 1C in a 5 : 95 CO2–air incubator.
The cells were cultured for 3 d then loaded onto a 35 mm
diameter glass-bottomed coverslip. The cells were incubated
with 10 mM 1 for 30 min, washed with PBS three times and
bathed in serum-free DMEM (2 mL) before imaging.
In summary, chemosensor 1 behaves as a colorimetric and
fluorescent chemosensor for copper ions under physiological
conditions. Although its fluorescence emission was quenched
while being exposed to copper ions, 1 is able to monitor the
variation of copper ions in living cells. In addition, combining
Confocal fluorescence microscopy
An Olympus FV-1000 laser scanning microscopy system
equipped with a 488 nm laser head was applied to confocal
image NIH 3T3 cells stained with 1. The emission was
collected at 560–610 nm. All of the images were gathered at
the same confocal microscope settings and processed with
Olympus FV10-ASWV0107A-1 software (Olympus, Japan).
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1242 | New J. Chem., 2010, 34, 1239–1242