High-Quantum-Yield Mitochondria-Targeting Near-Infrared Fluorescent Probe for Imaging Native Hypobromous Acid in Living Cells and in Vivo

Article abstract of DOI:10.1021/acs.analchem.6b04094

The discovery that hypobromous acid (HOBr) can regulate the activity of collagen IV has attracted great attention. However, HOBr as an important reactive small molecule has hardly ever been studied using a detection method suitable for organisms. Herein, a high-quantum-yield mitochondria-targeting near-infrared (NIR) fluorescent probe for HOBr, RhSN-mito, was designed. RhSN-mito was easily obtained by the Suzuki cross-coupling reaction. The test results show that RhSN-mito can rapidly respond to HOBr with ultrasensitivity and high selectivity. The achievement of ultrasensitivity lies in the high signal-to-noise ratio and the highest fluorescence quantum yield of the reaction product (φ_F = 0.68) in the near-infrared region, as far as we know. RhSN-mito is successfully applied to image native HOBr in mitochondria of HepG2 cells and zebrafish. Thus, RhSN-mito is a powerful tool for detecting native HOBr in vivo and is expected to provide a method to further study the physiological and pathological functions related to HOBr.

Full text of DOI:10.1021/acs.analchem.6b04094

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Article
A High-quantum-yield Mitochondria-targeting Near-infrared Fluorescent
Probe for Imaging Native Hypobromous Acid in Living Cells and in Vivo
Xiaojun Liu, Aishan Zheng, Dongrui Luan, Xiaoting Wang, Fanpeng Kong, Lili Tong, Kehua Xu, and Bo Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04094 • Publication Date (Web): 05 Jan 2017
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Analytical Chemistry
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A High-quantum-yield Mitochondria-targeting Near-infrared Fluo-
rescent Probe for Imaging Native Hypobromous Acid in Living Cells
and in Vivo
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Xiaojun Liu, Aishan Zheng, Dongrui Luan, Xiaoting Wang, Fanpeng Kong, Lili Tong, Kehua Xu*,
and Bo Tang*
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functional-
ized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Min-
istry of Education, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China.
Email: tangb@sdnu.edu.cn; xukehua@sdnu.edu.cn. Tel.: +86 0531-86180010. Fax: +86 0531-86180017.
ABSTRACT: The discovery that hypobromous acid (HOBr) can regulate the activity of collagen IV has attracted great
attention. However, HOBr as an important reactive small molecule has hardly ever been studied using a detection meth-
od suitable for organisms. Herein, a high-quantum-yield mitochondria-targeting near-infrared (NIR) fluorescent probe
for HOBr, RhSN-mito, was designed. RhSN-mito was easily obtained by the Suzuki cross-coupling reaction. The test
results show that RhSN-mito can rapidly respond to HOBr with ultrasensitivity and high selectivity. The achievement of
ultrasensitivity lies in the high signal-to-noise ratio and the highest fluorescence quantum yield of the reaction product
(
Φ = 0.68) in the near-infrared region, as far as we know. RhSN-mito is successfully applied to image native HOBr in
F
mitochondria of HepG2 cells and zebrafish. Thus, RhSN-mito is a powerful tool for detecting native HOBr in vivo and is ex-
pected to provide a method to further study the physiological and pathological functions related to HOBr.
1
9-21
Mitochondria are vital intracellular organelles, involved in
many biological processes, including cell signaling, cellu-
lar differentiation, cell growth, and triggering of cell
veloped in our group has been reported lately.
The two
reported reversible fluorescent probes are not competent
for the specific detection of HOBr. BPP is the first specific
fluorescent probe for native HOBr, yet cannot target mi-
tochondria in living cells. This means none of the report-
ed fluorescent probes for HOBr are suitable tools to inves-
tigate whether HOBr is generated in mitochondria. Con-
sequently, it is of great significance and pressing need to
design mitochondria-targeting fluorescent probe to moni-
tor native HOBr in vivo.
