A mitochondria-targeted ratiometric fluorescence sensor for the detection of hypochlorite in living cells

Article abstract of DOI:10.1016/j.dyepig.2019.107708

A new fluorescence probe based on phenothiazine-coumarin platform was rationally designed and developed for the detection of hypochlorite in a ratiometric manner in aqueous solution and living cells. The probe possessed a large pseudo Stokes shift (209 nm) and lager gap between two emissions (462/629 nm), which should avail to perform more accurate detection. The probe could respond to hypochlorite with selectivity, sensitivity and celerity. Moreover, the probe was successfully used for imaging endogenous hypochlorite in mitochondria of RAW264.7 macrophage cells with high sensitivity.

Full text of DOI:10.1016/j.dyepig.2019.107708

Accepted Manuscript
A mitochondria-targeted ratiometric fluorescence sensor for the detection of
hypochlorite in living cells
Wei Wang, Jun-Ya Ning, Jin-Ting Liu, Jun-Ying Miao, Bao-Xiang Zhao
PII:
S0143-7208(19)31253-7
DOI:
https://doi.org/10.1016/j.dyepig.2019.107708
Article Number: 107708
Reference:
DYPI 107708
To appear in: Dyes and Pigments
Received Date: 31 May 2019
Revised Date: 4 July 2019
Accepted Date: 9 July 2019
Please cite this article as: Wang W, Ning J-Y, Liu J-T, Miao J-Y, Zhao B-X, A mitochondria-targeted
ratiometric fluorescence sensor for the detection of hypochlorite in living cells, Dyes and Pigments
(2019), doi: https://doi.org/10.1016/j.dyepig.2019.107708.
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A mitochondria-targeted ratiometric fluorescence sensor for the
detection of hypochlorite in living cells
Wei Wanga, , Jun-Ya Ningb, , Jin-Ting Liua, Jun-Ying Miaob, Bao-Xiang Zhaoa*
aInstitute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan
250100, PR China
bInstitute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
*Corresponding author. Tel.: +86 531 7862972268; fax: +86 531 7862972268.
E-mail address: bxzhao@sdu.edu.cn.
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Abstract
A new fluorescence probe based on phenothiazine-coumarin platform was rationally designed
and developed for the detection of hypochlorite in a ratiometric manner in aqueous solution and
living cells. The probe possessed a large pseudo Stokes shift (209 nm) and lager gap between two
emissions (462/629 nm), which should avail to perform more accurate detection. The probe could
respond to hypochlorite with selectivity, sensitivity and celerity. Moreover, the probe was
successfully used for imaging endogenous hypochlorite in mitochondria of RAW264.7 macrophage
cells with high sensitivity.
Keywords: Fluorescence; FRET; Stokes shift; Ratiometric; Endogenous hypochlorite;
Mitochondria-targeted.
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1. Introduction
As one of reactive oxygen species (ROS) [1, 2], hypochlorous acid/hypochlorite (HOCl/OCl−)
plays an essential role in our daily life. HOCl/OCl− is usually related to various diseases such as lung
injury, asthma, arthritis and even some cancers [3-6]. Endogenous HOCl/OCl− is generated from
chloride ion and hydrogen peroxide in the myeloperoxidase (MPO)-catalyzed reaction and plays an
important role in resisting multifarious pathogens bacteria and pathogens in organism [7-9]. Hence,
developing reliable methods to detect HOCl/OCl− quantitatively is urgent. Fortunately, fluorescent
probes have many prominent advantages in quantitative detection such as low cost, high sensitivity,
excellent selectivity and fast response [10-17]. In the past few years, various fluorescence probes
have been designed on different fluorophores such as cyanine [18], BODIPY [19, 20], coumarin [21,
22], rhodamine [23, 24], phenothiazine [25, 26]. But most of them were intensity-based fluorescence
probes which were usually affected by many factors such as instrumental deviation and environment
variations. On the contrary, ratiometric fluorescence probes could sufficiently overcome these
shortcomings by means of the fluorescence intensity ratios of two emission peaks [27, 28]. Therefore,
ratiometric fluorescence probes based on different platforms, including FRET [29-32], TBET [33,
34], and ICT [35-37], have remained a focus of extensive research activity. Moreover,
organelle-targeted fluorescence probes for the selective detection of chemical species were another
intriguing direction. So far, some ratiometric and organelle-targeted fluorescence probes for the
detection of HOCl/OCl− have been reported [38, 39], but most of them existed widespread
drawbacks in Stokes shifts, response time and organelle-targeted capacity. Therefore, rational design
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of ratiometric and organelle-targeted fluorescence probes for HOCl/OCl− based on new platforms
and fluorophores is still challenging.
