Rapid detection of hydrogen peroxide based on aggregation induced ratiometric fluorescence change

Article abstract of DOI:10.1021/ol4000845

In surfactant solution, probe D-BBO can detect H₂O₂ with an enhanced reaction rate (k_obs = 1.83 × 10^-2 s^-1) and a large bathochromic shift of 105 nm. Furthermore, D-BBO displays a highly selective response to H₂O₂ over other reactive oxygen species under identical conditions.

Full text of DOI:10.1021/ol4000845

ORGANIC
LETTERS
2013
Vol. 15, No. 4
924–927
Rapid Detection of Hydrogen Peroxide
Based on Aggregation Induced
Ratiometric Fluorescence Change
Guoping Li,†,‡ Dongjian Zhu,†,‡ Qing Liu,†,‡ Lin Xue,*,† and Hua Jiang*,†
Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of
Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190
P. R. China, and University of Chinese Academy of Sciences, Beijing, P. R. China
hjiang@iccas.ac.cn; zinclin@iccas.ac.cn
Received January 11, 2013
ABSTRACT
In surfactant solution, probe D-BBO can detect H2O2 with an enhanced reaction rate (kobs = 1.83 ꢀ 10ꢁ2 sꢁ1) and a large bathochromic shift of
105 nm. Furthermore, D-BBO displays a highly selective response to H2O2 over other reactive oxygen species under identical conditions.
Fluorescent sensing is a very important research topic of
supramolecular chemistry and provides an efficient meth-
od for analyte reporting.1 Hydrogen peroxide (H2O2) is a
common chemical and widely used for bleaching, cleaning,
and disinfection.2 It is also an important biological mole-
cule connected to many physiological processes, such as
oxidative damage, defense response, and cellular signal
transduction.3 Owing to the development of molecular
probes in the past few years, fluorescent visualizing and
monitoring of H2O2 has been realized with high sensitivity
and selectivity.4 Up to now, most of these probes are
designed according to a chemical-reaction-based ap-
proach, which takes advantage of a specific chemical
group, such as benzenesulfonyl ester, benzil, or boronate
ester to react with H2O2.4,5 However, these reactions
mostly need a long reaction time to achieve signal output,
which may lead to a potential influence on real-time
monitoring and quantification. Therefore, it is still challeng-
ing to design efficient probes that can rapidly respond
to H2O2.
It is well-known that the amphiphilic compounds, con-
sisting of a hydrophilic headgroup and a hydrophobic tail,
€
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† Beijing National Laboratory for Molecular Sciences.
‡ University of Chinese Academy of Sciences.
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r
10.1021/ol4000845
Published on Web 02/07/2013
2013 American Chemical Society

