A near-IR reversible fluorescent probe modulated by selenium for monitoring peroxynitrite and imaging in living cells

Article abstract of DOI:10.1021/ja202582x

We have developed a near-IR reversible fluorescent probe containing an organoselenium functional group that can be used for the highly sensitive and selective monitoring of peroxynitrite oxidation and reduction events under physiological conditions. The probe effectively avoids the influence of autofluorescence in biological systems and gave positive results when tested in both aqueous solution and living cells. Real-time images of cellular peroxynitrite were successfully acquired.

Full text of DOI:10.1021/ja202582x

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pubs.acs.org/JACS
A Near-IR Reversible Fluorescent Probe Modulated by Selenium for
Monitoring Peroxynitrite and Imaging in Living Cells
Fabiao Yu,† Peng Li,† Guangyue Li, Guangjiu Zhao, Tianshu Chu, and Keli Han*
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
S Supporting Information
b
Scheme 1. Structures of Cy-PSe and Its Oxidized Product
Cy-PSeO and the Fluorescence Enhancement Mechanism
ABSTRACT: We have developed a near-IR reversible fluo-
rescent probe containing an organoselenium functional
group that can be used for the highly sensitive and selective
monitoring of peroxynitrite oxidation and reduction events
under physiological conditions. The probe effectively avoids
the influence of autofluorescence in biological systems and
gave positive results when tested in both aqueous solution
and living cells. Real-time images of cellular peroxynitrite
were successfully acquired.
eroxynitrite (ONOOÀ) performs as a strong oxidizing agent
As an overall strategy, cyanine (Cy), an NIR fluorescent dye
with a high extinction coefficient,13 was selected as a signal trans-
ducer, while 4-(phenylselenyl)aniline (PSe) was selected as a
modulator because it can respond sensitively to ONOOÀ.11,14
Following the ping-pong mechanism,12 we designed and synthe-
sized a new NIR reversible fluorescent probe (Cy-PSe) for
detection of ONOOÀ in living cells through a fast photoinduced
electron transfer (PET) process. The fluorescence of Cy-PSe is
quenched as a result of PET between the modulator and the
transducer, but Se oxidation prevents the PET, causing the
fluorescence emission to be “switched on” (Scheme 1). Notably,
there is no significant change in the absorption spectrum in going
from the “off” state to the “on” state (Figure 1a). These spectral
properties indicate that the PET has been successfully regulated
in the molecule.15 On the basis of the enzymatic catalytic cycle,
selenoxide can be effectively and rapidly reduced to selenide by
GSH, at which point the probe begins to function.
Furthermore, the photophysical properties of Cy-PSe and Cy-
PSeO were investigated using time-dependent density functional
theory calculations (BP86 functional with TZVP basis sets as
implemented in the Gaussian 09 package16) to confirm the PET
mechanism. To evaluate the influence of the oxidation reaction
on the fluorescence properties, excitation energies and oscillator
strengths (f) were defined for Cy-PSe and Cy-PSeO using the
COSMO solvent model for water. The results showed that the
process of PET from the PSe moiety to the cyanine moiety
occurs in the S1 state of Cy-PSe [Figure S5 in the Supporting
Information (SI)]. The small oscillator strength of the S0 f S1
excitation (f = 0.013) implies a forbidden transition, indicating
that the S1 state is a dark state rather than an emissive state. In
other words, this cannot be accessed directly by excitation from
the S0 state. However, it can be populated by internal conversion
P
in physiological and pathological processes.1À3 In vivo,
abnormally high concentrations of ONOOÀ are formed from
the fast reaction between nitric oxide (NO) and superoxide anion
(O2À), which requires no enzymatic catalysis.4 The peroxynitrite
anion is relatively stable, but the acid form (ONOOH) rapidly
decays to nitrate. Although the half-life of ONOOH is ∼1 s at pH
7.40,5 the oxidative species contributes to signal transduction,
homeostasis regulation, and oxidative damage, which forms a
unique biological oxidationÀreduction cycle indicating human
health and disease.6 Therefore, the development of reversible
detection technology for peroxynitrite would have important
biomedical significance. In comparison with other technologies,
fluorescence microscopy provides greater sensitivity, less inva-
siveness, and more convenience.7 Especially the use of near-IR
(NIR) light (650À900 nm) allows deep penetration into tissues
and efficaciously avoids the influence of bioautofluorescence.
