A selenium-contained aggregation-induced "turn-on" fluorescent probe for hydrogen peroxide

Article abstract of DOI:10.1039/c4ob00206g

A selenium-contained fluorescent 'turn-on' probe D-HMSe was developed for monitoring hydrogen peroxide. The probe D-HMSe is highly selective to hydrogen peroxide over other reactive oxygen species (ROS). An aggregation-induced enhancement (AIE) phenomenon was involved in the sensing process.

Full text of DOI:10.1039/c4ob00206g

Organic &
Biomolecular Chemistry
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A selenium-contained aggregation-induced “turn-
on” fluorescent probe for hydrogen peroxide†
Cite this: Org. Biomol. Chem., 2014,
12, 3004
Ye-Xin Liao, Kun Li,* Ming-Yu Wu, Tong Wu and Xiao-Qi Yu*
Received 26th January 2014,
Accepted 12th March 2014
DOI: 10.1039/c4ob00206g
www.rsc.org/obc
A selenium-contained fluorescent ‘turn-on’ probe D-HMSe was glucose and glucose oxidase8 and others.9 Though many fluo-
developed for monitoring hydrogen peroxide. The probe D-HMSe rescent probes of H2O2 have been developed, new types of sen-
is highly selective to hydrogen peroxide over other reactive oxygen sitive and selective fluorescent probes for monitoring
species (ROS). An aggregation-induced enhancement (AIE) hydrogen peroxide are still greatly in demand.
phenomenon was involved in the sensing process.
The aggregation-induced enhancement (AIE) phenomenon,
first reported by Tang et al. in 2001,10 was widely studied for
its advantages to overcome the shortcomings of the aggrega-
tion-caused quenching (ACQ) phenomenon in the aggregated
state.11 It involves a process of restricting the intramolecular
rotation of single bonds to block the nonradiative relaxation
pathway. Fluorescent probes for monitoring H2O2 involving an
AIE process are seldom reported in the literature. Jiang et al.
had developed an aggregation-involved boronate based fluo-
rescent probe for monitoring H2O2 assisted by forming micel-
lar systems with cetyltrimethylammonium bromide (CTAB).12
As an excellent probe, it should exhibit suitable solubility in
aqueous solution without any auxiliary reagents. Herein, we
Hydrogen peroxide (H2O2) is a major member of the reactive
oxygen species (ROS) family, which can cause oxidative stress
and damage nucleic acid, protein and lipid in cells.1 Homeo-
stasis of H2O2 and other ROS relates to organisms’ aging and
age-related diseases, including cardiovascular diseases,2a neu-
rodegenerative diseases2b like Alzheimer disease, immune-
system aging2c and cancer.2d On the other hand, H2O2 acts as
a second messenger in growth and proliferation processes of
cells when it is under normal physiological concentrations.3,4
In the food industry, H2O2 is used to replace the chloride-con-
taining bleaching and sterilizing agents. H2O2 can keep dairy
food products away from microbial contamination. On the
other hand, excessive residual H2O2 in foods can induce food
poisoning.5 Because of the biological importance and food
security significance of H2O2, considerable attention has been
devoted to developing new types of fluorescent probes for
monitoring H2O2.
Fluorescent probes have become excellent tools in modern
analytical method for their advantages such as high temporal–
spatial resolution, high selectivity and sensitivity, long absorp-
tion and emission wavelengths, as well as large absorption
coefficient.6 Up to now, several types of small molecular fluo-
rescent probes have been developed to monitor H2O2, includ-
ing boronate group cleavage based probes which were
developed by Chang et al.,7 and a further application of
sensing glucose based on the boronate group cleavage reaction
caused by H2O2 which was generated by the reaction between
present
a novel selenium-containing aggregation-induced
“turn-on” fluorescent probe for hydrogen peroxide. An alkyl
tail introduced into the probe will increase the hydrophobicity,
so that it could aggregate in the micellar systems; then the
ESIPT process performed well for the restriction of intramole-
cular rotation in the aggregation state. In addition, introducing
bulky steric hindrance groups into probes would enhance the
AIE as those groups can suppress the π–π interaction between
probe molecules which can cause the ACQ.13
Selenium is a fundamental micronutrient, which is in a
main state of selenocysteine at the active site of selenoproteins
such as glutathione peroxidase in organisms.14 Selenium can
react with ROS such as hydrogen peroxide15 and keep the
homeostasis of redox-cycles in organisms, so it plays an impor-
tant role in antioxidant defense systems. Photoinduced elec-
tron transfer (PET) is widely used in the fluorescent probe
design strategy.16 The PET process would quench the fluo-
rescence of a fluorophore in most cases, and fluorescence
recovers when the PET is blocked. Some selenium-containing
PET fluorescent probes have been reported.16b,17 This type of
probes usually involve a Se oxidation that would inhibit the
PET process. Thus, we introduced selenium into the target
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College
of Chemistry, Sichuan University, Chengdu, 610064, P.R. China.
