Efficient two-photon-sensitized luminescence of a novel europium(iii) β-diketonate complex and application in biological imaging

Article abstract of DOI:10.1039/c1cc14968g

A novel europium(iii) β-diketonate complex exhibiting bright two-photon-sensitized luminescence is synthesized and applied as a two-photon-sensitized luminescent probe to stain DNA in live cells.

Full text of DOI:10.1039/c1cc14968g

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Efficient two-photon-sensitized luminescence of a novel europium(III)
b-diketonate complex and application in biological imagingw
Zhang-Jun Hu,ab Xiao-He Tian,c Xiang-Hua Zhao,a Peng Wang,a Qiong Zhang,a
Ping-Ping Sun,a Jie-Ying Wu,*a Jia-Xiang Yanga and Yu-Peng Tian*ad
Received 10th August 2011, Accepted 14th October 2011
DOI: 10.1039/c1cc14968g
A novel europium(III) b-diketonate complex exhibiting bright two-
photon-sensitized luminescence is synthesized and applied as a two-
photon-sensitized luminescent probe to stain DNA in live cells.
whereas they provide a new challenge to make new two-
photon-sensitizable Ln(III) probes for bioimaging.7 Mean-
while, how to obtain the improved long lifetime cell-permeable
Ln(III)-based two-photon probes is also one of the essential
research directions in biological science,8 to supply a vision for
the analysis of biological information.
Many studies have been focused on the design of metal complexes
as cellular image probes and the application in therapy because
of their many advantages over other traditional cellular
probes, such as high sensitivity and membrane permeability.1
Among others, the luminescent lanthanide(III) (Ln(III)) complexes
have attracted intensive research efforts,2 due to their unique
photophysical properties such as sharp emission bands, long
luminescence lifetimes and insensitivity to environmental
quenching and oxygen.3 However, ultra-violet light is usually
used for the sensitization of Ln(III), which limits the investigation
depth and presents some phototoxicity due to inherent
features of this high energy excitation. Acknowledgedly, the
two-photon fluorescence imaging technique is desirable to
overcome this obstacle. The light located in the far visible/
near-IR range penetrates biological media most effectively and
provides higher resolutions, lower photodamage and photo-
bleaching in imaging.4 Therefore, developing the two-photon-
sensitizable Ln(III) complexes could combine the above advantages
into one probe precisely. To achieve effective two-photon-
sensitized luminescent probes, the ‘‘antennae’’ with efficient
two-photon absorption (TPA) for light-harvesting are needed
to overcome the poor extinction coefficients of the Ln(III) ions
caused by the symmetry-forbidden nature of the inner-shell f–f
transition.5 Initially, the ‘‘antenna’’ effects were utilized to
sensitize the luminescence of Eu(III) and Tb(III), which were
directly linked to proteins, nucleic acids, and biologically
relevant chromophores.6 However, these intrinsic biological
fluorophores chelated to Ln(III) generally give low TPA activities,
Ln(III) emission can be enhanced when the lanthanides are
bound to chelate ‘‘antennae’’ due to the shielding of the cations
from the quenching effects of water,7,8 of which the chelate ligands
based on b-diketonate are extensively used. They provide a smaller
energy gap between the lowest S1 state and the T1 state, resulting in
effective energy transfers from ‘‘antennae’’ to Ln(III) ions for highly
efficient emissions under the excitation.9 Herein, we describe a
novel b-diketonate derivative HTHA based on a carbazole unit
which is frequently used in TPA dye tailoring.10 The experimental
results show that the THAÀ chelateprovides an efficient two-
photon-sensitization of Eu(III) luminescence in the neutral complex
Eu(THA)3Phen (Phen: 1,10-phenanthroline) (Scheme 1). The
complex displays both efficient two-photon-sensitized and high-
purity red emission. Furthermore, Eu(THA)3Phen was used as a
two-photon fluorescence molecular probe to explore the cellular
uptake and localization characteristics in live cells.
In order to obtain accurate structure information, single
crystals of HTHA and Eu(THA)3Phen were grown and the
molecular structures were determined by single crystal X-ray
diffraction (in ESIw). The result shows that Eu(THA)3Phen is
mononuclear and the Eu(III) ion eight-coordinates with six
oxygen atoms from three bidentate THA anions and two nitrogen
atoms from a Phen without any water molecules. Photophysical
a Key Laboratory of Functional Inorganic Materials of Anhui
Province, Anhui University, Department of Chemistry, 230039 Hefei,
P. R. China. E-mail: jywu1957@163.com, yptian@ahu.edu.cn;
Tel: +86-551-5018151
b State Key Laboratory of Pollution Control and Resource Reuse,
Tongji University, 200092 Shanghai, P. R. China
c Department of Biomedical Science, University of Sheffield, Sheffield, UK
d State Key Laboratory of Coordination Chemistry, Nanjing University,
Nanjing 210093, P. R. China
w Electronic supplementary information (ESI) available. CCDC
[CCDC NUMBER(S)]. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c1cc14968g
Scheme 1 Synthetic route to HTHA and Eu(THA)3Phen.
