Synthesis, molecular docking calculation, fluorescence and bioimaging of mitochondria-targeted ratiometric fluorescent probes for sensing hypochloritein vivo

Article abstract of DOI:10.1039/d0tb02735a

Mitochondria are the main sites for the production of hypochlorite (OCl^?). The protein adenine nucleotide translocase (ANT) is located in the inner mitochondria membrane, which is mainly participated in the transportation of ions and metabolites. At the cellular organelle level, overexpression of ANT is associated with enhanced production of OCl^?, however, abnormal levels of OCl^?cause redox imbalance and loss of function of mitochondria. Herein, a novel mitochondria-targeted ratiometric fluorescent probeMi-OCl-RPhas been developed. Molecular docking calculation suggested a potential molecular target for the probe in the ANT, and the high binding energy (?8.58 kcal mol^?1) may explain the high mitochondria selectivity ofMi-OCl-RP. The unique probe exhibits excellent spectral properties including ratiometric fluorescence response signals to OCl^?(within 7 s), high selectivity and sensitivity, and a large Stokes shift (278 nm). In addition, the colocalization coefficient confirms thatMi-OCl-RPcan effectively target mitochondria. Furthermore,Mi-OCl-RPhas low toxicity and good permeability, and was successfully employed in ratiometric imaging of OCl^?in vivo, affording a robust molecular tool for investigating the biological functions of OCl^?in living systems.

Full text of DOI:10.1039/d0tb02735a

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Materials Chemistry B
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PAPER
Synthesis, molecular docking calculation,
fluorescence and bioimaging of
mitochondria-targeted ratiometric fluorescent
probes for sensing hypochlorite in vivo†
Cite this: J. Mater. Chem. B, 2021,
9, 2666
Ling Huang,
Wanting Su, Yuping Zhao, Jingting Zhan
and Weiying Lin
*
Mitochondria are the main sites for the production of hypochlorite (OClÀ). The protein adenine
nucleotide translocase (ANT) is located in the inner mitochondria membrane, which is mainly
participated in the transportation of ions and metabolites. At the cellular organelle level, overexpression
of ANT is associated with enhanced production of OClÀ, however, abnormal levels of OClÀ cause redox
imbalance and loss of function of mitochondria. Herein, a novel mitochondria-targeted ratiometric
fluorescent probe Mi-OCl-RP has been developed. Molecular docking calculation suggested a potential
molecular target for the probe in the ANT, and the high binding energy (À8.58 kcal molÀ1) may explain
the high mitochondria selectivity of Mi-OCl-RP. The unique probe exhibits excellent spectral properties
including ratiometric fluorescence response signals to OClÀ (within 7 s), high selectivity and sensitivity,
and a large Stokes shift (278 nm). In addition, the colocalization coefficient confirms that Mi-OCl-RP can
effectively target mitochondria. Furthermore, Mi-OCl-RP has low toxicity and good permeability, and
was successfully employed in ratiometric imaging of OClÀ in vivo, affording a robust molecular tool for
investigating the biological functions of OClÀ in living systems.
Received 23rd November 2020,
Accepted 3rd February 2021
DOI: 10.1039/d0tb02735a
rsc.li/materials-b
functions.10–13 Nevertheless, abnormal levels of OClÀ have been
implicated in a variety of diseases, including cardiovascular
Introduction
Mitochondria, as one of the most important subcellular orga- diseases, neurodegenerative conditions, arthritis, and cancer.14–19
nelles, are involved in a variety of key cellular processes,
Additionally, the active mitochondria of mammals are the
including the production of reactive oxygen species (ROS), main part of ROS production, with a pH range of weak alkaline
regulation of programmed cell death, and maintenance of (B8.0), and are vulnerable to ROS attack and damage, resulting
cellular protein homeostasis.1,2 The protein adenine nucleotide in mitochondria dysfunction.20–22 Compared with other analytical
translocase (ANT) is among the most abundant proteins in the methods, fluorescence imaging is one of the most favorable
inner mitochondria membrane, which is mainly participated techniques to detect biological molecules and biological
in the transportation of ions and metabolites through the parameters in living systems because of its high sensitivity
mitochondria inner membrane.3,4
and selectivity, non-interference detection, real-time imaging,
Overexpression of ANT is associated with enhanced production convenient sample preparation and spatial resolution. So far,
of ROS, which mediates cell death via ROS-dependent some fluorescent probes for the detection of OClÀ in mitochondria
upregulation.5 ROS are a family of molecular oxygen derivatives have been made.23–26 However, most of the reported probes exhibit
and free radicals with redox activity, which are produced in a relatively short Stokes shifts, and the potential mitochondria-
kind of physiological process.6–9 Hypochlorite (OClÀ) is a vital targeted protein obtained by molecular docking calculations is
member of ROS and plays a crucial role in a range of physio- not studied (Table S1, ESI†). Therefore, it is critical to study
logical and pathological processes in the human body, such as potential mitochondria-targeted proteins by molecular docking
antibacterial and anti-inflammatory physiological defence calculations and development of a ratiometric fluorescent probe
with a large Stokes shift for response to OClÀ in mitochondria.
