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684 Chem. Commun., 2012, 48, 684–686 This journal is c The Royal Society of Chemistry 2012
Cite this: Chem. Commun., 2012, 48, 684–686
A near-infrared fluorescent probe for monitoring ozone and imaging in
living cellsw
Kehua Xu, Shuxia Sun, Jing Li, Lu Li, Mingming Qiang and Bo Tang*
Received 21st September 2011, Accepted 8th November 2011
DOI: 10.1039/c1cc15844a
A near-infrared fluorescent probe (Trp-Cy) for endogenous
ozone is presented, which exhibited a large stokes shift about
140 nm and a rapid fluorescence response to ozone with high
selectivity and sensitivity.
The investigation of reactive oxygen species (ROS) in living
cells has accelerated the process of understanding the relation-
ship of oxidative stress to diseases, along with the development
of imaging technology. However, it is predestined that new
species of ROS generated from living cells will be constantly
discovered due to the complexity of cells. For example,
Wentworth and associates recently observed that antibodies can
catalyze the generation of previously unknown oxidants, including
dihydrogen trioxide (H2O3) and ozone (O3) in neutrophils
(PMNs) isolated from human peripheral blood,1 and reported
on ozone formation in human atherosclerotic arteries,2 although
ozone (O3) as an oxidizer harmful to human health has been
widely studied.3,4 Zhang et al. subsequently found that the
cholesterol ozonolysis products existed in clinical brain samples
and suggested that they can likely trigger misfolding of protein in
sporadic amyloid disease.5 Thus, we think that a fascinating area
of study on O3 has been opened up, while at the same time we also
realize that the lack of ozone probes suitable for monitoring O3 in
living cells will suppress its development.
Indigo carmine is a sensitive probe for O3 detection in
aqueous systems,6 but it is not a specific one.1 Recently,
Garner and coworkers synthesized a fluorescent molecular
probe (lex/lem = 497/523 nm) to successfully detect ozone in
both biological and atmospheric samples and visualize the
level of O3 in human bronchial epithelial cells when these cells
were exposed to ozone.7 However, to the best of our knowledge,
rapid and specific methods for the detection of endogenous
ozone have not yet been reported.
Ozone is a highly reactive oxygen species and has a very
short half-life of 66 s and very low concentration in living cells.
Thus, in order to effectively detect ozone derived from cells, a
new probe should be developed. Near-infrared (NIR) fluo-
rescent probe, as is well-known, is an excellent sensor for
biomolecules, being capable of affording high spatial resolution
and deep tissue imaging, and of minimizing tissue auto-
fluorescence.8 Therefore, our strategy of probe design was to
choose tricarbocyanine (Cy), a near-infrared (NIR) fluorescent
dye with a high extinction coefficient,9–11 as a signal transducer
and to choose L-tryptophan (Trp), as an O3-indicator12 to
synthesize a new optical probe, Trp-Cy, for O3 detection
(Scheme 1), based on a twisted intramolecular charge transfer
(TICT) mechanism (see ESI, Fig. S1w).13 The synthesized
probe was successfully applied for the first time to detecting
and imaging O3 derived from living cells.
Trp-Cy was easily synthesized through a simple one-step reaction
and was purified by column chromatography. The structure of
Trp-Cy was characterized with 1H-NMR, 13C-NMR and MS
(see ESIw). The spectral properties of Trp-Cy and Cy were
compared, as shown in Fig. 1a. Trp-Cy has a larger stokes shift
about 140 nm and its lmax of fluorescence excitation and emission
lies at 630 nm and 770 nm, respectively, which is a very desired
property in probe design because it can improve the detection
sensitivity and reduce photobleaching. In addition, the oxidized
Trp-Cy from the reaction of Trp-Cy with O3 was characterized by
MS (see ESIw).The response of Trp-Cy to O3 was tested and the optimal
conditions were determined (Figs S2–S4). As expected, upon
the reaction with different concentrations of O3, a gradual
increase in the fluorescence intensity was observed in Fig. 1b
and there was a good linearity between the relative fluores-
cence intensity (DF) and O3 concentration in the range
0.05–7.0 mM. The regression equation was DF = 654.3 +
1784.8 � [O3] mM with a linear coefficient of 0.9953. The limit
Scheme 1 The synthesis of Trp-Cy and reaction of Trp-Cy with O3.