Chem. Commun., 2011, 47, 12467–12469 12467
c
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Table 1 Photophysical data for HTHA and Eu(THA)3Phen measured
at room temperature in dichloromethane (DCM)
Compound
la
eb Â104
lc
Fd
te
sf
HTHA
Eu(THA)3Phen
360
364
2.37
6.57
449
613
0.36
0.08
4.19 ns
678 ms
—
80
a
b
lmax of the maximum linear absorption spectra in nm. The molar
absorption coefficient in L molÀ1 cmÀ1
. lmax of the emission spectra
c
d
e
in nm. Fluorescence quantum yield (Æ15%). Luminescence life-
f
time (Æ5%). Maximum TPA cross-section in GM (at 720 nm).
Fig. 2 TD-DFT computed frontier orbitals of HTHA obtained at the
B3LYP level.
luminescence of Eu(III) is observed, confirming that energy
transfer takes place from the THA anions to Eu(III). As shown
in Fig. 1b, the bands at lower energy are the emission bands of
Eu(III), which are typical of europium centered transitions
5
7
from the D0 levels to the lower F0–4 levels of the ground-
state multiplet. The sharp emission at 613 nm corresponds to
the hypersensitive transition of 5D0 - 7F2, which is forbidden
as an electric dipole for Eu(III) in the strict D4d symmetry. The
Eu(III) cation factually has a geometrical environment between
a square antiprism and a dodecahedron (in ESIw).12 This coordi-
nation configuration around Eu(III) leads to a low structural
symmetry. In addition, other important factors cannot be
ignored, such as the aromatic electronic clouds and polarization
effects13 caused by strong bonding of Eu(III) to the three
THAÀ and the disorders in the crystal.14 They may also result
in the effective crystal electric field symmetry at the Eu(III) site
being lower than the ca. D4d adopted by the coordination
polyhedron or trigger 5D0 - 7F2 transition.13 Notably, it is found
that the intensity of the short-wavelength emission (B440 nm)
from the THA anions nearly disappears and 5D0 - 7F0–4 are well
resolved when excited in the solid state, which means that only a
minute fraction of the energy of the THA anions is given out as
their own emissions rather than transferred to Eu(III). It can be
presumed that the presence of solvent oscillators in solution could
well serve to affect the local symmetry of the Eu(III) site or
deactivate the ligand and Eu(III) excited states, so that the energy
transfer to Eu(III) becomes less efficient.15 The luminescent quantum
yield is rather modest (0.08) at room temperature, however, due to
the emission being concentrated in the hypersensitive DJ = 2
narrow band (613 nm), the emitted red light is still intense. The
measured 5D0 - 7F2 luminescence decays of Eu(THA)3Phen can
be described by monoexponential kinetics and the measured
luminescence lifetime (t) is 678 ms at room temperature, which
matches the need of long lifetime sensor systems.
Fig. 1 (a) Absorption spectra of HTHA and Eu(THA)3Phen in DCM
(1 Â 10À5 mol LÀ1); (b) photoluminescence spectra of Eu(THA)3Phen
in DCM and solid sate (insets: photographs of solid powder and
solution of Eu(THA)3Phen under UV light (365 nm)); (c) TPA cross
sections (s) for Eu(THA)3Phen; (d) Two-photon-excited fluorescence
of HTHA and two-photon-sensitized luminescence of Eu(THA)3Phen
in DCM (1 Â 10À3 mol LÀ1).
data for HTHA and Eu(THA)3Phen are provided in Table 1. As
shown in Fig. 1a, HTHA mainly presents a broad absorption
band centered at 360 nm and a shoulder around 278 nm in
absorption spectra. The TD-DFT {[6-31G(d)]} calculations
(Fig. 2) indicate that the low energy transition presents marked
charge transfer (CT) character, with the HOMO and the LUMO
being mainly located on the carbazole donor and trifluoro-
methane acceptor parts of the molecule, respectively. The most
intense transition (367.8 nm) arises from HOMO - LUMO and
HOMO À 1 - LUMO transition, it has a p–p* character. The
4.23 eV (293.4 nm) transitions receive contributions from excitations
involving several molecular orbitals, with a predominant weight
of excitations from HOMO À 1, HOMO À 2 and HOMO
toward LUMO and LUMO À 1. These transitions are assigned
to two classes of transitions. One corresponds mainly to CT
transition from the alkyl group to the p-conjugated bridge part,
the other one can arise from a locally excited (LE) transition as
monoelectronic transition localized in the p-conjugated system.