1
-5
apoptosis. These processes have a close relationship
6
with reactive oxygen species (ROS). As the primary con-
sumers of cellular oxygen, mitochondria are a major
source of cellular reactive ROS including known superox-
•
-
7
8
ide anion (O ) , hydrogen peroxide (H O ) , hydroxyl
2
2
2
9
2
radical (•OH) and hypochlorous acid (HOCl) . However,
it is still unknown whether HOBr is generated in mito-
chondria.
In 2014, B.G. Hudson et al. reported that HOBr can regu-
late the activity of collagen IV, based on a coupled reac-
tion of hydroxylysine and methionine in the presence of
HOBr is an important ROS very similar to HOCl, both in
its structure and formation. In organelles, HOBr and
-
-
22
HOCl are generated from a halogen anion (Br or Cl ) and
HOBr. Inspired by their work, we design a high-
10
H O under the catalysis of heme peroxidase. Recently,
quantum-yield mitochondria-targeting near-infrared (NIR)
fluorescent probe (RhSN-mito) for imaging native HOBr
in vivo. The synthesis process and reaction response to
HOBr are displayed in Scheme 1. In the design of the
probe structure, we chose rhodamine 110 as a fluorophore
and the mitochondria-targeting group. This dye possesses
high fluorescence quantum yield, good water-solubility
and a vital property of targeting mitochondria, avoiding
the introduction of additional mitochondria-targeting
functional groups in the probe molecule, such as a tri-
phenylphosphonium (TPP) moiety or quaternized pyri-
2
2
more and more mitochondria-targeting fluorescent
11-17
probes for HOCl have been reported, such as the rho-
damine thiolactone triphenylphosphonium cation (RSTPP)
11
fluorescent probe and two-photon fluorescent probes
12
that target mitochondria and lysosomes . However, the
-
low plasma level of Br , at least 1,000-fold lower than that
-
of Cl , greatly limits the establishment of detection meth-
18
od for HOBr. To date, there are only two reversible fluo-
rescent probes for HOBr/ Vc (ascorbic acid) and HOBr/
H S, and a visible fluorescent probe (BPP) for HOBr de-
2
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dine moiety. RhSN-mito was easily obtained by a Suzu-
ki cross-coupling reaction, and the response of RhSN-
mito to HOBr, as expected, was rapid, ultrasensitive and
highly selective. The achievement of ultrasensitivity (limit
of detection was 20 pM) should be attributed to the struc-
tural advantages of the probe and the reaction product: 1)
the large emission-wavelength red shift (~100 nm) be-
tween RhSN-mito and the product RhSN improves the
signal-to-noise ratio of the detected signal, because the
fluorescence signal from RhSN-mito itself cannot be
found in the noise region, that is to say, the large red-shift
can reduce the signal noise; 2) RhSN possesses the high-
Rhodamine 110 (200 mg, 0.54 mmol) was introduced in a
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flask bottle and dissolved in DMF (2 mL). To the resulting
solution was added dropwise NBS (86.5 mg, 0.486 mmol)
in DMF (1 mL) in an ice bath. The mixture was stirred in
an ice bath for approximately 1 h, then heated to room
temperature and stirring was continued for another 6 h
and monitored by thin layer chromatography (TLC). After
completion, the reaction mixture was washed with dry
acetonitrile three times, and the filtrate was concentrated
to give Compound 1 as red solid which was used directly
in the next step. HRMS: m/z calcd. for C H BrN O :
0
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+
409.0182; found: 409.0155 [M+H] .
est fluorescence quantum yield (Φ = 0.68) in the near-
F
A mixture of Compound 1 (100 mg, 0.25 mmol), 2-
methylthiophenylboronic acid (126 mg, 0.75 mmol), tet-
rabutyl ammonium bromide (8.1 mg, 0.025 mmol) and 1,1'-
bis(diphenylphosphino) ferrocene-palladium (II) chloride
dichloromethane complex (20 mg, 0.025 mmol) were dis-
solved in 15 mL toluene. To the resulted mixture were
added ethanol (8 mL) and K CO (4 mL, 4 M). The result-
ing solution was heated to 80 C and stirred for 24 h un-
der Ar atmosphere, and monitored by thin layer chroma-
tography (TLC). After completion, the reaction mixture
was cooled to room temperature, filtered and concentrat-
ed. The crude product was purified by neutral alumina
column chromatography (methanol: dichloromethane =
infrared region, as far as we know, compared to the prod-
ucts generated by all the other NIR probes reported in
23-27
literature.
