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: klwong@hkbu.edu.hk;Tel: +852-34112370

bDepartment of Chemistry, Durham University, Durham,UK DH1 3LE. E-mail: s.l.cobb@durham.ac.uk;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: lizc@szpku.edu.cn; Tel: +86 2603 2701

d School of Biomedical Sciences, The Chinese University ofHong Kong, Shatin, New Territories, Hong Kong SAR.E-mail: tcklau@cuhk.edu.hk

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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