A near-infrared reversible fluorescent probe for peroxynitrite and imaging of redox cycles in living cells

Article abstract of DOI:10.1039/c1cc12994e

BzSe-Cy is a small-molecule fluorescent probe containing Se, which can respond reversibly to changes in ONOO^- or reduced ascorbate and exhibit high sensitivity and selectivity for ONOO^-.

Full text of DOI:10.1039/c1cc12994e

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COMMUNICATION
A near-infrared reversible fluorescent probe for peroxynitrite and
imaging of redox cycles in living cellsw
Kehua Xu, Huachao Chen, Jiangwei Tian, Baiyu Ding, Yanxia Xie, Mingming Qiang and
Bo Tang*
Received 22nd May 2011, Accepted 12th July 2011
DOI: 10.1039/c1cc12994e
BzSe-Cy is a small-molecule fluorescent probe containing Se,
which can respond reversibly to changes in ONOOꢀ or reduced
ascorbate and exhibit high sensitivity and selectivity for
ONOOꢀ.
probes that can respond reversibly to changes in concentration
of ONOOꢀ would be much more valuable for studying the
generation, metabolism of ONOOꢀ and its dynamic damaging
of living cells. Selenium, long known to be an important
dietary ‘‘antioxidant’’, is now recognized as an essential
component of the active sites of many enzymes.11 Moreover,
depending on the redox cycling of selenium, many organo-
selenium compounds are capable of simulating catalytic
functions demonstrated by natural enzymes.12 One of the
important biochemical functions of these compounds is the
protection against ONOOꢀ.13 Although there is an increasing
amount of evidence showing that selenium redox cycling can
enhance the protective effects of organoselenium compounds
against ONOOꢀ, integrating selenium into a fluorescent probe
to detect ONOOꢀ and monitor the redox cycles in living cells
has not been reported to date.
Redox homeostasis is critical for proper cellular function.1
The reduction–oxidation state of the cell is primarily a
consequence of the precise balance between the levels of
reactive oxygen species (ROS) and reducing equivalents.2
Unregulated production of ROS results in potentially
cytotoxic ‘‘oxidative stress’’.3 Many diseases (e.g. Alzheimer’s
disease) are linked with ROS damage as a result of an
imbalance between radical-generating and radical-scavenging
systems.4 Thus, the study of intracellular redox cycles has an
important physiological and pathological significance. However,
only a few reversible fluorescent probes based on the modified
fluorescent proteins for redox activity5 and one small-molecule
fluorescent probe designed for monitoring redox cycles in
living cells have been described in the past several years.6
Nowadays, new fluorescent probes which can not only respond
reversibly to redox events but also exhibit high selectivity for
one kind of ROS should be more useful for studying dynamic
redox chemistry in biosystems.
In this report, we chose tricarbocyanine, a near-infrared
(NIR) fluorescent dye, as a fluorophore and divalent selenium
as a redox-responsive group to synthesize a new reversible
organoselenium probe benzylselenide-tricarbocyanine (BzSe-Cy,
Scheme S1, ESIw) for the selective detection of ONOOꢀ and
monitoring redox cycles in living cells. The structure of
BzSe-Cy was characterized with 1H NMR, 13C-NMR, IR
and ESI-MS (see ESIw). The redox cycling of the probe—
whereby the oxidized probe produced from BzSe-Cy by
ONOOꢀ (Scheme 1) is then recycled back to the BzSe-Cy by
reduced ascorbate (ASCH2)—can be repeated at least 8 times,
and the real-time imaging of cellular redox cycles was achieved
successfully in living RAW 264.7 cells. In addition, another
benefit of this probe is its NIR emission wavelength, which can
lead to minimize photodamage and cell autofluorescence.14
Peroxynitrite (ONOOꢀ), a powerful oxidizing agent formed
ꢀ
ꢁ
from a diffusion-controlled reaction between NO and O2 in
cells, plays an important role in biological processes.7 Depending
on the concentration, ONOOꢀ can be either ‘‘friend’’ or ‘‘foe’’.
