A charge transfer assisted fluorescent probe for selective detection of hydrogen peroxide among different reactive oxygen species

Article abstract of DOI:10.1039/c2cc30932g

A charge transfer assisted fluorescent probe is synthesized which undergoes a change in fluorescence emission at two different wavelengths in the presence of hydrogen peroxide.

Full text of DOI:10.1039/c2cc30932g

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COMMUNICATION
A charge transfer assisted fluorescent probe for selective detection of
hydrogen peroxide among different reactive oxygen speciesw
Manoj Kumar,*a Naresh Kumar,a Vandana Bhalla,a Parduman Raj Sharmab and
Yasrib Qurishib
Received 9th February 2012, Accepted 20th March 2012
DOI: 10.1039/c2cc30932g
A charge transfer assisted fluorescent probe is synthesized which
undergoes a change in fluorescence emission at two different
wavelengths in the presence of hydrogen peroxide.
The addition of H2O2 changes the fluorescence spectra of
probe 3 from the twisted intramolecular charge transfer state
to the delocalized excited (DE) state. The removal of the low
energy TICT band and the appearance of the high energy
emission band (DE) permit us to observe the change in the
emission intensities at two different wavelengths. Furthermore,
biological application of probe 3 is evaluated for in vitro
detection of H2O2 in prostate cancer (PC3) cell lines with a
change in the fluorescence emission from red to blue.
Among various reactive oxygen species (ROS), hydrogen peroxide
(H2O2) is one of the important species in living systems1 which is
produced during the activity of almost all oxidases2 and is involved
in regulating biological processes.3 Any increase in the level of
cellular H2O2 is associated with the DNA damage, aging and
cardiovascular disorders.4 Apart from this, a significant concen-
tration of H2O2 is also present in the atmosphere, marine environ-
ment and is widely used in industries as a result of which a large
quantity of it is released into the environment.5 Keeping in view
the role played by H2O2 in day to day life, simple and rapid
detection of H2O2 in biological as well as environmental systems is
very important. However, most of the reported fluorescent probes
show fluorescence turn-on changes in the presence of H2O2 which
is not as sensitive as required for desired analytical purposes.6 Thus,
for high sensitivity and simplicity, it is extremely advantageous to
develop a novel fluorescent probe that exhibits a significant spectral
shift in the presence of H2O2.7 The most fundamental approach to
assure such spectral changes is to design a molecule exhibiting an
intramolecular charge transfer (ICT) mechanism which involves
the observation of changes in the ratio of the intensities of the
absorption or emission at two wavelengths which in turn is
independent of defects such as probe concentration, instru-
mental error and thus, improving the sensitivity of the method.
In continuation of our research work on the design, synthesis
and evaluation of fluorogenic receptors,8 in the present manuscript
we have designed and synthesized a dimethylaminocinnamaldehyde
linked arylboronate based probe 3 which undergoes a selective
spectral shift in the fluorescence emission with hydrogen peroxide
among various reactive oxygen species. Probe 3 exhibits a fluores-
cence emission at 566 nm in aqueous media which is ascribed
to the twisted intramolecular charge transfer (TICT) state.
Condensation of N,N-dimethylaminocinnamaldehyde 1 with
boronic ester 28b furnished compound 3 in 74% yield (Scheme 1,
see ESIw, S5 and S6). The structure of compound 3 was
confirmed from its spectroscopic and analytical data (see ESIw,
S15–S18). The molecular recognition behavior of compound 3
was studied toward different reactive oxygen species (ROS)
such as hydroxyl radical (HOꢀ), hydroperoxide radical (HOOꢀ),
hydrogen peroxide (H2O2), hypochlorite (ClOÀ), and hydro-
peroxide (ROOH) by UV-vis and fluorescence spectroscopy.
The absorption spectrum of probe 3 (5 mM) in ethanol shows
an absorption band at 400 nm (Fig. 1A). Upon addition of
H2O2 (10 mM) to the solution of receptor 3, the band centered
at 400 nm shows apparent decrease in absorbance along with
the appearance of a new absorption band at longer wavelength
(522 nm) which is visible to the naked eye with a clear color
change from pale yellow to pink (inset of Fig. 1A). The variation
in the absorption spectrum of probe 3 after treatment with H2O2
led to the formation of a clear isosbestic point at 464 nm. The
addition of other reactive oxygen species did not alter the
absorption spectrum of probe 3 (see ESIw, S7). The fluorescence
a Department of Chemistry, UGC Sponsored Centre for Advanced
Studies-1, Guru Nanak Dev University, Amritsar-143005, Punjab,
India. E-mail: mksharmaa@yahoo.co.in; Fax: +91 (0)183 2258820;
Tel: +91 (0)183 2258802 9 ext. 3205
b Department of Cancer Pharmacology, Indian Institute of Integrative,
Medicine, Canal Road, Jammu-180001, India
w Electronic supplementary information (ESI) available: Experimental
details, NMR and mass spectra. See DOI: 10.1039/c2cc30932g
Scheme 1 Synthesis of compound 3.
c
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Fig. 1 (A) UV-vis spectra of 3 (5 mM) in EtOH in the presence of H2O2
(10 mM). Spectra were recorded at 25 1C after the addition of H2O2 from
0 to 20 min at an interval of every one min. (B) Fluorescence response of
3 (5 mM) in EtOH; lex = 400 nm; in the presence of H2O2 (5 mM).