E-mail: kli@scu.edu.cn, xqyu@scu.edu.cn
†Electronic supplementary information (ESI) available: 1H NMR, 13C NMR and
HRMS of all compounds. See DOI: 10.1039/c4ob00206g
3004 | Org. Biomol. Chem., 2014, 12, 3004–3008
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treated with 20 eq. H2O2 (quantum yield: 0.002). The UV/Vis
spectrum of D-HMSe (20 μM) shows three absorption peaks at
238 nm, 274 nm and 354 nm. It also exhibits very weak fluo-
rescence in a quantum yield of 0.003 in DMSO–water (1/99,
v/v). However, after D-HMSe was treated with H2O2 (20 eq.) in
DMSO–water (1/99, v/v) at ambient temperature for 1 h, the
quantum yield of the solution increased to 0.096.
Fluorescence spectra of 10 μM D-HMSe before and after
treating with 200 μM H2O2 in different solvents, including
water, DMSO, CH3OH, CH3CN, THF and DMF, were measured
(all of these solvents include 1% DMSO, v/v). As shown in
Fig. S2,† D-HMSe showed very weak fluorescence in the
absence of H2O2 in different solvents. After treating with
200 μM H2O2, D-HMSe in different solvents also exhibited
weak fluorescence except in DMSO–water (1/99, v/v). Thus, we
chose DMSO–water (1/99, v/v) buffered by HEPES (20 mM, pH
7.40) as the test solvent.
Scheme 1 Synthetic procedures of compounds HMSe and D-HMSe. (a)
Ethanol, reflux. (b) C12H25Br, 33% NaOH, DMF, 60 °C. (c) H2, Pd/C,
CH3OH, r.t. (d) SOCl2, DMF, reflux. (e) NEt3, DCM, −20 °C.
Subsequently, the reaction between D-HMSe and H2O2 was
studied. We treated the probe D-HMSe with H2O2 overnight at
room temperature, and a white solid was collected as the
major product which was named D-HMSeO (Scheme 2). With
the oxidation of the Se atom, the PET process was inhibited.
The structure of D-HMSeO was confirmed by 1H NMR, 13C
NMR and high-resolution mass spectrometry (HRMS).
Through the HRMS result of D-HMSeO, we also found a
[M + H]+ peak at m/z 576.2126 (Fig. S5†), when the m/z value of
[M + H]+ was calculated as 576.2124. Fluorescence spectra of
D-HMSe and D-HMSeO in the solid state were recorded
(Fig. S3†). D-HMSe exhibited very weak fluorescence emission
in the solid state (the quantum yield of D-HMSe in the solid
state was too low to be detected), but D-HMSeO exhibited
strong fluorescence emission in the solid state (also see
Scheme 2, inset image) at 493 nm (quantum yield: 0.1139). We
also treated D-HMSe (10 μM) with 20 equiv. H2O2 in different
probe molecule and expected that it could be the reactive site
of H2O2. We chose 2-phenyl-1H-benzimidazole as a fluoro-
phore and introduced a dodecyl chain to substitute the hydro-
gen atom of the secondary amine of benzimidazole and hoped
that it could act as a hydrophobic and bulky group that could
block the π–π interaction between adjacent probe molecules in
aggregation form. As expected, the compound D-HMSe exhi-
bits very weak fluorescence in water for the PET process, and
displays strong fluorescence enhancement after treating with
H2O2 in water at ambient temperature. As controlled, a com-
pound without an alkyl chain named HMSe was also syn-
thesized and we found that HMSe exhibited no notable change
in fluorescence intensity after treating with H2O2.
