A new europium chelate-based phosphorescence probe specific for singlet oxygen

Article abstract of DOI:10.1039/b503980k

The first Eu³⁺ chelate-based phosphorescence probe specific for singlet oxygen has been designed, synthesized and characterized. The probe is highly sensitive, selective and water soluble for time-resolved luminescence detection of singlet oxygen with a detection limit of 2.8 nM. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b503980k

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A new europium chelate-based phosphorescence probe specific for
singlet oxygen{
Bo Song, Guilan Wang and Jingli Yuan*
Received (in Cambridge, UK) 18th March 2005, Accepted 2nd June 2005
First published as an Advance Article on the web 24th June 2005
DOI: 10.1039/b503980k
The first Eu3+ chelate-based phosphorescence probe specific for
the probe, which makes it rather unsuitable for use with
biosystems.
singlet oxygen has been designed, synthesized and character-
ized. The probe is highly sensitive, selective and water soluble
for time-resolved luminescence detection of singlet oxygen with
a detection limit of 2.8 nM.
Time-resolved fluorometry combined with the use of lanthanide
chelate-based luminescence probes has provided an excellent way
for developing highly sensitive bioaffinity assays. The applications
of lanthanide luminescence probes for time-resolved fluoroimmu-
noassay, DNA hybridization assay, and luminescence microscopy
bioimaging have been extensively investigated.8 Different from
organic fluorescence probes, lanthanide luminescence probes have
the properties of long luminescence lifetime, large Stokes shift and
sharp emission profile, which makes them suitable for use in
microsecond time-resolved luminescence measurement to minimize
the interference caused by background noises associated with
biological samples, scattering lights (Tyndall, Rayleigh and Raman
scatterings) and the optical components (cuvettes, filters and
lenses).9
Singlet oxygen (1O2), an excited state of molecular oxygen, is a
useful oxidant in organic synthesis and has an important biological
reactivity. As an intermediate species in the detrimental oxidation
of biomolecules, it can react with many kinds of biomolecules,
such as DNA, protein and lipids.1 Furthermore, it plays an
important role in the cell cascade and induction of gene
expression.2 Since oxygen is ubiquitous and efficiently quenches
1
electronically excited states, O2 is likely to be formed following
irradiation in countless situation and involved in various chemical,
biological, and several disease processes.3
Due to the outstanding importance of 1O2 in photochemical and
photobiological processes, several methods for 1O2 detection have
been developed. Monitoring the direct emission of 1O2 at 1270 nm
is a specific and noninvasive method, but this method suffers from
weak signal, and quantitative detection of very small amounts of
1O2 is currently not possible in any medium.4 Chemical trapping
by spectroscopic probes is also found to be specific and much more
sensitive than the detection of the 1270 nm luminescence. A
commonly used 1O2 trap, 9,10-diphenylanthracene (DPA), can
react specifically with 1O2 to form a thermostable endoperoxide at
In the present work, a novel Eu3+ chelate-based phosphores-
cence probe specific for time-resolved luminescence detection of
1O2, [49-(9-anthryl)-2,29:69,20-terpyridine-6,60-diyl]bis(methyleneni-
trilo) tetrakis(acetate)-Eu3+ (ATTA-Eu3+) was designed and
synthesized. In this chelate, the 9-anthryl group was used as a
specific reactive moiety for 1O2,5–7 and (2,29:69,20-terpyridine-
6,60-diyl)bis(methylenenitrilo) tetrakis(acetate)-Eu3+, as a fluoro-
phore. The almost non-luminescent chelate can specifically react
1
with O2 to yield its endoperoxide (EP-ATTA-Eu3+) with a great
increase of the phosphorescence intensity, which can be used for
time-resolved luminescence measurement (Scheme 1).
a rate of k 5 1.3 6 106 M21 21
accompanied by the decrease in
s
1
absorbance at 335 nm as a signal of O2 production.5 However,
this method is less sensitive because the detection is based on the
measurement of absorbance. Nagano’s group has synthesized two
fluorescence probes for 1O2 by conjugating fluorophore of
fluorescein with 9,10-diphenylanthracene or 9,10-dimethylanthra-
cene.6 These probes can react with 1O2 to yield the corresponding
endoperoxides giving sensitive fluorescence responses. Recently, a
chemiluminescence probe for 1O2 by incorporating an electron-rich
tetrathiafulvalene unit into a reactive luminophore of anthracene
has been developed.7 This probe exhibits strong chemilumines-
cence response and high selectivity for 1O2 with a detection limit of
76 nM. The main drawback of this probe is its lower water
solubility, a buffer containing 50% THF is necessary to dissolve
The new ligand ATTA was synthesized following the eight-step
reaction (S1 in supporting information{). The corresponding
endoperoxide of its Eu3+ chelate was synthesized by reacting
ATTA-Eu3+ with chemically generated 1O2 (MoO422/H2O2),10
and the production of EP-ATTA-Eu3+ was confirmed by mass
spectrum detection (S2 in supporting information{). The phos-
phorescence properties of ATTA-Eu3+ and EP-ATTA-Eu3+ are
listed in Table 1. The excitation and emission maximum
{ Electronic supplementary information (ESI) available: synthesis, experi-
mental detail and characterization of ATTA and EP-ATTA-Eu3+. See
http://dx.doi.org/10.1039/b503980k
Department of Analytical Chemistry, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, 116023, P. R. China.
