Highly selective fluorescent chemosensor for Fe3+ imaging in living cells

Article abstract of DOI:10.1016/j.inoche.2010.11.025

2-Hydroxybenzyl dansyl cadaverine (DNSCH) was designed as a fluorescent chemosensor for Fe³⁺. DNSCH exhibited significant FL quenching with increasing concentrations of Fe³⁺. The presence of 15 M equiv of Fe³⁺ significantly quenched the FL intensity of DNSCH. The binding constant of DNSCH towards Fe³⁺ was determined and found to be 1.5 ± 0.4 × 10^-4 M^-1. DNSCH was employed as an intracellular chemosensor for Fe³⁺.

Full text of DOI:10.1016/j.inoche.2010.11.025

Inorganic Chemistry Communications 14 (2011) 351–354
Contents lists available at ScienceDirect
Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
Highly selective fluorescent chemosensor for Fe3+ imaging in living cells
b
b
Srung Smanmoo a, , Weerachai Nasomphan , Pramuan Tangboriboonrat
⁎
a
Bioresources Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang,
Pathumthani 12120, Thailand
Department of Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Phyathai, Bangkok 10400, Thailand
b
a r t i c l e i n f o
a b s t r a c t
2-Hydroxybenzyl dansyl cadaverine (DNSCH) was designed as a fluorescent chemosensor for Fe3+. DNSCH
exhibited significant FL quenching with increasing concentrations of Fe3+. The presence of 15 M equiv of Fe3+
significantly quenched the FL intensity of DNSCH. The binding constant of DNSCH towards Fe3+ was
determined and found to be 1.5 0.4×10−4 M−1. DNSCH was employed as an intracellular chemosensor for
Article history:
Received 24 August 2010
Accepted 18 November 2010
Available online 25 November 2010
Fe3+
.
Keywords:
© 2010 Elsevier B.V. All rights reserved.
Fluorescent chemosensor
Fe3+ sensor
Dansyl cadaverine
Fluorescence quenching
Bioimaging
Molecular recognition of metal ions is one of active areas in
supramolecular chemistry. Based on de Silva et al., the design of
fluorescent (FL) chemosensor is simply achieved by the incorporation
of metal-binding receptor and fluorophore via the spacer [1]. FL
chemosensor receives much research attention in the field of
bioanalytical science due to its high selectivity, real-time detection
and simplicity [2–6]. The recognition of metals at the receptor is
achieved through various modes of interactions, e.g., hydrogen
bonding, van der Waals and electrostatic forces [7–9]. The binding
of metal ions at the receptor initiates the communicating event with
the fluorophore in which the signal obtained can be expressed as an
energy transfer. Particularly, in the FL quenching type chemosensor,
the quenching process involves the formation of an exciplex with
charge transfer or photo-induced electron transfer.
The design of FL chemosensors exhibiting selective recognizing of
Fe3+ receives much research attention due to Fe3+ is the most abundant
ion in intracellular compartments. Recently, several FL chemosensors
have been designed for the recognition of Fe3+. Jang et al. designed a
benzimidazole-based FL sensor in which the imidazole moiety presents
in the sensor serves as the binding part for Fe3+ [10]. The sensor exhibits
good binding constant with Fe3+. Zheng and Huang reported an on/off
chemosensor for Fe3+ controlled by protonation–deprotonation
processes [11]. The sensor is designed based on calix[4]arene with
two 3-alkoxy-2-naphthoic acids pending on the upper rim. The binding
of Fe3+ is controlled by the deprotonation of phenolic moieties of calix
[4]arene and dicarboxylic group of napthoic acid. On the contrary, the
unbound state of calix[4]arene towards Fe3+ is driven by the
protonation of calix[4]arene's phenol. Noh and his co-workers have
designed an excellent chemosensor for Fe3+. The sensor is based on a
ferrocenyl chalcone structure which is capable in discriminating Fe3+
and Fe2+ via the selective oxidation [12]. Up to date, Fe3+ chemosensors
for cell applications are scarcely reported and challenging for the design.
We have designed a new FL chemosensor, DNSCH, based on dansyl
cadaverine. DNSCH adopts the fluorophore–spacer–receptor format.
The dansyl fluorophore was attached via a pentylene spacer to the
2-hydroxybenzaldehyde receptor. DNSCH was evaluated against various
metal ions and found to exhibit selective FL quenching in the presence of
Fe3+. DNSCH was employed as an intracellular chemosensor for Fe3+
.
Dansyl derivatives were extensively used as chromophores to
construct a chemosensor platform [13–15]. The incorporation of
2-hydroxybenzaldehyde is expected to serve as the metal ion binding
moiety and to affect the intramolecular charge transfer which induces
the emission change of the sensor. 2-Hydroxybenzyl dansyl cadaverine
(DNSCH) was synthesized in one step from the condensation reaction
between dansyl cadaverine and 2-hydroxybenzaldehydes (Scheme 1).
After reaction mixture was left stirring under nitrogen atmosphere for
24 h, DNSCH was isolated and crystallized from ethyl acetate/hexane to
afford DNSCH in 81% (the spectroscopic identification for DNSCH is
accomplished and can be found in Supplementary Material). The C=N
group formed is aimed to provide the chelation to metal ions since it is
known that C=N exhibits good degree of affinity towards transition
and post-transition metal cations due to its electronic structure [16,17].
The selectivity of DNSCH towards metal ions was investigated
(Fig. 1). Of all metal ions, DNSCH exhibited a significant degree of FL
quenching in the presence of Fe3+. In the presence of 15 M equiv of
⁎
Corresponding author. Tel.: +66 2 564 6700x3560; fax: +66 2 564 6632.
E-mail address: srung.sma@biotec.or.th (S. Smanmoo).
1387-7003/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.inoche.2010.11.025

