Nonlinear fluorescence response driven by ATP-induced self-assembly of guanidinium-tethered tetraphenylethene

Article abstract of DOI:10.1039/c2cc33262k

Nonlinear fluorescence response, which is particularly important to attain the high signal-to-background ratio, was realized by the aggregation-induced fluorescence increase of guanidinium-tethered tetraphenylethene with ATP.

Full text of DOI:10.1039/c2cc33262k

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Nonlinear fluorescence response driven by ATP-induced self-assembly of
guanidinium-tethered tetraphenylethenew
Takao Noguchi,a Tomohiro Shiraki,b Arnab Dawn,a Youichi Tsuchiya,b Le Thi Ngoc Lien,c
Tatsuhiro Yamamotob and Seiji Shinkai*abd
Received 7th May 2012, Accepted 25th June 2012
DOI: 10.1039/c2cc33262k
Nonlinear fluorescence response, which is particularly important to
attain the high signal-to-background ratio, was realized by the
aggregation-induced fluorescence increase of guanidinium-tethered
tetraphenylethene with ATP.
be realized by a modular-type structure composed of an emissive
moiety, a spacer and a recognition site, and the aggregation is
triggered by the target-specific association. Most importantly, the
AIE molecules are endowed to show the ‘‘turn-on’’ switching in
response to self-assembly with a target molecule. Despite their
remarkable advantages, a limited number of examples have so
far been reported to apply the AIE molecules as sensors for
DNA,7 protein8 and heparin9 and as a chiral recognition host.10
It is clear that the wide challenging field still remains toward
development of a new class of AIE-based bioprobes.
The development of fluorescent sensing systems that recognize
biologically important metals, molecules and polymers has
currently been an active research area because the fluorescence
technique is highly simple and sensitive, facilitating their
potential applications as bioprobes, and in imaging and
monitoring.1 As fluorophores, small organic dyes have been
mainly used, in which recognition sites specific to concerned
targets are integrated. The organic dyes are allowed therein to
optimize their wavelength window, brightness and photo-
stability by appropriate chemical modifications.2 However,
most of the organic dyes are intrinsically emissive and
quenched at the higher concentration due to their aggregation
(self-quenching).3 On the basis of this basic principle, various
fluorescent probes have been designed.4 However, this approach
(i.e. turn-off switching) inevitably causes low response as well as
low signal-to-background contrast in the fluorescence detection
because simultaneous satisfaction of the efficient emission and the
efficient quenching is rather difficult. As an alternate approach, a
few examples have demonstrated target-specific molecular
probes, in which the recognition-induced disassembling of
the probes switches them ‘‘turn-on’’.5 In contrast, fluoro-
phores exhibiting aggregation-induced emission (AIE) have
been increasingly attracting much attention because AIE
molecules behave in an opposite manner to intrinsic self-
quenching dye molecules.6 The design of AIE molecules can
In order to elicit full potential of AIE properties, we have
designed a novel fluorescent molecule, that is, the marriage of
AIE with an allosteric function attained by a self-assembly
system. This strategy was inspired from our previous work on
the highly selective molecular recognition attainable via the
allosteric control.11 Since the allosteric control realizes nonlinear,
sigmoidal information transduction, a small change in the input
signal can be amplified into a large change in the output signal.12
If such a system is accomplished in the fluorescent sensing, a
steep turn-on fluorescent transition will be realized in a nonlinear
fashion, leading to a significant enhancement of the signal-to-
background contrast. In this context, the present system is
intrinsically different from the linearly transmitted examples
generally observed for conventional fluorescent sensing systems.
We have thus focused on tetraphenylethene known as an AIE
molecule, whose emission mechanism is rationalized in terms of
the restriction of C–C bond rotation.13 Fig. 1 shows our probe
design for target ATP, one of the most significant biomolecules
involving a universal energy source as well as an extracellular
signalling mediator.4 The designed probe, TPE, has the following
three unique features: (1) multiple recognition sites to be required
a Institute for Advanced Study, Kyushu University, 744 Moto-oka,
Nishi-ku, Fukuoka 819-0395, Japan.