On the other hand, the high selectivity to
HOBr should be attributed to the difference between
HOBr and HOCl in the coupled reaction of the amino
group and S-methyl group. According to B.G. Hudson et
al., HOCl cannot catalyze the cyclization reaction of the
2
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o
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amino group and S-methyl group in RhSN-mito. As
expected, only under the condition of excess RhSN-mito
was a non-cyclic sulfoxide compound formed, but this
didn't affect the detection of HOBr. The probe was also
successfully applied to trace the changes in the HOBr
level in the mitochondria of living cells, and we found
that the probe can visualize native HOBr in HepG2 cells
and zebrafish. Collectively, we believe that RhSN-mito is
a powerful tool for detecting native HOBr in vitro and in
vivo, and that it will provide a way to further study the
physiological and pathological functions related to HOBr.
1
:10, v/v) to provide the desired product as red yellow sol-
id (33.9 mg, 30%). HRMS: m/z calcd. for C H N O S:
2
0
20
2
3
+
1
4
53.1267; found: 453.1248 [M+H] . H NMR (400 MHz,
DMSO-d ): δ 2.36 (s, 3H), 5.18 (s, 2H), 5.91 (s, 2H), 6.11 (s,
6
1
H), 6.34-6.57 (m, 3H), 6.45 (d, J = 8 Hz, 1H), 7.21-7.32 (m,
H), 7.46 (s, 2H), 7.69-7.79 (m, 2H), 7.99 (d, J = 8 Hz, 1H).
3
13
C NMR (100 MHz, DMSO-d ): δ 15.1, 99.5, 100.5, 110.8,
6
1
1
11.9, 125.7, 126.0, 128.3, 128.9, 129.2, 130.3, 131.4, 135.2, 139.9,
50.7, 152.6, 153.6, 169.3.
Scheme 1. a) Synthesis route of RhSN-mito and b)
proposed reaction between RhSN-mito and HOBr
-
Preparation of HOBr and OBr.
Preparation of HOBr: Liquid bromine (100 μL) was dis-
solved in ultrapure water (15 mL), then titrated with Ag-
NO solution in an ice bath to the colorless endpoint and
3
then filtered. Lambert-Beer's law (Equation (1)) was used
-
1
to calculate the concentration of HOBr (ε = 160 L mol
cm ).
260
-
1
-
Preparation of OBr: Sodium hydroxide (1.1700 g) was dis-
solved in ultrapure water (10 mL), and then to this solu-
tion cooled in an ice bath was added liquid bromine
(386.66 μL), dropwise. The Lambert-Beer's law (Equation
-
(1)) was used to calculate the concentration of OBr (ε
332 L mol cm ).
=
29
3
-
1
-1
Preparation of 50 μM HOBr in specified pH: to 2 mL of
HEPES solution of specified pH value (2, 4, 6, 8, 10 and 12)
was added a certain amount of the stock solution of HOBr,
ensuring the absorbance value of which equaled that of 50
μM HOBr, according to the Lambert-Beer's law (Equation
-
1
-1
(
1), ε260=160 L mol cm ). To evaluate the effect of pH on
fluorescence intensity of RhSN-mito toward HOBr, a
final concentration of 6 μM RhSN-mito was added to the
EXPERIMENTAL SECTION
Synthesis of RhSN-mito.
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served as the blank control. With the supernate removed,
solution of 50 μM HOBr in 2 mL of HEPES of specified pH
value and the fluorescence intensities were tested.
Lambert-Beer's law: A = εbc
−
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100 μL MTT solutions (0.5 mg mL in PBS) were added to
each well away from light. After 4 h, the remaining MTT
solution was removed, and 150 μL DMSO was added to
each well to dissolve the formed formazan crystals. The
absorbance was measured at 490 nm with the microplate
reader. The experiment was repeated three times, and the
data are shown as the mean ± SD.
