Fluorescence imaging of intracellular cadmium using a dual-excitation ratiometric chemosensor

Article abstract of DOI:10.1021/ja803429z

We described here a coumarin-based dual-excitation ratiometric probe for cadmium, CadMQ. This fluorescence sensor has high quantum yields of 0.59 and 0.70 in the metal-free and Cd²⁺-bound forms, respectively, and has a dissociation constant of 0.16 nM for Cd²⁺. CadMQ is cell permeable and locates within the acidic compartments of the cells. We further show that CadMQ is a useful tool to ratiometrically probe the change in the intracellular Cd²⁺ levels with the use of two excited wavelengths. Copyright

Full text of DOI:10.1021/ja803429z

Published on Web 08/30/2008
Fluorescence Imaging of Intracellular Cadmium Using a Dual-Excitation
Ratiometric Chemosensor
Masayasu Taki,*,†,‡ Mika Desaki,† Akio Ojida,§ Shohei Iyoshi,† Tasuku Hirayama,† Itaru Hamachi,§
and Yukio Yamamoto†
Graduate School of Human and EnVironmental Studies and Graduate School of Global EnVironmental Studies,
Kyoto UniVersity, Yoshida, Sakyo-ku, Kyoto 606-8501, and Graduate School of Engineering, Kyoto UniVersity,
Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan
Received May 8, 2008; E-mail: taki@chem.mbox.media.kyoto-u.ac.jp
Scheme 1
Cadmium (Cd), an inessential element for life, has been
recognized as a highly toxic heavy metal and is listed by the U.S.
Environmental Protection Agency as one of 126 priority pollutants.
Humans are exposed to Cd2+ through the ingestion of contaminated
food or water and inhalation of cigarette smoke. Cd2+ causes a
number of lesions in many organs and tissues such as the kidney,
liver, gastrointestinal tract, brain, and bone.1 In addition, chronic
exposure to Cd2+ has been implicated as a cause of cancers of the
lung, prostate, pancreas, and kidney;2 therefore, the International
Agency for Research on Cancer (IARC) has classified cadmium
and cadmium compounds as category I carcinogens.3 Although
many experimental results have been obtained on the ability of Cd2+
to adversely affect cellular functions, the molecular mechanisms
of Cd2+-uptake by cells as well as of carcinogenesis due to Cd2+
in humans and other mammals remains unclear.1
bound to the benzopyrone moiety by a single bond, and the
absorption energy has a higher value than it does in a polar solvent.9
We speculate that if the chelator, which can allow metal-ion binding
with the 7-amino group on the coumarin ring, is incorporated into
C120, the ICT structure may change into a nonplanar structure and
therefore a blue shift of the excitation spectrum will be observed.10
Fluorescent sensors that can aid in the visualization of specific
metal ions of interest in living cells have been indispensable tools
for understanding of biological phenomena.4,5 Many synthetic
sensors that exhibit a fluorescence response to Cd2+ have been
demonstrated; however, most of these sensors can respond to Zn2+
as well because the two metal cations often exhibit similar
coordination properties.6 Hence, it has been a significant challenge
to develop a Cd2+-selective fluorescent sensor that can discriminate
Cd2+ from Zn2+ under physiological conditions.7 Recently, the
research groups of Peng and of Wang have reported confocal images
of Cd2+ in living cells using BODIPY- and fluorescein-based
sensors, respectively; however, they have not shown the reversible
binding of Cd2+ in these cells.7a,b Furthermore, sensor molecules
with higher affinity for Cd2+ than either BODIPY- or fluorescein-
based sensors are required because cell growth and DNA synthesis
would be significantly stimulated by Cd2+ concentrations as low
as 100 pM,8 a level that lies beyond the detection limits of either
of these sensors. Herein, we report a new Cd2+-selective fluorescent
sensor, CadMQ. The excitation wavelength of this molecule exhibits
a significant blue shift under physiological conditions when bound
to Cd2+, thereby enabling dual-excitation ratiometric imaging of
Cd2+, which can, in principle, provide accurate and quantitative
measurements of metal ion concentrations in cells.
Spectroscopic measurements of CadMQ were performed under
physiological conditions (50 mM HEPES, pH 7.20, 0.1 M KNO3).