A well-controlled level of ONOOꢀ drives important cellular
functions such as keeping cellular integrity and organ homeo-
stasis, whereas a high level of ONOOꢀ can induce DNA
damage and initiate lipid peroxidation in biomembranes.8
Among the reported methods for detecting ONOOꢀ, the use
of fluorescent probes had its apparent advantages over other
methods. For example, a few fluorescent probes such as
HKGreen9 and [Eu3+/Tb3+(DTTA)]10 have been developed for
the selective detection of ONOOꢀ. Furthermore, fluorescent
Key Laboratory of Molecular and Nano Probes, Engineering Research
Center of Pesticide and Medicine Intermediate Clean Production,
Ministry of Education, College of Chemical Engineering and
Materials Science, Shandong Normal University, Jinan, 250014,
China. E-mail: tangb@sdnu.edu.cn
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc12994e
Scheme 1 The redox cycle of BzSe-Cy.
c
9468 Chem. Commun., 2011, 47, 9468–9470
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Fig. 1 (a) Fluorescence responses of 5 mM BzSe-Cy to different
concentrations of ONOOꢀ. Inset: a linear correlation between emission
intensity and concentrations of ONOOꢀ. (b) Fꢀluorescence responses
of BzSe-Cy to ROS and RNS (5 mM for ONOO , 50 mM for ꢁOH, O3
and ClOꢀ, 0.5 mM for others). Bars represent the final (F) over the
initial (F0) integrated emission. Black bars represent the addition of
one of these bioanalytes to a 5 mM solution of BzSe-Cy. White bars
represent the addition of ONOOꢀ plus one of these interferences to
the probe solution. All spectra were acquired in 30 mM phosphate
buffer with pH 7.4 at 37 1C (lex/lem = 770/800 nm).
Fig. 2 Redox cycling of BzSe-Cy in 30 mM phosphate buffer with pH
7.4 at 37 1C (lex/lem 770/800 nm), showing reversible fluorescence
quenching upon alternating addition of ONOOꢀ and ASCH2. A
solution of 20 mM BzSe-Cy was treated with 15 mM ONOOꢀ and
then reduced with 40 mM ASCH2.
Spectroscopic evaluation of BzSe-Cy was performed
under simulated physiological conditions (phosphate buffer,
pH 7.4) and the optimal measurement conditions were
established (Fig. S1–S2, ESIw). Initially, we tested the fluores-
cence reponses of the probe to ONOOꢀ. As expected,
BzSe-Cy exhibits apparent fluorescence in phosphate buffer
(e = 28 778 Mꢀ1 cmꢀ1, FF = 0.106, t = 2.21 ns). Upon the
reaction of BzSe-Cy with ONOOꢀ, the probe was converted
to the oxidized BzSe-Cy (BzSeO-Cy), which is weakly fluorescent
upon 770 nm excitation due to a photo-induced electron-
transfer process. The structure of BzSeO-Cy was confirmed
by mass spectrometry and 77SeNMR (Fig. S3–S4, ESIw). As
shown in Fig. 1a, the addition of ONOOꢀ triggered 5-fold
decrease of the fluorescence intensity. When BzSe-Cy reacted
with different concentrations of ONOOꢀ, there was a good
linearity between relative fluorescence intensity and concen-
trations of ONOOꢀ, the equation of the regression analysis
was F = 570.36 ꢀ 91.40 [ONOOꢀ] (mM) with a correlation
coefficient of 0.9975, which indicates that the probe could
detect ONOOꢀ quantitatively in the linear range.
buffer solution with pH 7.40 at 37 1C. Upon treatment of
BzSe-Cy with ONOOꢀ, a remarkable fluorescence decrease
was observed. And then ASCH2 was added into the above
solution, the fluorescence intensity of which increased rapidly
and reached a constant value after only o15 s. This reversible
redox cycle can be repeated at least 8 times under the same
conditions. The experiment results show that BzSe-Cy is a
sensitive redox probe, which is ideal for monitoring changes in
the intracellular ONOOꢀ levels.
The monitoring of the redox cycles in living cells by using
BzSe-Cy was further undertaken by confocal laser scanning
microscopy. RAW 264.7 cells incubated with 20 mM BzSe-Cy
for 30 min at 37 1C showed strong fluorescence (Fig. 3a),
indicating that the probe can be easily internalized by the
living cells. Next, a marked fluorescence decrease in Fig. 3b
appeared after stimulating the BzSe-Cy-loaded cells with 20 mM
SIN-1 (ONOOꢀ donor)16 for 1 h (the incubation time was
To study the selectivity of BzSe-Cy toward ONOOꢀ, we
tested its responses toward other bioanalytes. Considering the
antioxidative function of selenium, the effect of interference of
other ROS and reactive nitrogen species (RNS) was detected.