1
)
.
(
-
Where A is the absorbance of HOBr or OBr, ε is molar
extinction coefficient, b is the path length, b=1 cm, c is the
concentration of HOBr or OBr.
-
Determination of fluorescence quantum yield.
Fluorescence quantum yield was determined by using
Cy5.5 (Φ = 0.28 in PBS) as a fluorescence standard. For
F
0
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measurement of the fluorescence quantum yield of RhSN,
the solution of the product was adjusted to an absorbance
of ca. 0.05. The emission spectra were recorded using the
maximal excitation wavelength, and the integrated areas
of the fluorescence-corrected spectra were measured. The
fluorescence quantum yield was obtained by comparing
the area under the emission spectrum of the test samples
with that of a solution of Cy5.5 (Φ = 0.28 in PBS). The
fluorescence quantum yield was calculated using Equa-
tion 2.
F
2
 A F

nX




S
X
ΦF( X ) = ΦF(S)

( )
2
AX F
S

nS
Where Φ is the fluorescence quantum yield, A is the
F
absorbance at the excitation wavelength, F is the area
under the corrected emission curve, and n is the refrac-
tive index of the solvents used. Subscripts S and X refer to
the standard and to the unknown, respectively.
Confocal fluorescence imaging.
HepG2 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) with 10% FBS under 10% CO , 1% peni-
cillin, and 1% streptomycin at 37 C (w/v) in a 5% CO /
2
o
2
95% air incubator. HepG2 cells and HL-7702 cells were
Figure 1. a) Excitation and emission spectra of RhSN-
mito (black) and the product of RhSN-mito with HOBr
(red). b) Fluorescence intensities of RhSN-mito (6 μM)
treated with increasing concentrations of HOBr in HEPES
(10 mM, pH 7.4, 0.3% DMSO as a cosolvent). The concen-
tration range of HOBr was 0 – 30 μM, slit widths: 10/20
nm. c) Fluorescence intensities of RhSN-mito (6 μM)
treated with increasing concentrations of HOBr in HEPES
(10 mM, pH 7.4, 0.3% DMSO as a cosolvent). The concen-
tration range of HOBr was 0 – 100 nM, slit widths: 20/20
nm. d) Linear correlation between fluorescence intensi-
ties and concentrations of HOBr in the range of 0 – 100
nM. e) Selectivity of RhSN-mito (6 μM) toward HOBr
against various ROSs: 0.5 mM for H O , t-BuOOH,
grown on confocal dishes. Cells were washed three times
with PBS buffer (pH 7.4). Then, the cells were imaged
immediately using a confocal microscope with an objec-
tive lens (× 40). Excitation of probe-treated cells at 633
nm was performed using an argon laser, and the emitted
light was collected with a META detector between 650
and 750 nm.
Static confocal images of zebrafish were collected on a
Leica Laser Scanning Confocal using 10 × objective.
Zebrafish embryos were grown in normal media, and were
divided into different groups. Embryos were visualized at
72 hours after fertilization. Excitation of probe-treated
fish at 633 nm was performed using an argon laser, and
the emitted light was collected with a META detector
between 650 and 750 nm.
2
2
-
•-
1
ONOO , O , O , •OH, NO, 200 μM for HOCl, and 30 μM
2
2
for HOBr, slit widths: 10/20 nm. f) Time course of the flu-
orescence intensities of RhSN-mito (6 μM) to HOBr (30
μM) in HEPES (10 mM, pH 7.4, 0.3% DMSO as a cosol-
vent), slit widths: 10/20 nm, operating voltage: 600 v.
λ /λ = 624/663 nm.
MTT assay.
6
HepG2 cells (1 × 10 cells/well) were dispersed within 96-
well microtiter plates with 200 μL per well. Plates were
maintained at 37 C in a 5% CO / 20% O air incubator for
o
ex
em
2
2
24 h. After the supernate was discarded, the HepG2 cells
RESULTS AND DISCUSSION
were incubated with RhSN-mito of different concentra-
tions (0, 10, 20, 100, 200, 300, 500 μM) for another 2 h.
