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8052 Chem. Commun., 2011, 47, 8052–8054 This journal is c The Royal Society of Chemistry 2011
Cite this: Chem. Commun., 2011, 47, 8052–8054
Two-photon induced responsive f–f emissive detection of Cyclin A with a
europium-chelating peptidew
Hoi-Kwan Kong,aFrances L Chadbourne,
bGa-Lai Law,
bHongguang Li,
cHoi-Lam Tam,
e
Steven L Cobb,*bChi-Kong Lau,*
dChi-Sing Lee*
cand Ka-Leung Wong*
a
Received 13th May 2011, Accepted 3rd June 2011
DOI: 10.1039/c1cc12811f
Responsive linear and two-photon induced europium emissive probes
have been synthesised with a tailor made peptide for the detection of
Cyclin A, the hypersensitive Eu emission (Eu-2) gave the real time
signalling and also enhanced the two-photon absorption cross section
from 12 GM to 68 GM after Cyclin A binding.
Since the pioneering work of Webb and his co-workers on two-
photon laser scanning fluorescence microscopy,1 multi-photon
microscopy has rapidly become an invaluable bioimaging tool
for the study of dynamic biochemical processes in cells, thick
tissues, and live animals.2 The advantageous features of multi-
photon bioimaging include a reduction in photobleaching and
photodamage to the imaging probes and cellular structures; the
capability to penetrate thick tissues; and the ability to bring
about precise three-dimensional localized photosensitization,
photolysis, ablation, and cutting at the sub-cellular level.
Examples of coordination and organometallic complexes with
two-photon emission properties are scarce, and the potential of
multi-photon luminescent lanthanide complexes for bioimaging
has not been fully explored.3 Rather than using commercial
organic dyes, we have undertaken a more advantageous
approach that uses lanthanide complexes as the emitter, with
energy transfer from the ligand to the metal, to generate
a fingerprint emission band and a longer emission lifetime
(ms to ms scale) so that the signals can be easily distinguished
from biological auto-fluorescence in the cell or culture medium.4
In fact, coordination and organometallic/organic-lanthanide
complexes are good candidates for multi-photon imaging lumino-
phores. Impressive two-photon induced f–f emission can be
achieved by the strong multi-photon antenna (lanthanide)
upon near-infrared photo-excitation.5
Dysregulation of the normal stem cell self-renewal process
could lead to the accumulation of stem cells with an accompanied
risk of malignant transformation.6 The proposed model that
cancer might be driven by a small population of cells has
brought a paradigmatic shift in cancer biology.7 Up till now,
the functional and molecular details involved in stem cell
self-renewal are largely unknown. Recent evidence suggests
that the cell cycle has tremendous influence on this process. It
was shown that Cyclin A function is essential for cell cycle
progression in stem cells.8 The most direct approach to
analyze cell cycle regulation within stem cells is to visualize
them. Observing long term cell cycle regulation in live stem
cells would markedly improve knowledge about the underlying
mechanisms of the stem cell self-renewal process. The most
common technique to study endogenous proteins using
fluorescence is labelling with a primary antibody followed by
amplification with a secondary antibody conjugated to organic
dyes. However, this technique is restricted to fixation and
permeabilization. Another method is genetic tagging, where
green fluorescent protein (GFP) can be covalently fused to any
cDNA of interest. Disadvantages of this technique are the
needs of ectopic expression, and transfection required to
deliver the GFP-tagged proteins into cells. However, the
transfection efficiencies of stem cells are extremely low.9 So
far, there is no single imaging approach that is ideal for
continuous observation of endogenous cell cycle regulators
in live stem cells. There is, so far, very limited development on
lanthanide probes targeting specific peptides or proteins
sensitized with two-photon induced emission. The only study
which characterized the specific Cyclin A core peptide
sequence tagged to a terbium complex, revealed absorption
and emission at the ultra-violet region, is not suitable for an
in vitro imaging study.10 Pazos et al. have demonstrated
intermolecular sensitization of lanthanide ions as a useful
strategy for the design of Cyclin A biosensors. Although their