As shown in Fig. 1b, BzSe-Cy exhibits a significant decrease in
fluorescence intensity upon reaction with ONOOꢀ, whereas
the response toward other bioanalytes is negligible. An error
of ꢂ5.0% in the relative fluorescence intensity was considered
to be tolerable. The experiments show that BzSe-Cy is highly
specific to ONOOꢀ. The change of fluorescence is due to the
interconversion between selenides and selenoxides, and the
oxidation potential of BzSe-Cy (0.622 V, see ESIw) is higher
than that of most other reported cyanine dyes,15 which largely
hampers the unwanted oxidation of BzSe-Cy to the corres-
ponding oxindole by ROS. These features made BzSe-Cy an
ONOOꢀ-specific probe. In addition, the selectivity of this
probe has been further identified in vivo (Fig. S5, ESIw).
The reversibility of the probe was then tested. Fig. 2 shows
the time courses of fluorescence intensity of the probe for
redox cycles mediated by ONOOꢀ and ASCH2 in 30 mM
Fig. 3 Live-cell imaging of redox cycles by confocal microscopy at
37 1C. (a) RAW 264.7 cells loaded with 20 mM BzSe-Cy for 30 min.
(b) Probe-loaded cells treated with 20 mM SIN-1 for 1 h. (c) Probe-
loaded, SIN-1-treated cells incubated with 50 mM ASCH2 for 30 min.
(d) Cells exposed to a second dose of 20 mM SIN-1 for an additional
1 h. (e) Cells in panel (d) incubated with 50 mM ASCH2 for another
30 min. (f) Brightfield image of live RAW 264.7 cells in panels (a)–(e).
Scale bar = 25 mm.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 9468–9470 9469

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DAPI in Fig. 4d and e, and the cells were viable and not
stained by PI as shown in Fig. 4f and g. These results show
that the probe has low cytotoxicity under these experimental
conditions.
To conclude, we have developed a NIR organoselenium
fluorescent probe for detecting ONOOꢀ and imaging of redox
cycles in living cells. BzSe-Cy is a unique small-molecule
indicator containing Se that responds reversibly to changes
in ONOOꢀ or ASCH2 concentration, and it exhibits high
sensitivity and selectivity for ONOOꢀ. Moreover, the new
probe was successfully applied to the imaging of multiple
cycles of oxidative stress and reductive repair in RAW 264.7
cells. This work provides a new approach to studying the
generation, metabolism of ONOOꢀ and its dynamic damage
process of living cells with related chemical tools.
Fig. 4 Confocal fluorescence images of living RAW 264.7 cells.
(a) Cells incubated with 20 mM BzSe-Cy at 37 1C for 30 min and then
incubated with 50 nM Mito Tracker Green FM for 10 min, using a
633 nm laser. (b) The above cells were excited by a 488 nm laser.
(c) One overlay image of (a) and (b). (d) Cells incubated with 20 mM
BzSe-Cy at 37 1C for 30 min and then incubated with 10 mg mLꢀ1
DAPI for 15 min, using a 405 nm laser. (e) One overlay image of
(d) and an image of the (d) cells excited by a 633 nm laser. (f) Cells
incubated with 20 mM BzSe-Cy at 37 1C for 30 min and then incubated
with 50 mg mLꢀ1 PI for 5 min, using a 543 nm laser. (g) One overlay
image of (f) and an image of the (f) cells excited by a 633 nm laser.
This work was supported by National Basic Research
Program of China (973 Program, 2007CB936000), National
Natural Science Funds (NNSF) for Distinguished Young
Scholar (No. 20725518), NNSF of China (No. 20875057),
National Key Natural Science Foundation of China (No.
21035003), Key Natural Science Foundation of Shandong
Province of China (ZR2010BZ001), and the Science and
Technology Development Programs of Shandong Province
of China (No. 2008GG30003012).
optimized in Fig. S6 (ESIw)), while a bright fluorescence was
again observed at the same field of view after addition of
50 mM ASCH2 into the medium and incubation for another
30 min at 37 1C (Fig. 3c). To further prove that BzSe-Cy has a
good reversibility, addition of a second aliquot of SIN-1
resulted in another decrease in intracellular fluorescence
(Fig. 3d), and when the same cells were incubated with ASCH2
again, the bright fluorescence appeared (Fig. 3e). The result
indicates that the fluorescence changes in the RAW 264.7 cells
are due to the changes in the intracellular ONOOꢀ levels, and
BzSe-Cy can also monitor the redox cycles in living cells. The
bright-field image (Fig. 3f) confirmed that the cells were
viable throughout the imaging experiments. In addition, the
internalization assay has been carried out both at 37 1C and
4 1C, proving that BzSe-Cy exhibits excellent cell membrane
permeability (Fig. S7, ESIw).