However, there exists a major obstacle to the design of novel
fluorescent probes for ONOOÀ, namely, the nitro group, which
is considered to be a strong quencher for fluorophores.3 To date,
only a few fluorescent probes for ONOOÀ detection have been
reported.8 Thus, we anticipate widespread interest in a redox-
reversible NIR fluorescent probe, which would exhibit much
more value for visualizing cycles of redox signaling and stress
caused by peroxynitrite.9 Here we report a redox-responsive NIR
fluorescent probe for continuous monitoring of ONOOÀ.
ONOOÀ is modulated by cellular antioxidant defense
systems,1,10 in which selenium (Se) plays an important role as
the active site of the antioxidant enzyme glutathione peroxidase
(GPx).11 GPx can catalyze the reduction of ONOOÀ by
glutathione (GSH) via a unique ping-pong mechanism.12 Tak-
ing the advantage of this, we mimicked the catalytic cycle and
developed an NIR fluorescent probe containing an organoselenium
moiety that can be used for reversible peroxynitrite detection.
Received: March 22, 2011
Published: June 27, 2011
r
2011 American Chemical Society
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Figure 3. Fluorescence recovery rate with various reducing materials.
Cy-PSe (10.0 μM) was oxidized by addition of 1 equiv of ONOOÀ, and
then the solution was treated with various reducing materials for 20 min:
1. L-cysteine (20.0 μM); 2, glutathione (GSH, 20.0 μM); 3, metallothio-
nein (100.0 μM); 4, vitamin C (100 μM); 5, vitamin E (100 μM); 6, uric
acid (100 μM); 7, tyrosine (100 μM); 8, histidine (100 μM); 9,
hydroquinone (100 μM). The fluorescence recovery percentage is
defined as (F À F0)/F Â 100%, where F is the fluorescence intensity
of Cy-PSeO (10.0 μM) and F0 is the fluorescence intensity of the probe
after addition of the reducing material.
Figure 1. (a) Absorption spectra of Cy-PSe buffer solution treated with
various amounts of 10.0 μM peroxynitrite in 0.1 M NaOH solution.
(b) Fluorescence response of 10.0 μM Cy-PSe to ONOOÀ concentra-
tions of 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 μM. Spectra
were acquired in 0.1 M PBS (pH 7.40) with excitation at 758 nm and
emission ranging from 710 to 850 nm. Inset: Relationship between the
fluorescence intensity at 775 nm and [ONOOÀ].
Figure 2. (a) Fluorescence responses of 10.0 μM Cy-PSe in 0.1 M PBS
(pH 7.40) to diverse reactive oxygen species (ROS). Bars represent
fluorescence responses to various compounds. In each group, the gray
bar shows the response to 10.0 μM ONOOÀ and the black bar the
response to another ROS: 1, blank; 2, NO (100 μM); 3, H2O2 (20À0
Figure 4. (a) Time course of Cy-PSe (10.0 μM) as measured by a
spectrofluorometer. Cy-PSe was oxidized for 1 h by 1 equiv of added
ONOOÀ, after which the solution was treated with 2 equiv of GSH for
another hour. (b) Fluorescence responses of Cy-PSe (10.0 μM) to redox
cycles. Cy-PSe was oxidized by 1 equiv of added ONOOÀ, and 8 min
later the solution was treated with 2 equiv of GSH. When the
fluorescence returned to the baseline level, another 1 equiv of ONOOÀ
was added to the mixture. The redox cycle was repeated five times
without an obvious change in fluorescence. All of the spectra were
acquired in 0.1 M PBS (pH 7.4) at λex = 758 nm. ONOOÀ was obtained
from a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1).
μM); 4, OH (200 μM); 5, ClOÀ (500 μM); 6, 1O2 (200 μM); 7, O2
3
(200 μM); 8, methyl linoleate hydroperoxide (MeLOOH, 200 μM); 9,
tert-butyl hydroperoxide (t-BuOOH, 200 μM); 10, cumene hydroper-
oxide (CuOOH, 200 μM). The probe was incubated with the various
ROS for 20 min before measurement. (b) Time courses of the responses
of Cy-PSe to the ROS in (a) for 60 min.
from the S3 state, in view of the maximum oscillator strength of
the S0 f S3 excitation (f = 1.617). In contrast to Cy-PSe, Cy-
PSeO is a more strongly fluorescent molecule because of the
absence of the PET process. The calculated results revealed that
the low-lying transition-dipole-allowed S0 f S1 excitation with
an oscillator strength f = 1.014 corresponds to the orbital
transition from the HOMO to the LUMO, both of which reside
on the cyanine moiety. These calculations are consistent with the
experimental results and rationalize the PET process (for details
of the calculations, see the SI).