can formmicellarsystems and offera microenvironment to
accelerate the observed rates of some chemical reactions.6
Molecular recognition and fluorescent sensing can greatly
benefit from the micellar systems as well.7 However,
fluorescent dyes or probes in the micelles may tend to
aggregate, generally leading to fluorescence quenching and
reduction of probe sensitivity.8,9 Thus, the photophysical
properties of the fluorophore need to be further elabo-
rated. We speculate that the combination of a traditional
fluorescent sensing mechanism with the surfactant effect
could set up an efficient and simple method for rapid
detection of H2O2.
Our strategy is to choose 2-(20-hydroxyphenyl)ben-
zoxazole (HBO) as a fluorophore and C9 alkyl chain as
the hydrophobic tail (Scheme 1). HBO analogs are classic
fluorescent compounds, exhibiting a unique aggregation-
induced emission enhancement (AIEE) phenomenon.10 In
the aggregated form, the intramolecular rotation is re-
stricted, resulting in the restriction of the nonradiative
pathway, so that they mostly exhibit an intensely pro-
nounced excited-state intramolecular proton transfer
(ESIPT) emission resulting from the keto form.11 In present
paper, the hydroxyl group was protected with the boronate-
based benzyl cleavable group to obtain probe D-BBO
based on the following considerations: (i) increasing the
hydrophobicity of D-BBO, (ii) inhibition of the ESIPT
process, and (iii) specificity toward H2O2. We envisioned
that probe D-BBO can easily aggregate into particles in
a surfactant solution and emit purple fluorescence due to
the absence of ESIPT. Under the surfactant acceleration,
the H2O2-triggered boronate cleavage would take place
more rapidly and liberate the ESIPT compound D-HBO,
giving rise to considerable red-shifted fluorescence emission.
Therefore, a rapid detection of H2O2 with ratiometric signal
output can be established.
Figure 1. (a) Fluorescence spectra of 4 μM D-HBO in DMSO
(1); H2O/DMSO = 1:1 (2); 4 μM A-HBO in DMSO (3); H2O/
DMSO = 1:1 (4), λex = 341 nm. Inset: photographs of the
solutions, λex = 365 nm. (b) Fluorescence spectra of D-HBO
and A-HBO in solid state. Inset: photographs of the solids,
λ
ex = 365 nm.
As shown in Figure 1a, both D-HBO and A-HBO
exhibit fluorescence emission around 405 nm in DMSO,
which strongly breaks the intramolecular hydrogen bond
leading to the inhibition of the ESIPT process.12 In a mixed
solution (H2O/DMSO = 1:1), the emission peak of
D-HBO is obviously red-shifted, whereas A-HBO shows
fluorescencequenching witha much smaller emission shift.
The distinct emission color of solutions can be easily
observed by the naked eye (Figure 1a, inset), whereas both
D-HBO and A-HBO exhibit strong fluorescence around
500 nm in the solid state (Figure 1b). This phenomenon
clearly suggests that these features are assignable to the
AIEE mechanism. However, the water molecules can
induce the aggregation of hydrophobic D-HBO molecules,
which reduce the fluorescence quenching caused by the
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Scheme 1. Synthesis of Compounds
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Shvadchak, V. V.; Klymchenko, A. S.; Duportail, G.; Pivovarenko,
The synthetic procedures are shown in Scheme 1. Com-
pound D-HBO can be easily prepared by coupling com-
pound 1 with decanoic acid. The hydroxyl group was
further alkylated by using 4-(bromomethyl)benzene boro-
nic acid pinacol ester under basic conditions giving D-BBO
in moderate yield. A control molecule with a short alkyl
chain, A-HBO, was also synthesized by direct acetylation
of compound 1.
ꢀ
V. G.; Mely, Y. J. Phys. Chem. A 2007, 111, 10435–10438.
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7680–7689.
Org. Lett., Vol. 15, No. 4, 2013
925

intramolecular rotation. As a result, D-HBO exhibits
apparent ESIPT emission in H2O/DMSO solution.10,11
Figure 3. (a) Time-course kinetic measurement of fluorescence
response of D-BBO to H2O2. Data were collected under pseudo-
first-order conditions (2 μM D-BBO, 1 mM H2O2) in HEPES
buffer (0.1ꢁ2.0 mM CTAB, λex = 341 nm, 25 °C, λem = 405 nm).
Data collected from 1 to 15 min were used to fit the rate constants
(red lines). (b) The observed rate constant change as a function of
CTAB concentration.
Figure 2. Fluorescence emission spectra of D-BBO (4 μM) upon
addition of 100 μM H2O2 for 0ꢁ11 min in HEPES buffer (20 mM
HEPES, 0.3 mM CTAB, pH = 7.4, 25 °C), λex = 341 nm.
prepared a series of surfactant solutions with various
CTAB concentrations (0.1ꢁ2.0 mM). The observed rate
constants of D-BBO for H2O2 were measured and shown
in Figure 3. The maximum value of 1.83 ꢀ 10ꢁ2 sꢁ1 is
found at the CTAB concentration of 0.3 mM. Under these
conditions, the CTAB molecules do not form micelles.
However, while the hydrophobic D-BBO was added into
the CTAB solution, the probe could be encapsulated by
CTAB molecules via the interactions of long alkyl chains,
resulting in formation of well dispersed cationic particles,
which was further confirmed by dynamic light scattering
(DLS) measurements(FigureS4). Theaggregatedparticles
have an average size of about 195 nm when the concentra-
tion of CTAB is 0.3 mM, which is apparently larger than
that in 2 mM CTAB solution (105 nm). In our system it
turns out that 0.3 mM CTAB solution has the best accel-
eration effect on H2O2-sensing, probably because of the
formation of aggregate with appropriate size and charge
distribution under these conditions. Since the boronate
cleavage highly depends on the nucleophilicity of H2O2,4k
the cationic particles can obviously accelerate the reaction
of boronate substrate with anionic HOOꢁ through an
electrostatic effect, as observed in other micelles systems.6
The aggregation of product D-HBO restricts intramolec-
ular rotation, which is beneficial to the intramolecular
hydrogen bond, and consequently turns on the ESIPT
emission (Figure 4). Additionally, neither anionic sodium
dodecylsulfate (SDS) nor nonionic Triton X-100 shows the
acceleration effect on the boronate cleavage (Figure S5).
The boronate group has been widely introduced to
design fluorescent probes for detection and imaging
H2O2 by Chang and other groups.4 The most notable
feature of these probes is the high selectivity to H2O2 over
other reactive oxygen species in aqueous solutions. In
order to check whether this chemoselectivity could be
preserved under surfactant conditions, we further per-
formed fluorimetric titration of D-BBO with different
reactive oxygen species (ROS) for 10 min in surfactant
buffer (20 mM HEPES, 0.3 mM CTAB, pH = 7.4). As
shown in Figure 5, only H2O2 induced a sharp and rapid
response in both fluorescence emission and the emission
Boronate-caged probe D-BBO is highly hydrophobic
and its fluorescence emission gradually decreased within
60 min in aqueous solution (Figure S1a). But the fluorescent
intensity of D-BBO displayed almost no change during the
same period in the cetyltrimethylammonium bromide
(CTAB) aqueous solution (20 mM HEPES, 0.3 mM
CTAB, pH = 7.4) (Figure S1b), suggesting that the probe
can be well dispersed in this solution. As shown in Figure 2,
D-BBO exhibits a strong fluorescence emission maximum
at405nm (Φ = 0.21), which isverysimilartothe enolform
emission of D-HBO in DMSO.13,7i Addition of 100 μM
H2O2 induces a rapid decrease of the emission at 405 nm
and concomitantly a dramatic increase of the emission
at 510 nm (Φ = 0.37) with a large bathochromic shift of
105 nm and an isoemissive point at 474 nm. These data
clearly suggested that H2O2 triggered the boronate clea-
vage process so as to generate D-HBO, which emits ESIPT
fluorescence. This process was also confirmed by the ESI-
MS analysis (Figure S3). As expected, the probe showed a
rapid optical response to H2O2. Using pseudo-first-order
kinetics (2 μM D-BBO, 1 mM H2O2), the observed rate
constant for H2O2-deprotection is1.83ꢀ 10ꢁ2 sꢁ1, which is
20ꢁ60-fold greater than that of reported H2O2-selective
probes.4c,d Thus, D-BBO can be an excellent ratiometric
probe for rapid detection of H2O2 in micellar systems with
a large emission shift and ratio changes.
With these data in hand, we next investigated the effect
of CTAB on the rates of detection. It is well-known that
CTAB is a cationic surfactant and widely used to construct
micellar systems for catalyzing chemical reactions. The
critical micelle concentration (CMC) is 0.9 mM in water.14
To further evaluate the acceleration effect of micelles, we
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926
Org. Lett., Vol. 15, No. 4, 2013