Guangxi Key Laboratory of Electrochemical Energy Materials, Institute of Optical
In this paper, a new ratiometric fluorescent probe Mi-OCl-RP
is constructed based on the mechanism of internal charge
transfer (ICT) processes. Molecular docking calculations are
Materials and Chemical Biology, School of Chemistry and Chemical Engineering,
Guangxi University, Nanning, Guangxi 530004, P. R. China.
E-mail: weiyinglin2013@163.com
used to identify possible binding sites of Mi-OCl-RP in mito-
† Electronic supplementary information (ESI) available: Supplementary spectra,
NMR and HRMS data. See DOI: 10.1039/d0tb02735a
chondria against a potential molecular target, that is, ANT.
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The molecular docking mode shows high affinity of the probe The last group, LPS (1.0 mg mLÀ1) for 3 h and then ABAH
to ANT in mitochondria, providing high feasibility for the (200.0 mM) for 1 h were incubated with 10.0 mM Mi-OCl-RP.
specific targeting ability of the probe in mitochondria. The Before imaging, the cells were washed three times with PBS
probe recognizes OClÀ with an ultrafast response, a large buffer. The A549 cells, MCF-7 cells and RAW264.7 cells were
Stokes shift, and high selectivity and sensitivity. In addition, tested under excitation at 405 and 588 nm, respectively.
the colocalization coefficient supports that Mi-OCl-RP can
Colocalization experiment
effectively target mitochondria. Finally, the imaging experiments
of living cells and zebrafish confirm that Mi-OCl-RP can be used
to monitor the level of OClÀ in vivo.
For confirming the intracellular localization of the probe, A549
cells were used to colocalize the probe and Mito Tracker Green.
The cells were treated with Mi-OCl-RP (10 mM) for further
incubation for 15 min at 37 1C in the incubator and then
treated with 100 nM Mito Tracker Green for another 5 min.
Then the cells were washed with PBS prior to imaging.
The A549 cells under excitation were tested at 405, 490 and
588 nm, respectively.
Experimental section
Molecular docking calculations
Atomic coordinates for ANT were obtained from the crystal-
lographic structure solved at the RCSB Protein Data Bank.
Molecular docking simulations were performed with the
program AutoDock 4.2. The ligands were first optimized for
an optimal structure with chembio3D Ultra 14.0. The rigid
conformation docking between the dehydrogenated ANT and
the probe was carried out. The grid maps were calculated by
using AutoGrid. The Lamarckian genetic algorithm was
adopted in this work. Other parameters set to default. The
binding energy was obtained for further analysis.
Imaging of OClÀ in zebrafish
Zebrafish were purchased from Nanjing Eze-Rinka Biotechnology
Co., Ltd. All animal experiments were carried out in compliance
with the guidelines for the requirements of Laboratory Animals of
Guangxi University. One group of zebrafish was incubated with
Mi-OCl-RP (10 mM) stock solution at room temperature for
30 min. Under the same condition, the other groups were
pre-treated with different concentrations of OClÀ (5, 8, and 10 mM)
for 10 min, and then 10 mM of Mi-OCl-RP was added for another
30 min. Then, the four groups were transferred to the new
glass chassis for imaging. The zebrafish were imaged by a
4 Â objective lens confocal microscope.
