A selective fluorescent sensor for imaging Cd2+ in living cells

Article abstract of DOI:10.1021/ja0643319

A selective sensor 1 for the fluorescent imaging of Cd²⁺ in living cells has been designed and synthesized based on an internal charge transfer (ICT) mechanism. It can distinguish Cd²⁺ from Zn²⁺ and can be used in both general fluorescence intensity microscopy and ratiometric fluorescence microscopy. Copyright

Full text of DOI:10.1021/ja0643319

Published on Web 01/19/2007
A Selective Fluorescent Sensor for Imaging Cd2+ in Living Cells
Xiaojun Peng,* Jianjun Du, Jiangli Fan,* Jingyun Wang, Yunkou Wu, Jianzhang Zhao,
Shiguo Sun, and Tao Xu
State Key Laboratory of Fine Chemicals, Dalian UniVersity of Technology, 158 Zhongshan Road,
Dalian 116012, P. R. China
Received June 19, 2006; E-mail: pengxj@dlut.edu.cn; fanjl@dlut.edu.cn
Cadmium is currently used in many processes such as electro-
plating, metallurgy, war industry, etc., and it is even found in
1
phosphate fertilizers. These sources lead to cadmium exposure,
and in certain areas, there is evidence of increasing cadmium content
in food, which poses severe harm for human health and the
2
2+
environment. There have been many reports on the toxicity of Cd
to procreation, bones, kidneys, nerve system, and tissues, conse-
quently resulting in renal dysfunction, calcium metabolism dis-
orders, and an increased incidence of certain forms of cancers.3
Since cadmium can be accumulated in organisms, there is a great
need for methods of detecting and monitoring cadmium levels in
living cells or tissue samples.
Fluorescent sensors are often used to detect many ions owing to
their simplicity and sensitivity. However, only a few examples of
Figure 1. The fluorescence spectra of 1 (5 µM) in the presence of different
metal ions (150 µM) in Tris-HCl (0.01 M) solution (acetone/water ) 9/1,
4
2+
2+
2
+
5
2+
v/v, pH 7.4), and nearly no response to some other metal ions (Ca , Mg ,
fluorescent sensors for Cd have been reported. Generally, Cd
+
+
2+
+
2+
2+
Na , K , Hg , Ag , Mn , Pb ).
and Zn2 have very similar chemical properties, so the discrimina-
tion between them is very difficult. Recently, Gunnlaugsson et al.
have reported the first example of a Cd -selective fluorescent
chemosensor, which can distinguish Cd2 from Zn2+ to some extent
by their different bathochromic shift of the fluorescence spectra.
+
6
bora-3a,4a-diaza-s-indacene) with 4-(bis(pyridin-2-ylmethyl)amino)-
benzaldehyde in 18% yield.
2+
+
The λmaxab in the absorption spectra of free 1 in the ICT band
7
2+
is near 600 nm (Figure S1a in Supporting Information). When Cd
Unfortunately, up to now, there is no report about a Cd2+-selective
sensor suitable in living cells. Herein we describe the first
intracellular emission fluorescent Cd2 sensor 1 based on the ICT
mechanism.
was added gradually, the λab showed a 29 nm blue shift with an
isosbestic point at 580 nm and the color of the solution turned from
light blue to bright pink (Figure S8 in Supporting Information).
In the fluorescence emission, free 1 exhibits λmaxem at 656 nm
+
with a quantum yield of 0.12. Upon addition of CdCl
undergoes a blue shift to 597 nm with a quantum yield of 0.59
Figure 1). A well-defined isoemission point at 673 nm is also ob-
2
, the λmaxem
(
served (Figure S1b). The emission intensity at 597 nm (F597, Figure
S2) and the intensity ratio, R (F597/F697, Figure S3), increased upon
the gradual addition of Cd2 , which allowed the detection of Cd
+
2+
by both normal fluorescence and ratiometric fluorescence methods.
From the sigmoidal curves in Figures S2 and S3, dissociation
-
5
-5
constants (4.8 ( 0.3) × 10 and (7.0 ( 0.3) × 10 M are
obtained, respectively. The fluorescence responses in both methods
fit a Hill coefficient of 1 (Figures S4 and S5); it is consistent with
8
The internal charge transfer (ICT) mechanism has been widely
9
10
exploited for ions sensing, molecular switching, and fluorescent
labeling11 due to the advantages of spectral shifts and quantitative
detection. When a fluorophore contains an electron-donating group
2+
the formation of a 1:1 stoichiometry for the 1-Cd complex.
The fluorescence titration of 1 with various metal ions (Figure
1) shows excellent selectivity to Cd2 . Physiologically important
+
(often an amino group) conjugating to a fluorophore, it undergoes
2
+
2+
+
ICT from the donor to the fluorophore upon light excitation, which
provides a red-shifted emission. Coordinated with an ion, the amino