The HepG2 cells incubated with the culture medium
RhSN-mito was synthesized according to Scheme 1, and
1
13
the structure was confirmed by HRMS, H NMR and
C
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NMR (see Experimental Section). As shown in Figure 1a, photo-bleaching test showed that the reaction product is
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the excitation and emission peaks of the probe are located
at 495 and 530 nm, respectively. The reaction product of
RhSN-mito with HOBr displays excitation and emission
maxima in the NIR region at 624 and 663 nm, respectively.
The addition of HOBr triggers the formation of sul-
filimine, resulting in both the excitation and emission
spectra exhibiting a large redshift of approximately 100
nm, and the fluorescence quantum yield of the reaction
product is up to 0.68, which greatly improves the signal-
to-noise ratio and the detection sensitivity.
highly resistant to photobleaching (Figure S6). Collective-
ly, these results indicate that RhSN-mito is an excellent
fluorescence probe for HOBr and is ideal for specifically
detecting native HOBr in living cells and in vivo.
To elucidate the response mechanism of RhSN-mito to
HOBr, the fluorescence changes of RhSN-mito in pres-
-
ence of HOBr, OBr and HOCl were particularly examined
(
Figure S7). HOBr solution was prepared with Br titrated
2
by AgNO , and NaOBr solution was obtained from NaOH
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titrated by Br . As shown in Figure S7, the product of
2
Subsequently, the fluorescence measurement conditions
were optimized, and the optimized concentrations of
RhSN-mito were 6 μM (Figure S1). Given that the pH has
a significant effect on the performance of the fluorescent
probe, the fluorescent intensities of the product in HEPES
buffers with different pH values ranging from 2 to 12 were
then evaluated. After HOBr was added, remarkable flu-
orescence enhancements were observed over a wide pH
range (pH 2−12) without significant variances (Figure S2),
implying that RhSN-mito could function properly in liv-
ing cells, especially in mitochondria.
RhSN-mito with HOBr exhibited a robust increase in the
fluorescence intensity at 663 nm, while OBr and HOCl
-
did not trigger any obvious fluorescence fluctuation at
663 nm. Accordingly, the reaction mechanisms for the
response of RhSN-mito to HOBr and HOCl were pro-
posed (Scheme 2 and S1), which were in accord with the
22
reported behaviors of HOBr and HOCl. It is HOBr ra-
-
ther than OBr or HOCl that catalyzes the cyclization re-
action of the amino group and the S-methyl group in
-
RhSN-mito. In detail, HOBr, converted from Br , formed
a bromosulfonium-ion intermediate that energetically
selects for this cyclization reaction.
The fluorescence responses of RhSN-mito (6 μM) to
HOBr were investigated in HEPES 7.4 (10 mM) at 37 °C
(
λex = 624 nm). Figure 1b and 1c show that the fluores-
Scheme 2. The proposed reaction mechanism for the
response of RhSN-mito to HOBr
cence intensity of the reaction product increased in a
dose-dependent manner over a wide concentration range
of HOBr. Figure 1d depicts the good linearity between the
fluorescence intensity and the concentration of HOBr in
the range of 0 to 100 nM. The regression equation was F =
0
.64 + 1.94 [HOBr] nM, with a linear coefficient of 0.9975.
The limit of detection (3S/m) was determined to be as low
as 20 pM, which makes the probe feasible for monitoring
the endogenous HOBr. To the best of our knowledge,
RhSN-mito is the first NIR fluorescent probe capable of
the ultrasensitive detection of HOBr at the picomolar
level (Table S1).