We also evaluated the effect of pH on the fluorescence. The
fluorescence of the probe was quenched at pH above 8.7. How-
ever, as shown in Figure S1, the pH of the medium had hardly any
effect on the fluorescence intensity over the pH range from 4.0 to
8.6. Thus, the probe was expected to work well under physiolo-
gical conditions (pH 7.40, 0.1 M PBS).
The fluorescence and absorbance spectral properties of the probe
were examined under simulated physiological conditions (pH 7.40,
0.1 M PBS, 10.0 μM Cy-PSe). The probe showed λmax for absorption
and emission at 758 nm (ε758nm = 203 700 MÀ1 cmÀ1) and 800 nm,
respectively, both of which lie in the NIR region. Upon addition of
1 equiv of ONOOÀ to the buffer solution containing Cy-PSe, the
fluorescence intensity increased by ∼23.3-fold and the quan-
tum yield increased from 0.05 to 0.12. Interestingly, the
emission profile showed a blue shift in λmax from 800 to
775 nm (Figure 1b). This phenomenon was induced by the
pushÀpull electronic effect of the modulator moiety.17 Upon
oxidization by ONOOÀ, the probe contained an electron-
withdrawing group (the PSeO moiety) instead of an electron-
donating group (the PSe moiety). Therefore, the fluorescence
spectrum underwent a slight blue shift. We studied the ability of
the probe to quantify peroxynitrite in water solution. Figure 1b
demonstrates that there is a linear dependence of the fluorescence
intensity on the peroxynitrite concentration (0.0À10.0 μM);
the regression equation was F =
10435, with r = 0.9901.
16799[ONOOÀ] À
775nm
To study the specificity of Cy-PSe toward peroxynitrite, an
important procedure was carried out to determine whether
reactive oxygen species other than ONOOÀ could potentially
introduce signal noise. As shown in Figure 2a, no significant
fluorescence intensity changes were observed in the emission
spectra except in the case of ONOOÀ. To check whether the
chemoselective selenide switch might turn on upon incubation
with other ROS over time, the probe’s time courses with various
ROS for 1 h were measured. As Figure 2b demonstrates, Cy-PSe
selectively responded to ONOOÀ by a turn-on fluorescence
switch and avoided a host of other ROS oxidants.
Because various types of antioxidants are contained in cells, an
additional important test of the probe was performed to deter-
mine whether other reducing species would act as interferents. As
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Figure 5. Confocal fluorescence images of oxidative stress in RAW264.7
cells. Macrophage cells were treated with various stimulants and then
incubated with Cy-PSe (10.0 μM) for 5 min. (a) Control. (b) LPS
(1 μg/mL) and IFN-γ (50 ng/mL) for 4 h then PMA (10 nM) for 0.5 h.
(c) AG (1 mM), LPS (1 μg/mL), and IFN-γ (50 ng/mL) for 4 h then
PMA (10 nM) for 0.5 h. (d) TEMPO (100 μM), LPS (1 μg/mL), and
IFN-γ (50 ng/mL) for 4 h then PMA (10 nM) for 0.5 h. (e) Cells treated
with 50 μM H2O2 for 9 min. (f) Image after the administration of 50 μM
NOC-5 for 3 min.
Figure 6. CFM images of RAW264.7 cells incubated with Cy-PSe
(10.0 μM) for 5 min and rhodamine 6G (1.0 μM) for 10 min.
(aÀc) Controls. (dÀf) Cells treated with LPS (1 μg/mL) and IFN-γ
(50 ng/mL) for 4 h and then with PMA (10 nM) for 0.5 h. (a, d) Cells
loaded with Cy-PSe; (b, e) overlay of images showing fluorescence from
Cy-PSe and rhodamine 6G; (c, f) overlay of bright-field, Cy-PSe, and
rhodamine 6G images.
shown in Figure 3, the probe Cy-PSeO displayed excellent
selectivity response to thiols, which are the main antioxidants
in vivo at an intracellular concentration of ∼5 mM.18 The average
fluorescence recovery rate was up to ∼97% for reduction by
L-cysteine and GSH.