Spectra were recorded at 25 1C after the addition of H2O2 from 0 to 8 min
at an interval of every 30 seconds. The inset shows the color/fluorescence
change (a) before (b) after the addition of H2O2.
Fig. 2 Fluorescence response of 3 (5 mM) in H2O : EtOH (8 : 2, v/v)
buffered with HEPES, pH = 7.0; lex = 400 nm in the presence of H2O2
(10 mM). Spectra were recorded at 25 1C after the addition of H2O2
from 0 to 15 min at an interval of every one minute. The inset shows the
normalized fluorescence intensity; blue, free 3; green, 3 + H2O2 and
fluorescence change (a) before (b) after the addition of H2O2.
the TICT band (66 nm) was observed. The appearance of the
red shifted emission band upon increasing the polarity of the
solvent system confirms that the emission of receptor 3 in
aqueous media is due to the twisted intramolecular charge
transfer state.9 However, after the addition of H2O2 (10 mM)
to the aqueous solution of receptor 3, the emission at 566 nm
undergoes fluorescence quenching with simultaneous appear-
ance of a new blue shifted emission band at 484 nm (Fig. 2).
Interestingly, the disappearance of emission at 566 nm and the
appearance of blue shifted emission at 484 nm were observed
immediately after the addition of H2O2. Furthermore, the
emission at 484 nm undergoes maximum enhancement in
emission intensity within 15 min with the appearance of blue
colored fluorescence. The changes in the fluorescence spectra of
probe 3 are ascribed to the protonation of the dimethylamino
moiety upon addition of H2O2 assisted by the conversion of the
arylboronate moiety into phenol resulting in the disappearance
of emission at 566 nm along with appearance of a higher energy
emission at 484 nm (Fig. 3). To confirm the protonation of the
dimethylamino moiety, we studied the fluorescence behaviour of
probe 3 at different pH values (see ESIw, S14). At pH 6.0, an
emission band corresponding to the DE state is observed at
484 nm and there is no band corresponding to the TICT emission
at 566 nm. The disappearance of TICT emission at 566 nm is due
to the protonation of the dimethylamino group which inhibits the
twisted intramolecular charge transfer from the dimethylamino
group to the imino moiety. On the other hand, at pH 6.5 a weak
TICT emission band is observed at 566 nm along with the DE
spectrum of 3 in ethanol exhibits a weak fluorescence emission
at 500 nm ascribed to the twisted intramolecular charge
transfer (TICT) state with a shoulder at shorter wavelength
(460 nm) corresponding to the delocalized excited (DE) state
(Fig. 1B). The origin of the TICT emission band is due to the
charge transfer from the nitrogen atom of the dimethylamino
moiety (donor) to the imino moiety (acceptor). However, the
addition of H2O2 (5 mM) results in the appearance of a blue
shifted emission band at 470 nm attributed to the formation of
delocalized excited state emission (Fig. 1B). The emission
enhancement at 470 nm is probably due to the protonation
of the dimethylamino group9 as a result of a proton released
by hydrogen peroxide during reaction with the boronate
moiety which trammels the twisted intramolecular charge
transfer state formation. No significant change in the fluores-
cence behaviour was observed in the presence of other reactive
oxygen species (see ESIw, S8).
In mixed aqueous media (H2O/EtOH; 8 : 2, v/v; buffered
with HEPES, pH = 7.0; at 25 1C), the fluorescence spectrum
of probe 3 (5 mM) exhibits a broad emission band at 566 nm
attributed to the twisted intramolecular charge transfer state
formation (Fig. 2; lex = 400 nm). The fluorescence emission of
3 at 566 nm is due to the extended conjugated system as well as
to the more polar nature of the solvent system which is
responsible for the red shifted fluorescence emission of probe 3.
To confirm that the emission observed for receptor 3 in
aqueous media is based on the twisted intramolecular charge
transfer, we studied the fluorescence behaviour of receptor 3
by varying the polarity of the solvent system used for the
fluorescence studies (see ESIw, S10). In dichloromethane,
receptor 3 shows fluorescence emission corresponding to the
delocalized excited (DE) state at shorter wavelength (460 nm)
with a shoulder at longer wavelength (484 nm) ascribed to the
twisted intramolecular charge transfer (TICT) state. Any
increase in the polarity of solvents did not affect the position
of the delocalized excited band. In comparison to the deloca-
lized excited band, the TICT band shows a large red shift upon
increasing the polarity of the solvent system. For example, in
ethanol a red shift of the TICT band (16 nm) was observed in
comparison to dichloromethane. Upon further increasing the
polarity of the system (aqueous media) a more red shift of
Fig. 3 Possible mechanism for the reaction of H2O2 with probe 3.
c
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compared to that of free 3 (0.16) at 566 nm which shows good
agreement with fluorescence spectra obtained for receptor 3 in
the presence of H2O2. Next, we investigated the fluorescence
response of 3 to the addition of various concentrations of
hydrogen peroxide (see ESIw, S12). Even the presence of only
2 mM of hydrogen peroxide changes the fluorescence behaviour
of 3 which in turn indicates the highly reactive nature of 3
towards the hydrogen peroxide.