Complexation to Eu(III) results in a slight shift of the CT
transition, which shows that the lanthanide Lewis acidity effect
is partially compensated by the tris-anionic nature of the complex.
As shown in Fig. 1a, the spectral shapes of the complex are similar
to HTHA, indicating that the coordination of the Eu(III) does not
significantly influence the energy of the singlet state of the b-
diketone ligand.11 Furthermore, it is accompanied by a threefold
escalation of the extinction coefficient of low energy transition.
Upon excitation of Eu(THA)3Phen at 364 nm (CT band
of the ligand), the characteristic bright-red long-lived
The excitation-energy dependence of integrated emission for
HTHA and Eu(THA)3Phen are present in a logarithmic scale,
respectively (Fig. S3, in ESIw). In both cases, the experimental
points fit nicely to a linear relationship with similar slopes of
2.06 Æ 0.03 and 2.09 Æ 0.04, respectively. These values indicate
that the emissions (Fig. 1d) arise from a two-photon process. The
c
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excitation power dependence was examined for 700–810 nm and
used in the subsequent determination of the TPA cross-sections
(s), which shows that the Eu(THA)3Phen is considered to have
high efficiency in two-photon sensitization (Fig. 1c). The
maximum s value is estimated to be 80 GM at 720 nm, which
is comparable to that of the two-photon-sensitized luminescent
Eu(III) complex reported.7a–f Notably, the experimental points of
TPA spectra agree with the wavelength-doubled linear absorption
spectra of HTHA, which indicates that the sensitized lumines-
cence at 614 nm is attributed to THA anions.6a
more it identifies the promising direction for the synthesis of
two-photon sensitized luminescent probes for less harmful
and better quality bioimaging. Further optimizations of the
‘‘antennae’’ structure for needs of biological imaging are
currently underway and will be reported in due course.
This work was supported by a grant from the National
Natural Science Foundation of China (21071001, 50873001),
Education Committee of Anhui Province (KJ2010A030), the
Team for Scientific Innovation Foundation of Anhui Province
(2006KJ007TD), the 211 Project of Anhui University, and the
Ministry of Education Funded Projects Focus on Returned
Overseas Scholar.
Our initial confocal microscopy studies reveal that
Eu(THA)3Phen functions as a luminescent cellular DNA stain
for MCF-7 cells being successfully taken up by live cells and
clearly displaying nucleus structure. In 400 mM of complex,
most of luminescence emerges from cellular cytoplasm
(in Fig. 3a); we presume that the luminescence from punctate
bright dots outside the nuclei region is because of the aggregation
of the complex inside the lysosome or mitochondria due to the
high concentration. Some of the charged biological macro-
molecules like peptides and liposomes accelerate this aggregation.
In this context, we lowered the concentration to 200 mM, and
then took an image (in Fig. 3b). The punctate luminescence
disappears, however, luminescence from the cell nucleus and
nucleoli is clearly observed. As we can see cellular DNA
undergoes both interphase and metaphase staining by
the complex; in other words, in lower complex concentration,
we propose that there is no aggregation in the cytosol hence
the cell nucleus and nucleoli uptake the complex without
affect. The imaging properties of the Eu(III) complex somehow
provide a great opportunity to develop a low toxicity, highly
sensitive and DNA-specific molecular two-photon probe for
cell biologists. We are also investigating the mechanism by
which the complex recognizes DNA and this will form the
basis of future researches.
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In conclusion, we have demonstrated an efficient two-
photon sensitization of Eu(III) luminescence in the novel
Eu(THA)3Phen complex. The TPA cross section values of
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Fig. 3 Live cellular image based on Eu(THA)3Phen, MCF-7 cells
were incubated with: (a) 400 mM and (b) 200 mM complex for 1 hour,
then imaged by two-photon microscopy (excitation wavelength l =
770 nm, emission wavelength l = 613 nm) without fixation. Note that
cell cytosol staining is clear emerging in higher concentration; in
contrast cell nucleus and nucleoli luminescence is more significant in
low concentration. All the scale bars represent 10 mm.
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