A highly selective fluorescent probe for cadmium ions in aqueous solution and living cells

Article abstract of DOI:10.1039/c3cc48668k

An organic salt based on double 1,3,4-oxadiazole derivatives as fluorophores and BAPTA as a receptor has been designed for detection of Cd ²⁺. The fluorescent probe exhibits high selectivity for Cd ²⁺ and a low detection limit of 20 nM in aqueous solution, making it useful for Cd²⁺ imaging in living MCF-7 cells.

Full text of DOI:10.1039/c3cc48668k

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A highly selective fluorescent probe for cadmium
ions in aqueous solution and living cells†
Cite this: Chem. Commun., 2014,
50, 2498
Qiaoling Liu,ab Lixia Feng,ab Caixia Yuan,a Lin Zhang,a Shaomin Shuang,a
Chuan Dong,*a Qin Huc and Martin M. F. Choi*c
Received 13th November 2013,
Accepted 10th January 2014
DOI: 10.1039/c3cc48668k
www.rsc.org/chemcomm
An organic salt based on double 1,3,4-oxadiazole derivatives as intermolecular hydrogen bonding,9 and fluorescence resonance energy
fluorophores and BAPTA as a receptor has been designed for transfer10 have been developed. Unfortunately, most of them suffer
detection of Cd2+. The fluorescent probe exhibits high selectivity from UV excitation,5,8,11 poor water-solubility11,12 or poor selectivity
for Cd2+ and a low detection limit of 20 nM in aqueous solution, between Cd2+ and Zn2+.13 Recently several water-soluble Cd2+ probes
making it useful for Cd2+ imaging in living MCF-7 cells.
have been introduced but their performances are still far from
satisfactory. For instance, there is a relatively small Stokes shift
Cadmium (Cd), one of the very important metallic elements, is (20 nm) for the Cd2+ sensor,4 or the detection limit is higher than
extensively used in many areas such as metallurgy, electroplating, the the level of the maximum limit in water stipulated by the US EPA and
war industry, nickel–cadmium batteries, pigments, and semi- WHO (4–40 nM).14 As such, it is highly desirable to develop a new Cd2+
conducting quantum dots and rods.1 The extensive use of Cd leads probe with a better performance in sensitivity and selectivity in an
to serious environmental and health problems including lung, aqueous environment.
prostatic, and renal cancers.2 The carcinogenic effect of Cd exposure
To our knowledge, fluorescent ICT-based probes have been
on laboratory rats showed that between 0.0 and 2.5 mM of CdCl2 widely exploited for the detection of cations owing to their strong
could induce tumour incidence and multiplicity; a dose of 2.5 mM emission intensity and large Stokes shift. These attributes arise from
could significantly elevate the prostatic tumour incidence; and a the p-electron conjugation system between the electron donor and
20 or 40 mM dosage would strongly induce Leydig cell tumour the electron acceptor, facilitating the ICT process. Once the ICT-
incidence.3a Moreover, Cd can accumulate in the human body and based probe has coordinated with metal ions, the ICT is inhibited or
cause renal dysfunction, calcium metabolism disorders and other reduced with concomitant changes in not only the energy gap
relevant forms of diseases. The Itai-Itai disease in Japan in the 1940s between the lowest unoccupied molecular orbital (LUMO) and the
is a typical example.3 Thus, there is a great demand for developing a highest occupied molecular orbital (HOMO) but also the absorption
facile method with high sensitivity and good selectivity to monitor and fluorescence intensity and spectral shifts.15 To date, there are
Cd in the environment and in biological samples.
only a few Cd2+ probes incorporating the ICT concept. Herein we first
Fluorescent probes based on metal ion induced changes in fluores- report a highly selective and sensitive probe for Cd2+ detection based
cence appear to be particularly advantageous owing to their simple on a novel bichromophoric and bifluorophoric molecule (OBO) and
operation and low detection limit. Cadmium ion (Cd2+) probes with the ICT processes.
different detection mechanisms such as photoinduced electron trans-
The Cd2+ probe (OBO) was designed by incorporating a
fer,4 intramolecular charge transfer (ICT),5 chelation-enhanced fluores- multifunctional ionophore 1,2-bis(2-aminophenoxy)ethane-
cence,6 metal–ligand charge transfer,7 excimer/exciplex formation,8 N,N,N0,N0-tetraacetic acid (BAPTA) as the Cd2+ receptor with
two 1,3,4-oxadiazole derivatives (OXD) to form a sandwich
compound OXD–BAPTA–OXD possessing an ICT capability.
