Fluorescent Probe HKSOX-1 for imaging and detection of endogenous superoxide in live cells and in vivo

Article abstract of DOI:10.1021/jacs.5b01881

Superoxide anion radical (O₂^-) is undoubtedly the most important primary reactive oxygen species (ROS) found in cells, whose formation and fate are intertwined with diverse physiological and pathological processes. Here we report a highly sensitive and selective O₂^- detecting strategy involving O₂^- cleavage of an aryl trifluoromethanesulfonate group to yield a free phenol. We have synthesized three new O₂^- fluorescent probes (HKSOX-1, HKSOX-1r for cellular retention, and HKSOX-1m for mitochondria-targeting) which exhibit excellent selectivity and sensitivity toward O₂^- over a broad range of pH, strong oxidants, and abundant reductants found in cells. In confocal imaging, flow cytometry, and 96-well microplate assay, HKSOX-1r has been robustly applied to detect O₂^- in multiple cellular models, such as inflammation and mitochondrial stress. Additionally, our probes can be efficiently applied to visualize O₂^- in intact live zebrafish embryos. These probes open up exciting opportunities for unmasking the roles of O₂^- in health and disease.

Full text of DOI:10.1021/jacs.5b01881

Article
pubs.acs.org/JACS
Fluorescent Probe HKSOX‑1 for Imaging and Detection of
Endogenous Superoxide in Live Cells and In Vivo
†
,∥
†,∥
†
‡
†
‡
†
Jun Jacob Hu, Nai-Kei Wong, Sen Ye, Xingmiao Chen, Ming-Yang Lu, Angela Qian Zhao,
§
§
§
,†
Yuhan Guo, Alvin Chun-Hang Ma, Anskar Yu-Hung Leung, Jiangang Shen, and Dan Yang*
†
‡
§
Morningside Laboratory for Chemical Biology and Department of Chemistry, School of Chinese Medicine, and Department of
Medicine, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China
*
W Web-Enhanced Feature *S Supporting Information
ABSTRACT: Superoxide anion radical (O2•−) is undoubtedly
the most important primary reactive oxygen species (ROS)
found in cells, whose formation and fate are intertwined with
diverse physiological and pathological processes. Here we
•
−
report a highly sensitive and selective O detecting strategy
2
•
−
involving O2 cleavage of an aryl trifluoromethanesulfonate
group to yield a free phenol. We have synthesized three new
•
−
O2 fluorescent probes (HKSOX-1, HKSOX-1r for cellular
retention, and HKSOX-1m for mitochondria-targeting) which
•−
exhibit excellent selectivity and sensitivity toward O2 over a
broad range of pH, strong oxidants, and abundant reductants found in cells. In confocal imaging, flow cytometry, and 96-well
•−
microplate assay, HKSOX-1r has been robustly applied to detect O2 in multiple cellular models, such as inflammation and
•−
mitochondrial stress. Additionally, our probes can be efficiently applied to visualize O in intact live zebrafish embryos. These
2
•−
probes open up exciting opportunities for unmasking the roles of O2 in health and disease.
probes for accurate O2• detection in biological systems has
−
INTRODUCTION
Superoxide anion radical (O •−) has long been recognized as a
vital cellular signaling molecule involved in a plethora of
physiological and pathological processes including innate
■
hampered mechanistic research on ROS signaling, mitochon-
dria biology, and related diseases.
for O2 detection, hydroethidine (HE) and its mitochondria-
targeting analogue MitoSOX have been extensively used,
despite that they suffer from limitations of nonspecific DNA-
staining and autoxidation, resulting in poor selectivity and
sensitivity, as well as inappropriate cellular localization.
major challenge in achieving reliable O2 sensing is to develop
2
18,19
Among all available tools
•
−
1
−3
20
immunity and metabolic homeostasis.
Aberrant production
of O2 can lead to oxidative damage of biomolecules such as
•−
4
−6
7
21
iron−sulfur (Fe−S) proteins and cysteine thiols, or directly
induce cell death in bacterial and mammalian cells alike. Its
8
21,22
A
secondary products, such as hydrogen peroxide (H O ),
•−
2
2
•
−
hydroxyl radical ( OH), peroxynitrite (ONOO ), and hypo-
chlorous acid (HOCl), are also involved in signal transduction
and diverse pathological conditions, including pulmonary
•−
small-molecule fluorescent probes that selectively detect O2
in the presence of other reactive species in cells, in particular,
highly reactive oxygen species (hROS; up to μM concen-
9
9
10
arterial hypertension, cardiomyopathy, atherosclerosis,
23,24
9
9
tration) and ubiquitous thiols (in mM concentrations).
ischemia-reperfusion injury, diabetes mellitus, rheumatoid9
•
−
9
11
12
Previously, several O2
fluorescent probes employing non-
arthritis, autism, Alzheimer’s disease, Parkinson’s disease,
9
13,14
amyotrophic lateral sclerosis, and cancer.