E-mail: shinkai_center@mail.cstm.kyushu-u.ac.jp;
Fax: +81-92-805-3814; Tel: +81-92-802-6990
b Nanotechnology Laboratory, Institute of Systems, Information
Technologies and Nanotechnologies (ISIT), 203-1 Moto-oka,
Nishi-ku, Fukuoka 819-0385, Japan
c Department of Materials Science and Technology,
Graduate School of Engineering, Kyushu University, Japan
d Department of Nanoscience, Faculty of Engineering, Sojo University,
4-22-1 Ikeda, Kumamoto 860-0082, Japan
w Electronic supplementary information (ESI) available: Details of
synthesis, spectroscopic analysis and fluorescent titration. See DOI:
10.1039/c2cc33262k
Fig. 1 Chemical structure of guanidinium-tethered tetraphenylethene
(TPE) for ATP detection.
c
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for a nonlinear response under the allosteric control, (2) guani-
dinium groups14 as a recognition site which is known to bind
with phosphate anions in a 1 : 1 molar ratio via the specific
hydrogen-bond-assisted electrostatic interaction in water and (3)
short spacers consisting of the propyl group between TPE and
guanidinium to avoid the background fluorescence arising from
micelle formation of TPE itself in water. We have confirmed that
our TPE probe realizes selective fluorescent detection of ATP
through the cooperative self-assembly that is characterized by the
sensitive, nonlinear fluorescence response together with the high
signal-to-background ratio.
Fig. 3 (a) pH-dependent fluorescence spectra (lex = 335 nm) of TPE
(6.0 mM) in the presence of ATP (8.0 mM) in HEPES (5.0 mM). (b) Plot
of the fluorescence intensity (463 nm) versus the medium pH value.
The core skeleton of TPE was synthesized by McMurry
coupling,15 which was decorated by introduction of the guanidine
groups.16 The TPE structure was confirmed by ESI-MS and
elemental analysis. We first examined the fluorescence response
of TPE upon addition of nucleotides (AMP, ADP, and ATP)
keeping the guanidinium/phosphate ratio at 1 : 1 (Fig. 2a). No
significant fluorescence enhancement was observed upon addition
of AMP and ADP ([TPE] = 6.0 mM, [AMP] = 24 mM, [ADP] =
12 mM). In contrast, a strong blue emission was observed at
463 nm only when TPE was mixed with ATP ([ATP] = 8.0 mM).
The emission intensity was increased by 60 fold compared with
that in the absence of ATP, and the bright emission was detectable
even by our naked eyes (Fig. 2b). In the excitation spectrum, the
maximum appeared at 335 nm (Fig. S1a, ESIw), which is in good
agreement with the absorption maximum of the TPE–ATP
mixture (Fig. S1b, ESIw), indicating that the blue emission is
originated from TPE. This result indicates that TPE can act as an
ATP-selective turn-on fluorescent probe. The most important
point of this finding is that the three sequential phosphate groups
as in ATP are essential for the fluorescence enhancement. This
suggests that the cooperative self-assembly takes place in the
aggregation process of TPE and ATP.
with the cationic guanidinium group in an electrostatic comple-
mentary fashion. The fluorescence enhancement is observed
only at the pH region where the phosphate group of ATP is
deprotonated, indicating that the electrostatic interaction is the
primary force for the association. The UV-Vis spectra showed
that the addition of ATP to TPE (final concentration: [TPE] =
6.0 mM and [ATP] = 8.0 mM) shifted the absorption maximum
from 310 to 335 nm and significantly broadened the peak
(Fig. S1b, ESIw). This change is indicative of the aggregate
formation. This fact was further confirmed by the measurements
of dynamic light scattering (DLS) and scanning electron micro-
scopy (SEM). In DLS measurements, the aqueous dispersion
showed the presence of aggregates with a mean diameter of
674 Æ 252 nm (Fig. 4a). The SEM measurements confirmed the
formation of cohesive aggregates with a bulk size of ca. 1 mm
(Fig. 4b), which is comparable with that obtained from the DLS
measurements. The SEM energy dispersive X-ray (EDX) micro-
analysis showed the presence of phosphine atoms, indicative of
the formation of a TPE–ATP complex (Fig. S2, ESIw). To
visualize the origin of the fluorescence emission, the aqueous
dispersion was subjected to fluorescence microscopy. As shown
in Fig. 4c, fluorescent aggregates with a mean size of ca. 1 mm
were directly observed, and their image was well overlapped with
that obtained from differential interference contrast microscopy
(Fig. 4d). These results consistently support the view that
the fluorescence enhancement is induced by ion-pairing-based
The relationship between the self-assembly properties and
fluorescence emission was further investigated under the various
measurement conditions. Fig. 3 shows the effect of pH on the
fluorescence intensity of the TPE/ATP complex. The fluorescence
intensity at 463 nm decreased gradually with lowering of the pH
value of aqueous media, and showed a sharp decrease at pH 6.5.
This pH value corresponds to the pKa2 of a phosphate group
(pH B 6.8). Below pKa2, the triphosphate group of ATP mainly
exists as its partially protonated species, which is unable to interact
Fig. 2 (a) Fluorescence spectra (lex = 335 nm) of TPE (6.0 mM) in
HEPES buffer (5.0 mM, pH 7.4) in the presence of AMP (24 mM), ADP
(12 mM) and ATP (8.0 mM). (b) Photograph of TPE (6.0 mM) in the
presence of the corresponding nucleotides in HEPES buffer (5.0 mM,
pH 7.4); left: AMP (24 mM), middle: ADP (12 mM) and right: ATP
(8.0 mM). The image was obtained under UV irradiation (lex = 365 nm).