methodology may be of general use to synthesize biosensors
for other biomolecular systems, the major drawback lies on
the excitation of Trp217 as the antenna at the ultra-violet
region which is not suitable for in vitro imaging. Herein, we
aDepartment of Chemistry, Hong Kong Baptist University,Kowloon Tong, Hong Kong SAR. E-mail: [email protected];Tel: +852-34112370
bDepartment of Chemistry, Durham University, Durham,UK DH1 3LE. E-mail: [email protected];Tel: +44 191 33 42086
c Laboratory of Chemical Genomics, School of Chemical Biology andBiotechnology, Peking University Shenzhen Graduate School,Shenzhen University Town, Xili, Shenzhen 518055, China.E-mail: [email protected]; Tel: +86 2603 2701
d School of Biomedical Sciences, The Chinese University ofHong Kong, Shatin, New Territories, Hong Kong SAR.E-mail: [email protected]
eDepartment of Physics, Hong Kong Baptist University,Kowloon Tong, Hong Kong SARw Electronic supplementary information (ESI) available: Experimentaldetails of synthesis, NMR spectra of new compounds, MADLI-MSspectra of ligands and their complexes Eu-1 and Eu-2, linear and two-photon induced spectroscopic data. See DOI: 10.1039/c1cc12811f
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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 8052–8054 8053
reported newly synthesized two-photon induced (near-infrared
excited) europium complexes as the potential Cyclin A tracer.
Our europium complexes were able to demonstrate significant
signal increase after conjugating the peptides with the Cyclin A
and also show the real time signal change through the hyper-
sensitive 5D0 - 7FJ (J = 2) by linear excitation in the UV
region and also near-infrared excitation through two-photon
absorption.
The design of the two europium complexes (Eu-1 and Eu-2)
as Cyclin A tracer has the following basic structure—an
amide-substituted 1,4,7,10-tetraazacyclododecane ligand and
its pendant arms with carboxylic groups, a highly conjugated
chromophore and three different peptides respectively. Carboxylic
groups help to improve the solubility of the complexes for the
detection of Cyclin A in vitro. Two peptides were proposed by
Pazos et al. as the tracker for Cyclin A and we have conjugated
such peptides with our chromophore which allows two-photon
absorption. The same cyclen skeleton and peptide sequence
were used in this complex (Fig. 1: R1–Eu-1). New Cyclin A
peptide sequences (Fig. 1: R2–Eu-2) were synthesized with
slight elongation compared to R1. Detailed synthetic procedures
of two europium complexes are shown in the ESIw with
essential photophysical measurement (UV absorption, linear
emission spectra). The conjugated chromophore demonstrated
strong two-photon absorption and appropriate triplet states
(B21739 cm�1, Gd analogues at 77 K in 2-methyl tetra-
hydrofuran; Fig. S20, ESIw) which provide an efficient pathway
for the energy transfer from the ligand to the excited state of
europium. Four structural red f–f (5D0 - 7FJ, J = 1–4)
emission bands can be obtained from complexes Eu-1 or Eu-2
upon excitation at 350 nm and 800 nm. The absolute quantum
yields (F) and emission lifetimes (t) of complexes Eu-1 and
Eu-2 are 0.02 (Eu-1), 0.03 (Eu-2) and 0.61 ms (Eu-1), 0.68 ms
(Eu-2), respectively. The two-photon absorption cross section
of complex Eu-1 is around 9 GM and Eu-2 is 12 GM
(GM = 10�50 cm4 s photon�1 molecule�1) (Table 1). The
overall shapes of the emission spectra of both Eu complexes in
the solution of HEPES are similar (Fig. 2, upper) suggesting a
similar chemical environment of the metal ions. However, with
the addition of Cyclin A to these two europium complexes,
the variation of f–f emission splitting and intensities is
observed (Fig. 3, upper). The ratio of 5D0 - 7F2 transition
to 5D0 -7F1 decreased with the addition of Cyclin A (Fig. 3,
upper; Eu-1 5D0 -7F2 :
5D0 -7F1 fromB0.7 toB0.5 and in
Eu-2, from B0.7 to 0.4) This alteration in the spectroscopic
signal may be due to the changes in the coordination environment
of the europium ion after binding with bulky Cyclin A and can
be used for real-time detection of proteins. The quantum
efficiency of two-photon absorption is less than 1% when
compared to the linear absorption process. The signal to noise
ratio in the two-photon process was lower than that of the
Fig. 1 The molecular structures of europium complexes Eu-1 and
Eu-2.