First, OXD was chosen as the bichromophore and bifluoro-
a Institute of Environmental Science, and School of Chemistry and Chemical
Engineering, Shanxi University, Taiyuan 030006, P. R. China.
phore owing to its large Stokes shift and high fluorescence
quantum yield. Second, BAPTA is well known for its high
selectivity to various metal ions. Third, OBO is a water-soluble
salt which can completely dissolve in water to selectively bind
with Cd2+ in aqueous and cellular environments. Another
major attribute of OBO is that it does not respond to most
divalent metal ions under visible light excitation.
E-mail: dc@sxu.edu.cn; Fax: +86-351-7018613; Tel: +86-351-7018613
b Department of Chemistry, Taiyuan Normal University, Taiyuan 030031, P. R. China
c Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road,
Kowloon Tong, Hong Kong SAR, P. R. China. E-mail: mfchoi@hkbu.edu.hk;
Fax: +86-852-34117348; Tel: +86-852-34117839
† Electronic supplementary information (ESI) available: Detailed synthetic pro-
cedures, supplementary figures, NMR spectra and the mass spectrum. See DOI:
10.1039/c3cc48668k
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image incorporated with the OBO-ester and Cd2+ under the confocal
fluorescence microscope (vide infra).
The 1H NMR spectrum of OBO–Cd2+ shows that the protons
(Ha and Hb) in N-methylene of acetate and the 3-position of benzene
display the upfield shifts when OBO binds to Cd2+ (Fig. S7 in the
ESI†), suggesting that the binding site is BAPTA. The quantum
chemical calculation also supports this conclusion. Fig. S8 (ESI†)
depicts the optimal geometries of free OBO and the OBO–Cd2+
complex, which indicate the structure and the binding site at the
BAPTA moiety of OBO. It clearly visualizes that the Cd2+ ion binds to
the oxygen and nitrogen atoms of BAPTA. The main coordinated
bond lengths at this binding site are summarised in Table S1 (ESI†).
The main coordinated bond lengths of Cd–O1, Cd–O2, Cd–O3, and
Cd–O4 are slightly shorter than the reported Cd–O bond length,
suggesting a strong interaction between the tetra-carboxyl groups
and Cd2+. By contrast, the Cd–O5 and Cd–O6 bond lengths are
longer and exceed the range of the bond force, inferring that the
phenoxyethane moieties have almost no interaction with Cd2+. The
Cd–N7 and Cd–N8 bond lengths are longer than the usual Cd–N
bond length, inferring a weaker interaction between the N atoms
and Cd2+.16 Tables S2 and S3 (ESI†) display the ground-state energies
and Cartesian coordinates of OBO and the OBO–Cd2+ complex in
water at the B3LYP/LanL2DZ+6-31G* level. These results again
corroborate the formation of OBO–Cd2+ complex with the binding
site at BAPTA.
Fig. 2 depicts the emission spectra of OBO upon the addition of
various concentrations of Cd2+ at an excitation wavelength of 405 nm.
Free OBO exhibits an emission maximum (lfml ax) of 594 nm with an
enormous Stokes shift of 189 nm. The quantum yield (j) is 0.024.
Upon addition of Cd2+, the lmfl ax undergoes a hypsochromic shift to
532 nm with an increase in fluorescence and j is 0.078. The inset
displays the changes in emission intensities at 532 and 594 nm,
respectively by plotting (F – Fo) against the concentration of Cd2+,
where F and Fo are the emission intensities with and without Cd2+,
respectively. It is clearly seen that the emission intensities at 532 and
594 nm increase with the increase in the concentration of Cd2+.
A good linear relationship is observed between (F – Fo) and [Cd2+]
(0.0–0.40 mM). The detection limit is found to be 20 nM (3s/slope),
demonstrating that OBO is a highly sensitive probe for Cd2+.
