Intermolecular energy transfer from Tb3+ to Eu3+ in aqueous aggregates and on the surface of human cells

Article abstract of DOI:10.1021/ol200328p

Efficient intermolecular energy transfer from carbostyril 124-sensitized Tb³⁺ to Eu³⁺ in aqueous aggregates is reported. This energy transfer was also recapitulated on the cell surface of a human kidney cell line (HEK-293T) and imaged by fluorescence microscopy as an example for the applicability of this energy transfer probe for imaging in biological systems.

Full text of DOI:10.1021/ol200328p

ORGANIC
LETTERS
Intermolecular Energy Transfer from Tb3þ
to Eu3þ in Aqueous Aggregates and on the
Surface of Human Cells
2011
Vol. 13, No. 11
2802–2805
Minhee Lee, Matthew S. Tremblay, Steffen Jockusch, Nicholas J. Turro,
and Dalibor Sames*
Department of Chemistry, Columbia University, 3000 Broadway, New York,
New York 10027, United States
sames@chem.columbia.edu
Received February 9, 2011
ABSTRACT
Efficient intermolecular energy transfer from carbostyril 124-sensitized Tb3þ to Eu3þ in aqueous aggregates is reported. This energy transfer was
also recapitulated on the cell surface of a human kidney cell line (HEK-293T) and imaged by fluorescence microscopy as an example for the
applicability of this energy transfer probe for imaging in biological systems.
Fluorescence resonance energy transfer (FRET) and
luminescence resonance energy transfer (LRET) are power-
ful tools for studying molecular interactions in solution and
in a cellular context.1 Extensive research has been carried out
using resonance energy transfer between various energy
transfer partners, such as small organic fluorophores,2
fluorescent proteins,3 quantum dots,4 and fluorescent/lumi-
nescent metal ions.5,6 In particular, long-lived luminescent
lanthanide ions as energy transfer donors6 have been shown
to offer many advantages, since light output can be measured
in a time domain that is essentially background-free.7 The use
of a second lanthanide ion as an energy transfer acceptor can
make the resonance energy transfer measurement more
sensitive because the emissions from a donor and an acceptor
are readily resolvable as sharply spiked peaks. Energy trans-
fer between two lanthanide ions in heterodinuclear com-
plexes and mixed lanthanide systems has long been known in
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r
10.1021/ol200328p
Published on Web 05/12/2011
2011 American Chemical Society