E-mail: jingliyuan@yahoo.com.cn; Fax: +86-411-84379660;
Tel: +86-411-84379660
1
*jingliyuan@yahoo.com.cn
Scheme 1 Reaction of ATTA-Eu3+ with O2.
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Table 1 Phosphorescence properties of ATTA-Eu3+ and EP-ATTA-Eu3+a
Chelate
lex,max (nm)
lem,max (nm)
e335 nm (cm21M21
)
w (%)
t (ms)
ATTA-Eu3+
289,335
289,335
615
615
17200
14500
0.58
10.0
989
1209
EP-ATTA-Eu3+
a
All data were obtained in 0.05 M borate buffer of pH 9.1.
wavelengths (lex,max, lem,max) are not changed (there is no
significant difference in the UV spectrum patterns between
ATTA-Eu3+ and EP-ATTA-Eu3+, which is similar to the
Nagano’s probe6), and molar absorption coefficients (e) and
luminescence lifetimes (t) are changed slightly from ATTA-Eu3+ to
EP-ATTA-Eu3+. However, the luminescence quantum yield (w) is
drastically increased after the formation of EP-ATTA-Eu3+. The
luminescence quantum yield of EP-ATTA-Eu3+ is 17 times higher
than that of ATTA-Eu3+, so it can be said that ATTA-Eu3+ itself
is almost non-luminescent, while EP-ATTA-Eu3+ is strongly
luminescent.
of the excited state of the Eu3+ ion. During photosensitised
luminescence of the Eu3+ ion by ligand, the triplet state of
terpyridine chromophore (CT1, E 5 22400 cm21 15
)
transfers the
5
energy to D0 energy level of Eu3+ (E 5 17374 cm21 16
)
after the
excitation of the terpyridine chromophore at 335 nm. However,
the strong triplet (CT1) – triplet (anthracene, AT1, E 5
14900 cm21) quenching blocks the effective energy transfer of
CT1
A
5D0,17 so the phosphorescence of ATTA-Eu3+ is very
weak. After the formation of EP-ATTA-Eu3+, the triplet–triplet
quenching between CT1 and AT1 disappears, thus the Eu3+ chelate
becomes strongly luminescent.18
When the EP-ATTA-Eu3+ complex was challenged with a five-
fold excess of ethylenediamine tetraacetic acid, a conditional
stability constant was measured to be y1020 by using the
Verhoeven’s method.11 Moreover, no decrease of the phosphor-
escence intensity of EP-ATTA-Eu3+ was observed after several
days at room temperature. Using the luminescence lifetimes
of ATTA-Eu3+ and EP-ATTA-Eu3+ in H2O and D2O buffers, the
average number (q) of water molecules in the first coordina-
tion sphere of Eu3+ ion was calculated from the equation of
q 5 1.2 (1/tH2O21/tD2O20.25) to be 0.05 and 0.04, respectively.12
These results show that the probe has a high kinetic and
thermodynamic stability, and the increase of the probe’s
phosphorescence intensity is not caused by the decrease of the
number of the coordinated water molecules.13
The effects of pH on the phosphorescence intensity and lifetime
of EP-ATTA-Eu3+ have been investigated (S3 in supporting
information{). In contrast to the rapid decrease of fluorescence
intensity of fluorescein-based probes at pH , 7,6 the phos-
phorescence intensity of EP-ATTA-Eu3+ is stable at pH . 3. This
result indicates that ATTA-Eu3+ is very useful as a luminescence
1
probe for O2 in weakly acidic, neutral and basic buffers.
