Two-photon excited fluorescent probes for calcium based on internal charge transfer

Article abstract of DOI:10.1039/b905571a

Two-photon excited (TPE) calcium fluorescent probes are designed and synthesized based on internal charge transfer (ICT) with high Ca²⁺ affinity and large two-photon action cross section, which can be used in living cells and detected with two-

Full text of DOI:10.1039/b905571a

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COMMUNICATION
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Two-photon excited fluorescent probes for calcium based on internal
charge transferw
Xiaohu Dong,a Yiyao Yang,a Jian Sun,b Zhihong Liu*ac and Bi-Feng Liu*b
Received (in Cambridge, UK) 19th March 2009, Accepted 13th May 2009
First published as an Advance Article on the web 8th June 2009
DOI: 10.1039/b905571a
Two-photon excited (TPE) calcium fluorescent probes are
designed and synthesized based on internal charge transfer
(ICT) with high Ca2+ affinity and large two-photon action cross
section, which can be used in living cells and detected with
two-photon microscopy (TPM).
high laser power. Most recently, Kim and co-workers reported
a two-photon calcium probe, ACa1, with enlarged TP cross
section, i.e., 110 GM at 780 nm.6 They have subsequently
developed a series of interesting ACa1 analogs, ACa2 and
ACa3.7 Like most fluorescent probes for metal ions, these
calcium indicators are designed on basis of the photoinduced
electron transfer (PET) principle, which presents enhanced
fluorescence upon chelation of metal ions.8–10 An example is
the probe ACa1, containing the calcium chelator (BAPTA)
and the chromophore derived from 2-acetyl-6-(dimethylamino)-
naphthalene which are functionally independent and were
structurally linked with an amide bond so as to enable the
signaling process.
Calcium fluorescent probes have been the focus of biological
and chemical researches for a long time owing to the vital role
of calcium in various cellular processes. Since the Ca2+
chelator BAPTA was prepared by Tsien in 1980,1 numerous
BAPTA derivates have been synthesized and used as calcium
probes. Early calcium probes, such as Indo-1, Quin-2 and
Fura-2, worked under ultraviolet light excitation.2 It has been
commonly recognized that there are some disadvantages when
these molecules are applied in biological samples, i.e., the
UV light-caused damage to living cells/tissue, the shallow
penetration depth, the autofluorescence of samples and photo-
bleaching of fluorophores.3 Although some visible light-
excited probes including Fluo-, Rhod- and Ca Green-series4
are able to partly resolve the above problems, new molecules
with the potential to circumvent such shortcomings are still
desired. Recently, two-photon (TP) excitation has attracted
much attention in biological applications due to its anti-Stokes
photoluminescence nature. TPE molecules can be excited in
the NIR region to give emission in the visible region, therefore
one can reasonably expect lowered photodamage to biological
samples and photobleaching of fluorophores, eliminated
autofluorescence and enhanced penetration depth in micro-
scopic imaging. In TP excitation applications, the TP cross
section is undoubtedly one of the most important and crucial
properties of TPE molecules. Some Ca2+ indicators, for
instance Oregon Green BAPTA-1,5 were utilized in TPM,
however, the TP cross sections were rather poor, which
required impractically high probe concentrations and/or a
Aiming at further developing TPE calcium fluorescent
probes with improved performance, and according to our
previous experiences in preparing TPE molecules as fluorescence
donors,11 we hereby designed and prepared two TPE calcium
probes on basis of the ICT principle with BAPTA as chelator
(namely TP-BAPTA and TP-CN-BAPTA, Scheme 1) in the
present work. The TP chromophores were derived from
1,2-diphenylethene, which is an efficient TPE fluorescent
motif. Two types of scaffold, i.e., D–p–D for TP-BAPTA
and D–p–A–p–D for TP-CN-BAPTA (where A refers to an
electron acceptor and D an electron donator),12 which have
different length of conjugate chain, different electronic effect
and consequently different TP cross section and quantum
yield, were constructed and compared. In the as-designed
molecules, part of the chelating domain (BAPTA) also belongs
to the fluorescence domain, which essentially differs from
PET-based probes. The ICT process occurs because of the
structural integration of the fluorophore and the chelating
domain.13 In such a process, fluorescence is quenched upon
metal ion chelation. One of the remarkable advantages of
fluorescence quenching is that it can overcome the background
signal and afford high sensitivity appropriate for quantitative
a College of Chemistry & Molecular Sciences, Wuhan University,
Wuhan 430072, China. E-mail: zhhliu.whu@163.com;
Fax: 86-27-68754067; Tel: 86-27-87218754
b The Key Laboratory of Biomedical Photonics of MOE-Hubei
Bioinformatics & Molecular Imaging Key Laboratory – Division of
Biomedical Photonics at Wuhan National Laboratory for
Optoelectronics, Department of Systems Biology, College of Life
Science & Technology, Huazhong Univ Sci & Technol,
Wuhan 430074, China. E-mail: bfliu@mail.hust.edu.cn;
Fax: 86-27-87792170; Tel: 86-27-87792203
c Key Laboratory of Analytical Chemistry for Biology and Medicine
(Wuhan University), Ministry of Education, China
w Electronic supplementary information (ESI) available: Synthesis
procedures, determination of quantum yield, TPA cross section and
apparent dissociation constant, cell culture and TPM imaging, cyto-
toxicity measurements, results of the interference of other metal ions
and pH change. See DOI: 10.1039/b905571a
Scheme 1 The structures of TP-BAPTA and TP-CN-BAPTA.
