A near-infrared reversible and ratiometric fluorescent probe based on Se-BODIPY for the redox cycle mediated by hypobromous acid and hydrogen sulfide in living cells

Article abstract of DOI:10.1039/c3cc42313a

We have developed a near-infrared (NIR) reversible and ratiometric fluorescence sensor based on Se-BODIPY for the redox cycle between hypobromous acid oxidative stress and hydrogen sulfide repair. Real-time imaging shows that the probe is able to monitor

Full text of DOI:10.1039/c3cc42313a

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A near-infrared reversible and ratiometric fluorescent
probe based on Se-BODIPY for the redox cycle
mediated by hypobromous acid and hydrogen sulfide
in living cells†
Cite this: Chem. Commun., 2013,
49, 5790
Received 30th March 2013,
Accepted 30th April 2013
DOI: 10.1039/c3cc42313a
Bingshuai Wang, Peng Li, Fabiao Yu, Junsheng Chen, Zongjin Qu and Keli Han*
www.rsc.org/chemcomm
We have developed a near-infrared (NIR) reversible and ratiometric
Due to the specificity and cumulative signaling effects of
fluorescence sensor based on Se-BODIPY for the redox cycle fluorescence technology, many fluorescent probes have become
between hypobromous acid oxidative stress and hydrogen sulfide important tools for detecting various species in bio-systems to study
repair. Real-time imaging shows that the probe is able to monitor physiological and pathological processes.12 The redox cycle between
intracellular HBrO/H2S redox cycle replacement.
HBrO and H2S is a very complicated process that requires reversible
fluorescent probes for visual detection of the intracellular HBrO/H2S
redox cycle. There has been increasing research interest in the
development of H2S fluorescent probes,13 however, only a few
fluorescent probes have been reported by our group for the redox
process between reactive oxygen species and H2S.14 Furthermore,
only limited fluorescent probes have been reported for HBrO detec-
tion.15 In this communication, we have developed a highly sensitive
reversible fluorescent probe diMPhSe-BOD for the redox cycle
between HBrO oxidative stress and H2S repair.
Hypobromous acid (HBrO) is an integral factor in biological host
defense systems, with very similar chemical and physical properties
to those of hypochlorous acid.1 Endogenous HBrO is formed by the
reaction of H2O2 with BrÀ catalyzed by eosinophil peroxidase (EPO).2
In vivo, HBrO is a powerful oxidizer with effective antibacterial
activity, however, excessive generation of HBrO will injure organisms
and lead to inflammatory tissue damage,3 and can result in many
diseases, such as arthritis, cardiovascular diseases, cancers, and
asthma.4 The correlation between serum EPO and clinical severity
in asthmatic patients provides major evidence that EPO levels in
serum increase by 300% in asthmatic patients than in healthy
individuals.4–6 In light of the potential role of HBrO in these
diseases, it is significant to explore the physiological functions of
HBrO in biological tissues.
Upon the occurrence of HBrO-oxidative damage in cells, the
antioxidative defense mechanisms of living organisms promptly
begin to protect the essential components of cells.7 Although hydro-
gen sulfide (H2S) has been recognized as the third gasotransmitter,8 it
also acts as an important endogenous antioxidant, which is enzyma-
tically generated in many organs (heart, lungs, kidneys, brain, nervous
system, etc.).9 The endogenous H2S can play an anti-inflammatory role
and can help in the recovery of the pulmonary function in asthma
pathogenesis.8–11 The H2S concentration in serum decreases by 60%
in asthmatic patients compared with healthy subjects.9 It is suggested
that endogenous H2S has the potential to serve as an inhibitor or a
repairer of HBrO-induced oxidative stress in vivo. Therefore, it is
important to investigate the redox process between HBrO and H2S
in cells.
In order to achieve the visualization of dynamic changes in the
equilibrium of HBrO/H2S redox coupling, a suitable chemical tool
was designed and synthesized for monitoring the intracellular redox
cycles. Our design concept for realizing our goal is based on the
oxidation and reduction processes of selenides.16 We chose the
BODIPY dye as the fluorophore because of its good photostability,
strong extinction coefficients, and high fluorescence quantum yield.
