Design of a Hypersensitive pH-Sensory System Created by a Combination of Charge Neutralization and Aggregation-Induced Emission (AIE)

Article abstract of DOI:10.1002/chem.201703560

In our bodies, a slight pH change causes remarkable activation or serious damage in the biological processes and continuously keeps biological homeostasis. Detection of such a slight pH change has been a constant demand in searching for unusual biological

Full text of DOI:10.1002/chem.201703560

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Accepted Article
Title: Design of a Hypersensitive pH Sensory System Created by a
Combination of Charge Neutralization and Aggregation-Induced
Emission (AIE)
Authors: Daisuke Yoshihara, Takao Noguchi, Bappaditya Roy, Junji
Sakamoto, Tatsuhiro Yamamoto, and Seiji Shinkai
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To be cited as: Chem. Eur. J. 10.1002/chem.201703560
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Design of a Hypersensitive pH Sensory System Created by a
Combination of Charge Neutralization and Aggregation-Induced
Emission (AIE)
Daisuke Yoshihara[a], Takao Noguchi[b], Bappaditya Roy[b], Junji Sakamoto[a], Tatsuhiro Yamamoto[a],
and Seiji Shinkai*[a, b]
Abstract: In our bodies, a slight pH change causes remarkable
activation or serious damage in the biological processes and
continuously keeps biological homeostasis. Detection of such a
slight pH change has been a constant demand in searching unusual
biological events. In this paper, we demonstrate a novel pH sensory
system that has been achieved through a combination of charge
neutralization by a slight pH change with aggregation-induced
emission (AIE). We selected a cyano-functionalized oligo(phenylene-
vinylene) (cyanoOPV) backbone for AIE and introduced ammonium-
tethered boronic acid groups as a pH-dependent function. The self-
assembling of these dyes (OPV-Cn) was readily achieved by pH-
dependent charge neutralization at the neutral pH region. This
sensory system showed unusually sensitive pH responsiveness in a
narrow pH range. Moreover, this pH change was observed in a
biologically important neutral pH region. We therefore believe that
this system is broadly applicable to detect the slight pH change
occurring in the biological events.
change) by the supramolecular stacking,[6a] but not by the
conventional structural change (i.e., configurational change).[7]
We reported a few novel fluorescence sensory systems by the
marriage of molecular recognition and AIE.[8] For example,
guanidinium-tethered cyano-functionalized oligo(phenylene-
vinylene) (cyanoOPV) recognized the structural difference
among anionic polysaccharides (heparin, chondroitin sulfate,
and hyaluronic acid) and resulted in the aggregate formation
with different fluorescence intensity.[8a] In addition, we explored a
D-glucose sensing system by the combination of AIE and
dynamic covalent bond formation between boronic acid and D-
glucose, resulting in ratiometric fluorescence sensing of D-
glucose.[8b] We considered that a very sensitive pH sensory
system might be explored by utilizing this AIE-based molecular
recognition.
There are a few reports about the relationship between a pH
change and an AIE phenomenon.[9] Apparently, some of them
showed a sharp fluorescence change in a narrow pH region,[9a]
and those results were obtained mostly in the polymeric system
where the cooperative actions were operative.[9b] Moreover, the
pH range in action is only in acidic or basic regions. Taking the
application to biological systems (e.g., to the healthcare
research field) into consideration, the sharp fluorescence
change occurring at neutral pH region has been a constant need
of demand.[10]
As described above, we already reported a sugar sensing
system through the combination of AIE and boronic acid.[8b] This
system worked well in the neutral pH region. Boronic acid is
well-known as a functional group that changes the charge
between neutral and anionic states by a pH change (Figure
1).[11] One can design the boronic acid group so that it may
change the charge state at the neutral pH region. This change in
the charge state should affect the aggregation properties of a
boronic acid-tethered AIE dye molecule. As the aggregation of
the AIE dye tends to occur in a cooperative fashion, this
cascade change, pH – charge state – aggregation – AIE should
lead to a novel pH-sensitive sensing system around neutral pH
region.
The pH value plays decisive roles in many biological events
and maintains the biological homeostasis at the moderate
conditions. As a matter of cause, therefore, it is highly important
to monitor a slight pH change occurring in our bodies.[1] In order
to attain this purpose, we usually use a pH meter or a chemical
pH indicator.[2] Recently, a fluorescence-based pH indicator has
been paid more attention because of its easy recognition by
naked eyes. In fact, various fluorescence-based pH indicators
have been explored so far.[3] In addition, the fluorescence-based
sensory system features the very high sensitivity,[4] so that one
may consider that this system is more advantageous in many
cases than the UV-vis absorption-based pH sensory system.