Phenothiazine and its derivatives as fluorophores showed many outstanding advantages such as
high fluorescence quantum yield, large pseudo Stokes shift, excellent photostability and
the ability of being chemical modified. So some fluorescence probes for the detection of HOCl/OCl−
based on phenothiazine derivatives have been reported recent years [25, 26]. However, almost all
these phenothiazine-based fluorescence probes for the detection of HOCl/OCl− were based ICT
platform and single emission. To date, the Förster resonance energy transfer (FRET) mechanism has
not been applied to phenothiazine-based fluorescence probes for the detection of HOCl/OCl−. As
a continuation of our efforts directed toward the development of FRET-based ratiometric
fluorescence probes [29-32], we designed a new phenothiazine-based ratiometric fluorescence probe
(named PPC) for the detection of HOCl/OCl− in aqueous solution. Probe PPC showed excellent
selectivity toward HOCl/OCl−. The limit of detection was calculated to be as low as 0.321 µM,
suggesting the high sensitivity. Importantly, probe PPC could respond to HOCl/OCl− shortly (within
40 s). What's more, probe PPC was successfully used for the fluorescence imaging of endogenous
HOCl/OCl− in mitochondria of RAW264.7 macrophage cells with high sensitivity.
2. Experimental
2.1 Equipment and materials
13
1H NMR (300 MHz) and C NMR (100 or 75 MHz) was recorded on a Bruker Avance 300
spectrometer or Bruker Avance 400 spectrometer with DMSO-d6 as a solvent and TMS as an internal
standard. Mass spectra were recorded using a Q-TOF6510 spectrograph (Agilent). Melting points
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were recorded by an XD-4 digital micro melting point apparatus. UV-vis spectra were obtained by a
Hitachi U-4100 spectrophotometer. pH was measured by a PHS-3C pH meter. All fluorescence
spectra were recorded by the excitation at 420 nm.
2.2 Preparation of test solutions
The probe was dissolved in DMSO to form a stock solution (10-3 M). The test systems were
prepared by adding 50 µl stock solution into a 10 ml volumetric flask and diluting it to 10 ml with
phosphate buffer (pH = 7.4). Various testing species were added with a micropipette to the above test
systems. All these test solutions would be measured immediately.
2.3 Preparation of ROS and RNS
Various ROS (0.125 mM) and RNS (0.125 mM) were prepared by the following methods. NaClO,
H2O2, t-BuOOH solutions were obtained by diluting their stock solution with twice-distilled water. F-,
2-
2-
2-
-
Cl-, Br-, I-, CO3 , SO4 , AcO-, HPO3 , NO2 , Na+, Al3+, Mg2+, Cu2+, Fe2+, Zn2+, Fe3+, Ca2+ solutions
were prepared by dissolving corresponding inorganic salts in twice-distilled water. Hydroxyl radical
(•OH) was produced by Fenton reaction on mixing FeSO4·7H2O with H2O2. Nitric oxide (NO) was
prepared by potassium nitroprusside. Tert-butoxy radical (t-BuO•) [30], peroxynitrite (ONOO-) [40],
superoxide (-O2) [41], single oxygen (1O2) [42] were produced as the reported methods.