Chem. Commun., 2009, 3883–3885 | 3883
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analysis, and that is why quenching-based chemosensors have
been extensively developed for analytical and biochemical
applications.14 With such molecular design, both the TP cross
section and the calcium affinity of the probe are significantly
increased, as compared to existing Ca2+ probes.
Both TP-BAPTA and TP-CN-BAPTA showed two-photon
induced fluorescence under 800 nm excitation (Fig. 1), which
could be easily confirmed with the fact that neither of the two
molecules has linear absorption around 800 nm. TP-BAPTA
has a maximum absorption at 370 nm and a maximum
emission at 440 nm. As a result of the enlarged conjugation
plane and facilitated flow of the electronic cloud, the maximal
absorption and fluorescence emission of TP-CN-BAPTA were
red-shifted to 434 and 564 nm, respectively, relative to
TP-BAPTA. The two-photon fluorescence of the molecules
matched exactly with their one-photon fluorescence, which
suggested that the excitation mode did not affect the excited
state of the molecules.
The Ca2+ ion-binding ability of the as-prepared probes
were tested in MOPS buffer (30 mM, pH = 7.2). With
TP-BAPTA as an example, the binding of metal ions resulted
in a decrease of fluorescence intensity (Fig. 2(a)), which is
explained by the fact that the aromatic amine lone pairs of
BAPTA are involved in both the chelation and fluorescence
emission. After binding to Ca2+ ions, the charge of the
aromatic amine electron-donator was transferred to the
cation, thus the electron donating ability of the nitrogen atom
was weakened, which resulted in the decrease of the electron
density on the conjugate plane and caused fluorescence
quenching. As is mentioned above, quenching-based assays
can always afford high sensitivity in quantitative analysis. The
Fig. 2 (a) Fluorescence titration of 1 mM TP-BAPTA (100 mM KCl,
30 mM MOPS, 10 mM EGTA, pH = 7.2) with free Ca2+ from
0 to 2.84 mM. (b) Hill plot for the complexation of TP-BAPTA with
free Ca2+ (0–2.84 mM). (c) fluorescence titration of 3 Â 10À5
M
TP-CN-BAPTA (100 mM KCl, 30 mM MOPS, 10 mM EGTA,
pH = 7.2) with free Ca2+ from 0 to 2.8 mM. (d) Hill plot for the
complexation of TP-CN-BAPTA with free Ca2+ (0–2.8 mM).
apparent dissociation constant (Kd) of TP-BAPTA determined
with fluorescence titration confirmed this hypothesis
(Fig. 2(b)). The Hill plot gave a straight line where the slope
indicated the combining ratio between TP-BAPTA and Ca2+
,
and the x-axis intercept represented the dissociation constant.
The result showed a 1 : 1 complex and a Kd value of 51 nM.
Compared to the Kd values of other calcium probes such as
Fura-2 (145 nM), Fluo-3 (390 nM), Ca Green-1 (190 nM) and
ACa1 (270 nM), the Ca2+ affinity of TP-BAPTA was notably
improved. It should be noted, however, that high Ca2+ affinity
is not always an advantage. Although it could be promising in
terms of quantitative determination of Ca2+ concentration, it
may make the probe unsuitable for measuring the calcium
wave in living cells because of irreversible binding (vide infra).
The interference of other metal ions was investigated
through measuring the fluorescence quenching of TP-BAPTA
with the co-existence of Ca2+ and various ions (Fig. S1, ESIw).