The synthetic procedures are shown in Scheme 1 (see ESI†
for details). Starting from o-phenylenediamine and 2-nitro-
benzaldehyde, we got 2-(2′-nitrophenyl)benzimidazole (1a).
Then a dodecyl chain was introduced into 1a by reacting with
bromododecane and 1b was obtained. Reducing compound 1
with H2 using Pd/C afforded compound 2. Compounds 318
and 419 were synthesized according to earlier literature reports
(see ESI† for details). Finally, compound 2 reacted with 4 to
generate target products HMSe and D-HMSe.
First, spectroscopic properties of D-HMSe and HMSe were
investigated. UV/Vis absorption (Fig. S1†) of HMSe and
D-HMSe were recorded. HMSe (20 μM) exhibits two absorption
peaks at 241 nm and 288 nm and a very weak fluorescence
emission in DMSO–water (1/99, v/v). The quantum yield of
HMSe in DMSO–water (1/99, v/v) is 0.002 (Table 1). No remarkable
change was observed in fluorescence intensity after HMSe was
Scheme 2 The reaction of the compound D-HMSe and H2O2. Inset:
image of solid state emission of compounds HMSe and D-HMSe under a
365 UV lamp.
Table 1 Photochemical properties of HMSe and D-HMSe in DMSO–water (1/99, v/v)
ε (L mol−1 cm−1
)
λex (nm)
λem (nm)
Φ1
Φ2
a
b
Compound
λmax (nm)
HMSe
D-HMSe
241, 288
238, 274, 354
3.30 × 104 (241 nm), 2.57 × 104 (288 nm)
350
330
436
476
0.002
0.003
0.002
0.096
1.94 × 104 (238 nm), 1.83 × 104 (274 nm), 0.52 × 104 (354 nm)
a Fluorescence quantum yields of HMSe and D-HMSe in solution were calculated using quinine bisulfate (5 × 10−6 M). b Fluorescence quantum
yields of HMSe and D-HMSe after treating with 20 eq. H2O2 for 1 h at ambient temperature in solution.
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volume ratios of H2O and DMSO (Fig. S4†). Weak fluorescence remarkably to about 3 μm. These results indicated that an
was found in DMSO solution. However, it exhibited strong aggregation process occurs after D-HMSe reacted with H2O2.
fluorescence as the water fraction rose to 99%. We attributed
The influence of the pH value on D-HMSe before and after
this phenomenon to the intrinsic AIE properties of the oxi- treating with H2O2 was also investigated (Fig. S7†). Variation of
dative product D-HMSeO. With a tail of dodecyl chain, the pH value from 3.01 to 11.91 does not cause any significant
D-HMSeO is strongly hydrophobic, and it aggregates in water changes in fluorescence intensity of 10 μM D-HMSe in HEPES
inducing restriction on intramolecular rotation which is a non- buffer (20 mM)–DMSO (99 : 1, v/v) at ambient temperature. But
radiative relaxation pathway, but with a flexible alkyl chain, it shows pH-dependency for 10 μM D-HMSe after treating with
probe molecules cannot aggregate to generate the ACQ 200 μM H2O2 in HEPES buffer (20 mM)–DMSO (99 : 1, v/v). It
phenomenon, thus resulting in
a notable fluorescence exhibits strong fluorescence when the pH value is between
enhancement together with blocking the PET process. It exhi- 4.51 and 7.40. It shows very weak fluorescence when the pH
bits almost no fluorescence in other solvents due to the dissol- value is above 8.56. So the pH 7.40 is suitable for the monitor-
ution and dispersion of probe molecules, which can free ing of H2O2. Further investigation of the pH influence on
intramolecular rotation as a nonradiative relaxation pathway.
D-HMSeO indicated that D-HMSeO showed strong fluorescence
Subsequently, high performance liquid chromatography intensity when the pH < 4.5. For this point, we supposed that
(HPLC) was used to investigate the reaction medium. The pH could affect the reaction between D-HMSe and H2O2.
results showed that when D-HMSe was treated with 20 equiv.