Table 1 Luminescence lifetimes (t, ms), number of inner-sphere water molecules (q), quantum yield (F) and two-photon absorption cross-section(s2) of Eu-1 and Eu-2 in the presence and absence of Cyclin A
ta (H2O) ta (D2O) t (Cyclin) qa Fb (H2O) Fb (Cyclin) s2c (H2O) s2
c (Cyclin)
Eu-1 0.61 1.05 0.82 0.82 0.02 0.035 9 GM 11 GMEu-2 0.68 1.11 0.98 0.68 0.03 0.08 12 GM 68 GM
a Derived hydration numbers, q (�20%) qEu = 1.2[(k(H2O) � k(D2O)) � (0.25 + 0.07x)] (x = number of carbonyl-bound amide NH oscillators),11
decay curve monitored at 616 nm (5D0 - 7F2, lex = 380 nm).9 b lem = 560–700 nm, lex = 350 nm; c Two-photon absorption cross-section
(GM = 10�50 cm 4 s photon �1 molecule�1, lem = 580–700 nm, lex = 800 nm).
Fig. 2 The UV (upper: lex = 350 nm) and two-photon (lower: lex =800 nm) induced f–f emission spectra of complexes Eu-1 and Eu-2
(10 mM) in HEPES exhibited real time signal change to Cyclin A
(47 nM Cyclin A, 10 mM HEPES buffer, pH 7.5, 100 mM NaCl,
lex = 350 nm). Lower inset: the power dependence experiment of
Eu-2 with the addition of 40 nM Cyclin A and excitation at 800 nm,
the slope = 1.9.
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8054 Chem. Commun., 2011, 47, 8052–8054 This journal is c The Royal Society of Chemistry 2011
titration experiments via linear absorption. The time-gated
system should be able to help improve this weakness in the
near coming future. Eu-2 demonstrated much stronger emission
enhancement than Eu-1 with the addition of same amount of
Cyclin A. The slight modification of peptide sequences in Eu-2
served for stronger binding affinity to Cyclin A. For two-
photon induced f–f emission, two-photon absorption cross
section of complex Eu-2 was increased from 12 GM to 68 GM.
The binding affinities between lanthanide-bound peptide
sequences in two complexes to Cyclin A were examined and
compared by titrating the complex with different concen-
trations of Cyclin A (Fig. 3). Complex Eu-2 (1685 � 175 nM)
demonstrated the stronger binding to Cyclin A than complex
Eu-1 and also shows the positive signal changes (Fig. 3, Eu-2
shows 16 times emission enhancement with addition of 40 nM
Cyclin A).
Multi-photon confocal laser scanning microscopy offers
excellent resolution for three-dimensional images of fluorescently
labelled live samples with specific excitation in the micrometre
range. During two-photon excitation, the simultaneous absorption
of the two infrared photons that are specific and intrinsic to
the fluorescent molecules used as specific labels for biological
structures, organelles and molecules gives emission in the
visible region for image construction. In vitro experiments
have been carried out in HeLa cells with linear and two-
photon microscopes. Eu-1 shows detectable europium emis-
sion with excitation at the near-infrared region, 800 nm (via
two-photon absorption through fs Ti:sapphire laser excitation).
Under the same incubation conditions (dosed concentration of
complexes, dosed time and excitation wavelength), Eu-2 shows
more promising in vitro emission in the nucleus, revealing the
high specificity of the probe to Cyclin A and the capability of
an in vitro imaging study (Fig. 4).
Moreover, no dark cytotoxicity was evident in HeLa cells
that were exposed to the complexes Eu-1 and Eu-2 in four
different dosed time durations (Fig. S3, ESIw) at 100 mM.