Next we integrated the modulator (4-methyoxylphenylselenide,
MPhSe) into the fluorescent platform through a styrene bridge. This
approach can extend the p-conjugation system of the fluorophore
and tune the fluorescent emission red-shift efficaciously.17 Moreover,
the strong electron-donating properties of the Se group further
facilitate the fluorescence of the probe to the near-infrared (NIR)
region. Compared with UV-visible light probes, NIR probes are
optimal for biological imaging because of their minimum photo-
damage, autofluorescence of biological objects.13d,18 Previously, we
have developed a selenium-containing fluorescent probe Cy-PSe for
monitoring cellular redox changes induced by peroxynitrite and
glutathione.19 The present probe, diMPhSe-BOD, only responds to
HBrO and then efficiently reacts with H2S in its reduced form. After
‘‘selenide’’ is oxidized to ‘‘selenoxide’’ by HBrO, the fluorescence of
the probe will blue-shift because of the electron-withdrawing effect of
‘‘selenoxide’’. Once ‘‘selenoxide’’ is reduced to ‘‘selenide’’ by H2S, the
probe recovers its original form and the fluorescence red shifts again
(Scheme 1). The changes in fluorescence wavelength result in the
State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical
Physics (DICP), Chinese Academy of Sciences (CAS), 457 Zhongshan Road, Dalian
116023, P. R. China. E-mail: klhan@dicp.ac.cn
† Electronic supplementary information (ESI) available: Experimental details and
supplementary data. See DOI: 10.1039/c3cc42313a
c
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Fig. 1 Fluorescence spectra of diMPhSe-BOD (10 mM) with HBrO in different
concentrations of 20 mM pH 7.4 PBS (acetonitrile 20%) (lex: 610 nm, HBrO
concentrations: 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, and
Scheme 1 Structure of diMPhSe-BOD and the detection mechanism of
diMPhSe-BOD for the HBrO/H2S induced redox cycle. Inset image: left: fluores-
cence image of 10 mM probe; right: fluorescence image of 10 mM probe + 100 mM
HBrO in 20 mM pH 7.4 PBS (20% acetonitrile).
100 mM). Inset: the relationship between the fluorescence intensity ratios (F635nm
/
F711nm) and the HBrO concentrations. F635nm and F711nm: fluorescence intensity of the
probe solution at 635 and 711 nm, respectively. (b) Fluorescence intensity ratio of
diMPhSe-BOD that reacts with ROS for 60 min. F635nm and F711nm: fluorescence
intensity of the probe solution at 635 and 711 nm, respectively (lex: 610 nm).
probe behaving as a ratiometric fluorescent probe, which can
circumvent the effects of various factors, including polarity, probe
molecule concentration, and its stability under photo-illumination.20
Given these advantages, the probe is expected to acquire positive
results for the HBrO/H2S redox cycle.
and HBrO concentrations in the given range. Therefore, the probe
diMPhSe-BOD can be applied in ratiometric detection of HBrO.
For assessing the specific nature of diMPhSe-BOD towards
HBrO, we investigated the effects of relevant physiological oxidants,
including cumene hÀydroperoxide (CuOOH), tert-butylhydroperoxide
The spectral properties of the probe were investigated under
simulated physiological conditions. diMPhSe-BOD exhibits the
maximum absorption at 672 nm (e = 22 770 MÀ1 cmÀ1) and a short-
wavelength shoulder at 618 nm (e = 16 300 MÀ1 cmÀ1) (Fig. S1a, ESI†).
Based on the normalized absorption spectra (Fig. S1b, ESI†), the
relative intensity of the short-wavelength shoulder increased, and the
maximum absorption and the short-wavelength shoulder blue-shifted
to 655 nm (e = 16 700 MÀ1 cmÀ1) and 603 nm (e = 13 800 MÀ1 cmÀ1),
respectively (Fig. S1a, ESI†). This process was accompanied by an
obvious color change in the probe solution from green to blue
(Fig. S1b, ESI† inset images). We selected the maximum emission
wavelength (711 nm) of diMPhSe-BOD as the testing wavelength.