The reaction of hydrogen peroxide disproportionation catalyzed
1
by molybdate ions was used as a chemical source of O2 for the
1
detection of O2 production using ATTA-Eu3+ as the probe. The
reaction was performed in 0.1 M carbonate buffer of pH 10.5,
since the MoO422/H2O2 system only works under basic condi-
tions.10 A series of H2O2 solutions were added to the buffer
solutions containing 10 mM of ATTA-Eu3+ and 1 mM of
Na2MoO4. After the reaction, the solutions were 10-fold diluted
(final probe concentration 5 1.0 mM) with 0.05 M borate buffer of
pH 9.1, and the excitation and emission spectra were measured
with a time-resolved mode. As shown in Fig. 1, the phosphores-
cence signals show a good response with the increase of H2O2
concentration, and the emission of the probe consists of several
discrete bands between 580 and 710 nm corresponding to the
Within a general paradigm, the overall luminescence quantum
yield (wtot) of a lanthanide complex upon exciting of the
chromophore of ligand is determined by the efficiency of the
sensitization (gsens) and by the quantum yield (wLn) of
the lanthanide luminescence step (wtot 5 gsenswLn).14 In the present
case, the excited state lifetime and the chemical surroundings of the
ion of the EP-ATTA-Eu3+ are very similar to the ones of ATTA-
Eu3+, so the significant increase of luminescence quantum yield can
be considered due to an increase in the efficiency of the population
7
5D0 A FJ (J 5 0–4) transitions of Eu3+. This emission feature
indicates that the emission of the probe can be monitored at
Fig. 1 Time-resolved excitation (A) and emission (B) spectra of ATTA-Eu3+ in the reaction with 1O2 generated from a MoO422/H2O2 system. The
conditions of delay time, 0.2 ms, gate time, 0.4 ms, cycle time, 20 ms, excitation slit, 10 nm, and emission slit, 5 nm were used for the measurements.
Excitation spectra were recorded with lem 5 615 nm and emission spectra with lex 5 335 nm.
3554 | Chem. Commun., 2005, 3553–3555
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the time-resolved luminescence microscopy imaging of 1O2 in
biological systems using the new probe would be a favorably useful
technique for visualizing the temporal and spatial distribution of
1O2 in biological samples.
Notes and references
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Fig. 2 Calibration curve for 1O2. The curve was derived from the
luminescence intensity of the H2O2/MoO422/ATTA-Eu3+ reaction in 0.1 M
carbonate buffer of pH 10.5 with 100 nM of ATTA-Eu3+, 10 mM of
Na2MoO4 and a series of standard H2O2 solutions.
several discrete points between 580 and 710 nm. The significant
increase of phosphorescence signal of the probe was also observed
1
by using the photosensitization of rhodamine B as a O2 source19
in a 0.05 M Tris-HCl buffer of pH 7.4. When azide, a quencher of
1O2,20 was added to MoO422/H2O2/ATTA-Eu3+ system, the
change of the probe’s phosphorescence intensity can not be
observed. These results distinctly indicate that the increase of
phosphorescence intensity is caused by the reaction of the probe
1
with O2.
1
Because of quantitative generation of O2 from MoO422/H2O2
system (one 1O2 molecule can be formed quantitatively by the
reaction of two H2O2 molecules),10 this system was used for the
quantitative detection of 1O2. As shown in Fig. 2, the dose-
dependence of phosphorescence intensity of the probe on 1O2
concentration shows a good linearity. The detection limit for 1O2,
calculated as the concentration corresponding to three standard
deviations of the background signal, is 2.8 nM, which is y28 times
lower than that of the chemiluminescence method.7
The reactions of ATTA-Eu3+ with different reactive oxygen
12 A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker,
L. Royle, A. S. de Sousa, J. A. G. Williams and M. Woods, J. Chem.
Soc., Perkin Trans. 2, 1999, 493.
species (H2O2, OH, O22, and O2) were investigated to examine
1
?
its selectivity. In the same buffer, the phosphorescence intensities
of 100 nM ATTA-Eu3+ upon reactions with 10 mM H2O2,
13 O. S. Wolfbeis, A. Du¨rkpo, M. Wu and Z. Lin, Angew. Chem. Int. Ed.,
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7
?
10 mM H2O2 + 10 mM ferrous ammonium sulfate ( OH), 10 mM
2 21
KO2 (O2
)
and 10 mM H2O2 + 10 mM Na2MoO4 (1O2) were
increased 46%, 76%, 6% and 1246%, respectively. These results
indicate that the probe ATTA-Eu3+ is highly specific for O2.
1
In summary, the first Eu3+ chelate-based phosphorescence probe
1
specific for time-resolved luminescence detection of O2 has been
designed, synthesized and characterized. The photophysical
characterization and the mechanism of the phosphorescence of
the probe were discussed. The properties of high sensitivity,
selectivity and water solubility of the new probe for 1O2 detection
suggest that the probe should be widely useful for the luminescence
detection of 1O2 in many chemical and biological systems. Perhaps,
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Chem. Commun., 2005, 3553–3555 | 3555