Accumulating
redox strategies were reported to afford respectable selectivity
over other ROS, though they still suffered clear interference
from cellular reductants such as cysteine, glutathione (GSH; in
mM concentrations) and Fe2+ (in μM concentrations), which
renders those probes unsuitable for cell imaging.
Here we report the development of three novel O2
fluorescent probes (HKSOX-1, HKSOX-1r, and HKSOX-
m) via a nonredox strategy for ultrasensitive and selective
detection of O2 in live cells without the interference of
cellular reductants and pH.
evidence suggests that imbalances in ROS production
frequently concur with mitochondrial dysfunction and disease
9
development. Nevertheless, due to great differences in
chemical properties of various ROS (such as half-life, reactivity,
and compartmental distribution) and biochemical complexity
of cellular milieu (such as pH, antioxidants, and detoxification
systems), specific detection of single ROS in live cells and
tissues should be of tremendous help to clarify controversial
25−28
•−
1
•
−
2
,8,14−17
effects of a specific ROS in biological phenomena.
In comparison to indirect measurements, development of
selective and sensitive fluorescent probes for O2• detection
enables direct approaches to quantitate, characterize and
predict ROS events. However, lack of reliable fluorescent
−
Received: February 20, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b01881
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
Figure 1. Design of new fluorescent probes HKSOX-1 and HKSOX-1r for O2•− detection.
RESULTS AND DISCUSSION
Reactivity and Selectivity of HKSOX-1 for Superoxide.
The reaction of HKSOX-1 (10 μM) with O2 (10 equiv, 100
■
•−
Design and Synthesis of Fluorescent Probes. Inspired
•
−
μM) generated by enzymatic reaction of xanthine (X; 300 μM)
by previous works on nonredox strategy for O2 sensing, we
designed an aromatic framework with phenol OH group
protected as trifluoromethanesulfonate group. On the one
hand, the triflate group is unreactive toward abundant cellular
reductants at neutral pH. On the other hand, the trifluor-
omethyl group is a small but strong electron-withdrawing group
that can activate the sulfonate ester toward nucleophilic attack
25,27
and xanthine oxidase (XO; 0.01 U/mL)
in potassium
phosphate buffer (0.1 M, pH 7.4, 0.1% DMF) at 25 °C yielded
a dramatic time-dependent fluorescence increase, which was
completed within 10 min (Figures S1 and S2). This suggests a
•
−
very rapid reaction between the probe and O , with a rate
2
5
−1 −1
constant of ca. 2.0 × 10
M
s
(see the Supporting
•
−
Information for details), which is comparable to that of HE (2.6
by O2 , yielding a free phenol. This nonredox reaction can be
exploited to tune the fluorescence properties of xanthenes, such
as fluorescein, rhodol, and naphthofluorescein. In particular, we
envisioned that installation of triflate ester groups at the 3′ and
5
−1 −1 31
×
10 M s ). Furthermore, the fluorescence intensity
remained unchanged within 60 min, indicating high chemo-
stability of the fluorescent product (Figure S2). The identity of
the product was proven to be 5-carboxy-2′,4′,5′,7′-tetrafluoro-
fluorescein, which was also obtained in 92% yield when
6
′ positions of xanthenes would quench their fluorescence due
to lactone ring formation, while the free xanthenes can be
released upon treatment with O2• , leading to turn-on
fluorescence (Figure 1).