Figure 4. Proposed mechanism for rapid detection of H2O2.
ratio (F510/F405 nm). But other ROS, including tert-butyl-
hydroperoxide (TBHP), hypochlorite (ClOꢁ), hydroxyl
radical ( OH), tert-butoxy radical ( OtBu), nitric oxide
3
3
(NO), and singlet oxygen (1O2), gave out no obvious
fluorescence emission ratio changes. Therefore, we
conclude that our probe bears excellent chemoselectiv-
ity for H2O2 over other competing ROS in surfactant
solution.
Figure 5. Fluorescence responses of 4 μM D-BBO to various
reactive oxygen species (ROS) at 100 μM. Bars represent emis-
sion intensity ratios F510/F405
addition of each ROS. Data were acquired in HEPES buffer (20 mM
at 0, 2, 4, 6, 8, 10 min after
nm
HEPES, 0.3 mM CTAB, pH = 7.4, 25 °C, λex = 341 nm).
In conclusion, we designed a new ratiometric fluorescent
probe for rapid detection of H2O2 in surfactant buffer
solution. The probe can be well encapsulated by surfactant
molecules leading to cationic aggregates, which apparently
accelerate the reaction rate of the probe with H2O2. More-
over, the unique AIEE and ESIPT photophysical proper-
ties of the boronate-leaved product provide a remarkable
fluorescence signal output with an extremely large emis-
sion red shift in the sensing process. Therefore, we have
established a new method for rapid detection of H2O2
based on aggregation induced ratiometric fluorescence
change. We also emphasize that this method can be cap-
able of facility and efficiency in the design of reaction-
based probes for other species.
Acknowledgment. We thank the National Natural
Science Foundation of China (21102148, 21125205,
212210017), National Basic Research Program of China
(2009CB930802, 2011CB935800), and the State Key Lab-
oratory of Fine Chemicals, Department of Chemical En-
gineering, Dalian University of Technology for financial
support.
Supporting Information Available. Synthetic proce-
dures, characterization of compounds, and additional
spectroscopic data. This material is available free of
charge via the Internet at http://pubs.acs.org.
The authors declare no competing financial interest.
Org. Lett., Vol. 15, No. 4, 2013
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