Key Laboratory of Molecular and Nano Probes, Engineering ResearchCenter of Pesticide and Medicine Intermediate Clean Production,Ministry of Education, College of Chemical Engineering andMaterials Science, Shandong Normal University, Jinan, 250014,China. E-mail: [email protected] Electronic supplementary information (ESI) available: Details ofsynthesis, characterization, fluorescence properties and imaging ofTrp-Cy. See DOI: 10.1039/c1cc15844a
ChemComm Dynamic Article Links
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This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 684–686 685
of detection was 17 nM (standard deviation 10.1, n = 11).
Furthermore, a fast response of Trp-Cy to O3 was confirmed
by a kinetics experiment, in which the fluorescence intensity
instantly increased by adding O3 into the probe solution and
the intensity was kept level for at least 25 min, as shown in
Fig. 2a.
The selectivity of Try-Cy was studied. Considering the
complexity of the intracellular environment, the interferences
of various bioanalytes were carried out, including other reactive
oxygen species (ROS), reactive nitrogen species (RNS) and
biological antioxidants, such as glutathione (GSH) and ascorbic
acid (Vc). Also, in consideration that L-tryptophan could bind
many metal ions in solution, an additional test was performed
in order to determine whether metal ions, such as K+,
Na+,Ca2+, Mg2+, Zn2+, Cd2+, Co2+, Ni2+ and Cu2+, are
potential interferences. The tolerance level was defined as a
relative error not exceeding �5% in the determination of the
analytes. The results showed that the Trp-Cy probe possessed
high selectivity towards O3, as shown in Figs 2b and S5aw. Totake into account the application of Trp-Cy in cell imaging, an
experiment on extending the reaction time to 30 min was
performed. Fig. S5b showed that these interferences did not
affect the detection of O3. In addition, the molecular mechanism
of this probe to specifically recognize ozone was discussed using
mass spectrometry (see Fig. S6 and Scheme S1w).With the above results, the application of Trp-Cy was firstly
operated in living A549 lung cancer cells because lung cells are
susceptible to ozone. The A549 cells were incubated with 10 mMTrp-Cy for 30 min at 37 1C; no obvious fluorescence appeared,
as shown in Fig. 3a. When the above cells were supplemented
with 6 mM O3 for 10 min, as expected, a bright fluorescence
was observed, as shown in Fig. 3b. The bright-field image of
(b) is shown in Fig. 3c. In order to confirm the selectivity of
Trp-Cy to ozone in living cells, probe-loaded cells were
incubated with SNP (NO donor), NaClO and O2�� generated
by X/XO, as illustrated in Figs 3d–f. The experimental results
displayed that the probe can selectively respond to the change
of O3 levels in living cells.
Secondly, we chose the mouse macrophage cell line
RAW264.7 to monitor endogenous ozone, since macrophage
cells can activate the generation of ozone after exposure to
phorbol 12-myristate 13-acetate (PMA).14 RAW264.7 cells
were stimulated by PMA, and then incubated with 10 mMTrp-Cy for 30 min. There was almost no fluorescence in the
absence of stimulant in Fig. 4a, while strong fluorescence
appeared after treatment with PMA (100 ng mL�1) for 20 min,
as shown in Fig. 4b. When the cells were pre-treated with ethyl
4-vinylbenzoate (0.1 mM) as a specific scavenger of ozone,15
much weak fluorescence similar to the control panel was
observed in the stimulated cells, as shown in Fig. 4c. The
experiment results illustrated that the strong fluorescence in
Fig. 4b was indeed induced by ozone, rather than other ROS.