To inspect whether RhSN-mito could specifically moni-
tor HOBr in the complicated intracellular environment,
we tested its ability to discriminate between HOBr and
various bioanalytes, such as competing ROS, RNS, and
relevant biological substances. As shown in Figure 1e,
RhSN-mito did not display an obvious fluorescence in-
crease in the presence of other ROS, including HOCL,
-
•-
1
H O , peroxynitrite (ONOO ), O , singlet oxygen ( O ),
2
2
2
2
•
OH, nitric oxide (NO) and TBHP. The metal ions and
amino acids present in the living cells had little interfer-
ences (Figure S3). Furthermore, after HOBr reacted with
RhSN-mito, the addition of various reductants caused
little decrease in the fluorescence intensities (Figure S4),
confirming the high stability of the reaction product. Fig-
ure 1f depicts the rapid response of RhSN-mito toward
HOBr. The fluorescence intensity increased instantly up-
on the addition of HOBr into the probe solution and was
stable for at least 20 min. Additionally, an MTT assay was
performed in HepG2 cells (Figure S5), and the IC50 value
was 650 μM, indicating the low toxicity of RhSN-mito. A
Figure 2. Fluorescence images of mitochondria in HepG2
cells. HepG2 cells were incubated with Mito Tracker Green
(500 nM) and RhSN-mito (50 μM), respectively, for 30 min
and subsequently incubated with HOBr (100 μM) for 30 min.
a) Emission from the green channel (Mito Tracker Green,
λex= 488 nm, λem: 500–600 nm), b) emission from the red
channel (RhSN-mito, λ = 633 nm, λem: 650–750 nm), c)
ex
merged image of images a and b, d) corresponding bright-
field image, e) intensity correlation plot of RhSN-mito and
Mito Tracker Green, f) intensity profile of ROIs across HepG2
cells. Scale bar = 25 μm.
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Then, RhSN-mito was used to detect HOBr in living cells
Next, we examine the mitochondria-targeting perfor-
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mance of RhSN-mito. The co-localization experiments
were conducted by co-staining HepG2 cells with Mito
Tracker Green (a typical commercially available mito-
chondrial tracker) and RhSN-mito. The fluorescence of
RhSN-mito from the co-stained cells in the presence of
HOBr (Figure 2b) overlaid well with that of Mito Tracker
Green (Figure 2a), as shown in the merged image (Figure
under physiological conditions. As shown in Figure 3b
and 3d, with Br and HOBr stimulation, the fluorescence
-
intensity of the HepG2 cells was greatly enhanced. And
the fluorescence response could largely be inhibited by
NAC (a scavenger of HOBr) (Figure 3c). These results
demonstrate that RhSN-mito is capable of monitoring
the exogenous HOBr in HepG2 cells. Additionally, it is
worth noting that a weak fluorescence signal was ob-
served in HepG2 cells treated only with RhSN-mito (Fig-
ure 3a and 3i). It was inferred that RhSN-mito due to its
ultrasensitivity could detect the native HOBr in living
cells without stimulation. Subsequently, an experiment by
pre-treating cells with and without NAC was carried out
to verify this inference. As shown in Figure 4, the fluores-
cence-inhibiting effect of NAC was observed in both
HepG2 and HL-7702 cells, further supporting the idea
that the fluorescence of the living cells incubated only
with RhSN-mito arises from the native HOBr. Moreover,
it was observed that the fluorescence signal from HepG2
cells was brighter than that of HL-7702 cells subjected to
the same treatment.
2c). The changes in the intensity profiles of the linear re-
gions of interest (ROIs) (RhSN-mito and Mito Tracker
Green co-staining) were synchronous (Figure 2e and 2f).
The co-localization was qualified using Pearson’s sample
correlation factors. A high Pearson's coefficient of 0.95
and an overlap coefficient of 0.94 are obtained, confirm-
ing that RhSN-mito was specifically located in the mito-
chondria of the living HepG2 cells.
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Figure 3. Fluorescence images of HepG2 cells incubated with
RhSN-mito (50 μM) for 30 min and subsequently incubated
for 30 min with a) nothing as a control, b) NaBr (100 μM), c)
NaBr (100 μM) plus NAC (20 μM) and d) HOBr (100 μM),
respectively. e)-h) are corresponding brightfield images of (a)
to (d). i) Normalized fluorescence intensity of cells in panels
a)-d). λex = 633 nm, λem: 650–750 nm, scale bar = 25 μm.
