Impressive europium red emission induced by two-photon excitation for biological applications

Article abstract of DOI:10.1021/ic102465j

Three triazine-based europium(III) complexes were synthesized that demonstrated strong two-photon induced europium emission with a high two-photon absorption cross-section. The modified triazine ligand of complex 3 initiated over 100% enhancement of the two-photon absorption cross-section (σ₂: 320 GM) when compared with complex 1 (σ₂: 128 GM) in a solution of DMSO. Europium complex 3 is also stable in vitro, and power-dependence curves were obtained in vitro to confirm the two-photon-induced f-f emission in HeLa cells.

Full text of DOI:10.1021/ic102465j

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pubs.acs.org/IC
Impressive Europium Red Emission Induced by Two-Photon Excitation
for Biological Applications
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Wai-Sum Lo,† Wai-Ming Kwok,† Ga-Lai Law,†, Chi-Tung Yeung,† Chris Tsz-Leung Chan,† Ho-Lun Yeung,†
Hoi-Kuan Kong,‡ Chi-Hang Chen,† Margaret B. Murphy,‡ Ka-Leung Wong,*,§ and Wing-Tak Wong*,†
†Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hum, Hong Kong SAR
‡Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Hong Kong SAR
§Department of Chemistry, Hong Kong Baptist University, Kowloon Tung, Hong Kong SAR
S Supporting Information
b
lanthanide ion. Therefore, when compared to the emission quan-
ABSTRACT: Three triazine-based europium(III) com-
plexes were synthesized that demonstrated strong two-
photon induced europium emission with a high two-photon
absorption cross-section. The modified triazine ligand of
complex 3 initiated over 100% enhancement of the two-
photon absorption cross-section (σ2: 320 GM) when
compared with complex 1 (σ2: 128 GM) in a solution of
DMSO. Europium complex 3 is also stable in vitro, and
power-dependence curves were obtained in vitro to confirm
the two-photon-induced fꢀf emission in HeLa cells.
tum efficiency to organic or organic-transition metal complexes,
the cross-sections of lanthanide materials should be relatively
diminished. Also, the two-photon absorption cross-sections in
this manuscript have been measured by a reference standard
method (with rhodamine 6G) which is based on the fꢀf emis-
sion.12,16,17 Notable exceptions are europium complexes with
dipicolinic acid derivatives reported by Maury et al., which have
two-photon absorption cross-sections up to 775 GM in dichloro-
methane.18,19 The demand for different two-photon lanthanide
materials for further development as time-resolved in vitro probes
is still very high; thus we hereby present three biological com-
patible europium complexes that are triazine-based ligands with
great potential as candidate probes. Complex 3, coordinated with
(2-(N,N-diethylanilin-4-yl)-4,6-bis-(pyrazol-1-yl)-1,3,5-triazine) and
2-thenoyl-trifluoroacetone, has the strongest two-photon absorp-
tion cross-section for triazine-based molecular europium com-
plexes in DMSO (320 GM). The stability of these europium
complexes has been examined, and they have demonstrated cell
permeability and low cytotoxicity and are potential candidates for
time-resolved molecular probes when conjugated with appro-
priate vectors such as peptides or specific antibodies.
In our previous studies, the triazine-based europium complex
1 showed promising two-photon-induced fꢀf emission.16
The modification of the triazine-based ligand in complex 1 here
shows an increased two-photon absorption of the triazine-based
europium materials from ∼100 GM to 320 GM. Complexes 1ꢀ3
(Figure 1) were synthesized by reacting Eu(tta)3 with the respec-
tive ligands (L2 and L3 are newly synthesized): 2-(N,N-diethy-
lanilin-4-yl)-4,6-bis(3,5-dimethyl-pyrazol-1-yl)-1,3,5-triazine (L1),
2-(N,N-diethylanilin-4-yl)-4,6-bis(indazole-1-yl)-1,3,5-triazine
(L2), and 2-(N,N-diethylanilin-4-yl)-4,6-bis(pyrazol-1-yl)-1,3,
5-triazine (L3). The newly synthesized complexes 2 and 3
are characterized by conventional methods (see Supporting
Information).