We then applied Trp-Cy to probe the subcellular locations
of endogenous ozone in RAW264.7 cells using confocal
fluorescence microscopy. PMA-stimulated RAW 264.7 cells
were incubated with 10 mM Trp-Cy for 10 min and then
incubated with a fluorescent nuclear stain (DAPI, 10 mg mL�1)
for 10 min. Fluorescence images were obtained, as shown in
Figs 4d–f. Co-staining with DAPI revealed the location of the
probe in the cytoplasm of RAW264.7 cells. Furthermore, con-
sidering that cyanine dyes16 can be selectively accumulated by the
mitochondria of living cells, a mitochondrial-targeted experiment
using the synthesized probe was supplemented. PMA-stimulated
RAW 264.7 cells were incubated with 10 mM Trp-Cy for 10 min
and then incubated with 50 nMMito Tracker Green FM (a green-
fluorescent mitochondrial stain) for 10 min. The fluorescence
Fig. 1 (a) Fluorescent spectra of 10 mM Cy and 10 mM Trp-Cy in
PBS at pH 7.4. (b) Fluorescence responses of 10 mM Trp-Cy to
different concentrations of O3. Inset: A linear correlation between
emission intensities and concentrations of O3 (lex/lem = 630/770 nm).
Fig. 2 (a) The time course of the fluorescence intensity of 10 mMTrp-Cy
to O3 (0 mM and 6 mM) in PBS at pH 7.4, measured at 770 nm with
excitation at 630 nm. (b) The relative fluorescence responses of Trp-Cy to
ROS, RNS and biological antioxidants (6 mM for O3 and ONOO�; 12 mMfor NO; 24 mM for 1O2 and NaClO; 30 mM for �OH, t-BuOOH; 120 mMfor H2O2 and O2
��; 0.3 mM for GSH and Vc). Black bars represent the
addition of one of these interferences to a 10 mM solution of Trp-Cy. Gray
bars represent the addition of O3 or O3 plus one of these interferences to
the probe solution. All data were acquired in 30 mM phosphate buffer
with pH 7.4 at 37 1C (lex/lem = 630/770 nm).
Fig. 3 Confocal fluorescence images of living A549 cells using a 633 nm
laser. (a) A549 cells incubated with 10 mMTrp-Cy at 37 1C for 30 min. (b)
Probe-loaded cells incubated with 6 mM O3 for 10 min. (c) A bright-field
image of (b). (d–f) Probe-loaded cells incubated with NO, NaClO and
O2�� (6 mM for each) for 10 min. All cells were rinsed three times with
0.1 M PBS buffer at room temperature before imaging.
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686 Chem. Commun., 2012, 48, 684–686 This journal is c The Royal Society of Chemistry 2012
images were obtained, as shown in Figs 5a–c. The overlay image
displayed that Trp-Cy is indeed a mitochondrial-targeted
O3 probe.
Finally, the cytotoxicity of Trp-Cy and photostability of
oxidized Trp-Cy were investigated. A methyl thiazolyl tetra-
zolium (MTT) assay was first performed in A549 cells (5 � 104
cell mL�1) dispersed within replicate 96-well microtiter plates
with probe concentrations of 10–500 mM. Absorbance was
measured at 490 nm in a TRITURUS microplate reader, as
shown in Fig. S7w. The result showed the cell viability was
90% under the experimental conditions. Then a photo-bleaching
test was carried out by means of time-sequential scanning of
the probe-loaded A549 cells incubated with 6 mMO3 for 10 min.