diMPhSe-BOD shows weak fluorescence due to the heavy
atom effect.20 Both the absorption and emission spectra of
diMPhSe-BOD lie in the NIR region. After the reaction of
10 mM diMPhSe-BOD and 100 mM HBrO, the fluorescence
maximum blue-shifted to 635 nm because the weaker electron-
donating ability of ‘‘selenoxide’’ than ‘‘selenide’’ shortened the
donor–acceptor p-conjugated system (Scheme 1). The fluores-
cence intensity at 635 nm was remarkably increased by 230-fold
because the heavy atom effect of Se was weakened by the binding
of an oxygen atom;21 the fluorescence quantum yield increased
from 0.00083 to 0.206. The detection limit of our probe was
determined to be 0.97 mM under the testing conditions. After
HBrO oxidation, the intensity of the emission spectra (675–
750 nm) of the probe would decrease (Fig. 1a), however, an
enhancement of the intensity of the fluorescence spectra in the
range of 675 nm to 725 nm was observed as the excitation
wavelength was 610 nm. Hence, we selected 670 nm (see Section
4 in ESI†) as the excitation wavelength, because the emission of
diMPhSeO-BOD was negligible at this excitation wavelength
(Fig. S3, ESI†). The fluorescence response of diMPhSe-BOD to
HBrO was collected in the fluorescence window from 685 nm to
850 nm. The emission intensities of diMPhSe-BOD decreased
slowly with the addition of HBrO (Fig. S3, ESI†). The emission
intensity ratio (F635nm/F711nm) increased by 118-fold as the HBrO
concentration increased from 0 mM to 50 mM (Fig. 1a, inset).
A linear relationship (R = 0.988) was obtained between the ratio
3+
À
1
(t-BuOOH), Fe , O2 , H2O2, ꢀOH, NO, ONOO , O2 and HClO. The
fluorescence intensity ratios of diMPhSe-BOD that reacted with
various oxidants for 60 min are shown in Fig. 1b and Fig. S4 (ESI†).
After adding HBrO into the testing solution, the fluorescence ratio of
our probe increased and the fluorescence ratio enhanced from
0.10 to 11.88 within 3 min. Obviously, the fluorescent noise was
much lower than the fluorescence signal induced by HBrO. These
selectivity profiles showed that diMPhSe-BOD had good selectivity
and sensitivity to HBrO under simulated physiological conditions.
The selectivity of diMPhSeO-BOD to the reductants was
tested within 30 min. As shown in Fig. S6 (ESI†), diMPhSeO-
BOD was quickly reduced (within 10 min) by H2S, and the
fluorescence intensity ratio recovered to the original level. By con-
trast, the ratios decreased slowly upon the addition of the other
reductants. The fluorescent ratios of diMPhSeO-BOD after reacting
with various reductants for 30 min are presented in Fig. 2a. H2S
reduced the fluorescent ratio from 13.2 to 0.66. However, the
maximum ratio decrease induced by Na2S2O4 among other reduc-
tants was only 22% of the blank value. Fig. S8 (ESI†) shows the
ꢀ
Fig. 2 (a) Comparison of the fluorescence intensity ratio of diMPhSeO-BOD (10 mM)
with the addition of different reductants after 30 min in 20 mM PBS (acetonitrile 20%)
at pH 7.4 (lex: 610 nm). (b) Fluorescence ratio responses of diMPhSe-BOD (10 mM) to
redox cycles. diMPhSe-BOD was oxidized by HBrO. After 10 min, the solution was
treated with H2S in an appropriate concentration. When fluorescence intensity at
635 nm returned to starting levels, another portion of HBrO was added. The redox
cycles were repeated five times. All spectra were acquired in 20 mM PBS pH 7.4, lex
=
610 nm. The representation of the letters in Fig. 2: a, 50 mM HBrO; b, 45 mM H2S; c,
100 mM HBrO; d, 45 mM H2S; e, 100 mM HBrO; f, 60 mM H2S; g, 100 mM HBrO; h, 60 mM
H2S; i, 100 mM HBrO; and j, 60 mM H2S.
c
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H2O2, EPO and KBr was added to the cells, the average ratio
increased again (Fig. 3d). All results demonstrate that our probe
diMPhSe-BOD can be applied in the ratiometric visualization of the
HBrO/H2S redox cycle continuously in living cells. Co-staining with
the nuclear counterstain Hoechst (Fig. 3e and f) revealed that the
subcellular location of the probe was in the cytoplasm of the living
RAW264.7 cells. Bright-field transmission images confirmed that the
cells are still viable (see ESI†).