Spectral measurements
The stock solution of 1 mM probe was fresh in dry methanol
and stored at 4 1C. The various testing analyte stock solutions
were prepared at 10 mM in twice distilled water. The test
solution contained the probe and an analyte, and was tested
in pH 7.4 PBS buffer (20 mM, 1% methanol). Then, the
absorption and fluorescence spectra were recorded.
Result and discussion
Design and synthesis of mitochondria-targeted ratiometric
fluorescence probe Mi-OCl-RP
Imaging of exogenous and endogenous OClÀ in living cells
Human lung cancer cell line (A549 cells), human breast cancer Cyanine is an important fluorescent dye with prominent spectral
cell line (MCF-7 cells), and macrophage (RAW264.7) were properties such as high fluorescence quantum yield and molar
seeded into 35 mm glass-bottom culture dishes and cultured absorption coefficient, which is used as a desirable fluorescent
for 24 h. For the imaging of exogenous OClÀ, A549 and MCF-7 platform for research and application extensively.27,28 Moreover,
cells were pre-treated with OClÀ (5, 8 and 10 mM) for 10 min, phenyl isothiocyanate is an electron-donating group, and could
and then incubated with Mi-OCl-RP (10 mM) for 20 min at 37 1C be easily oxidized by OClÀ to provide sulfoxide atoms with
in the incubator. For the imaging of endogenous OClÀ, electron-donating properties.29–31 Thus, we selected cyanine
RAW264.7 cells were divided into four groups. In one group, dye as the fluorescent emitter, and sulfur atoms of the phenyl
the cells were treated with Mi-OCl-RP (10 mM) for 20 min at isothiocyanate group as a specific recognition group for OClÀ
37 1C in the incubator. Another two groups, LPS (1.0 mg mLÀ1
)
based on the fluorescence mechanism of intramolecular charge
for 3 h or ABAH (200.0 mM) for 1 h were incubated with 10.0 mM transfer (ICT) to design the probe Mi-OCl-RP (Scheme 1).
Mi-OCl-RP in culture media for another 20 min at 37 1C. The cyanine moiety was used to provide absorption and emission.
Scheme 1 Rational design of the probe Mi-OCl-RP reporting OClÀ.
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Table 1 Binding energy of Mi-OCl-RP and ANT
pocket of ANT by forming intermolecular forces (p–p, p–cation,
hydrophobic and van der Waals’ interactions). The binding
energy of conformation 1 reaches À8.58 kcal molÀ1, which may
be caused by p–p interactions between the phenyl moiety of the
positively charged cyanine structure and the amino acid PHE81
(Fig. 1). The bonding processes of conformations 2 and 3 have
also p–cation or p–p interaction. Compared with the binding
energy of conformation 1, the affinity energies of conformations 2
and 3 are also up to À8.28 and À8.01 kcal molÀ1, with the potential
result of the interaction between the phenyl moiety of the isothio-
cyanate group and the amino acid residues (LYS271 and TRP70,
respectively) (Fig. S1 2a and 3a, ESI†). Similarly, conformation 9 has
p–p interactions. Nevertheless, the affinity energy of conformation
9 is only À6.69 kcal molÀ1. This may be due to the phenyl moiety of
the non-positively charged cyanine structure and the amino acid
PHE271 interaction (Fig. S1 9a, ESI†). The results show that the p–p
bonds may play a significant role in the high binding energy
(À8.58 kcal molÀ1) between Mi-OCl-RP and ANT.
Affinity
Affinity
Conformation
(kcal molÀ1
)
Conformation
(kcal molÀ1
)
1
2
3
4
5
À8.58
À8.28
À8.01
À7.66
À7.51
6
7
8
9
10
À7.51
À7.42
À6.96
À6.69
À6.13
It is expected that nucleophilic substitution of the phenyl iso-
thiocyanate group by OClÀ would release a cyanine unit of
secondary amines, which undergoes primary–secondary amine
tautomerization to form Cy-azyl. This tautomerization would
shift emission and absorption distinctly, showing ratiometric
fluorescence and photoacoustic response to OClÀ. The probe
was prepared via reaction of the corresponding phenyl isothio-
cyanate group with cyanine. The details of the synthetic procedure
of the probe are described in the ESI.† The chemical structures are
verified by 1H NMR, 13C NMR and HRMS analysis.