MTT assays on these cells exposed to the same concentration
of complexes Eu-1 and Eu-2 for prolonged periods revealed a
gradual decrease of viable cells. The IC50 values of two
europium complexes are B1 mM (Eu-1) and B1.3 mM
(Eu-2) respectively.
In conclusion, we have synthesized europium complexes
with modified synthetic peptides and chromophores which demon-
strated highly specific and responsive europium emission for
Cyclin A through linear and nonlinear (near-infrared) excitation.
Based on these findings, we can further establish a new Cyclin
A tracer that is less phototoxic and that can visualize them
within the ‘‘biological window’’ of excitation (700 nm–1000 nm);
the utility of the Cyclin A tracer in biological systems will be
confirmed both in vitro and in vivo. Our results demonstrate
the great potential of our lanthanide complexes (Eu-2) as a
new generation of bio-tracer for Cyclin A and have applications
both for Cyclin A detection and inhibition of the cell division
for research purposes as well as in the marketplace.
This work was funded by grants from The Hong Kong
Research Grants Council, ESPRC, Hong Kong Baptist
University (FRG 1/10-11/037), The Hong Kong Chinese
University, Peking University Shenzhen Graduate School
and Durham University.
Notes and references
1 W. Denk, J. H. Strickler and W. W. Webb, Science, 1990, 248, 739.2 N. Maindron, S. Poupart, M. Hamon, J.-B. Langlois, N. Ple,L. Jean, A. Romieu and P.-Y. Renard, Org. Biomol. Chem., 2011,9, 2357; K. M. L. Taylor-Pashow, J. D. Rocca, R. C. Huxford andW. Lin, Chem. Commun., 2010, 46, 5832; W. R. Zipfel,R. M. Willams and W. W. Webb, Nat. Biotechnol., 2003,21, 1369, and the references therein.
3 S. Das, A. Nag, D. Goswami and P. K. Bharadwaj, J. Am. Chem.Soc., 2006, 128, 402.
4 G.-L. Law, K.-L. Wong, C. W.-Y. Man, W.-T. Wong, S.-W. Tsao,M. H.-W. Lam and P. K.-S. Lam, J. Am. Chem. Soc., 2008,130, 3714.
5 J. C. G. Bunzli, Chem. Rev., 2010, 110, 2729; P. A. Tanner andC.-K. Duan, Coord. Chem. Rev., 2010, 254, 3026; G.-L. Law,K.-L. Wong, Y.-Y. Yang, Q.-Y. Yi, W.-T. Wong andP. A. Tanner, Inorg. Chem., 2007, 46, 9754.
6 T. Reya, S. J. Morrison, F. Clarke and I. L. Weissman, Nature,2001, 414, 105.
7 T. Lapidot, C. Sirard, J. Vormoor, B. Murdoch and T. Hoang,Nature, 1994, 367, 645.
8 I. Kalaszczynska, Y. Geng, T. Iino, S. Mizuno, Y. Choi,I. Kondratiuk, D. P. Silver, D. J. Wolgemuth, K. Akashi andP. Sicinski, Cell, 2009, 138, 352.
9 Y. Ma, A. Ramezani, R. Lewis, R. G. Hawley andJ. A. Thompson, Stem Cells, 2003, 21, 111.
10 E. Pazos, D. Torrecilla, M. V. Lopez, L. Castedo, J. L. Mascarenas,A. Vidal and M. E. Vazquez, J. Am. Chem. Soc., 2008, 130,9652.
11 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, 1993, 493.
Fig. 3 The europium luminescence enhancement to Cyclin A
(0.1–50 nM, lex = 350 nm) of Eu-1 (left) and Eu-2 (right); (inset)
the plot of the 5D0 -7F2 emission intensity change to the increasing
concentration of Cyclin A with the best fitting binding curve.
Fig. 4 Two-photon (lex = 800 nm) induced in vitro imaging in live
HeLa cells after 3 hours incubation with complex Eu-2 (10 mM).
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