Scheme 1
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2.4 Synthesis of
10-(3-(4-(7-(diethylamino)-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)propyl)-10H-phenothiazi
ne-3-carbaldehyde (PPC).
Compound 2-4, PPC-S, Donor and Acceptor were synthesized according to literature [43, 44]
(Scheme S1).
10-(3-(4-(7-(Diethylamino)-2-oxo-2H-chromene-3-carbonyl)piperazin-1-yl)propyl)-10H-phenot
hiazine-3-carbaldehyde (PPC-S) (300 mg, 0.50 mmol) and malononitrile (33 mg, 0.50 mmol) were
dissolved in absolute ethanol (30 ml). The reaction mixture was heated and kept refluxing for 2 h.
The solvent was removed under reduced pressure and the crude product was purified by column
chromatography on silica gel (CH2Cl2: CH3OH= 10: 1) to give PPC (205.3 mg) in 63.68% yield
1
(Scheme 1). Red solid, m.p.: 142-144 ; H NMR (300 MHz, DMSO-d6): δ = 1.13 (t, J = 6.9 Hz,
6H), 1.83 (t, J = 6.3 Hz, 2H ), 2.34 (s, 4H), 2.41-2.51 (m, 2H), 3.24 (s, 2H), 3.41-3.48 (m, 6H), 4.04
(t, J = 6.9 Hz, 2H), 6.54 (d, J = 2.4 Hz, 1H), 6.73 (dd, J1 = 5.7 Hz, J2 = 3.3 Hz, 1H), 7.02 (t, J = 7.4
Hz 1H), 7.16 (d, J = 7.8 Hz, 2H), 7.21-7.27 (m, 2H), 7.48 (d, J = 9 Hz, 1H), 7.68 (d, J = 2.1 Hz, 1H),
7.82 (dd, J1 = 5.4 Hz, J2 = 3.3 Hz 1H), 7.92 (s, 1H), 8.23 (s, 1H). 13C NMR (100 MHz, DMSO-d6):
164.34, 159.21, 158.81, 157.02, 151.66, 150.49, 143.91, 142.57, 132.36, 130.49, 129.21, 128.59,
127.74, 125.87, 124.53, 123.40, 122.14, 117.22, 116.65, 116.17, 115.41, 114.55, 109.82, 107.59,
96.80, 76.47, 54.64, 46.80, 45.55, 44.63, 23.88, 12.76. HRMS: m/z calculated for [C37H36N6O3S +
H]+: 645.2642, found 645.2458 (Fig. S1-4).
Acceptor: 1H NMR (300 MHz, CDCl3): δ = 2.09-2.19 (m, 2H), 3.76 (t, J = 6.3 Hz, 2H), 4.13 (t,
J = 6.8 Hz, 2H), 7.02-7.06 (m, 1H), 7.16-7.29 (m, 4H), 7.71 (d, J = 2.4 Hz, 1H), 7.85 (dd, J1 = 3.3
Hz, J2 = 5.4, 1H), 8.27 (s, 1H). 13C NMR (75 MHz, DMSO-d6): 158.80, 149.79, 141.97, 131.70,
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128.94, 128.16, 127.40, 125.62, 124.19, 123.40, 121.97, 116.64, 115.73, 114.84, 113.96, 76.46, 44.14,
42.42, 28.83 (Fig. S5, 6).
2.5 Culture and imaging of HOCl/OCl− in RAW264.7cells
RAW264.7 macrophage cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
o
containing 10% fetal bovine serum in an atmosphere of 5% CO2 and 95% air at 37 C. RAW264.7
macrophage cells were stimulated by 1 µg/ml lipopolysaccharides (LPS) for 12 h and then incubated
with probe PPC (2 µM) for 30 min. After cells were washed three times with PBS, images were
obtained by a confocal microscope (LSM 700) at 405 nm excitation. The images of cells were
collected at emission channels of 405-555 nm (blue channel) and 560-700 nm (red channel).