CadMQ exhibits an intense absorption band at 356 nm (ꢀ ) 1.77 ×
105 M-1 cm-1), suggesting the formation of the ICT structure in the
excited state. Upon addition of Cd2+, a decrease in the absorbance of
this band and a concomitant increase in that of a new band at 333 nm
(ꢀ ) 1.42 × 105 M-1 cm-1) were observed with a distinct isosbestic
point at 340 nm. A significant hypsochromic shift of 23 nm of the
absorption wavelength indicates coordination of the 7-amino group to
Cd2+ to form the nonplanar structure (Scheme 1). The absorption bands
at 356 and 333 nm linearly decreased and increased, respectively, up
to a 1:1 [Cd2+]/[CadMQ] ratio, which is consistent with a 1:1 complex
stoichiometry. The binding of Cd2+ resulted in a bathochromic shift
of the emission maximum of only 4 nm; this wavelength shift is much
less than that observed in the absorption titration. The quantum yields
of the free ligand and Cd2+-bound forms were determined to be 0.59
and 0.70, respectively. Therefore, it can be concluded that CadMQ
enables the calculation of [Cd2+
]free using the ratio of the fluorescence
CadMQ consists of coumarin-120 (C120: 7-amino-4-methyl-1,2-
benzopyrone), and a derivative of N,N,N′,N′-tetrakis(2-pyridylm-
ethyl)ethylenediamine (TPEN) as the chelator. It has been proposed
that C120 exists in an intramolecular charge transfer (ICT) structure
in higher polarity solvents, where the bond between the 7-amino
group and the 1,2-benzopyrone moiety attains a substantial double
bond character.9 In a nonpolar solvent, however, NH2 nitrogen is
intensities at the different two wavelengths in the excitation spectrum.
Figure 1a shows a set of excitation spectra for CadMQ in calibration
buffers with various [Cd2+
]
free. The apparent dissociation constant Kd
for Cd2+ was determined to be 0.16 nM at pH 7.20 by plotting the
fluorescence intensities at 328 or 368 nm against log[Cd2+
free (Figure
1b). This nonlinear fitting analysis reveals that CadMQ is suitable to
determine [Cd2+
between 40 and 660 pM. The fluorescence
]
]
free
intensity of CadMQ changed with pH and reached a maximum at pH
6.3 (Supporting Information, Figure S2). However, a significant shift
of the fluorescence wavelength was not observed; accordingly, the
† Graduate School of Human and Environmental Studies.
‡ Graduate School of Global Environmental Studies.
§ Graduate School of Engineering.
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12564 J. AM. CHEM. SOC. 2008, 130, 12564–12565
10.1021/ja803429z CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
of the cells (Figure S7). Subsequent experiments of Cd2+-imaging in
living cells were performed with a ratio imaging system. Cells were
exposed to 5 µM Cd2+ for 3 h at 37 °C, washed with PBS containing
200 µM EDTA to remove extracellular Cd2+, and further incubated
with 5 µM CadMQ for 10 min. The fluorescence ratio between the
intensities of the 340 and 387 nm bands remained unchanged for 330 s;
however, this immediately changed (within 30 s) upon the addition of
the heavy metal chelator TPEN (200 µM) to the medium (Figure 2).
A decrease and increase in the fluorescence intensities at 340 and 387
nm, respectively, induced by TPEN treatment indicate that CadMQ
can probe the change in the intracellular Cd2+ levels (Figure S9). In
the control experiment, by contrast, initial fluorescence intensity ratio
for cells grown without Cd2+ was lower than that for Cd2+-exposed
cells (Figure S10). In fact, a negligible change in the ratio was observed
upon the addition of TPEN.
In conclusion, we have developed a ratiometric fluorescent sensor
for Cd2+, CadMQ. This probe exhibits excellent Cd2+-selectivity over
other transition metal ions in aqueous media, including Zn2+. It also
has high quantum yields in both the apo and Cd2+-bound forms, a
strong affinity for Cd2+, and membrane permeability. The ratio-
imaging experiments demonstrate that CadMQ will be a useful tool
for detecting changes in Cd2+ concentrations in living mammalian
cells.
Figure 1. (a) Excitation spectra of CadMQ (2.5 µM) monitored at 440
nm in Cd2+/Mg2+/EDTA buffered system (50 mM HEPES, pH 7.20, 0.1
M KNO3; 1 mM EDTA, 10 mM MgSO4, 0-0.9 mM CdCl2) and in 50
mM HEPES buffer (pH 7.20) containing ∼20 µM CdCl2. (b) Plots of
fluorescence intensities at 328 (circle) and 368 (triangle) nm with best-fit
curves for the dissociation constant 1.62 × 10-10 M.
Acknowledgment. We thank Professor Y. Mori and Dr. S.
Kiyonaka at Kyoto University for helpful advice. This work was
financially supported by Grant-in-Aid for Young Scientists (B) (No.
17750155 to M.T.) from JSPS.
Supporting Information Available: Synthesis and characterization
of CadMQ, UV-vis titration for Cd2+, pH-titration, confocal images,
and experimental details. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 2. Ratio images of Cd2+ in HeLa cells exposed to 5 µM of Cd2+
for 3 h at 37 °C, then further incubated with 5 µM CadMQ for 10 min at
37 °C. Images were taken every 30 s: (a) bright-field transmission image;
(b) ratio image (340 nm/387 nm) of CadMQ-stained cells prior to TPEN
treatment; (c) ratio image of the same cells after treatment with 200 µM
TPEN. The image is of the system at 360 s. (d) Average ratio of cells (340
nm/387 nm, N > 10) at the corresponding time.