In conclusion, we have successfully developed an NIR rever-
sible and ratiometric fluorescent probe diMPhSe-BOD for the
HBrO/H2S redox cycle based on the oxidation and reduction
processes of selenides in solution and in living cells. Our probe
diMPhSe-BOD is highly sensitive and specific for the HBrO/H2S
redox cycle detection. Real-time imaging of the HBrO/H2S redox
cycle was successfully conducted with our probe diMPhSe-BOD
in macrophage cells. The experimental results show that the
Fig. 3 Confocal fluorescence images of the redox cycles between HBrO and H2S in
RAW264.7 cells. Macrophage cells were incubated with diMPhSe-BOD (5 mM) for 10 min
and then treated with various stimulants at 37 1C. Cell images were obtained at 635 nm
excitation wavelength and 640 nm to 670 nm and 680 nm to 770 nm emission bands.
(a) Control; (b) probe-loaded cells incubated with H2O2 (20 mM), EPO (105 U mLÀ1), and
KBr (50 mM) for 30 min; (c) cells in (b) incubated with H2S (50 mM) for 20 min; (d) (c) was
treated with a second dose of H2O2 (20 mM), EPO (105 U mLÀ1) and KBr (50 mM) for
30 min; (e) overlay of images showing fluorescence from diMPhSe-BOD and Hoechst probe has good permeability in living cells, and can monitor
dye; (f) Overlay of bright-field, diMPhSe-BOD, and Hoechst dye images.
intracellular HBrO/H2S redox cycle replacement continuously.
This work was supported by NSFC No. 21273234, 21203192,
and 2013CB834604.
fluorescence profile of diMPhSeO-BOD at different H2S concentra-
tions. Both the fluorescence intensities at 635 and 711 nm returned
Notes and references
to their original levels. It is indicated that diMPhSeO-BOD can be
1 D. Pattison and M. Davies, Biochemistry, 2004, 43, 4799.
reduced to diMPhSe-BOD by H2S. The fluorescence lifetime
decay of the reduction product overlapped completely with that
of diMPhSe-BOD (Fig. S7, ESI†). This result indicates that the
fluorescence signal returned to the original state. The reduced
probe still possessed the same reactivity with HBrO as before.
The redox cycles could be repeated at least five times (Fig. 2b)
with only an 18% decrement in the ratio (F635nm/F711nm). These
results suggest that our probe diMPhSe-BOD can be used to
selectively monitor the redox cycles between HBrO and H2S
continuously and quantitatively. The detection limits for HBrO
and H2S were determined to be 50 nM and 0.1 mM.
2 S. Weiss, S. Test, C. Eckmann, D. Roos and S. Regiani, Science, 1986,
234, 200.
3 A. Lane, J. Tan, C. Hawkins, A. Heather and M. Davies, Biochem. J.,
2010, 430, 161.
4 (a) J. Davies, D. Horwitz and K. Davies, Free Radical Biol. Med., 1993,
15, 637; (b) R. Aldridge, T. Chan, C. Van Dalen, R. Senthilmohan,
M. Winn, P. Venge, G. Town and A. Kettle, Free Radical Biol. Med.,
2002, 33, 847.
5 S. Mitra, A. Slungaard and S. Hazen, Redox Rep., 2000, 5, 215.
6 M. Sanz, A. Parra, I. Prieto, I. Dieguez and A. Oehling, Allergy, 1997,
52, 417.
´
7 I. Juranek and S. Bezek, Gen. Physiol. Biophys., 2005, 24, 263.
8 H. Kimura, Y. Nagai, K. Umemura and Y. Kimura, Antioxid.
Redox Signaling, 2005, 7, 795.
9 B. Predmore, D. Lefer and G. Gojon, Antioxid. Redox Signaling, 2012,
17, 119.
Based on the positive results, we next studied the bio-imaging
applications of diMPhSe-BOD for detecting the HBrO/H2S redox 10 P. Nagy and C. Winterbourn, Chem. Res. Toxicol., 2010, 23, 1541.