3. Results and discussion
Scheme 2
3.1. Selectivity of probe PPC toward HOCl/OCl− by fluorescence method
The selectivity of probe PPC toward HOCl/OCl− was verified in aqueous solution (10 mM PBS
with 1 mM Triton X-100, pH = 7.4). The emission spectra of probe PPC in the presence of various
species (vacant, H2O2, KO2, NO, 1O2, t-BuOOH, t-BuOO•, •OH, ONOO-, Na+, Al3+, Zn2+, Ca2+, Fe3+,
2-
2-
-
2-
Fe2+, Cu2+, Mg2+, F-, Cl-, Br-, I-, CO3 , SO4 , NO2 , AcO-, HPO3 , HOCl/OCl−, Cys, GSH, Hcy, H2S
-
and HSO3 were obtained (Fig. 1). After the addition of HOCl/OCl−, the emission peak at 629 nm
decreased obviously and the emission peak at 462 nm increased remarkably, suggesting that the
FRET process of probe PPC was suppressed. However, after the addition of other ROS/RNS, cations,
anions and bio-thiols, hardly any obvious changes were observed. The reason for this phenomenon is
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the destruction of the “C=C” double bond in probe PPC by HOCl/OCl−. In addition, the
anti-interference ability of probe PPC for the detection of HOCl/OCl− in the presence of other
species (Zn2+, Na+, K+, Cu2+, Ca2+, H2O2, NO, •OH, KO2, t-BuOO•) was evaluated (Fig. S7).
Compared with the emission band of probe PPC alone, in the presence of HOCl/OCl− and other ROS,
obvious changes of the emission band of probe PPC at 629 nm and 462 nm could be observed. The
decrease in fluorescence intensity at 629 nm and the increase in fluorescence intensity at 462 nm
gave rise to obvious enhancement of the ratio of fluorescence intensity (I462/I629). Therefore, probe
PPC showed outstanding selectivity and anti-interference ability, which should offer a powerful tool
for the specific detection of endogenous HOCl/OCl− in complex physiological environment.
Fig. 1
3.2. Time, pH and fluorescence lifetimes dependence of probe PPC toward HOCl/OCl−
The response time of probe PPC toward HOCl/OCl− was evaluated (Fig. S8). Upon addition of
HOCl/OCl− to the solution of the probe, the color of solution changed from red to pale yellow
immediately. The reaction could finish within 40 s. The outstanding response of probe PPC to
HOCl/OCl− was among the fastest ones (Table S1) [45-55], which meets well the requirement of
real-time detection. The impacts of pH on the detection of HOCl/OCl− are very important, especially
for cell image. Therefore, fluorescence of the probe and its response toward HOCl/OCl− in different
pH were measured (Fig. S9 a). The fluorescence intensity ratios (I462/I629) of the probe remained
stable over a wide range of pH (pH = 4-9). In addition, in the presence of HOCl/OCl−, the
fluorescence intensity ratios (I462/I629) of the solution were no obvious change in the range of pH 5-8.
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The mild surroundings should suit bioimaging in living cells. Fluorescence lifetimes of probe PPC
and PPC-S were respectively evaluated and showed 2.50 ns and 1.03 ns (Fig. S9 b).
3.3 Fluorescence titration of HOCl/NaOCl with PPC
Titration experiments were conducted by adding HOCl/OCl− (0-6.5 equiv.) into the solution of the
probe (5 µM) (Fig. 2a). As HOCl/OCl− varied from 0 to 6.5 equiv., the fluorescence intensity at 629
nm decreased and the fluorescence intensity at 462 nm increased gradually. Compared with other
fluorescent probes (Table S1) [45-55], the larger pseudo Stokes shift (209 nm) from 629 nm to 420
nm of the probe and the lager gap between two emissions implied that the more detection accuracy
could be achieved. What’s more, an excellent linearity was obtained between the fluorescence
intensity ratios (I462 nm/I629 nm) and the concentrations of HOCl/OCl− in the range of 0 to 6.5 equiv.