References
(1) (a) Waisberg, M.; Joseph, P.; Hale, B.; Beyersmann, D. Toxicology 2003,
192, 95–117. (b) Zalups, R. K.; Ahmad, S. Toxicol. Appl. Pharmacol. 2003,
186, 163–188. (c) Bridges, C. C.; Zalups, R. K. Toxicol. Appl. Pharmacol.
2005, 204, 274–308.
(2) (a) Waalkes, M. P. J. Inorg. Biochem. 2000, 79, 241–244. (b) Waalkes,
M. P.; Coogan, T. P.; Barter, R. A. Crit. ReV. Toxicol. 1992, 22, 175–201.
(3) Monographs on the EValuation of the Carcinogenic Risk of Chemicals to
Humans; Beryllium, Cadmium, Mercury, and Exposures in the Glass
Manufacturing Industry, Vol. 58; International Agency for Research on
Cancer: Lyon, France, 1993.
(4) (a) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;
McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515–
1566. (b) Valeur, B. Molecular Fluorescence: Principles and Applications;
Wiley-VCH: Weinheim, NY, 2002. (c) Domaille, D. W.; Que, E. L.; Chang,
C. J. Nat. Chem. Biol. 2008, 3, 168–175.
(5) Hinkle, P. M.; Shanshala, E. D.; Nelson, E. J. J. Biol. Chem. 1992, 267,
25553–25559.
(6) (a) Nolan, E. M.; Ryu, J. W.; Jaworski, J.; Feazell, R. P.; Sheng, M.;
Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 15517–15528. (b) Goldsmith,
C. R.; Lippard, S. J. Inorg. Chem. 2006, 45, 555–561. (c) Komatsu, K.;
Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2005,
127, 10197–10204. (d) Aoki, S.; Kagata, D.; Shiro, M.; Takeda, K.; Kimura,
E. J. Am. Chem. Soc. 2004, 126, 13377–13390. (e) Maruyama, S.; Kikuchi,
K.; Hirano, T.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 10650–
10651. (f) Taki, M.; Wolford, J. L.; O’Halloran, T. V. J. Am. Chem. Soc.
2004, 126, 712–713. (g) Henary, M. M.; Wu, Y. G.; Fahrni, C. J.
Chem.sEur. J. 2004, 10, 3015–3025.
(7) (a) Peng, X. J.; Du, J. J.; Fan, J. L.; Wang, J. Y.; Wu, Y. K.; Zhao, J. Z.;
Sun, S. G.; Xu, T. J. Am. Chem. Soc. 2007, 129, 1500–1501. (b) Liu, W. M.;
Xu, L. W.; Sheng, R. L.; Wang, P. F.; Li, H. P.; Wu, S. K. Org. Lett.
2007, 9, 3829–3832. (c) Choi, M.; Kim, M.; Lee, K. D.; Han, K. N.; Yoon,
I. A.; Chung, H. J.; Yoon, J. Org. Lett. 2001, 3, 3455–3457. (d)
Gunnlaugsson, T.; Lee, T. C.; Parkesh, R. Org. Lett. 2003, 5, 4065–4068.
(8) Vonzglinicki, T.; Edwall, C.; Ostlund, E.; Lind, B.; Nordberg, M.; Ringertz,
N. R.; Wroblewski, J. J. Cell Sci. 1992, 103, 1073–1081.
fluorescence ratios between 333 and 356 nm changed only slightly
over the wide range of pH.
A large hypsochromic shift in the excitation spectrum observed
with Cd2+ is unaffected by the presence of high concentrations of
Na+, K+, Mg2+, and Ca2+ (>5 mM), indicating that this probe
will be useful in a wide range of biological and microscopic
applications. Some of the transition metal ions including Cu2+
,
Zn2+, and Hg2+ interfered with Cd2+ binding (Figure S4), which
suggests competing binding of these cations.11 It should be noted
that the addition of Zn2+ resulted in a slight hypsochromic shift of
CadMQ in the excitation spectrum, and little affected the fluores-
cence ratio. Such the difference in the fluorescence response of
CadMQ toward Zn2+ and Cd2+ can be supported by 1H NMR
spectra in D2O (pD 7.6, Figure S6). Complexation with Cd2+
induced downfield shifts of the coumarin moiety whereas slight
upfield shifts were observed for Zn2+ complex. This observation
strongly demonstrates that the 7-amino group of the coumarin
interacts with Cd2+, but not with Zn2+
.
Incubation of HeLa cells with 5 µM CadMQ for 30 min at 37 °C
revealed a bright punctate staining pattern, indicating that this molecule
is membrane permeable. To determine the intracellular distribution of
CadMQ, cells were coincubated with LysoTracker Red. A complete
overlap of the images between CadMQ and LysoTracker Red revealed
that our sensor molecule was located within the acidic compartments
(9) Pal, H.; Nad, S.; Kumbhakar, M. J. Chem. Phys. 2003, 119, 443–452.
(10) Mizukami, S.; Nagano, T.; Urano, Y.; Odani, A.; Kikuchi, K. J. Am. Chem.
Soc. 2002, 124, 3920–3925.
(11) Kd for Zn2+ was determined to be 44.6 pM. See Supporting Information.
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