ANT in mitochondria may be a suitable candidate that
serves as a molecular target.32,33 Potential interactions of
Mi-OCl-RP and ANT are investigated by molecular docking
calculations. Table 1 shows the binding energy of interaction
between Mi-OCl-RP and ANT in the various conformations. The
binding sites and interactions are shown in Fig. 1 and Fig. S1
(ESI†), and the various conformations are held in an active
In this binding model, the intermolecular forces played a major
role in the interaction between Mi-OCl-RP and the amino acid
residues of ANT. The results suggest that strong intermolecular
forces promote Mi-OCl-RP targeting to mitochondria, and the high
binding energy (À8.58 kcal molÀ1) may explain the high mito-
chondria selectivity of Mi-OCl-RP. Thus, we hypothesize that as a
molecular target of Mi-OCl-RP, ANT may translocate it into the
mitochondria by intermolecular forces.
Spectral properties of Mi-OCl-RP
The UV-vis absorption response of Mi-OCl-RP to OClÀ was
examined in pH 7.4 PBS buffer (20 mM, 1% methanol), which
exhibited similar changes after reaction with OClÀ. NaOCl was
selected as a OClÀ source. As shown in Fig. 2a, the absorption
spectrum of Mi-OCl-RP (10 mM) showed a strong absorption
band at 580 nm and a middle band at 380 nm. After reaction
with OClÀ (0–10 mM), Mi-OCl-RP not only showed an absorption
band with the peak at 580 nm reduced, but a new band
centered at 380 nm increased prominently with an isosbestic
point at 440 nm, which could be attributed to the formation of
Cy-azyl.
Fig. 1 Molecular binding modeling of conformation 1. Tertiary structure
of ANT, active site and residues of (1a) ANT (PDB: 1OKC). The p–p
interaction is indicated by a golden block.
The emission spectrum of Mi-OCl-RP exhibited a main
emission band centered at 651 nm and a minor broad band
Fig. 2 (a) UV-vis absorption and (b and c) fluorescence spectra of Mi-OCl-RP (10 mM) in the presence of 0–10 mM OClÀ in pH 7.4 PBS (20 mM, 1%
MeOH). ((b): lex = 373 nm; (c): lex = 588 nm.) (d) Ratios of fluorescence intensity (I471/I651) of the probe (10 mM) to various relevant species (10 mM): (1)
blank; (2) OClÀ; (3) hydroxyl radicals; (4) Cys; (5) Hcy; (6) glutathione; (7) CH3COOOH; (8) H2O2; (9) t-butylhydroperoxide; (10) NO; (11) Ca2+; (12) Zn2+
;
(13) Co2+; (14) Cu2+; (15) Fe2+; (16) Mg2+; (17) FÀ; (18) ClÀ; (19) BrÀ; (20) IÀ; (21) HCO3À; (22) SO32À; (23) HSO3À; (24) NO2À; and (25) ONOOÀ in pH 7.4 PBS
buffer. Inset: Photographs showing the color of 10 mM Mi-OCl-RP before and after the addition of OClÀ (10 mM) to the solution. ((d): lex = 373 nm.).
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centered at 471 nm (lex = 373 nm), as shown in Fig. 2b.
Meanwhile, under excitation at 588 nm, it showed a strong
emission band at 651 nm (Fig. 2c). Therefore, the Stokes shift
value is calculated to be 278 nm (under excitation at 373 nm,
the emission band of Mi-OCl-RP is concentrated at 651 nm).
With the concentration increase of OClÀ, the strong fluorescence
intensity of Mi-OCl-RP at 471 nm increased and an emission
band at 651 nm decreased, the color of the solution changes
from violet-blue to light yellow and could be detected even by
the naked eyes. These results allow for ratiometric (I471/I651
)
detection of OClÀ. A linear correlation (R2 = 0.99055) between
the emission intensity ratios (I471/I651) and concentration of OClÀ
was found in the range of 0–10 mM (Fig. S2a, ESI†). The detection
limit was calculated to be 0.35 mM (according to 3s/k, where s is
the standard deviation of 11 blank measurements, and k is the
slope of the linear equation).