(Fig. 2b). Then the limit of detection was calculated with the established formula (LOD = 3σ/slope).
The limit of detection for HOCl/OCl− was evaluated to be as low as 0.321 µM, suggesting probe
PPC had excellent sensitivity toward HOCl/OCl−. Therefore, the probe could be used to detect low
concentrations of endogenous HOCl/OCl− in living cells.
Fig. 2
3.4 Absorption spectra of probe PPC titrated with HOCl/OCl− and excitation spectra of probe PPC
The absorption spectra of probe PPC showed two peaks at 412 nm and 466 nm, which could be
attributed to the donor (coumarin) and the acceptor (phenothiazine) moiety, respectively (Fig. S10
a). With addition of increasing amount of HOCl/OCl− (0-6 equiv.), the absorption band of the
acceptor moiety at 466 nm decreased gradually and that of the donor moiety remained unchanged.
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These results suggested that the structure of the acceptor unit was destroyed and the donor unit was
unchanged in the presence of HOCl/OCl−. The excitation spectra of probe PPC also be measured
and showed a main excitation scope from 350 nm to 550 nm (Fig. S10 b).
3.5 Calculation of energy transfer efficiency (ETE) and fluorescence quantum yield (Φ)
To evaluate energy transfer efficiency of probe PPC, an absorption spectrum of the acceptor
and a fluorescence spectrum of the donor were measured (Fig. S11). The results showed that the
absorption spectrum of the acceptor overlaid well with the fluorescence spectrum of the donor,
implying high energy transfer efficiency could be expected. To further calculate energy transfer
efficiency of probe PPC quantitatively, fluorescence intensities of probe PPC and the donor were
measured in the same condition. Energy transfer efficiency (ETE) was calculated according to the
following equation:
ηETE =1-FD/FDA
Where, FD denotes the maximum of fluorescence intensity of lone donor, and FDA denotes the
maximum of fluorescence intensity of the donor moiety in the probe. ηETE = 0.91 implied high energy
transfer efficiency of probe PPC.
The fluorescence quantum yield (Φ) was assessed by quinine sulfate as a standard and
calculated by following equation:
Φ=Φs(IAs/IsA)(η2/ηs2)
Where, A denotes the absorbance, I denotes the integrated fluorescence intensity, η denotes the
refractive index of the solvent. Φ of PPC and PPC-S were determined to be 0.233 and 0.392
respectively at room temperature.
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3.6 Bioimaging of probe PPC
Encouraged by the excellent fluorescence properties of probe PPC for HOCl/OCl−, we further
studied the detection of intracellular HOCl/OCl−. Firstly, we evaluated the practicability of probe
PPC for the detection of HOCl/OCl− in living cells (Fig. 3). After RAW264.7 macrophage cells were
incubated with probe PPC (2 µM) for 30 min, strong fluorescence in the red channel and weak
fluorescence in the blue channel were observed. After further incubation with various concentrations
of HOCl/OCl− for 30 min, the fluorescence in the red channel decreased and the fluorescence in the
blue channel increased remarkably in a low concentrations range (0-5 µM). This proved that probe
PPC could detect HOCl/OCl− in living cells in a ratiometric fluorescence manner.
Fig. 3
Next, we investigated the suitability of probe PPC toward endogenous HOCl/OCl− in
RAW264.7 macrophage cells (Fig. 4).