solution8 and in the solid state.9 However, there are rare
examples of intermolecular energy transfer between Tb3þ and
Eu3þ in aqueous solution,10 as well as its application for
biological sensing or cellular imaging.11 As a part of a broad
program aimed at the development of optical probes for
imaging metabolic and signaling events in cells and tissues,12
we became interested in exploring energy transfer between
lanthanide(III) ions and its applicability. Herein, we report
intermolecular energy transfer from carbostyril 124-sensi-
tized Tb3þ to Eu3þ in aqueous aggregates and show the first
example of imaging this phenomenon on the cellular mem-
brane of HEK-293T cells.
Scheme 1. Synthesis of C12Ln
Figure 1. Structure of C12Ln and C1Ln (Ln = Tb or Eu).
Two sets of lanthanide complexes, C12Ln and C1Ln
(Ln = Tb or Eu, Figure 1), were prepared after 11- and
4-step sequences from 3-nitroaniline (Scheme 1) and 7-ami-
no-4-methylquinolin-2(1H)-one (see Supporting In-
formation), respectively, followed by purification by pre-
parative reversed-phase HPLC. The amphiphilic C12Ln
with a polar Ln:DOTA (1,4,7,10-tetraazacyclododecane-
N,N0,N00,N000-tetraacetic acid) head and a nonpolar dode-
cane chain was designed to have intermolecular proximity in
water by forming micelle-like aggregates. On the other
hand, the control compound, C1Ln, does not form aggre-
gates in aqueous solution but exists as free solutes.
4-Methylcarbostyril (Cs124) was chosen as the organic
photon antenna because it is known to sensitize terbium
luminescence much more efficiently than europium lumi-
nescence,13 so the energy transfer from terbium to europium
can be readily observed as quenched terbium emission and
enhanced europium emission with minimum background.
Toexamine the potentialofC12Ln toformaggregates in
aqueous solution, the concentration dependence of time-
resolved emission profiles of C12Tb and C1Tb was exam-
ined (Figure 2A and 2B). Luminescence decay curves of
C12Tb from 2 to 40 μM are biexponential, indicating that
C12Tb exists as two distinct species with different lifetimes
(1.31 ( 0.06 and 0.27 ( 0.04 ms) in aqueous solutions,
presumably monomeric C12Tb with a longer lifetime and
C12Tb aggregates with a shorter lifetime mainly due to
(10) Li, W.; Li, W.; Yu, G.; Wang, Q.; Jin, Y. J. Alloys Compd. 1993,
191, 107–110.
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Halim, M.; Sames, D. J. Am. Chem. Soc. 2007, 129, 7570–7577. (c)
Froemming, M. K.; Sames, D. J. Am. Chem. Soc. 2007, 129, 14518–
14522. (d) Tremblay, M. S.; Lee, M.; Sames, D. Org. Lett. 2008, 10, 5–8.
(e) Halim, M.; Yee, D. J.; Sames, D. J. Am. Chem. Soc. 2008, 130, 14123–
14128. (f) Gubernator, N. G.; Zhang, H.; Stall, R. G. W.; Mosharov,
E. V.; Pereira, D. B.; Yue, M.; Balsanek, V.; Vadola, P. A.; Mukherjee,
B.; Edward, R. H.; Sulzer, D.; Sames, D. Science 2009, 324, 1441–1444.
(13) Parker, D.; Williams, J. A. G. J. Chem. Soc., Perkin Trans. 2
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Figure 2. Tb3þ luminescence decay curves of (A) C12Tb and (B)
C1Tb at different concentrations (2, 5, 10, 20, and 40 μM); (C) 20
μM C12Tb and a cocktail of 20 μM C12Tb þ 20 μM C12Eu (in
H2O, λex = 340 nm, λem = 544 nm). (D) Steady-state lumines-
cence spectra of 20 μM C12Tb, 20 μM C12Eu, and a cocktail of
20 μM C12Tb þ 20 μM C12Eu (in H2O, λex = 340 nm).
Org. Lett., Vol. 13, No. 11, 2011
2803

self-quenching.14 Indeed, the composition of these two
species changes in a direction to favor shorter lifetime
species (from 8% to 75%, see Supporting Information,
Figure S2) as the concentration increases from 2 to 40 μM.
In contrast, C1Tb exhibits monoexponential emission
decay curves with a luminescence lifetime of 1.56 ( 0.02
ms regardless of the concentration, indicating its homo-
geneous distribution in water. Addition of 20 μM C12Eu
to an aqueous solution of 20 μM C12Tb quenched terbium
emission selectively from the short-lived, presumably ag-
gregated component, while not affecting the lifetime of the
long-lived component (Figure 2C). Moreover, the hetero-
metallic cocktail of C12Tb and C12Eu exhibits signifi-
cantly decreased emission from terbium, whereas the
emission from europium enhances by 2-fold at 700 nm
(Figure 2D). These results together suggest that energy
transfer takes place from Cs124-sensitized terbium to
europium when the two complexes are brought into proxi-
mity by forming mixed aggregates in aqueous solution
(Figure 3).
Figure 4. Emission ratio of (A) C12Tb and a cocktail of C12Tb
þ C12Eu (blue diamond), C1Tb and a cocktail of C1Tb þ C1Eu
(red triangle) at 544 nm; (B) C12Eu and a cocktail of C12Tb þ
C12Eu (blue diamond), C1Eu and a cocktail of C1Tb þ C1Eu
(red triangle) at 700 nm in water with increasing % (v/v) of DMSO.
[C12Ln] or [C1Ln] = 20 μM16 in all measurements. Error bars:
s.d. (n = 3).
as free solutes in aqueous solution and the low efficiency of
energy transfer by simple diffusional collisions.15
The efficiency of energy transfer was investigated as a
function of solvent polarity. When the emission was
monitored at 544 nm, the magnitude of the reduction in
Intermolecular energy transfer from Tb3þ to Eu3þ was
recapitulated on the cell surface and imaged by fluores-
cence microscopy as a preliminary study for bioimaging
applications. As the dodecane hydrocarbons are reported
tobesufficientlylipid-like,17 the lipophilic chains of C12Ln
should guide the compounds to be inserted into the cellular
Figure 3. Schematic representation of intermolecular energy
transfer from Cs124-sensitized Tb3þ to Eu3þ in amphiphilic
C12Tb þ C12Eu cocktail.
terbium emission resulting from the presence of C12Eu
decreased as DMSO content was increased (Figure 4A,
blue diamonds). A similar polarity effect was also observed
on the europium emission at 700 nm (Figure 4B, blue
diamonds) to give a smaller difference in emission inten-
sities between C12Eu and the cocktail at lower polarity.
This implies that the energy transfer efficiency is solvent-
polarity-dependent and that it is stronger in pure water
than in solvent mixtures of lower polarity, presumably
because the propensity for the C12Ln complexes to form
aggregates in water by hydrophobic interaction is driven by
solvent polarity. In contrast to C12Ln, emissions at 544 and
700 nm from C1Tb and C1Eu, respectively, are not affected
by each other’s copresence at varying solvent polarity
(Figure 4A and B, red triangles), suggesting their existence
Figure 5. Luminescence and bright field images of HEK-293T
cells incubated with 20 μM C12Tb (AꢀC), 20 μM C12Eu (DꢀF),
and a cocktail of 20 μM C12Tb þ 20 μM C12Eu (GꢀI) for 15
min. Emissions from terbium and europium were imaged using
a fluorescence microscope mounted with two filter sets, one with
a 350 ( 25 nm exciter and 550 ( 20 nm emitter (for terbium
emission) and the other with a 350 ( 25 nm exciter and 705 ( 20
nm emitter (for europium emission).
(14) Prieto, M. J. E.; Castanho, M.; Coutinho, A.; Ortiz, A.; Aranda,
ꢀ
ꢀ
F. J.; Gomez-Fernandez, J. C. Chem. Phys. Lipids 1994, 69, 75–85.
(15) The bimolecular energy transfer rate constant from C1Tb to
C1Eu in H2O was determined to be 3 ꢁ 106 Mꢀ1 sꢀ1 which is more than 3
orders of magnitude smaller than the diffusion limit (for more details, see
Supporting Information, Figure S3).
2804
Org. Lett., Vol. 13, No. 11, 2011