In general, the principal of the traditional unimolecular pH
sensor is based on dissociation or neutralization of a functional
group integrated in the concerned dye molecule. Hence, its color
change is basically same as the pH titration curve, which can be
analyzed by
a
Henderson–Hasselbalch equation.[5] This
manifests the limitation of this method: that is, the unimolecular
pH sensory system is not suitable for sensitive detection of a
slight pH change. To monitor the slight pH change, one must
explore some new pH sensory systems on the basis of the
mechanism different from the traditional unimolecular pH
sensory mechanism.
Aggregation-induced emission (AIE) has recently been raised
as an active target of research.[6] The phenomenon is caused by
the suppression of the molecular motion (i.e., conformational
[a]
Dr. D. Yoshihara, Dr. J. Sakamoto, Dr. T. Yamamoto, Prof. S. Shinkai
Nanotechnology Laboratory, Institute of Systems Information
Technologies and Nanotechnologies (ISIT) 4-1 Kyudaishinmachi, Nishi-
ku, Fukuoka 819-0388 (Japan)
Figure 1. Molecular structure of OPV-Cn (n = 3 or 4) and neutral and anionic
states of the boronic acid group.
E-mail: shinkai_center@mail.cstm.kyushu-u.ac.jp
Dr. T. Noguchi, Dr. A. Dawn, Prof. S. Shinkai
[b]
Institute for Advanced Study, Kyushu University 744 Moto-oka, Nishi-ku,
Fukuoka 819-0395 (Japan)
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In this paper, we propose a novel approach toward a
fluorescence-based pH sensing system around neutral pH
region through a combination of charge neutralization and AIE.
We have found that a very sharp fluorescence response
emerges from charge pH-dependent neutralization followed by
self-assembling of AIE molecules. Once the self-assembling
action starts, it proceeds cooperatively. This auto-accelerative
behavior leads to the sharp increase in the fluorescence
intensity. Hence, one can expect that this system is useful to
sense a very slight pH change, compared with that obtained
from the conventional unimolecular pH sensors.
As a reference experiment, we measured the pH dependence of
2-naphthaleneboronic acid which should show the unimolecular
pH responsive behavior (Figure 2, triangle). The fluorescence
intensity for 2-naphthaleneboronic acid was changed by the
addition of OH- to boronic acid, leading to the emergence and
the inhibition of intramolecular charge transfer through pH
change.[13] As shown in Figure 2, the fluorescence intensity
decreases as the pH increases. Basically, this curvature is
equivalent to the unimolecular titration curve.
To estimate the sensitivity against the pH change
quantitatively, we tried to calculate apparent pKa values and n
We selected a cyanoOPV backbone for AIE[12] and introduced
ammonium-tethered boronic acid groups as a pH-dependent
function. A sharp fluorescence change was observed around pH
6-7, indicating that a pH change can be amplified through the
molecular assembling. Moreover, the pH responsive range was
easily adjustable by the spacer length between the cyanoOPV
and the ammonium group. We also investigated the correlation
between the medium pH and the resultant morphology. The
results revealed that an interesting relation exists among
medium pH, fluorescence, and morphology. To the best of our
values from a linear relationship in modified Henderson-
Hasselbalch equation [Eq. (1)],[14] where α is the degree of
dissociation.
⁄
푝퐻 = 푝퐾푎 − 푛푙표푔 {(1 − 훼) 훼}
(1)
This equation is frequently used to estimate apparent pKa value
of pH responsive polymer systems instead of unimolecular pKa
value.[14] The feature of this equation is that “n” is appended to
the usual Henderson-Hasselbalch equation. When this n value is
1, the molecule shows the unimolecular pH-dependent
dissociation behavior (normal Henderson-Hasselbalch equation)
(see n=1 line in Figure 3). When the n value is larger than 1, the
molecule shows lower pH responsiveness than the unimolecular
behavior. In general, most pH responsive polymer systems have
the n values larger than 1.[14a] In contrast, when the n value is
smaller than 1, the molecule shows higher pH responsiveness
than the unimolecular behavior (see n<1 line in Figure 3).[14b] We
evaluated apparent pKa values and n values of three compounds
from the fluorescence spectra. As summarized in Table 1, the
apparent pKa values for OPV-C3, OPV-C4, and 2-
naphthaleneboronic acid were 6.92±0.12, 6.22±0.04, and
8.41±0.21, respectively. These results clearly indicate that being
different from conventional boronic acids, those in OPV-C3 and
OPV-C4 can work around neutral pH region. We also evaluated
n values from Equation 1, the n values for OPV-C3, OPV-C4,
knowledge, this is the first report on
a hypersensitive
fluorescence pH sensory system around neutral pH region.
We prepared two cyanoOPV derivatives (OPV-C3, OPV-C4)
bearing ammonium-tethered phenylboronic acid groups (Figure
1). In order to examine their pH dependence, the fluorescence
spectra were measured at various pH conditions (Figure 2).