Fig. 4 (a) Size distribution of the aggregates measured by DLS.
(b) SEM image of the aggregates prepared by mixing TPE and ATP.
(c) Fluorescence and (d) differential interference contrast image of the
solution obtained by the addition of ATP to TPE. Arrows indicate the
fluorescent aggregates. Conditions: [TPE] = 6.0 mM, [ATP] = 8.0 mM,
[HEPES] = 5.0 mM (pH 7.4), 25 1C.
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impacts on the molecular self-assembly. Here, the subtle balance
(the difference of nucleobase structures) was embodied with the
fluorescence responses. The recognition of triphosphate along
with the discrimination between nucleobase structures could be a
striking feature of AIE-based bioprobes that utilize the molecular
self-assembly.
In conclusion, we have demonstrated that the AIE-based
guanidinium bioprobe shows the nonlinear fluorescence response
accompanying self-assembly of TPE with the aid of ATP. The
modular design of the AIE-based probe will open wide possibilities
for the detection of other biomolecules and polymers. We now
envisage that this system would be applicable to sensitive detection
of DNA sequences, as expected from the favourable binding of
purine-rings over pyrimidine-rings. Also, applications to other
biologically essential molecules bearing carboxylate and sulfate
anionic groups would be possible. We consider that these future
potentials will be realized by the synergistic marriage of molecular
self-assembly with AIE-based bioprobes.
Fig. 5 (a) Fluorescence titration curve (lex = 335 nm) of TPE (6.0 mM)
upon the addition of AMP (blue), ADP (green) and ATP (red) in HEPES
buffer (5.0 mM, pH 7.4) at 25 1C. (b) Magnified figure at the lower ATP
concentration region (0–5 mM). DF/F0 means the fluorescence intensity
change (DF = F À F0) and F0 is the fluorescence intensity of TPE in the
absence of nucleotides.
complexation of TPE and ATP followed by the formation of
the larger aggregates (Fig. S3, ESIw).
We are grateful to Dojindo Molecular Technologies, Inc. for
helpful discussions. This work was financially supported by the
Ministry of Education, Culture, Sports, Science and Technology
of Japan, Grant-in-Aid for Scientific Research on Innovative
Areas ‘‘Emergence in Chemistry’’ (20111011).
Next, we performed the fluorescence titration of TPE by AMP,
ADP and ATP. The addition of AMP or ADP exhibited no
significant fluorescence increase. On the other hand, the fluores-
cence intensity was increased by the incremental addition of ATP
(Fig. 5 and Fig. S4, ESIw). A change in the fluorescence intensity
was saturated at around 16 mM. Most importantly, the plot
of fluorescence intensity against ATP concentration showed a
sigmoidal curve, which means that the ATP recognition monitored
by fluorescence proceeds according to the nonlinear relationship.
Such a nonlinear response has been reported for the conversion of
molecular information,17 and utilized to improve the chiral
recognition efficiency: that is, the high enantioselectivity can
be attained through the combination with the allosteric effect
even though the enantioselectivity obtained in the conventional
1 : 1 stoichiometric system is low.11 In the present fluorescence
system, the nonlinear response realized by a steep turn-on
switching leads to the high signal-to-background ratio, which
is the most critical point in a sensing system.
Notes and references
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As mentioned above, the effect of ATP on the fluorescence
increase was the most conspicuous. It is undoubted that the
electrostatic interaction between guanidinium and triphosphate
groups plays the primary role in the aggregation leading to the
fluorescence enhancement (Fig. S5, ESIw). Then, does the adenine
moiety play any role to facilitate the aggregation? To answer this
question, the effects of triphosphate and nucleobase structures on
the fluorescence enhancement were investigated and compared
with that of ATP. Triphosphate gave the fluorescence intensity
comparable with that of ATP at the high concentration (60 mM),
whereas at the low concentration (0–16 mM) the fluorescence
intensities were generally lowered and the sigmoidal dependence
was rather indistinct (Fig. S5, ESIw). This difference is in contrast to
the effect of ATP which afforded a steep transition of the fluores-
cence response even at the low concentration region. This result
indicates that the collective p–p stacking in and the hydrophobic
interaction with the adenine moiety facilitate cooperative self-
assembly to produce the nonlinearly steep fluorescence response.
More remarkably, the difference of nucleobase structures was
manifested by the fluorescence response (Fig. S6 in ESIw, GTP
Z ATP 4 CTP 4 UTP). It is generally known that a subtle
balance of noncovalent intermolecular interactions predominantly
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