group loses its donating ability. Consequently, the ICT is inhibited
and the emission blue shifts. Fluorescence quantum yields always
change in the processes. In sensor 1, we chose boradiazaindacene
metal ions which exist in living cells, such as Ca , Mg , Na ,
+
3+
K , and Fe , do not give any responses at 30-fold excess
concentration. Most heavy and transition metal ions, such as Hg
Ag , Mn , and Zn , also have no interference. Only Cr induces
very slight fluorescence enhancement. Ni and Cu obviously
quench the fluorescence to some extent, which always meet in the
2+
,
+
2+
2+
3+
2+
2+
(BODIPY) as the fluorophore because it absorbs and emits in the
13
visible region with high quantum yield, large extinction coefficient,
other metal ion sensors.
The competition experiments of Cd2+ mixed with the metal ions
show no significant variation in the ratio fluorescence intensity (F597
12
and good photostability and N,N-bis(pyridin-2-ylmethyl)benzen-
amine as Cd2 receptor (and ICT donor). A vinyl group between
the receptor and the BODIPY fluorophore can induce longer
wavelengths in absorption and fluorescence spectra.
+
/
+
2+
F
697, Figure 2), although Cu2 and Ni have some fluorescence
quenching in normal fluorescence intensity at 597 ( 15 nm (F597
Figure S6). Another Zn titration experiment suggests that Zn
,
Sensor 1 was synthesized according to the general route10 by
condensation of BODIPY (4,4-difluoro-1,3,4,5,7-quantmethyl-4-
2+
2+
cannot induce any response of 1 even at high concentrations
1500
9
J. AM. CHEM. SOC. 2007, 129, 1500-1501
10.1021/ja0643319 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. Ratio fluorescence (F597/F697) images of Cd2 in DC cells (Leica
TCS-SP2 confocal fluorescence microscope, 20× objective lens). (a) DC
cells incubated with 1 (5 µM). (b) DC cells incubated with 1 and then further
incubated with 5 µM CdCl2.
+
Figure 2. The ratio fluorescence responses (F597/F697) of sensor 1 containing
distinguish Cd2+ from Zn2+ and especially it can be used in both
general fluorescence microscopy and ratiometric fluorescence
microscopy.
2
+
2
50 µM Cd to the selected metal ions (250 µM) in Tris-HCl (0.01 M)
solution (acetone/water, 9/1, v/v, pH 7.4). The concentration of 1 was 5
µM, and excitation wavelength was 580 nm.
Acknowledgment. This work was supported by the National
Science Foundation of China (20376010 and 20472012).
Supporting Information Available: Synthesis, experimental de-
tails, and additional spectroscopic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
Figure 3. Confocal fluorescence images of Cd2+ in DC cells. The excited
light is 543 nm, and the emission is centered at 597 ( 15 nm (Leica TCS-
SP2 confocal fluorescence microscope, 20× objective lens). (a) Bright-
field transmission image of DC cells incubated with 1 (5 µM). (b)
Fluorescence image of DC cells incubated with 1 (5 µM). (c) Fluorescence
image of DC cells incubated with 1 for 30 min, washed three times, and
then further incubated with 5 µM CdCl2 for 30 min.
(
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Figures S7 and S8). To the best of our knowledge, this is the first
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1
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Similarly to PC12 cells, DC cells were incubated with 1 (5 µM)
and then further incubated with 5 µM CdCl
2
. The fluorescence
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images of intracellular Cd2 were observed under a Leica TCS-
SP2 confocal microscope. The single-channel confocal fluorescence
at 597 ( 15 nm (Figure 3) shows more clear images than that of
a general microscope (Figure S10). The double-channel fluorescence
images at 597 ( 15 and 697 ( 15 nm are shown in Figure S11.
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fluorescence images were obtained (Figure 4).
+
2
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2
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The results suggest that 1 can be used to image intracellular Cd2+
in living cells in both general fluorescence and ratio fluorescence
ways. It should therefore be potentially useful for the study of the
toxicity or bioactivity of Cd2+ in living cells.
(
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In conclusion, we have reported that fluorescent sensor 1 can
be used for selective imaging of Cd2 in living cells. It can
+
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