The potential biological application of probe 3 was evaluated
for in vitro detection of H2O2 in prostate cancer (PC3) cell lines
(see ESIw, S4). The prostate cancer (PC3) cell lines were
incubated with probe 3 (5 mM) in an RPMI-1640 medium
for 20 min at 37 1C and washed with phosphate buffered saline
(PBS) buffer (pH 7.4) to remove excess of receptor 3. Microscope
images showed intracellular fluorescence in the red channel
which indicated the existence of the twisted intramolecular
charge transfer state (Fig. 5a–d).
Fig. 4 Fluorescence response of 3 (5 mM) in H2O : EtOH (8 : 2, v/v)
buffered with HEPES, pH = 7.0 (lex = 400 nm) to various reactive
oxygen species (10 mM each). Bars represent selectivity (I484/I566) of 3
upon addition of different reactive oxygen species. Data were given
after incubation with the appropriate ROS at 25 1C from 0 to 15 min at
an interval of every 3 min. The inset shows the variation of fluorescence
of 3 (5 mM) in H2O : EtOH (8 : 2, v/v) buffered with HEPES,
pH = 7.0; lex = 400 nm with H2O2 (10 mM) at 566 and 484 nm from
0–15 min at an interval of every one minute.
The cells with receptor 3 (5 mM) were then treated with H2O2
(5 mM) in the RPMI-1640 medium and incubated again for
20 min at 37 1C and washed with PBS buffer. After treatment
with H2O2 the cells show fluorescence in the blue channel (Fig. 5e)
while the fluorescence in the red channel almost gets quenched
(Fig. 5f–h). These results suggest that probe 3 is cell permeable
and an effective hydrogen peroxide imaging agent with the change
in fluorescence emission from red to blue attributed to the
transformation of the twisted intramolecular charge transfer
state to the delocalized excited (DE) state within the cells.
In conclusion, we synthesized a fluorescent probe 3 which
exhibits disappearance of twisted intramolecular charge transfer
emission and the appearance of high energy emission (DE) in the
presence of hydrogen peroxide. Furthermore, 3 can also be used
as a fluorescent probe for intracellular imaging of H2O2 with a
change in fluorescence emission (red to blue) which will help in
the understanding of biological processes at the molecular level.
Fig. 5 Fluorescence and brightfield images of PC3 cells lines. (a) and
(b) fluorescence images of cells in the blue and red channels, respec-
tively, treated with probe 3 (5 mM) for 20 min at 37 1C; (c) brightfield
image of (a) and (b); (d) overlay image of (a) and (b); (e) and (f)
fluorescence images of cells in the blue and red channels, respectively,
upon treatment with probe 3 (5 mM) and then H2O2 (5 mM) for 20 min
at 37 1C; (g) brightfield image of (e) and (f); (h) overlay image of (e)
and (f); lex = 405 nm; fluorescence images are recorded at both blue
(470 Æ 20 nm) and red channels (570 Æ 20 nm).
Notes and references
emission band at 484 nm. However, upon addition of H2O2 to this
solution the TICT emission disappeared completely, while the
fluorescence intensity of the DE band is significantly enhanced.
Thus, from these studies we may conclude that the fluorescence
changes of probe 3 at pH 7.0 in the presence of H2O2 (Fig. 2) are
due to the protonation of the dimethylamino group responsible
for the hampering of TICT state formation and appearance of DE
state emission. Thus, probe 3 undergoes a spectral shift of about
82 nm in fluorescence emission from the twisted intramolecular
charge transfer state to the delocalized excited state in aqueous
media upon addition of H2O2, allowing us to observe the change
in fluorescence (from orange to blue) at two different wavelengths.
Under the same conditions as used for H2O2 in aqueous
media, we also carried out the fluorescence studies of probe 3
toward other biologically relevant reactive oxygen species (ClOÀ,
HOꢀ, HOOꢀ and ROOH; see ESIw, S11). As shown in Fig. 4,
no significant change was observed with other reactive oxygen
species in comparison to the hydrogen peroxide. Further, by
considering the ratio of the fluorescence intensity of DE state
emission at 484 nm (I484) to that of TICT state emission at
566 nm (I566), we observed 9.5-fold fluorescence enhancement
in the case of 3–H2O2 system. The fluorescence quantum yield10
(see ESIw, S3) of the 3–H2O2 system is 0.22 (at lem = 484 nm) as
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