These two compounds showed a very sharp fluorescence
change around pH 6-7. The results clearly support the view that
our system working at neutral pH region is suitable for the
biological purposes. The fluorescence increased as the increase
in the medium pH, indicating that the charge state is changing
from more soluble cationic species to less soluble zwitterionic
species. Interestingly, the pH responsive range was different
between OPV-C3 and OPV-C4. This finding implies that one can
adjust the pH responsive range by the spacer length. As the
fluorescence intensity in the present system is associated with
the aggregation properties, one may consider that the change in
the pH responsive range is tuned by the hydrophobic effect
and/or the odd-even number effect of the alkyl chain.
Figure 2. Plots of the normalized fluorescence intensity vs. pH in MeOH-
aquaous MES buffer solution (0.10 M, 1:3 v/v) at 25 °C with 365 nm (OPV-Cn)
and 278 nm (2-naphthaleneboronic acid) excitation. Circle; I465/Imax in OPV-C3,
square; I465/Imax in OPV-C4, triangle; I345/Imax 2-naphthaleneboronic acid. The
error bars indicate 95 % confidence interval.
Figure 3. Schematic diagram for comparing the difference by the n value in
Modified Henderson-Hasselbalch equation.
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Table 1. Comparison of the parameters calculated by modified Henderson-
Hasselbalch equation.
our system this transition occurs cooperatively, which leads to a
hypersensitive pH sensory system.
and 2-naphthaleneboronic acid being 0.58±0.10, 0.50±0.04, and
1.04±0.22, respectively. The n value for 2-naphthaleneboronic
acid was about 1. In contrast, the n values for OPV-C3 and
OPV-C4 were considerably smaller than 1, indicating that the
pH-dependence of these two compounds is much sharper than
that of 2-naphthaleneboronic acid.
We also checked the reversibility of the pH-dependent
fluorescence change in OPV-C3 and OPV-C4. These two
compounds clearly displayed a reversible fluorescence change
between pH 5.3 and pH 7.3 (Figure 4).
Figure 5. TEM images of OPV-C4 at (a) pH 5.2, (b) pH 5.9, and (c), (d) pH 7.3.
Concentration: [OPV-C4] = 10 μM. All images were taken after staining with
4.0 wt% phosphotungstic acid. Black lines are scale bars.
In summary, we have succeeded in designing a novel highly
sensitive pH sensory system with the sensible combination of an
AIE-core with the concept of charge neutralization between
ammonium and pH-responsive boronic acid. The design concept
utilized herein is totally different from that utilized in the
traditional unimolecular sensors. This sensory system showed
the hypersensitive pH response useful around neutral pH region.
The relative sensitivity can be compared quantitatively by
applying
modified
Henderson-Hasselbalch
equation.
Furthermore, the responsive pH region can be easily adjusted
by changing the spacer length. We believe, therefore, that the
present system has a large potential to detect biological events
that may accompany a slight pH change.
Figure 4. Recycle tests of the pH-responsive OPV-Cn in MeOH-aquaous MES
buffer solution (0.10 M, 1:3 v/v) at 25 °C with 365 nm (OPV-Cn) excitation.
The pH value of the solution was varied reversibly between 5.3 and 7.3.
The morphological change was evaluated through observation
with a transmission electron microscope (TEM). The size of the
aggregates was increased with the increase in pH (Figure 5).
The result is highly compatible with the pH-dependent
fluorescence change. The trend supports the view that the
fluorescence increase observed with the pH increase is
originated from the aggregate formation.
From the foregoing results, we can propose a consistent
relationship among fluorescence, pH, and aggregate size
(Figure 6). In a pH region lower than pKa, OPV-C3 and OPV-C4
behave as cationic species. Their higher solubility does not allow
them to form aggregates and the solutions are nonfluorescent.
As the pH increases, boronic acids are changed to anionic state,
and the molecules form less soluble zwitterionic species by the
intermolecular charge neutralization. This triggers the aggregate
formation. In a pH region higher than pKa, the aggregates are
fully formed and the solutions become strongly fluorescent. In
Figure 6. Schematic illustration of proposed our pH responsive fluorescence
sensory system.
Acknowledgements
We thank analytical center at Fukuoka Industry-Academia
Symphonicity for providing the help to use NMR and TEM.
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Keywords: aggregation-induced emission • self-assembly •
boronic acid • pH sensor • charge neutralization
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
A novel pH sensory system can be
designed through a unique
combination of charge neutralization
by a slight pH change with
Daisuke Yoshihara, Takao Noguchi,
Bappaditya Roy, Junji Sakamoto,
Tatsuhiro Yamamoto, and Seiji
Shinkai*
aggregation-induced emission (AIE).
This sensory system showed
unusually sensitive pH
responsiveness in a narrow pH
range. Moreover, this pH change
was observed in a biologically
important neutral pH region.
Page No. – Page No.
Design of a Hypersensitive pH
Sensory System Created by a
Combination of Charge
Neutralization and Aggregation-
Induced Emission (AIE)
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