Furthermore, we assessed the ability of BzSe-Cy to target
the mitochondria in living cells. RAW 264.7 cells incubated
with 20 mM BzSe-Cy for 30 min at 37 1C showed bright
fluorescence in discrete subcellular locations as determined
by confocal microscopy (Fig. 4a). A co-staining experiment
with Mito Tracker Green FM (a commercially available
mitochondrial green-fluorescent probe) has been carried out
to establish that BzSe-Cy is localized to the mitochondria
(Fig. 4b). The overlay image confirms that the probe is
retained in the mitochondria of these living cells (Fig. 4c).
Finally, to further prove that BzSe-Cy has a low toxicity to the
cultured cell lines, the MTT assay (Fig. S8, ESIw) and nuclear
staining with 40,6-diamidino-2-phenylindole (DAPI, which
can stain both live and dead cells) and propidium iodide
(PI, which can only stain dead cells) were performed. The
experiments showed that the cell morphology was scatheless as
determined by comparison with the image of cells dyed with
Notes and references
1 M. Torresa and H. J. Forman, BioFactors, 2003, 17, 287.
2 W. Davis, Jr, Z. Ronai and K. D. Tew, J. Pharmacol. Exp. Ther.,
2001, 296, 1.
3 T. Lu, Y. Pan, S. Y. Kao, C. Li, I. Kohane, J. Chan and
B. A. Yankner, Nature, 2004, 429, 883.
4 J. K. Andersen, Nat. Med. (N. Y.), 2004, 10(suppl), S18.
5 (a) G. T. Hanson, R. Aggeler, D. Oglesbee, M. Cannon,
R. A. Capaldi, R. Y. Tsien and S. J. Remington, J. Biol. Chem.,
2004, 279, 13044; (b) V. V. Belousov, A. F. Fradkov,
K. A. Lukyanov, D. B. Staroverov, K. S. Shakhbazov,
A. V. Terskikh and S. Lukyanov, Nat. Methods, 2006, 3, 281.
6 (a) E. W. Miller, S. X. Bian and C. J. Chang, J. Am. Chem. Soc.,
2007, 129, 3458; (b) D. Srikun, A. E. Albers and C. J. Chang,
Chem. Sci., 2011, 2, 1156.
7 R. Radi, G. Peluffo, M. N. Alvarez and A. Cayota, Free Radical
Biol. Med., 2001, 30, 463.
8 J. S. Beckman, T. W. Beckman, J. Chen, P. A. Marshall and
B. A. Freeman, Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 1620.
9 (a) D. Yang, H. Wang, Z. Sun, N. Chung and J. Shen, J. Am.
Chem. Soc., 2006, 128, 6004; (b) T. Peng and D. Yang, Org. Lett.,
2010, 12, 4932.
10 C. Song, Z. Ye, G. Wang, J. Yuan and Y. Guan, Chem.–Eur. J.,
2010, 16, 6464.
11 G. Mugesh, W. W. du Mont and H. Sies, Chem. Rev., 2001,
101, 2125.
12 H. Sies and H. Masumoto, Adv. Pharmacol., 1997, 38, 229.
13 (a) C. W. Nogueira, G. Zeni and J. B. T. Rocha, Chem. Rev., 2004,
104, 6255; (b) S. W. May, L. Wang, M. M. Gill-Woznichak,
R. F. Browner, A. A. Ogonowski, J. B. Smith and S. H. Pollock,
J. Pharmacol. Exp. Ther., 1997, 283, 470; (c) S. Padmaja,
G. L Squadrito, J. N. Lemercier, R. Cueto and W. A. Pryor, Free
Radical Biol. Med., 1996, 21, 317.
14 D. Wu, A. B. Descalzo, F. Weik, F. Emmerling, Z. Shen, X. You
and K. Rurack, Angew. Chem., Int. Ed., 2008, 47, 193.
15 D. Oushiki, H. Kojima, T. Terai, M. Arita, K. Hanaoka, Y. Urano
and T. Nagano, J. Am. Chem. Soc., 2010, 132, 2795.
16 C. Q. Li, L. J. Trudel and G. N. Wogan, Chem. Res. Toxicol., 2002,
15, 527.
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