To verify the selectivity of Mi-OCl-RP for OClÀ, the specificity
of Mi-OCl-RP was tested in the presence of various relevant
Fig. 3 Theoretical modeling of Mi-OCl-RP response to OClÀ calculated
analytes. As shown in Fig. 2d, a large ratiometric signal by DFT using the optimal structures of the ground and the excited states.
(I471/I651) of 24.5 was observed with Mi-OCl-RP in the presence
of 10 equiv. of ClOÀ. By contrast, with some common anions,
including other biological amino acids, ROS and RNS, the
spectrum. Mi-OCl-RP possessed one electron-withdrawing group
(a positive nitrogen moiety of the cyanine group) and two
electron-donating groups (no positive nitrogen moiety of the
ratiometric signal is minimal. Fluorescence intensity of
Mi-OCl-RP in the presence of OClÀ is much weaker than other
cyanine group and the –NH– moiety), featuring a ‘‘push–pull’’
analytes under the excitation at 588 nm (Fig. S3b, ESI†). The
system to block the ICT process, which led to fluorescence.34 The
results show that Mi-OCl-RP has excellent selectivity for OClÀ.
ICT process also contributed to the lighting behavior. HOMO,
HOMO+1 and LUMO in Mi-OCl-RP were located on the fluoro-
phore, the –NH– moiety, and the whole molecular, respectively.
Then to test the photostability, Mi-OCl-RP was irradiated for
40 minutes in the absence and presence of OClÀ, and there was
no significant change at 471 and 651 nm (lex = 373 and 588 nm)
The highest-energy transition of Mi-OCl-RP came from the
(Fig. S4 and S5, ESI†), indicating that Mi-OCl-RP has good anti-
HOMO–LUMO and HOMO+1–LUMO orbital transitions. The
orbital transition of the HOMO–LUMO (HOMO+1–LUMO) in
excited state 1 (excited state 2) indicated a transfer process of
photobleaching ability.
As shown in the ESI,† Video S1, it only takes less than 7
seconds to complete the reaction, suggesting that Mi-OCl-RP
can be used for the real-time ultrafast monitoring of OClÀ.
one electron from the excited fluorophore unit (the –NH–
moiety) to the LUMO of the electron-deficient unit (ICT), leading
Finally, the pH effect of Mi-OCl-RP on the fluorescence
to fluorescence lighting. However, after the response to OClÀ,
properties of OClÀ in the pH range of 4.0–9.0 was also
the band gap energy of Mi-OCl-RP is 3.123 eV, which indicates
investigated (lex = 373 and 588 nm). The fluorescence ratio-
one charge transfer character (emission state). The results
display that Mi-OCl-RP has only one emission wavelength, which
is similar to the fluorescence spectrum (471 nm).
metric I471/I651 at pH 4.0–7.0 remained stable, and increased at
pH 7.0–9.0, when OClÀ was added, reaching the highest at
pH 8.0 (Fig. S6a, ESI†). Similarly, Mi-OCl-RP in the absence and
presence of OClÀ (10 mM) at pH 4.0–9.0 showed fluorescence
Fluorescence imaging in living cells
intensity signal under excitation at 588 nm (Fig. S6b, ESI†).
These data reveal that Mi-OCl-RP could be employed to detect Encouraged by the favorable spectral response of the probe to
OClÀ under neutral and alkaline conditions.
OClÀ, we further assessed the potential application of Mi-OCl-
RP to detect OClÀ in living cells by dual-color and ratiometric
fluorescence imaging. Before that, the cell cytotoxicity of
Mi-OCl-RP (0–40 mM) to A549 cells, MCF-7 cells and RAW264.7
The energy changes of the Mi-OCl-RP response to OClÀ
according to DFT
Density functional theory (DFT) quantum chemical calculation cells was evaluated by the standard CCK8 assay and the results
on Mi-OCl-RP and its OClÀ adduct was carried out. The show that Mi-OCl-RP shows a low cytotoxicity to A549 cells,
energies of the lowest unoccupied molecular orbital (LUMO), MCF-7 cells and RAW264.7 cells for 24 h (Fig. S7, ESI†), indicating
the highest occupied molecular orbital (HOMO) and the band that the probe could be further applied in cell imaging
gaps (HOMO–LUMO gaps) are shown in Fig. 3. It has been experiments. As shown in Fig. 4 1a-4b, the ratio of green and
found that the band gap energies of Mi-OCl-RP have two values red fluorescence exhibited different fluorescence signals. A549
(2.554 eV and 1.127 eV, respectively), which showed two charge cells incubated with only Mi-OCl-RP (10 mM) showed strong
transfer characters. The results indicate that Mi-OCl-RP exhibits green and red fluorescence signals (Fig. 4 1b-1c). However,
two excited states, which are the same as the UV-Vis absorption when the cells were treated with different concentrations of
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Mitochondrial-specific dye (Mito Tracker Green is used to
determine the presumed mitochondria-targeted property of
the probe). As shown in Fig. 5 3a-3b, the fluorescence image
of Mi-OCl-RP (Fig. 5 2a-2b) almost completely overlaps with that
of the mitochondria probe (Mito Tracker Green) (Fig. 5 1a-1b).