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S. Smanmoo et al. / Inorganic Chemistry Communications 14 (2011) 351–354
Scheme 1. Synthesis of DNSCH.
Fig. 3. FL ratio (I0-I/I0) of DNSCH (2.5×10−6 M) upon the addition of 15 M equiv
(3.75×10−5 M) of metal guests in CH3CN-H2O (50:50, v/v); λex =342 nm.
Fig. 1. Emission spectra of DNSCH (2.5×10−6 M) upon the addition of different metal
ions (3.75×10−5 M) in CH3CN-H2O (50:50, v/v); λex =342 nm.
Fe3+, the FL intensity of DNSCH was significantly quenched. The
design of Fe3+ chemosensor with good discrimination between the iron
valence states (Fe2+ and Fe3+) is necessary for better understanding of
various biological processes and Fenton reaction [18]. It was clearly
shown that DNSCH is a selective Fe3+ chemosensor with the good
discrimination for iron valence states. The presence of the 2-hydroxyl
group in DNSCH is thought to facilitate the binding of Fe3+ near to the
dansyl fluorophore prior to initiate the quenching process.
Fig. 2 represented the color and FL images of DNSCH in the
presence of various metal ions. In the absence of metal ions, DNSCH
exhibited strong yellow (λem =525 nm) FL when excited at 342 nm.
The emission of DNSCH at 525 nm is the characteristic emission of
dansyl fluorophore (λem =520 nm) [19]. In the addition of other
metal ions except Fe3+, DNSCH exhibited no color and FL changes.
However, the addition of Fe3+ induced a slight change in color from
clear to pale yellow [Fig. 2(a)], associated with the strong FL
quenching [Fig. 2(b)].
Fig. 4. FL spectra of DNSCH (2.5×10−6 M) titrated with (a) 0.5–15 M equiv of Fe3+, in
CH3CN-H2O (50:50, v/v); λex =342 nm.
Fig. 2. (a) Images and (b) FL images of DNSCH (2.5×10−6 M) in the presence of various metal ions (3.75×10−5 M) in CH3CN-H2O (50:50, v/v); λex =342 nm.

S. Smanmoo et al. / Inorganic Chemistry Communications 14 (2011) 351–354
353
Fig. 5. FL responses of DNSCH (2.5×10−6 M) containing Fe3+ (15 M equiv) and other
co-existing metal ions (15 M equiv) in CH3CN-H2O (50:50, v/v); λex =342 nm.
Fig. 7. FL images of (a) DNSCH (2.5×10−6 M). (b) DNSCH (2.5×10−6 M) in the presence
of 15 M equiv of Fe3+ (3.75×10−5 M). (c) DNSCH (2.5×10−6 M) in the presence of 15 M
equiv Fe3+ with the addition of excess EDTA.
In order to determine the degree of FL quenching of DNSCH by Fe3+
the FL ratio was employed for this investigation. It was clearly shown in
Fig. 3 that the degree of FL quenching for DNSCH was mostly
pronounced in the presence of Fe3+. There were also slight changes in
FL of DNSCH in the presence of Cd2+, Co2+, Mn2+, Pb2+ and Zn2+ ions.
Comparing to other metal ions, DNSCH is highly selective in its response
,
To determine the selectivity range of DNSCH towards Fe3+, the
titration experiment was conducted. The FL quenching of DNSCH was
investigated in the presence of various concentrations of Fe3+. Fig. 4
shows that with increasing concentrations of Fe3+, the FL emission
intensity of DNSCH at λmax 525 nm was gradually decreased. The FL
to Fe3+
.
Fig. 6. (a) Stern–Volmer plot for DNSCH in the presence of various Fe3+ concentrations indicating 1:1 stoichiometry. (b) Benesi–Hilderbrand to determine the binding constant
between DNSCH and Fe3+
.