To gain further insight into the spectral properties, the quantum
chemical calculations are conducted by the Gaussian 09 program.
The geometric parameters of OBO and OBO–Cd2+ are optimised by
the hybrid Becke three-parameter Lee–Yang–Parr exchange correla-
tion functional (B3LYP) method17 with LanL2DZ+6-31G* mixed basis
set (LanL2DZ18 for Cd and 6-31G* for other atoms). Fig. S9 (ESI†)
shows that the electrons of free OBO are localised on one side in the
HOMO and the ethoxyphenyl group does not participate in the
rearrangement of electrons. A total electron transfer from one side
to another is observed between the HOMO and the LUMO. When
OBO binds with Cd2+, the BAPTA-Cd moiety is formed which
functions as a bridge to connect the two OXO fluorophores. Electrons
are localised on the whole molecule of the HOMO, making the
p-conjugated system larger and preserving the ICT process. However,
the calculated energy gap between the LUMO and the HOMO of
OBO–Cd2+ (3.171 eV) is larger than that of free OBO (2.567 eV). This is
in agreement with the hypsochromic shift of the absorption and
Scheme 1 Reaction mechanism of OBO with Cd2+
.
The detailed synthetic routes and confirmation of the
chemical structures of the OBO-ester (the OBO precursor) and
1
OBO by H and 13C nuclear magnetic resonance spectroscopy,
mass spectrometry, and elemental analysis are provided in the
ESI† (Fig. S1–S5). Scheme 1 illustrates the chemical structure
and reaction mechanism of OBO with Cd2+
.
The photophysical properties of OBO are assessed under physio-
logical conditions (50 mM HEPES, 0.10 M KCl, pH 7.2). Fig. 1 depicts
the absorption spectra of OBO in the presence of various concentra-
tions of Cd2+. Free OBO exhibits two absorption bands at 304 nm
(e = 3.82 ꢀ 104 Mꢁ1 cmꢁ1) and 380 nm (e = 5.82 ꢀ 104 Mꢁ1 cmꢁ1),
corresponding to the p–p* transition and ICT bands, respectively.
Upon addition of Cd2+, the p–p* transition band decreases whereas
the ICT band exhibits a hypsochromic shift to a new band at 354 nm
(e = 7.18 ꢀ 104 Mꢁ1 cmꢁ1), accompanied by two isosbestic points at
313 and 370 nm. The ratio of absorbance at 354 nm to that at
405 nm (A354/A405) increases with the addition of Cd2+ concentrations
(Fig. S6 in the ESI†), indicating the formation of complex between
OBO and Cd2+. Similar results were obtained by taking the ratio of
absorbance at 354 nm to that at 380 nm. The formation of the OBO–
Cd2+ complex is further confirmed by 1H NMR spectra and the
quantum chemical calculations. Herein, the choice of 405 nm is in
line with the use of laser-induced excitation to capture the living cell
Fig. 1 Absorption spectra of 10.0 mM OBO in 50 mM HEPES (pH 7.2)
containing 0.10 M KCl at various concentrations (1–11: 0.0, 1.0, 2.0, 3.0,
4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 mM) of Cd2+ ion. The bottom inset
displays images of OBO with (left) and without (right) Cd2+
.
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Fig. 2 Fluorescence emission spectra of 1.0 mM OBO in 50 mM HEPES
(pH 7.2) containing 0.10 M KCl at various concentrations (1–16: 0.0, 0.025,
0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mM) of
Cd2+ at an excitation wavelength of 405 nm. The inset displays (F – Fo)
against concentration of Cd2+, where F and Fo are the fluorescent
intensities at 532 and 594 nm with and without Cd2+, respectively.
emission spectra of OBO upon the formation of an OBO–Cd2+
complex. In summary, the p-conjugated system could enhance the
fluorescence intensity and the ICT spectrum of OBO is hypso-
chromically shifted upon complexation with Cd2+.
Fig. 3 Confocal fluorescent images of MCF-7 cells stained with 5.0 mM
OBO-ester at an excitation wavelength of 405 nm: (a) bright field trans-
mission image of MCF-7 cells incubated with OBO-ester. (b) Fluorescence
image of MCF-7 cells incubated with OBO-ester at emission 570–610 nm.