After 300 s of continuous irradiation with a 633 nm laser, no
obvious changes were observed in fluorescence brightness of
oxidized Trp-Cy (Figs S8 and S9w).In summary, we developed a novel NIR fluorescent probe
(Trp-Cy) for ozone. The synthesized probe has a large stokes
shift about 140 nm and possesses low cytotoxicity. The
response of Trp-Cy to O3 was rapid, highly sensitive and
selective, and the fluorescence imaging of O3 in living cells
was successfully achieved using confocal laser scanning micro-
scopy. Our probe should provide valuable tools with which to
better understand ozone’s role in human health.
This work was supported by the National Key Natural
Science Foundation of China (No. 21035003), National Natural
Science Funds for Distinguished Young Scholar (No.20725518),
National Natural Science Foundation of China (No.20875057),
Key Natural Science Foundation of Shandong Province of
China (Nos. ZR2010BZ001 and ZR2011BZ006), the Science
and Technology Development Programs of Shandong Province
of China (No. 2008GG30003012), and Program for Changjiang
Scholars and Innovative Research Team in University.
Notes and references
1 P. Wentworth Jr., J. E. McDunn, A. D. Wentworth, C. Takeuchi,J. Nieva, T. Jones, C. Bautista, J. M. Ruedi, A. Gutierrez,K. D. Janda, B. M. Babior, A. Eschenmoser and R. A. Lerner,Science, 2002, 298, 2195.
2 P. Wentworth Jr., J. Nieva, C. Takeuchi, R. Galve,A. D. Wentworth, R. B. Dilley, G. A. DeLaria, A. Saven,B. M. Babior, K. D. Janda, A. Eschenmoser and R. A. Lerner,Science, 2003, 302, 1053.
3 G. C. Chuang, Z. Yang, D. G. Westbrook, M. Pompilius,C. A. Ballinger, C. Roger White, D. M. Krzywanski,E. M. Postlethwait and S. W. Ballinger, Am. J. Physiol.: LungCell. Mol. Phys., 2009, 297, 209.
4 S. A. Jorge, C. F. Menck, H. Sies, M. R. Osborne, D. H. Phillips,A. Sarasin and A. Stary, DNA Repair, 2002, 1(5), 369.
5 Q. Zhang, E. T. Powers, J. Nieva, M. E. Huff, M. A. Dendle,J. Bieschke, C. G. Glabe, A. Eschenmoser, P. Wentworth Jr.,R. A. Lerner and J. W. Kelly, Proc. Natl. Acad. Sci. U. S. A., 2004,101(14), 4752.
6 K. Takeuchi and I. Takeuchi, Anal. Chem., 1989, 61, 619.7 A. L. Garner, M. S. C. Claudette, R. P. Bruce, D. L. George,A. Shin and K. Kazunori, Nat. Chem., 2009, 1, 316.
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Fig. 4 Confocal fluorescence images of living RAW 264.7 cells. (a)
Cells incubated with 10 mM Trp-Cy at 37 1C for 30 min. (b) Cells
incubated with 100 ng mL�1 PMA for 20 min and then incubated with
10 mM Trp-Cy at 37 1C for 30 min. (c) Cells incubated with 0.1 mM
ethyl 4-vinylbenzoate and then treated according to (b). (d) PMA-
stimulated cells incubated with 10 mM Trp-Cy at 37 1C for 10 min and
then incubated with DAPI (10 mg mL�1) for 10 min, using a 633 nm
laser. (e) The image of the (d) cells using a 405 nm laser. (f) One
overlay image of (d) and (e). All cells were rinsed three times with
0.1 M PBS buffer at room temperature before imaging.
Fig. 5 Confocal fluorescence images of living mice macrophages
(RAW 264.7). (a) PMA-stimulated cells incubated with 10 mMTrp-Cy at 37 1C for 10 min and then incubated with 50 nM Mito
Tracker Green FM for 10 min, using a 633 nm He–Ne laser. (b) The
above cells were excited by a 488 nm argon laser. (c) One overlay
image of (a) and (b). All cells were rinsed three times with 0.1 M PBS
buffer at room temperature before imaging.
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