here are two main problems with most of the commercially
T
available molecular imaging probes—requiring excitation by
a UV source and autofluorescence1ꢀ3—which limit the quality of
in vitro imaging.4,5 Lanthanide materials, with their distinctive
features, such as long-lived luminescence lifetimes, large Stokes
shifts, and sharp emission peaks, for molecular imaging, diag-
nosis, or therapy, offer a solution to the latter problem.5ꢀ7 The
development of time gating technology combined with the char-
acteristic microsecond lifetimes of lanthanide complexes help to
eliminate nanosecond autofluorescence in order to achieve better
quality imaging in vitro/in vivo.8 For these reasons, lanthanide
materials have developed rapidly in the past two decades.9
Lanthanide materials can provide long lifetime emissions for
time-resolved imaging, but the source of excitation used is a
major obstacle.6,10ꢀ12 The fꢀf transitions of lanthanide ions are
forbidden by selection rules, and thus the resulting emissions
from direct excitation of the metal are very weak.7 The use of
organic ligands as suitable antennae allows energy from the
excited antennae to be transferred to the lanthanide ions first
by intersystem crossing (singlet to triplet state) of the ligand and
then to the excited states of the lanthanide ions.13,14 This energy
transfer pathway remarkably enhances emission. In general, most
organic ligands are only effectively excited in the UV region.15 In
aiming to develop nonphototoxic time-resolved imaging tools,
lanthanide chemists have been urged to look for organic chro-
mophores that can absorb numerous near-infrared photons and
efficiently transmit to lanthanide ions to induce fꢀf emissions.
However, even though the energy transfer pathway between
the organic antenna and lanthanide by linear and two-photon
absorption is the same, it is inevitable that energy is lost during
the transfer from the ligand (antenna) to the excited state of the
The solution state electronic excitation spectra of the europium
complexes display band shapes similar to their absorption spectra
in a solution of DMSO (Figures S9, Supporting Information). The
excitation bands of complexes 1ꢀ3 have maxima at ∼400 and
330 nm (attributed to the intraligand charge transfer and π f π *
transitions of ligands L1ꢀL3 in complexes 1ꢀ3) and ∼350 nm
Received: December 9, 2010
Published: May 16, 2011
r
2011 American Chemical Society
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Inorganic Chemistry
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Figure 1. The molecular structures of europium complexes (1ꢀ3) with
triazine-based ligands.
Figure 3. Results of the power-dependence experiments showing
emission intensity and incident power on log scales.
Figure 2. Two-photon (upper, λex = 750 nm) and linear (lower, λex
=
350 nm) induced europium emission in a solution of DMSO.
Table 1. Quantum Yields, Emission Lifetimes, and Two-
Photon Absorption Cross Sections of 1ꢀ3 in DMSO
Φa
τ (ms)b
σ2 (GM)c
1
2
3
0.48
0.33
0.32
1.34
1.12
1.09
128
98
Figure 4. (a) Two-photon induced in vitro imaging of HeLa cells
incubated with europium complexes 1 (ai), 2 (aii), and 3 (aiii) for
1 h. (b) The MTT assay results of complexes 1ꢀ3 for 24 h at 10 times
the dosed concentration used for the molecular imaging in a. (c) The
in situ power dependence curve (europium emission and incident power
on log scales) of complex 3; in situ emission spectra for the power
dependence curve were obtained via lambda scan from aiii—BP filter
550ꢀ650 nm, λex = 750 nm.
320
a λem = 560 ꢀ 700 nm, λex = 350 nm. b Decay curve monitored at 620 nm
(5D0 f F2, λex = 350 nm). c Two-photon absorption cross-section
7
(GM = 10ꢀ50 cm4 s photonꢀ1 moleculeꢀ1, λem = 560 ꢀ 700 nm,
λex = 750 nm).
(attributed to π f π* transitions of thenoyltrifluoroacetonate).