Furthermore, the intensity scatter plots and line profiles of
Mi-OCl-RP and Mito-Tracker Green showed high correlation
(Fig. 5 4a-5b), indicating that Mi-OCl-RP is mainly localized in
mitochondria of living cells, and the Pearson’s co-localization
coefficients were calculated to be 0.87 and 0.86, respectively.
The colocalization experiment suggests that Mi-OCl-RP targeted
mitochondria, which is consistent with the theoretical results
of the potential mitochondria-targeted protein obtained by
molecular docking calculation. The results establish that
Mi-OCl-RP is cell membrane permeable and could be used as a
fluorescent probe to image OClÀ in mitochondria of living cells.
Fig. 4 Confocal fluorescence images of Mi-OCl-RP responding to OClÀ
in A549 cells. (1a-1c) A549 cells incubated with Mi-OCl-RP (10 mM,
20 min); (2a-4c) A549 cells incubated with OClÀ (5, 8 and 10 mM) for
10 min and then incubated with Mi-OCl-RP (10 mM, 20 min). First column:
images for the ratios of excitation at 405 and 588 nm; second column:
excitation at 588 nm; third column: excitation at 405 nm. (5a-5c) Quanti-
fied relative fluorescence intensity of images 1a-4c. Scar bar: 10 mm.
Ratiometric fluorescence imaging of endogenous OClÀ
To further demonstrate that Mi-OCl-RP is used for the detection
of endogenous OClÀ, the fluorescence variation of Mi-OCl-RP in
OClÀ (5, 8 and 10 mM), the green fluorescence enhanced as monocyte-macrophages (RAW264.7) was investigated, considering
shown in Fig. 4 2b-4b and simultaneously the red fluorescence that this immune cell can be stimulated to produce high levels of
diminished as shown in Fig. 4 2c-3c, which is consistent with intracellular ROS. In order to produce high concentrations of
the results of responding spectra in aqueous solution. We can OClÀ in macrophages, the cells were used for pre-treatment with
more intuitively observe the changes in fluorescence signal as lipopolysaccharide (LPS) and then incubated with Mi-OCl-RP.
shown in Fig. 4 5a-5c. At the same time, as shown in Fig. S8 Compared with untreated cells (Fig. 6 1a-1c), the fluorescence
(ESI†), when MCF-7 cells were incubated with the probe and intensity of the green signal was significantly increased, and the
different concentrations of OClÀ, the change of fluorescence red signal was significantly decreased in the LPS-treated RAW
intensity was approximately consistent with that of A549 cells. 264.7 (Fig. 6 2a-2c). Subsequently, treatment of LPS caused an
These results illustrate that Mi-OCl-RP can be applied for increased level of endogenous OClÀ, which resulted in the
monitoring the level of OClÀ by ratiometric fluorescence elevated fluorescence signal, as apparently visualized by imaging.
imaging in living cells.
As shown in Fig. 6 4a-4c, the cells were incubated with LPS and
Mitochondria are important sites to produce ROS in living when 4-aminobenzoic acid hydrazide (ABAH, which is a specific
cells. The colocalization experiments were performed in inhibitor of MPO that suppressed the generation of HOCl) is
A549 cells using Mi-OCl-RP and a commercially available added, the fluorescence signal was approximately the same as that
Fig. 5 Confocal fluorescence images of A549 cells pre-stained with Mi-OCl-RP and subsequently co-incubated with Mito Tracker Green. (1a, 1b)
Images of Mito Tracker Green (lex = 490 nm, lem = 500–550 nm); (2a) images of Mi-OCl-RP (lex = 405 nm, lem = 420–520 nm); (2b) images of Mi-OCl-
RP (lex = 588 nm, lem = 600–700 nm); (3a) merge of 1a and 2a; (3b) merge of 1b and 2b; (4a) the overlap coefficients of excitation at 490 and 405 nm;
(4b) the overlap coefficients of excitation at 490 and 588 nm; (5a, 5b) the intensity profile of the ROI in merge images. Scar bar: 10 mm.