(c) Fluorescence image of MCF-7 cells incubated with OBO-ester for
30 min and then further incubated with 5.0 mM CdCl2 for 30 min at
emission 510–550 nm. (d) Images (a) and (b) are overlapped. (e) Images
(a) and (c) are overlapped. (f) Comparison of the average intracellular
fluorescence intensity with OBO at 570–610 nm and OBO–Cd2+ at
510–550 nm. Data are expressed as mean ꢂ standard derivation of 10 cells.
The stoichiometric ratio of OBO–Cd2+ complex is determined as
1 : 1 using Job’s method (Fig. S10 in the ESI†). Hill’s plot is applied to
determine the dissociation constant of the OBO–Cd2+ complex to be
116.0 ꢂ 27.0 nM (Fig. S11 in the ESI†), suggesting that the affinity of
OBO to Cd2+ is much larger than other reported Cd2+ probes.4,5b,6,12b
The fluorescence lifetimes of the free OBO and OBO–Cd2+ complex
determined from time-resolved fluorescence spectroscopy are 1.41
and 1.48 ns at an excitation wavelength of 405 nm, respectively.
The fluorescence titration of OBO with various metal ions is
depicted in Fig. S12a (ESI†). Among these metal ions, OBO displays after they have been exposed to OBO-ester, suggesting the penetra-
excellent selectivity to Cd2+ with a strong fluorescence enhancement tion of OBO-ester into the cell. The cells are changed to bright green
while other bivalent metal ions exhibit slight fluorescence quenching after they have been incorporated with Cd2+, demonstrating that
on OBO. Most importantly, OBO has almost no response to Zn2+ and Cd2+ can permeate into the cells and bind with OBO to form OBO–
is thus superior to other Cd2+ probes which often suffer from Zn2+ Cd2+. Fig. 3d and e presents the fluorescence images of the MCF-7
interference.13 In addition, the co-existence of other bivalent metal incorporated with OBO-ester in the absence and presence of Cd2+,
ions do not affect the measurement of Cd2+ (Fig. S12b in the ESI†), respectively. It is clear that OBO can stain the cells and also indicate
demonstrating that OBO possesses excellent selectivity to Cd2+ over the presence of Cd2+. Moreover, Fig. 3f shows the mean fluorescent
other bivalent metal ions. Fluorescent pH titration in the biologically intensities of each cell captured at 570–610 and 510–550 nm,
relevant pH range (5.5–9.0) shows that the optimal working pH of respectively. The emission intensity at 510–550 nm is about three
OBO or the OBO–Cd2+ complex is 7.0 (Fig. S13 in the ESI†) which times larger than that at 570–610 nm, corroborating that the
should meet their uses under a physiological environment.
emission intensity of OBO is enhanced in the presence of Cd2+ as
In order to examine the ability of OBO to track the intracellular shown in Fig. 2. Our results demonstrate that OBO can be potentially
Cd2+ level, OBO-ester was employed for the cell permeability. MCF-7 useful for imaging Cd2+ in living cells under a confocal laser
cells were incubated with OBO-ester (5.0 mM) for 30 min at 37 1C, scanning microscope.
and then the ester was converted to the active form of OBO inside
In summary, we have successfully designed an organic salt
the cells by intracellular esterase, allowing the intracellular Cd2+ to be as a fluorescent probe for Cd2+ based on the ICT principle. This
deciphered in the cytosol. Then exogenous Cd2+ was further intro- probe exhibits a large Stokes shift of 189 nm and a low
duced via incubation with 5.0 mM CdCl2 for another 30 min. The detection limit of 20 nM Cd2+ under visible excitation. More-
images were recorded at 570–610 and 510–550 nm under the over, its high sensitivity and selectivity to Cd2+ in an aqueous
excitation wavelength of 405 nm (Fig. 3b and c). Fig. 3a depicts environment facilitate the possible application of OBO in
the bright field transmission image of the cells under a confocal imaging Cd2+ in living cells, further demonstrating its potential
microscope. A very clear cell image is observed. The cells turn to red for Cd2+ detection in biological systems.
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This work is supported by the Hundred Talent Programme
of Shanxi Province, and the National Natural Science Founda-
tion of China (21175086 and 21175087).
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