−
HKSOX-1 was treated with KO at 25 °C for 10 min in neutral
2
potassium phosphate buffer (Supporting Information). The
We selected a special fluorescein, 5-carboxy-2′,4′,5′,7′-
excellent stability of HKSOX-1 toward pH changes in the range
−1
−1
29
•−
tetrafluorofluorescein (ε = 78 100 cm M , Φ = 0.59), as
of 2.2−8.8 further confirms its reliability for cellular O
2
fl
the fluorophore template because of its lower pK (3.7) and
detection (Figure S3).
a
2•
−
much greater photostability compared with fluorescein (pK =
Next, different concentrations of O
(0−100 μM)
a
6
.5). The lower pK (3.7) ensures application of the probe even
generated by X/XO were added to the testing solutions
containing 10 μM HKSOX-1 (Figure 2a). The fluorescence
intensity of the resulting solution was found to increase linearly
a
under quite acidic conditions (pH = 4.0) in cells, whereas
fluorescein cannot turn on efficiently under low pH. By one-
step triflation, we prepared HKSOX-1, which was then coupled
with dimethyl iminodiacetate to yield HKSOX-1r for better
•
−
with the concentrations of O2 (0−13 μM; Figure 2b), and the
detection limit was estimated to be as low as 23 nM (signal/
noise: S/N = 3). As shown in Figure 2a, the fluorescence
30
cellular uptake and retention. To prepare mitochondria-
targeting probe HKSOX-1m, tetrafluorofluorescein derivative 1
was conjugated with a positively charged triphenylphospho-
nium moiety, followed by MOM deprotection with acid and
triflation (Scheme 1).
•−
intensity became saturated when 2 equiv of O2 was added.
This result is in good agreement with the reaction
32
stoichiometry in our proposed mechanism (Figure 1).
The specificity of HKSOX-1 was examined by measuring its
fluorescence response after exposure to various analytes in
potassium phosphate buffer (0.1 M, pH 7.4). As shown in
Figure 2c, a >650-fold enhancement in fluorescence intensity
a
Scheme 1
•−
was observed toward 4 equiv of O2 , while 10 equiv (100 μM)
•
1
•
of other strong oxidants (H O , NO, O , ROO , TBHP,
2
2
2
•
−
2+
OH, ONOO , and HOCl), reductants (Fe , ascorbic acid,
and 1,4-hydroquinone) and esterase (0.4 U/mL) could only
give negligible fluorescence increase. Strikingly, the fluores-
cence response of HKSOX-1 to high concentration of
glutathione (GSH; 5 mM, 500 equiv) was only slightly higher
than that of other analytes. Thus, the probe exhibited at least
•
−
6
2
6−100 fold selectivity toward O2 over other analytes (Figure
•
−
c and Table S1). In addition, when incubated with O
2
•−
scavenger TEMPOL (40 μM), O2 decomposition catalyst
FeTMPyP (40 μM) or superoxide dismutase (SOD; 40 U/
mL), the fluorescence intensity could be reduced to basal level.
a
Synthesis of HKSOX-1, HKSOX-1r, and HKSOX-1m. Reagents and
conditions: (a) Tf O, pyridine, DCM, −78 to 25 °C, 20 min, 41%; (b)
2
These results demonstrate the excellent selectivity of HKSOX-
DMF, SOCl , 75 °C, 1 h, 100%; (c) dimethyl iminodiacetate, K CO ,
2
2
3
•−
1
for O2 over other much stronger oxidants, reductants, or
DCM, 25 °C, 12 h, 72%. (d) 3, EEDQ, DCM, rt, 12 h, 81%; (e) 4 M
HCl in 1,4-dioxane, rt, 30 min; Tf O, DCM, pyridine, −78 °C to rt, 20
even abundant GSH, thus suggesting its potential utility as a
2
•
−
min; Amberlite IRA-400 (Cl), 10 min, 78%.
molecular probe for detecting O2 in cells.
B
DOI: 10.1021/jacs.5b01881
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Journal of the American Chemical Society
Article
rapid, reaching a plateau in about 10 min in RAW264.7 cells
(
Figure 3d).
Encouraged by its excellent sensitivity in cell imaging, we
next attempted the use of HKSOX-1r in 96-well microplate
assay as a quantitative platform. HKSOX-1r showed dose-
dependent fluorescence response (concentration range: 0.05−
1
0 μM) toward FCCP, antimycin A, and the complex V
inhibitor oligomycin A in RAW264.7 cells (Figure 4), in
agreement with confocal imaging results (Figure S6). To our
best knowledge, this is the first example of small-molecule
•
−
probes for sensitive detection of endogenous O2
quantitative microplate assays.
in
•
−
As a definitive quantitative application for detecting O2 in
live cells, flow cytometry (FACS) analysis with HKSOX-1r was
explored. Again, HKSOX-1r showed outstanding sensitivity in
fluorescence response toward antimycin A challenge (50 nM to
•
−
1
0 μM) in RAW264.7 cells (Figure S7a). O2 signal induced
by antimycin A (1 μM) was efficiently reduced by the general
antioxidant NAC (N-acetylcysteine; 10 mM) (Figure 5). In
trials with PMA (phorbol 12-myristate 13-acetate) as an
oxidative burst stimulant, similarly robust response was elicited
Figure 2. HKSOX-1 reacts with O • to give a turn-on fluorescence
−
2
(
1
Figure S7b). Overall, our FACS results suggest that HKSOX-
r has strong potential applicability in this quantitative
platform.