Kinetic assays of the probe for 2 h were also done. They
showed that the probe solution was stable toward the medium,
light, and air. Figure 4a indicates that the probe responded to the
change in ONOOÀ within 8 min and was reduced by GSH within
5 min. Figure 4b shows that the reversible oxidationÀreduction
cycle could be repeated at least five times with no loss of fluo-
rescence intensity. These data indicate that Cy-PSe is suitable for
reversible peroxynitrite monitoring.
Since the Cy-PSe probe showed high redox sensitivity, selec-
tivity, and reversibility with respect to the redox environment,
we next asked whether it could monitor reversible ONOOÀ
redox cycles in living cells under physiological conditions.
We chose the mouse macrophage cell line RAW264.7 as a bio-
assay model, since the macrophage cells activate the generation
of reactive oxygen and nitrogen species after exposure to
lipopolysaccharide (LPS)/interferon-γ (IFN-γ) and phorbol 12-
myristate 13-acetate (PMA).19 RAW264.7 cells were stimulated
by various reagents, incubated with Cy-PSe (10.0 μM) for 5 min,
and then washed three times with PBS buffer. There was almost
no fluorescence in the absence of stimulant (Figure 5a). However,
strong fluorescence appeared after treatment with LPS (1 μg/mL)
and IFN-γ (50 ng/mL) for 4 h followed by additional stimulation
with PMA (10 nM) for 0.5 h (Figure 5b). When the cells were
pretreated with aminoguanidine (AG), an inhibitor of nitric
oxide synthase,20 much weaker fluorescence was detected in
the stimulated cells (Figure 5c). The amount of hydroxyl
Figure 7. Confocal fluorescence images of reversible redox cycles in
living RAW264.7 cells. (a) RAW264.7 cells loaded with 10.0 μM Cy-PSe
for 5 min. (b) Dye-loaded cells treated with 10.0 μM SIN-1 for 10 min.
(c) Dye-loaded, SIN-1-treated cells incubated with GST (125 units/mL)
for 10 min. (d) Cells exposed to a second dose of SIN-1 for an additional
10 min. (e) Merged images of red (b) and bright-field (f) channels.
(f) Bright-field image of (a).
scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO),20 no
fluorescence from the stimulated cells was observed (Figure 5d).
Live RAW264.7 cells loaded with 50 μM H2O2 for 9 min and
50 μM 3-aminopropyl-1-hydroxy-3-isopropyl-2-oxo-1-triazene
(NOC-5) for 3 min also showed faint fluorescence (Figure 5e,f).
These results indicate that Cy-PSe can be used to visualize ONOOÀ
selectively in cells.
We applied Cy-PSe to probe the subcellular locations of
endogenous peroxynitrite in RAW264.7 cells using confocal
fluorescence microscopy (CFM). Weak fluorescence in the cyto-
plasm of RAW264.7 cells was detected when the cells were
loaded with Cy-PSe (10.0 μM, incubated for 5 min; Figure 6a).
Co-staining with rhodamine 6G (1.0 μM, incubated for 10 min)
radical ( OH) produced in the stimulated cells should not
3
be decreased by the presence of AG. Therefore, the weaker
fluorescence in Figure 5c illustrated that the strong fluores-
cence in Figure 5b was induced by peroxynitrite rather than
OH. Additionally, after pretreatment of cells with the superoxide
3
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revealed the location of the probe in the cytoplasm of these living
RAW264.7 cells (Figure 6b,c).21 After treating the cells with the
same stimulants used in Figure 5b, we observed greater fluo-
rescence enhancement (Figure 6d) than with the control cells,
indicating an increase in the peroxynitrite level in the cytoplasm.
By co-staining with rhodamine 6G and overlaying with bright-
field images, we confirmed that the probe was retained in the
cytoplasm (Figure 6e,f).
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In summary, we have developed an NIR reversible fluorescent
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conditions in aqueous solution and living cells. The probe
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probe can be used to visualize intracellular peroxynitrite levels
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’ AUTHOR INFORMATION
Corresponding Author
klhan@dicp.ac.cn
Author Contributions
†These authors contributed equally.
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’ ACKNOWLEDGMENT
This work was supported by NSFC (20833008) and NKBRSF
(2007CB815202).
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