11 M. Tian, Y. Wang, Y. Lu, M. Yan, Y. Jiang and D. Zhao, Mol. Med.
Rep., 2012, 6, 335.
12 D. Quang and J. Kim, Chem. Rev., 2010, 110, 6280.
cycle in a biological system. We chose the mouse macrophage cell
line RAW264.7 as a bioassay model because the macrophage cells
activated the generation of HOBr after exposure to EPO, hydrogen 13 (a) H. Peng, Y. Cheng, C. Dai, A. King, B. Predmore, D. Lefer and
peroxide (H2O2), and bromide ions (BrÀ).22 Macrophage cells were
incubated with 5 mM probe for 10 min, cell images were taken
B. Wang, Angew. Chem., Int. Ed., 2011, 50, 9672; (b) C. Liu, J. Pan,
S. Li, Y. Zhao, L. Wu, C. Berkman, A. Whorton and M. Xian, Angew.
Chem., Int. Ed., 2011, 50, 10327; (c) A. Lippert, E. New and C. Chang,
through two collection channels ranging from 640 nm to 670 nm
and 680 nm to 770 nm. The fluorescence intensity in the shorter
channel (FS) was weaker than that in the longer channel (FL) (Fig. S9a
and b, ESI†). As mentioned previously, ratiometric detection can
efficiently eliminate the interference of environmental factors in the
complicated biological systems. The ratiometric cell image was
J. Am. Chem. Soc., 2011, 133, 10078; (d) F. Yu, P. Li, P. Song, B. Wang,
J. Zhao and K. Han, Chem. Commun., 2012, 48, 2852; (e) Y. Qian,
J. Karpus, O. Kabil, S. Zhang, H. Zhu, R. Banerjee, J. Zhao and C. He,
Nat. Commun., 2011, 2, 495; ( f ) R. Wang, F. Yu, L. Chen, H. Chen,
L. Wang and W. Zhang, Chem. Commun., 2012, 48, 11757.
14 (a) B. Wang, P. Li, F. Yu, P. Song, X. Sun, S. Yang, Z. Lou and K. Han,
Chem. Commun., 2013, 49, 1014; (b) Z. Lou, P. Li, Q. Pan and K. Han,
Chem. Commun., 2013, 49, 2445.
obtained using Olympus Micro FV10-ASW software with an average 15 F. Yu, P. Song, P. Li, B. Wang and K. Han, Chem. Commun., 2012, 48, 7735.
16 A. Mukherjee, S. Zade, H. Singh and R. Sunoj, Chem. Rev., 2010,
ratio of FS/FL = 0.6 to 0.8 (Fig. 3a). Upon the addition of H2O2
110, 4357.
(20 mM), EPO (105 U mLÀ1), and KBr (50 mM) to the probe-loaded
17 W. Zhao and E. Carreira, Chem.–Eur. J., 2006, 12, 7254.
cells for 30 min, FS increased and FL decreased. Hence, FS became 18 T. Ueno and T. Nagano, Nat. Methods, 2011, 8, 642.
19 F. Yu, P. Li, G. Li, G. Zhao, T. Chu and K. Han, J. Am. Chem. Soc.,
much stronger than FL (Fig. S10a and b, ESI†) and the average ratio
2011, 133, 11030.
of FS/FL enhanced to 3.5–4.5 (Fig. 3b), which is in accord with the
20 Y. Kurishita, T. Kohira, A. Ojida and I. Hamachi, J. Am. Chem. Soc.,
ratio changes in solution. When the cells were incubated with 50 mM
H2S for 20 min, the average ratio decreased to approximately
the starting level (Fig. 3c), indicating that the oxidation product
diMPhSeO-BOD was reduced to diMPhSe-BOD. After another dose of
2010, 132, 13290.
21 Y. Koide, M. Kawaguchi, Y. Urano, K. Hanaoka, T. Komatsu, M. Abo,
T. Terai and T. Nagano, Chem. Commun., 2012, 48, 3091.
22 H. Spalteholz, O. Panasenko and J. Arnhold, Arch. Biochem. Biophys.,
2006, 445, 225.
c
5792 Chem. Commun., 2013, 49, 5790--5792
This journal is The Royal Society of Chemistry 2013