It is known that HOCl/OCl− could be produced in cells by
stimulating with LPS. In the experimental groups, macrophage cells were stimulated by LPS (1
µg/ml) for 12 h and then incubated with probe PPC (2 µM) for 30 min, the fluorescence from the red
channel decreased and the fluorescence from the blue channel increased. In the control groups,
without addition of LPS, obvious fluorescence in the red channel and faint fluorescence in the blue
channel were obtained. The ratio of fluorescence intensity of blue channel and red channel (Iblue/Ired)
increased remarkably in the presence of LPS. Therefore, probe PPC could successfully be used for
the fluorescence imaging of endogenous and exogenous hypochlorite in RAW264.7 macrophage
cells.
Fig. 4
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The development of novel fluorescence probes for the selective detection of HOCl/OCl− inside
specific organelles is significant in biology and medical science. We evaluated the feasibility of
probe PPC for sub-cellular localization. The fluorescence areas of probe PPC overlaid well with that
of Mito Tracker Deep Red, and the co-localization coefficient is 0.94 (Fig. 5). Therefore, probe PPC
could target mitochondria in RAW264.7 macrophage cells. The preferential distribution of probe
PPC in mitochondria was unexpected. As we know, almost all mitochondria-targeted fluorescence
probes had positive charge groups such as triphenylphosphonium (TPP) [38, 56] or pyridinium [57,
58]. Whereas, some neutral fluorescence probes for mitochondria had been focused on and
developed [59-61]. Therefore, probe PPC probably was a second neutral mitochondria-targeted
fluorescence probe for ratiometric detection of endogenous HOCl/OCl−. Furthermore, probe PPC
showed an excellent photostability (Fig. S12) and little cytotoxicity for living cells (Fig. S13).
Fig. 5
3.7 Investigation of the detecting mechanism
According to literature, the carbon-carbon double bond was more likely attacked by HOCl/OCl−
than the co-existed thioether group of phenothiazine [44]. Therefore, the decreased fluorescence of
the acceptor moiety and the enhanced fluorescence of the donor unit may be attributed to the
1
destruction of the carbon–carbon double bond (Scheme 2). To confirm our speculation, H NMR
titration of the reaction of probe PPC with HOCl/OCl− were recorded (Fig. S14). With addition of
increasing amount of HOCl/OCl−, a signal peak of H1 of probe PPC gradually decreased until
disappeared and a signal peak of H2 of PPC-S presented and reached the maximum. Therefore,
PPC-S should be the product of probe PPC and HOCl/OCl−. To further confirm our speculation,
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three reaction solutions of probe PPC with 0, 3 and 7 equiv. HOCl/OCl− were evaluated by MS (Fig.
S15). The strong peak at 645.2458 (m/z) corresponded to [PPC + H]+ in MS spectrometry for the
solution of probe PPC alone. Three peaks at 597.2414 (m/z), 619.2220 (m/z) and 645.2523 (m/z)
corresponded to [PPC-S + H]+, [PPC-S + Na]+ and [PPC + H]+ in MS spectrometry for the solution
of probe PPC with 3 equiv. HOCl/OCl−, respectively. A prominent peak at 597.2438 (m/z) and a
minor peak at 619.2237 (m/z) corresponded to [PPC-S + H]+ and [PPC-S + Na]+ in MS
spectrometry for the solution of probe PPC with 7 equiv. HOCl/OCl−, respectively. Above data
implied the gradual transformation of the probe PPC to PPC-S.
Conclusion
In summary, a ratiometric fluorescence probe PPC based on FRET mechanism with a large
pseudo Stokes shift (209 nm) was developed for selective and sensitive detection of HOCl/OCl−.
The probe was successfully applied to the detection of exogenous and endogenous HOCl/OCl− in
living RAW264.7 macrophage cells with good photostability and neglectable toxicity. Because
of the excellent mitochondria-targeted ability, probe PPC can be competent in tracking of
exogenous and endogenous HOCl/OCl− in mitochondria of living cells.
Acknowledgment
This study was supported by the National Key Research and Development Program of China
(2017YFA0104604), the National Natural Science Foundation of China (No.31871407, 31741083，
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31870831 ), the Natural Science Foundation of Shandong Province (ZR2018MB042).