Based on its parent molecule HKSOX-1, HKSOX-1m, a
mitochondria-targeting probe, was synthesized for visualizing
response. Fluorescence spectra of HKSOX-1 (10 μM) in potassium
phosphate buffer (0.1 M, pH 7.4, 0.1% DMF) at 25 °C for 30 min. (a)
Incubation with different concentrations of O2• (10, 20, 30, 40, 50,
and 100 μM) by enzymatic reaction of xanthine (X) and xanthine
oxidase (XO) at 25 °C for 30 min. (b) Linear relationship between
fluorescence intensity of HKSOX-1 (10 μM) and concentration of
−
•
−
mitochondrial O2 dynamics. In confocal imaging, HKSOX-
•−
O2 (0−13 μM). (c) Incubation with various ROS/RNS and other
biological compounds at 25 °C for 30 min. The O
TEMPOL and SOD) and O decomposition catalyst (FeTMPyP)
were added to remove O2 generated by X/XO. The fluorescence
1m (10 μM) sensitively captured signals for basal and
•−
scavengers
•−
2
antimycin A (1 μM)-stimulated mitochondrial O2
in
•−
(
2
differentiated human THP-1 cells (Figure S8), at very low
laser power output (1% intensity at Ex 514 nm on Zeiss LSM
•−
intensity was measured at 534 nm by excitation at 509 nm.
5
10 Meta). By using this setting, rapid time-lapse monitoring of
•−
mitochondrial O2 in both resting and antimycin A-challenged
THP-1 cells can be easily performed without any signs of
photobleaching (Videos 1 and 2 of time-series imaging are
available).
Evaluation of HKSOX-1r/1m for Endogenous Super-
oxide Detection in Live Cells and In Vivo. To establish
applicability of HKSOX-1r in cellular studies, we first employed
mitochondrial stress as a metabolic model. Mitochondria play
crucial roles in a plethora of cellular processes such as energy
homeostasis, lipid metabolism, innate immunity, aging and cell
Inhibition of mitochondrial electron transfer chain
is a well-defined method to induce mitochondrial O
production. By using four mitochondrial respiratory inhib-
itors, rotenone (complex I inhibitor; 5 μM), FCCP (complex
II inhibitor; 5 μM), malonic acid (endogenous complex II
inhibitor; 500 μM), and antimycin A (complex III inhibitor; 5
μM), we tested the performance of HKSOX-1r in confocal
imaging in different cell types: HCT116 (human colon cancer
cells), BV-2 (mouse microglia), and RAW264.7 (mouse
macrophages). Within 30 min of inhibitor challenge, a robust
fluorescence response was reported in all three cell types
Having established HKSOX-1r as a facile cell imaging tool,
we proceeded to examine its practical utility in an oxidative
burst model, which mimics aspects of inflammation. Oxidative
burst or rapid production of ROS is characteristic of
mammalian phagocytes as a first-line defense in innate
2
,33,34
death.
•
−
2
3
5
37−39
immunity against a broad range of microbial pathogens.
3
6
•−
In particular, phagocyte O
production by enzymatic systems
2
such as NADPH oxidases (NOX) is a critical event in the
complex cascades leading to cellular redox reprogramming,
production of microbicidal secondary ROS, and eventual
3
7
•−
pathogen clearance. However, the dynamics of O
production has not been fully characterized.
2
To address this, we set out to verify the selectivity of
HKSOX-1r in LPS/IFN-γ stimulated mouse macrophages, in
•
−
(
Figure 3a and b). Interestingly, an order of efficacy was
which NOX is a well-documented O
2
source during
34
revealed: antimycin A > FCCP > rotenone > malonic acid.