Figure 5. In vivo fluorescence imaging of HOBr in zebrafish
embryos developed to 71 h. a) Zebrafish fed with RhSN-mito
-
for 30 min (50.0 µM), b) zebrafish fed with Br (100 μM) for
30 min and then fed with RhSN-mito (50.0 µM) for 30 min
and c) zebrafish incubated with NAC (20.0 µM) for 30 min
and then with RhSN-mito (50.0 µM) for 30 min. λex = 633
nm, λem: 650–750 nm, scale bar = 500 μm.
Finally, RhSN-mito was applied to image HOBr in living
animals. Zebrafish was chosen as the bioassay model. Fig-
ure 5b shows that the fluorescence intensity significantly
-
increased with the feeding of Br . A comparison between
Figure 5a and Figure 5c indicates that RhSN-mito could
Figure 4. Fluorescence imaging of native HOBr. (a) HepG2
cells incubated with RhSN-mito (50 μM) for 30 min, (b)
HepG2 cells incubated with NAC (20.0 µM) for 30 min and
then with RhSN-mito (50.0 µM) for 30 min, (c) HL-7702
cells incubated with RhSN-mito (50 μM) for 30 min, (d) HL-
visualize native HOBr in zebrafish. These fluorescence
-
images prove that HOBr can be generated from Br in
zebrafish, which is in accord with previously reported
29
results. Thus, RhSN-mito is a powerful tool for detect-
ing HOBr in vivo.
7702 cells incubated with NAC (20.0 µM) for 30 min and then
with RhSN-mito (50.0 µM) for 30 min. e)-h) are the corre-
sponding bright-field images of a)-h). i) Normalized fluores-
CONCLUSIONS
In conclusion,
a high-quantum-yield mitochondria-
cence intensities of cells in panels a)–d). λex = 633 nm, λem
:
targeting NIR fluorescent probe for HOBr, RhSN-mito,
has been developed based on a specific cyclization reac-
tion. The probe can respond promptly to HOBr with ul-
650–750 nm, scale bar = 25 μm.
5
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Analytical Chemistry
trasensitivity, and the limit of detection is as low as 20 pM.
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(17) Li, D.; Feng, Y.; Lin, J.; Chen, M.; Wang, S.; Wang, X.; Sheng,
This probe is able to target mitochondria and monitor the
native HOBr in HepG2 cells and zebrafish. Overall,
RhSN-mito is an ideal tool for the further study of the
physiological and pathological roles of HOBr.
H.; Shao, Z.; Zhu, M.; Meng, X. Sens. Actuators, B 2016, 222, 483-
4
91.
18) Hawkins, C. L.; Davies, M. J. Chem. Res. Toxicol. 2005, 18,
1600-1610.
19) Yu, F.; Song, P.; Li, P.; Wang, B.; Han, K. Chem. Commun.
2012, 48, 7735-7737.
20) Wang, B.; Li, P.; Yu, F.; Chen, J.; Qu, Z.; Han, K. Chem. Com-
mun. 2013, 49, 5790-5792.
21) Xu, K.; Luan, D.; Wang, X.; Hu, B.; Liu X.; Kong F.; Tang, B.
(
(
ASSOCIATED CONTENT
(
SUPPORTING INFORMATION
(
Materials and instruments, supporting figures and images.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Angew. Chem. Int. Ed. 2016, 55, 12751-12754.
(22) McCall, A. S.; Cummings, C. F.; Bhave, G.; Vanacore, R.; Page-
McCaw, A.; Hudson, B. G. Cell 2014, 157, 1380-1392.
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23) Beija, M.; Afonso, C. A. M.; Martinho, J. M. G. Chem. Soc. Rev.
009, 38, 2410-2433.
24) Tang, B.; Yu, F.; Li, P.; Tong, L.; Duan, X.; Xie, T.; Wang, X. J.
Am. Chem. Soc. 2009, 131, 3016-3023.
25) Yuan, L.; Lin, W.; Yang, Y.; Chen, H. J. Am. Chem. Soc. 2012,
134, 1200-1211.
26) Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N.; Gao, Y.; Xie, Y.;
Kong, F.; Tang, B. Chem. Sci. 2013, 4, 1079-1086.
(27) Wang, F.; Zhou, L.; Zhao, C.; Wang, R.; Fei, Q.; Luo, S.; Guo,
Z.; Tian, H.; Zhu, W. Chem. Sci. 2015, 6, 2584-2589.