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Legend of figures
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Scheme 1 Synthesis of probe PPC
Scheme 2 The proposed detection mechanism of probe PPC for HOCl/OCl−.
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Fig. 1 (a) Fluorescence spectra of probe PPC (5 µM) in the presence of HOCl/OCl− (6 equiv.) and
other analytes (25 equiv., vacant, H2O2, KO2, NO, 1O2, t-BuOOH, t-BuOO•, •OH, ONOO-, Na+,
2-
2-
-
2-
Al3+, Zn2+, Ca2+, Fe3+, Fe2+, Cu2+, Mg2+, F-, Cl-, Br-, I-, CO3 , SO4 , NO2 , AcO-, HPO3 ,
-
HOCl/OCl−, Cys, GSH, Hcy, H2S and HSO3 ) in PBS (pH = 7.4, 10 mM with 1 mM Triton X-100).
(b) Response (I462/I629) of probe PPC toward analytes in PBS (pH = 7.4, 10 mM with 1 mM Triton
X-100). 1. Vacant; 2. H2O2; 3. KO2; 4. NO; 5. 1O2; 6. t-BuOOH; 7. t-BuOO•; 8. •OH; 9. ONOO-; 10.
Na+; 11. Al3+; 12. Zn2+; 13. Ca2+; 14. Fe3+; 15. Fe2+; 16. Cu2+; 17. Mg2+; 18. F-; 19. Cl-; 20. Br-; 21. I-;
2-
2-
-
2-
22. CO3 ; 23. SO4 ; 24. NO2 ; 25. AcO-; 26. HPO3 ; 27. HOCl/OCl−, 28. Cys, 29. GSH, 30. Hcy, 31.
-
H2S, 32. HSO3 . λex = 420 nm.
Fig. 2 (a) Fluorescence spectra of probe PPC (5 µM) in the presence of different concentrations of
HOCl/OCl− (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 equiv.) in PBS (pH = 7.4, 10 mM with 1
mM triton X-100), (λex = 420 nm, slit: 15 nm/7 nm). Inset: A visual fluorescence changed from red to
blue under illumination using a 365 nm UV lamp. (b) I462/I629 changes with the addition of
HOCl/OCl− (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 equiv.). Inset: lg(I462/I629) changes with
the addition of HOCl/OCl− (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5 equiv.)
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Fig. 3 (a) Confocal fluorescence images of RAW 264.7 cells pretreated with different concentrations
of HOCl/OCl−(0, 1, 5 µM) and then incubated with probe PPC (2 µM) for 30 min. (b) The relative
ratio (blue/red) of fluorescence intensity.
Fig. 4 (a) The first row (horizontally): Fluorescence and bright field images of RAW264.7 cells
incubated with probe PPC (2 µM) for 30 min. The second row (horizontally): Fluorescence and
bright field images of RAW264.7 cells stimulated by LPS (1µg/ml) for 12 h and then incubated with
probe PPC (2 µM) for 30 min. (b) Ratio (blue/red) of fluorescence intensity.
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Fig. 5 RAW264.7 cells were incubated with probe PPC (2 µM) for 30 min, followed by Mito
Tracker Deep Red for 30 min. (a) The cell image of probe PPC, λex = 405 nm, λem = 405-550 nm. (b)
The cell image of Mito Tracker Deep Red, λex = 639 nm, λem = 640-700 nm. (c) Merged image. (d)
The co-localization coefficient (Pearson’s coefficient) of probe PPC and Mito Tracker Deep Red was
0.94.
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Highlights
‹ A novel ratiometric fluorescent probe (named PPC) for hypochlorite based on
FRET platform was developed.
‹ PPC showed a large pseudo Stokes shift (209 nm), high selectivity, sensitivity
and fast response.
‹
‹
PPC could target well mitochondria in cell image.
PPC could image endogenous hypochlorite in RAW264.7 cells.