Fluorescence responses of the probe D-HMSe to H2O2 were
H2O2 in CH3CN at ambient temperature for 1 h, the product evaluated in detail (Fig. 3). We investigated the emission
D-HMSeO was generated (Fig. 1). Dynamic light scattering responses of D-HMSe treated with different concentrations of
(DLS) results showed that before treating with H2O2, D-HMSe H2O2 for 1 h in HEPES buffer (20 mM)–DMSO (99 : 1, v/v) at
disperses in water at a size of 141 nm (PDI: 0.202), while on
treating with H2O2 for 1 h, the size increases to 1921 nm (PDI:
0.420) (Fig. S6†). More than 10 fold increment in size had been
observed after D-HMSe was treated with H2O2. This aggrega-
tion trend was also observed in the SEM. As shown in Fig. 2,
D-HMSe could form spheres with diameter around 500 nm.
After treating with H2O2, the diameter of the spheres enlarged
Fig. 1 HPLC results of D-HMSe (red line, a), D-HMSe treated with H2O2
for 1 h (blue line, b) and D-HMSeO (black line, c).
Fig. 3 (a) Fluorescence responses of 10 μM D-HMSe toward different
concentrations of H2O2 (final concentration: 0, 10, 20, 30, 40, 50, 60,
65, 70, 75, 80, 90, 100, 110, 120, 150, 200, 250, 300, 400, 500, 700 μM)
after 1 h at ambient temperature in HEPES (20 mM, pH 7.40)–DMSO
(99/1, v/v). Inset: relative fluorescence intensity of D-HMSe with increas-
ing concentrations of H2O2 at 460 nm. (b) The kinetic response of 10 μM
D-HMSe to 200 μM H2O2. Spectra were acquired at 0 to 120 min after
Fig. 2 (a) SEM images of the compound D-HMSe, (b) after D-HMSe was
H2O2 was added at ambient temperature in HEPES (20 mM, pH 7.40)–
treated with 20 eq. H2O2 at ambient temperature.
DMSO (99/1, v/v). λex = 330 nm, slit widths: ex/em = 5/5 nm.
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room temperature, and fluorescence spectra were recorded in exhibited high selectivity toward H2O2 over other ROS in
Fig. 3a. Fluorescence intensity exhibited no notable changes aqueous solution. It might serve as a good chemical sensor for
with increasing the concentration of H2O2 when it is less than further use in food or environmental science.
50 μM. But fluorescence intensity exhibited a great enhance-
ment when the concentration of H2O2 increased from 50 μM
to 150 μM and reached a maximum at 150 μM. So we used
200 μM, which is a little higher than 150 μM, as the test con-
Acknowledgements
centration of H2O2 for other experiments to ensure a complete This work was financially supported by the National Program
reaction between D-HMSe and H2O2. Kinetic characteristics on Key Basic Research Project of China (973 Program,
were investigated as well, and fluorescence and absorption 2012CB720603 and 2013CB328900), the National Science
spectra are shown in Fig. 3b and Fig. S8.† After the addition of Foundation of China (no. 21232005, 21321061, J1310008 and
200 μM H2O2, no remarkable changes were observed until J1103315), and the Specialized Research Fund for the Doctoral
15 minutes. After 30 min, the fluorescence intensity increases Program of Higher Education in China (20120181130006). We
notably and reaches a maximum at 50 min. So we chose also thank the Analytical & Testing Center of Sichuan Univer-
1 hour as the reaction time.
sity for NMR analysis.
The high selectivity of D-HMSe for H2O2 was acquired from
screening its response to different ROS under mimetic physio-
logical conditions (20 mM HEPES buffer, pH 7.40, 1% DMSO).
As shown in Fig. 4, more than 110 fold fluorescence enhance-
ment was observed after treatment with 200 μM H2O2 for 1 h
at ambient temperature. No notable fluorescence enhance-
ment was observed in the presence of other ROS, including
ClO−, tert-butyl hydroperoxide (TBHP), ONOO−, 1O2, •OH,
tBuOO• and O2−•. We also investigated the responses of HMSe
to various ROS. There were no notable changes after treating it
with H2O2 or other ROS. We supposed this is because the
hydrophobicity of HMSe is not strong enough, so it cannot
aggregate to exhibit the AIE phenomenon.
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In summary, we have developed a new selenium-contained
“turn-on” fluorescent probe D-HMSe for monitoring hydrogen
peroxide. The reaction mechanism involves a PET-blocked
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product D-HMSeO, and then an AIE process occurs exhibiting
a fluorescence ‘turn-on’ phenomenon. The probe D-HMSe
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