2
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AUTHOR INFORMATION
Corresponding Author
(
*
8
tangb@sdnu.edu.cn. Tel.: +86 0531-86180010. Fax: +86 0531-
6180017.
(
*
xukehua@sdnu.edu.cn.
ORCID
Bo Tang: 0000-0002-8712-7025
(
28) Shao, X.; Kang, R.; Zhang, Y.; Huang, Z.; Peng, F.; Zhang, J.;
Wang, Y.; Pan, F.; Zhang, W.; Zhao, W. Anal. Chem. 2015, 87, 399-
405.
Author Contributions
All authors have given approval to the final version of the
manuscript.
(
29) Fidler, A. L.; Vanacore, R. M.; Chetyrkin, S. V.; Pedchenko, V.
K.; Bhave, G.; Yin, V. P.; Stothers, C. L.; Rose, K. L.; McDonald, W.
H.; Clark, T. A.; Borza, D. B.; Steele, R. E.; Ivy, M. T.; Hudson, J. K.;
Hudson, B. G. Proc. Natl. Acad. Sci. U S. A. 2014, 111, 331-336.
ACKNOWLEDGMENT
This work was supported by 973 Program (2013CB933800)
and National Natural Science Foundation of China (21390411,
21535004, 21275092, 21575081 and 21507075).
REFERENCES
(
1
1) Xiao, H.; Li, P.; Zhang, W.; Tang, B. Chem. Sci. 2016, 7, 1588-
593.
(2) Hoye, A. T.; Davoren, J. E.; Wipf, P.; Fink, M. P.; Kagan, V. E.
Accounts Chem. Res. 2008, 41, 87-97.
(
1
(
3) Kroemer, G.; Galluzzi, L.; Brenner, C. Physiol. Rev. 2007, 87, 99-
63.
4) Hajnóczky, G.; Csordás, G.; Das, S.; Garcia-Perez, C.; Saotome,
M.; Sinha Roy, S.; Yi, M. Cell Calcium 2006, 40, 553-560.
(5) Green, D. R. Cell 1998, 94, 695-698.
(6) Fulda, S.; Galluzzi, L.; Kroemer, G. Nat. Rev. Drug Discovery
2
(
010, 9, 447-464.
7) Li, P.; Liu, L.; Xiao, H.; Zhang, W.; Wang, L.; Tang, B. J. Am.
Chem. Soc. 2016, 138, 2893-2896.
8) Dickinson, B. C.; Lin, V. S.; Chang, C. J. Nat. Protoc. 2013, 8,
(
1249-1259.
(9) Zhuang, M.; Ding, C.; Zhu, A.; Tian, Y. Anal. Chem. 2014, 86,
1
(
829-1836.
10) Weiss, S. J.; Test, S. T.; Eckmann, C. M.; Roos, D.; Regiani, S.
Science 1986, 234, 200-203.
11) Zhou, J.; Li, L.; Shi, W.; Gao, X.; Li, X.; Ma, H. Chem. Sci. 2015,
, 4884-4888.
(
6
(12) Yuan, L.; Wang, L.; Agrawalla, B. K.; Park, S.; Zhu, H.; Sivara-
man, B.; Peng, J.; Xu, Q.; Chang, Y. J. Am. Chem. Soc. 2015, 137,
5
930-5938.
13) Xiao, H.; Xin, K.; Dou, H.; Yin, G.; Quan, Y.; Wang, R. Chem.
Commun. 2015, 51, 1442-1445.
14) Xiao, H.; Li, J.; Zhao, J.; Yin, G.; Quan, Y.; Wang, J.; Wang, R.
J. Mater. Chem. B 2015, 3, 1633-1638.
15) Hou, J.; Wu, M.; Li, K.; Yang, J.; Yu, K.; Xie, Y.; Yu, X. Chem.
Commun. 2014, 50, 8640-8643.
16) Li, G.; Lin, Q.; Ji, L.; Chao, H. J. Mater. Chem. B 2014, 2, 7918-
(
(
(
(
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