Mg2+, Zn2+, Mn2+ and Cd2+ led to little interference,
but Co2+ and Ni2+ caused a rather serious fluorescence
decrease. The influence of pH change was also tested. In the
biologically relevant pH range, TP-BAPTA was pH-insensitive
(Fig. S2, ESIw), which could be favorable in biological
applications. Generally, the TP action cross section (F Â d),
which is the product of the TP-cross section (d) and quantum
yield (F), is regarded as the most important characteristic of
potential TPE fluorophores. TP-BAPTA had a satisfying
quantum yield in both DMF (F = 0.35) and MOPS buffer
(F = 0.27, for determination details see ESIw). However the
TP cross section was rather poor, i.e., d = 36 GM under 800 nm
excitation (1 GM = 1 Â 10À50 cm4 s photonÀ1, ESIw).
Considering to improve the TP action cross section,
TP-CN-BAPTA was designed and synthesized. In addition
Fig. 1 (a) Fluorescence emission of TP-BAPTA under one-photon
excitation (dotted line, excited at 370 nm) and two-photon excitation
(solid line, excited at 800 nm). (b) fluorescence emission of
TP-CN-BAPTA under one-photon excitation (dotted line, excited
at 434 nm) and two-photon excitation (solid line, excited at
800 nm). Spectra were recorded in DMF, and the fluorescence
intensities are given in normalized form.
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detected, although varying levels of ATP stimulation were
tried. Nonetheless, the overall results still strongly suggested
the feasibility of designing ICT-based TPE probes for
calcium, which could be applicable in biological imaging or
determination.
In summary, we prepared two TPE calcium probes on the
basis of ICT, with D–p–D and D–p–A–p–D scaffolds,
respectively. Large TP cross section and high Ca2+ affinity
were obtained, and the TP action cross section was tunable
through adjusting the structure of the molecules. The probe
could transfer across the cell membrane, hydrolyze in the
cytoplasm, and combine with free cytoplasmic calcium. The
very high Ca2+ affinity together with the TP excitation nature
make such probes suitable in quantitative determination of
Ca2+ in biological samples. However, when the calcium wave
in living cells/tissue is to be measured, further modification to
the molecular structure would be needed so as to achieve
moderated Ca2+ affinity.
Financial support from the National Natural Science
Foundation of China (No. 20675059) and the Science
Fund for Creative Research Groups (No. 20621502) is
acknowledged.
Fig. 3 (a) Bright field and (b) fluorescent images of HeLa cells loaded
with 5 mM TP-CN-BAPTA-ESTER under two-photon microscopy,
(c) quenching of intracellular fluorescence after stimulating the
probe-loaded cells with 20 mM ATP. Excitation wavelength = 800 nm.
Notes and references
to a larger conjugated plane, cyano groups were added to the
conjugated system to make a quadrupole molecule with a
D–p–A–p–D scaffold. As expected, the TP cross section of
TP-CN-BAPTA was dramatically increased to 917 GM.
Compared to TP-BAPTA, although the introduction of cyano
groups reduced the probe’s quantum yield to 0.12 (in DMF)
(still acceptable in fluorescence assays), the TP action cross
section was significantly enhanced. The Kd value of
TP-CN-BAPTA was calculated as 39 nM (Fig. 2(c) and (d)).
The interference of other metal ions on this probe was
also studied, which showed similar results to TP-BAPTA
(Fig. S3, ESIw).
The cytotoxicity of both TP-BAPTA and TP-CN-BAPTA
was investigated by MTT assay, which revealed that the
probes were nontoxic to HeLa cells at a concentration as high
as 24 mM (Fig. S4, ESIw). TP-CN-BAPTA was then chosen for
intracellular experiments owing to its satisfactory TP action
cross section. After incubating HeLa cells with TP-CN-BAPTA
tetraethyl ester (TP-CN-BAPTA-ESTER) and exhaustive
washing, uniform and bright green fluorescence was clearly
seen with TP fluorescence microscopy in living cells
(Fig. 3), which showed that TP-CN-BAPTA-ESTER was
membrane-permeable. After stimulating HeLa cells with
ATP to release free Ca2+, the fluorescence of the probe was
completely quenched (Fig. 3(c)), which demonstrated that
TP-CN-BAPTA-ESTER was successfully hydrolyzed by
intracellular esterase and could combine with free cytoplasmic
calcium. As mentioned above, the binding, unfortunately, was
nearly irreversible due to the inappropriately high Ca2+
affinity. As a consequence, the calcium wave could not be
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