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Fig. 6 Confocal fluorescence images of Mi-OCl-RP responding to endogenous OClÀ in RAW264.7 cells. (1a-1c) Macrophages treated with Mi-OCl-RP
(10 mM) for 20 min; (2a-2c) macrophages pretreated with LPS (1.0 mg mLÀ1) for 3 h after preincubation with Mi-OCl-RP (10 mM) for another 20 min;
(3a-3c) macrophages pretreated with 200.0 mM ABAH for 60 min after preincubation with Mi-OCl-RP (10 mM) for another 20 min; (4a-4c) macrophages
pretreated with LPS (1.0 mg mLÀ1) for another 3 h after stimulation with 200.0 mM ABAH for 60 min, and then incubated with Mi-OCl-RP (10 mM) for
20 min (1a-4b, ratio of two emission images; 1a-4b, lex = 588 nm, lem = 600–700 nm; 1c-4c, lex = 405 nm, lem = 420–520 nm); (5a, 5b) Quantified
relative fluorescence intensity of images. Scar bar: 10 mm.
Fig. 7 Confocal fluorescence images of Mi-OCl-RP responding to OClÀ in zebrafish. (1a-1c) Zebrafish incubated with Mi-OCl-RP (10 mM, 30 min);
(2a-4c) Zebrafish incubated with OClÀ (5, 8 and 10 mM) for 10 min and then incubated with Mi-OCl-RP (10 mM, 30 min). First column: images of ratios of
excitation at 405 and 588 nm; second column: excitation at 588 nm; third column: excitation at 405 nm. (5a-5c) Quantified relative fluorescence
intensity of images 1a-4c. Scale bar: 500 mm.
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of the signal of the control group (the cells were stained with the Notes and references
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further investigate the efficacy of Mi-OCl-RP monitoring OClÀ in
zebrafish. As shown in Fig. 7 1a-1c, the zebrafish were incubated
with only Mi-OCl-RP (10 mM), which showed bright green and red
fluorescence signals. After the treatment with 5 mM or 8 mM OClÀ
for 10 min, the green fluorescence was significantly enhanced,
and dim red fluorescence was obtained at the same time (Fig. 7
2a-3c). At the same time, when zebrafish were treated with a
higher concentration of OClÀ (10 mM), the green fluorescence
relatively strengthened, and the red fluorescence faded (Fig. 7
4a-4c). It can be seen that the green fluorescence intensity
was gradually increased and the red fluorescence intensity was
evidently decreased. Similarly, the ratios of fluorescence intensity
(Fgreen/Fred) showed enhancement (Fig. 7 5a-5c). The ratiometric
fluorescence of zebrafish imaging is also consistent with the
results of living cells imaging. The results indicate that
Mi-OCl-RP is applicable in detecting OClÀ in vivo, effectively.
¨
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Conclusion
In summary, a novel mitochondria-targeted ratiometric fluo-
rescent probe Mi-OCl-RP was successfully engineered. Molecular
docking calculation indicated that ANT could be the potential
molecular target of Mi-OCl-RP. The high binding energy
(À8.58 kcal molÀ1
) may explain the high mitochondria
selectivity of Mi-OCl-RP. The new probe possesses highly
favorable properties, including ratiometric fluorescence
response signals to OClÀ (within 7 s), high selectivity and
sensitivity, and a large Stokes shift (278 nm), which could be
easily observed by the naked eye. In addition, the colocalization
coefficient suggested that Mi-OCl-RP can effectively target mito-
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toxicity, and was effectively employed in real-time and ratio-
metric imaging of OClÀ in vivo. We expect that the probe could
have great effect on the study of mitochondria-targeted protein
and biological functions of OClÀ in the living system.
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