Two-photon confocal imaging indicated that maximal super-
oxide production induced by antimycin A was only found in the
cytoplasm (Figure S4), suggesting that the primary source of
superoxide was cytoplasmically localized. By addition of a
gradient of mito-TEMPO (a mitochondria-targeting superoxide
scavenger), superoxide signal was dose-dependently ablated
inflammation. RAW264.7 cells were challenged overnight
(14 h) with bacterial endotoxin lipopolysaccharide (LPS, from
Salmonella typhimurium; 500 ng/mL) and proinflammatory
cytokine interferon-γ (IFN-γ, from mouse; 50 ng/mL) as
described previously, in the presence or absence of various
NOX inhibitors and O2 scavengers (Figure 6a). LPS/IFN-γ
treatment induced a maximal level of O2 production as
reflected by the fluorescence response of HKSOX-1r, which
was potently inhibited with the addition of the nonspecific
NOX inhibitor DPI (dibenziodolium chloride; 50 nM) and
40
•−
•
−
(
Figure S5). Remarkable sensitivity of HKSOX-1r was further
demonstrated in antimycin A dose-dependency imaging, in
which as little as 50 nM antimycin A could yield a detectable
fluorescence change (Figure 3c). Kinetically, O2• formation
induced by antimycin A (5 μM) was shown to be unexpectedly
−
•−
specific NOX inhibitor gp91ds-tat (2.5 μM), or O2 scavenger
FeTMPyP (50 μM). Hence, it is confirmed that HKSOX-1r
C
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Journal of the American Chemical Society
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Figure 3. HKSOX-1r detects mitochondrial respiratory inhibitor-induced O2•− formation in a highly sensitive and rapid manner. (a) Representative
images of HCT116 human colon cancer cells, BV-2 mouse microglia or RAW264.7 mouse macrophages coincubated with HKSOX-1r (2 μM) and
mitochondrial respiratory inhibitor rotenone (5 μM), FCCP (5 μM), malonic acid (500 μM), or antimycin A (5 μM) for 30 min, before confocal
imaging. Scale bar = 10 μm. (b) Quantified relative fluorescence intensities of cells as used in (a). Data are mean ± SEM; n = 13−22 cells; ***p <
0
.001 versus untreated cells. (c) Representative images of RAW264.7 cells coincubated with HKSOX-1r (2 μM) and a dose gradient of antimycin A
50 nM to 5 μM) for 30 min, before confocal imaging at high magnification (315×) (below). Relative mean fluorescence levels of cells in the same
groups imaged at lower magnification (63×) were quantified (above). Data are mean ± SEM, n = 50−100 cells; *p < 0.05; ***p < 0.001 versus
(
•
−
untreated cells. Scale bar = 10 μm. (d) RAW264.7 cells were first preloaded with HKSOX-1r (2 μM) for 30 min before confocal imaging. Basal O
2
levels were monitored for 10 min, followed by antimycin A (5 μM) challenge (arrow), and further observation (35 min) for fluorescence changes at
min intervals, at adjacent locations of the same dish of cells. Data are mean ± SEM; n = 46−73 cells. Scale bar = 10 μm. Results are representative
of three independent experiments.
5
can be used to selectively detect endogenous O • produced
−
(Figure 6b). These results are consistent with literature
2
•
−
enzymatically during inflammation. As oxidative burst has been
described in the literature to occur quite rapidly in phagocytes
such as neutrophils and macrophages in response to certain
PAMPs (pathogen-associated molecular patterns), we
assessed how RAW264.7 cells respond to a range of established
oxidative burst stimulants including PMA, f MLP (N-formyl-
Met-Leu-Phe), CytoD (cytochalasin D), and zymosan (from
Upon 30 min challenge, PMA, a
nonspecific PKC (protein kinase C) activator, induced an
increase in O2 related HKSOX-1r response, which can be
effectively attenuated by the addition of DPI (50 nM) or a PKC
descriptions that NOX-derived O
by PKCs.
production is regulated
2
44
Interestingly, the TLR2 (Toll-like receptor 2) ligand
3
5
•−
zymosan rapidly triggered O2 formation which was highly
localized (Figure S9). Other immunostimulatory molecules
such as f MLP, a bacterial oligopeptide that induces phagocyte
chemotaxis, and CytoD, a fungal alkaloid with inhibitory
activity against phagocytosis, also dose-dependently induced
41−44
Saccharomyces cerevisiae).