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S. Smanmoo et al. / Inorganic Chemistry Communications 14 (2011) 351–354
Fig. 8. Living-cell imaging of intracellular Fe3+ by FL microscopy: (a) Bright field transmission image of Vero cells incubated with 50 μM of DNSCH. (b) FL image of Vero cells
supplemented with 50 μM of DNSCH in the growth media for 15 min at 25 °C. (c) Vero cells incubated with 50 μM of DNSCH in the growth media for 15 min at 25 °C, washed three
times, and then further incubated with 100 μM FeCl3 for 5 min at 25 °C.
quenching of DNSCH was mostly pronounced after the addition of Fe3+
at 15 M equiv (3.75×10−5 M). The addition of Fe3+ at 15 M equiv
significantly quenched the FL of DNSCH.
In summary, a novel FL chemosensor based on dansyl cadaverine,
DNSCH, was synthesized and evaluated for its sensing ability towards
metal ions. DNSCH exhibited high selectivity towards Fe3+ among
other screened metal ions with good discrimination between different
iron oxidation state (Fe2+ and Fe3+). The 2-hydroxyl group in DNSCH
facilitates the binding of Fe3+ prior to LMCT mechanism, which was
initiated to induce the FL quenching. DNSCH exhibited a selective
recognizing mode with high selectivity towards Fe3+. DNSCH shows
no interference from other metal ions. DNSCH was evaluated as a
FL responses of DNSCH towards Fe3+ were investigated in the
presence of co-existing metal ions, e.g., Co2+, Fe2+, Hg2+, Ni2+, Pb2+
,
Zn2+, Ag+, Mn2+, Cd2+ and Cu2+. It was shown in Fig. 5 that the
presence of other metals did not interfere the FL quenching of DNSCH
by Fe3+ and the FL changes caused by the addition of Fe3+ were not
affected by the co-existing metal ions.
In order to determine the binding ratio between DNSCH and Fe3+
,
cell-permeable chemosensor for intracellular detection of Fe3+
.
the Stern–Volmer plot was used and found the good fit in the
concentration range between 1 and 25 μM which indicated the binding
ratio between DNSCH and Fe3+ at 1:1 stoichiometry [Fig. 6(a)]. On the
basis of this binding ratio, the binding constant for DNSCH and Fe3+ was
then calculated using the Benesi–Hilderbrand equation and found to be
1.5 0.4×10−4 M−1 [Fig. 6(b)] [20]. The reversible binding mode of
DNSCH towards Fe3+ was investigated. It was clear that DNSCH was a
reversible FL chemosensor (Fig. 7). In the presence of 15 M equiv Fe3+
(3.75×10−5 M), DNSCH (2.5×10−6 M) exhibited strong FL quenching.
However, the FL of DNSCH was recovered after the addition of excess
EDTA. This demonstrated the potential application of DNSCH as a
reversible FL chemosensor.
Acknowledgments
The Thailand Research Fund/Commission on Higher Education's
research grant to S.S. (DIG5180025) and P.T. (RTA5180003) and the
Development and Promotion of Science and Technology Talents Project's
scholarship to W.N. are gratefully acknowledged. We are also grateful to
the partial support from National Center for Genetic Engineering and
Biotechnology (BIOTEC).
Appendix A. Supplementary Material
The mechanism for FL quenching of DNSCH by Fe3+ is proposed
from ligand metal charge transfer (LMCT) in which the transition of
the charge transfer is initiated by the binding of Fe3+ to the ligand
(DNSCH). The chelation between DNSCH and Fe3+ is facilitated by the
extra coordination from i) C=N group which is known for the strong
binding affinity to transition and post-transition metals [16,17] and ii)
the 2-hydroxyl group. These functional groups are in the positions
where Fe3+ is held close enough to the dansyl fluorophore. The
electronic charge of DNSCH transfers to Fe3+ (d-orbital) results in the
distribution of charge at the excited state. This finally causes the FL
quenching of DNSCH.
Supplementary data to this article can be found online at
doi:10.1016/j.inoche.2010.11.025.
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