plasma membrane. When HEK-293T cells were treated
with 20 μM C12Tb for 15 min at 37 °C, a luminescence
image of the cell surface was observed (350 ( 25 nm
excitation and 550 ( 20 nm emission, Figure 5A) which
indicatesthatthecellular plasmamembrane was selectively
labeled with C12Tb. In contrast, cells incubated with
20 μM C12Eu did not afford any detectable luminescence
signals (350 ( 25 nm excitation and 705 ( 20 nm emission,
Figure 5E) because of the inability of Cs124 to efficiently
sensitize europium. Importantly, upon incubating the cells
with a cocktail of 20 μM C12Tb and 20 μM C12Eu,
strikingly enhanced Eu3þ luminescence (Figure 5H) and
correspondingly quenched Tb3þ luminescence (Figure 5G)
were observed, demonstrating the energy transfer taking
place on the cell surface from Cs124-sensitized terbium to
europium. This represents the first example of cellular
imaging based on intermolecular energy transfer between
two lanthanide ions, which could be the basis for novel
optical sensors that extend the scope of FRET/LRET-
based techniques to include the measurement of two long-
lived emission signals.
In summary, we present support for the intermolecular
energy transfer from Cs124-sensitized Tb3þ to Eu3þ in
aqueous aggregates as shown by steady-state and time-
resolved luminescence studies and demonstrate that
this effect is sensitive to solvent polarity. We also image
this phenomenon on the cell surface, suggesting its
potential use for the development of novel optical
biosensors.
Acknowledgment. Financial support for this work was
provided by the G. Harold & Leila Y. Mathers Foundation.
S.J. and N.J.T. thank the National Science Foundation for
financial support through Grant NSF-CHE-07-17518. We
thank Dr. Julien Massue (University of Strasbourg, France)
for his intellectual and editorial support.
Supporting Information Available. Detailed synthetic
procedures, characterization of new compounds, ex-
perimental details for photophysical studies, and fluor-
escence microscope imaging protocols. This material is
available free of charge via the Internet at http://pubs.
acs.org.
(16) At this concentration, 60% of C12Ln exists as aggregates in
water (see Supporting Information, Figure S2).
(17) Shynkar, V. V.; Klymchenko, A. S.; Kunzelmann, C.; Duportail,
G.; Muller, C. D.; Demchenko, A. P.; Freyssinet, J.-M.; Mely, Y. J. Am.
Chem. Soc. 2007, 129, 2187–2193.
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