The room-temperature emission spectra of complexes 1ꢀ3 under
350 nm laser excitation in the solution state (DMSO) are
displayed in Figure 2 (lower), which show characteristic 5D0 f
7FJ (J = 0ꢀ4) transitions of Eu3þ. As expected, the emission
spectra of complexes 1ꢀ3 are very similar, particularly with regard
to the band energies (Figure 2 upper).
quantum yield (measured using an integrating sphere) of the
europium complexes were monitored between 560 and 700 nm.
The triazineꢀeuropium complex 3, without the electron donat-
ing group or electron withdrawing groups borne by L1 and L2,
featured 1.5-fold larger two-photon-induced fꢀf emissions and a
2.5-fold greater two-photon absorption cross-section (Table 1).
Power-dependence experiments have been carried out to con-
firm that the energy of two near-infrared femtosecond photons
are absorbed by the triazine ligands, then transferred to the
europium excited state for their emission (Figure 3). No
significant cytotoxicity was observed for the three europium
complexes (dosed concentration = 30 μM, 24 h; Figure 4b), and
In our previous studies, we defined complex 1 as having
undergone a direct energy transfer from the ligand singlet to
5
5D0 and D1 of the europium ion.16 The two-photon induced
photophysical properties of triazineꢀeuropium complexes were
examined in a solution of DMSO with femtosecond excitation at
750 nm. The two-photon absorption cross-section and absolute
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the IC50 values for HeLa cells were found to be around 150 μM.
All three europium complexes do not have any specific localiza-
tion profile, and strong two-photon induced fꢀf emissions in the
in vitro emission spectra can be observed (Figure 4). There are
numerous studies on two-/three-photon-induced fꢀf in vitro
imaging with lanthanide materials, but in situ power dependence
experiments are rare. The in situ power dependence experiments
with lanthanide complexes were conducted initially on cells with
complex 3. Only complex 3 showed an impressively high two-
photon absorption cross-section among europium complexes in
the power dependence experiments (Figure 4c). Variations in
laser power were recorded with a power meter for the coherent
(II) laser output from a confocal microscope, and the emission
intensities were monitored after adjusting the laser power for 5
min. Complex 3 demonstrated an in situ power dependence
curve which confirms that the europium signal originated from
within the HeLa cells (Figure 4aiii) was induced by a two-photon
process (slope = ∼2.3) rather than scattering or the SHG of the
laser line. The stability of the three europium complexes in vitro
was examined via titration of various biologically meaningful
small molecules, such as citrate, bicarbonates, and HSA (Figure
S12 and S13, Supporting Information). Slight significant lumi-
nescent enhancement/quenching was observed.
In conclusion, a new motif of triazine-based europium com-
plex 3 has been synthesized, and it demonstrates a strong two-
photon absorption cross-section (320 GM). The dramatic
change of two-photon photophysical properties of these three
europium complexes has been induced by slight modification of
the triazine ligand. Europium complex 3 has demonstrated low
cytotoxicity, cell permeability, and fast cellular uptake without a
specific localization profile, and therefore it has the potential to
become a new time-resolved imaging probe that is excitable at a
wavelength within the “biological window”—in which light
penetration through biological tissue is maximal.
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details of synth-
b
eses, photophysical and in vitro studies. 1H and 13C NMR spectra
of ligands and complexes. UVꢀvis absorption and in vitro
emission spectra of complex 3. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 852 3411 2370 (K.-L.W.), 852 3400 8789 (W.-T.W.).
E-mail: klwong@hkbu.edu.hk (K.-L.W.), bcwtwong@polyu.edu.
hk (W.-T.W.).
Present Addresses
Department of Chemistry, University of California Berkeley,
Berkeley, California, 94720-1460
’ ACKNOWLEDGMENT
This work was funded by grants from The Hong Kong
Research Grants Council (PolyU 503510), the Hong Kong
Polytechnic University (1BD07), City University of Hong Kong,
and Hong Kong Baptist University (FRG 1/10-11/037). This
work was also supported by the Area of Excellence Scheme of the
University of Grants Committee (Hong Kong).
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