•
−
O2 production in RAW264.7 cells (Figures S10 and S11),
•−
suggesting that HKSOX-1r can be used to selectively detect
•
−
O2 in diverse cellular contexts. In addition, the cytotoxicity of
inhibitor (Go
̈
6983, Go
̈
6976 100, or Ro32-0432; 100 nM)
HKSOX-1/1r/1m were assessed in RAW264.7 or THP-1 cells
D
DOI: 10.1021/jacs.5b01881
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Journal of the American Chemical Society
Article
Figure 4. HKSOX-1r sensitively detects O • induced by FCCP,
−
2
antimycin A and oligomycin A in 96-well microplate fluorometric
measurement. RAW264.7 cells were preloaded with HKSOX-1r (2
μM in 50 μL HBSS) for 30 min. A dose gradient of FCCP, antimycin
A or oligomycin A (dissolved in 50 μL HBSS buffer with 2 μM
HKSOX-1r) was added to achieve the required drug concentrations
(
0.05−10 μM) for further incubation for another 30 min, before
fluorescence reading. Data are presented, without subtraction of
background fluorescence, as mean ± SEM for fluorometric measure-
ment with replicates (n = 4) in at least three independent experiments;
Figure 6. HKSOX-1r selectively detects O2•− under two types of
immune stimulation. (a) Representative images of RAW264.7 mouse
macrophages stimulated with LPS (500 ng/mL) and IFN-γ (50 ng/
mL) for 14 h, in the absence or presence of DPI (50 nM), gp91ds-tat
*p < 0.05; **p < 0.01; ***p < 0.001 versus untreated cells.
(
2 μM), or FeTMPyP (50 μM). Cells were costained with
mitochondrial dye MitoTracker Red (50 nM), nuclear DNA dye
Hoechst 33342 (150 ng/mL), and HKSOX-1r (2 μM) for 30 min
before confocal imaging. Merged: all fluorescence images merged. (b)
Representative confocal images of RAW264.7 cells preloaded with
HKSOX-1r (2 μM) for 30 min, and briefly challenged with PMA (200
ng/mL) in the absence or presence of DPI (50 nM) or a PKC
inhibitor (Go
μm.
̈
6983, Go
̈
6976, or Ro32-0432; 100 nM). Scale bar = 10
Figure 5. Quantitative application of HKSOX-1r in flow cytometry
(
(
FACS) analysis. RAW264.7 cells were coincubated with HKSOX-1r
4 μM), antimycin A (50, 100, 500, or 1000 nM) in the presence or
absence of the antioxidant NAC (10 mM) for 30 min. Results are
representative of at least three independent experiments. Representa-
tive dot plots (left) and histograms (right) of RAW264.7 cell response,
as reported by HKSOX-1r. The unstained group (cells only) and
untreated group (cells with 4 μM probe alone, 30 min) were included
as controls.
Figure 7. Confocal imaging of endogenous O2• in 72 h
postfertilization (hpf) zebrafish embryos. At 72 hpf, zebrafish embryos
were first preloaded with HKSOX-1r (10 μM) for 20 min, and briefly
challenged with PMA (200 ng/mL) or antimycin A (500 nM) for 15
min, before confocal imaging (at 10× magnification). Representative
confocal images are shown. Merge: fluorescence and phase images
merged. Scale bar = 200 μm.
−
(Figure S12), which suggested that theses probes are virtually
nontoxic to cells when used at up to 10 μM after 24 h
incubation.
To explore the potential of HKSOX-1r as an in vivo imaging
tool, we applied it to O2• detection in live zebrafish embryos
−
loading. Embryos were then subjected to challenge of PMA or
(
Figures 7 and S13). Briefly, 72 hpf (hours postfertilization)
zebrafish embryos were anaesthetized (50 mg/L tricaine) and
immersed in E3 buffer with HKSOX-1r (10 μM) for probe
antimycin A. Compared to the untreated control, significantly
•
−
increased O2 levels were observed in PMA- or antimycin A-
treated zebrafish, with distinct fluorescence distribution (Figure
E
DOI: 10.1021/jacs.5b01881
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
). These results suggest that HKSOX-1r penetrates tissues
Article
7
Foundation Professor in Haematology and received funding
from its endowment.
•−
efficiently, and has potential utility in detecting O2 in vivo.
CONCLUSION
■
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■
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•−
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-
2
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•
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Corresponding Author
yangdan@hku.hk
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The authors declare the following competing financial
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ACKNOWLEDGMENTS
■
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This work was supported by The University of Hong Kong, the
University Development Fund, and Hong Kong Research
Grants Council under General Research Fund Scheme (HKU
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