A mitochondria-targeted fluorescent probe for fast detecting hypochlorite in living cells

Article abstract of DOI:10.1016/j.dyepig.2020.108192

A fast-responsive fluorescent probe PI-Py was designed and synthesized for sensitive and selective detecting hypochlorite (ClO^?). The probe PI-Py was intrinsically non-fluorescent due to the PET effect of the phenothiazine moiety. After reacting with ClO^?, the PET effect was blocked, and a strong green emission of the BODIPY unit was recovered. The probe PI-Py showed an excellent selectivity towards ClO^? in comparison of other ROS. The limit of detection for ClO^? was calculated to be 1.7 nM, proving the probe's high sensitivity. Moreover, the probe PI-Py has been proved to be effective in visualizing the variations of exogenous ClO^? in living HeLa cells. A co-localization study employing Mito Tracker red reveals that the PI-Py is specifically located in the mitochondria.

Full text of DOI:10.1016/j.dyepig.2020.108192

Journal Pre-proof
A mitochondria-targeted fluorescent probe for fast detecting hypochlorite in living cells
Hongfang Li, Yu Miao, Zhixue Liu, Xue Wu, Chunxiang Piao, Xin Zhou
PII:
S0143-7208(19)32875-X
DOI:
https://doi.org/10.1016/j.dyepig.2020.108192
DYPI 108192
Reference:
To appear in: Dyes and Pigments
Received Date: 8 December 2019
Revised Date: 6 January 2020
Accepted Date: 6 January 2020
Please cite this article as: Li H, Miao Y, Liu Z, Wu X, Piao C, Zhou X, A mitochondria-targeted
fluorescent probe for fast detecting hypochlorite in living cells, Dyes and Pigments (2020), doi: https://
doi.org/10.1016/j.dyepig.2020.108192.
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Hongfang Li: Formal analysis; Data curation; Investigation; Writing – original draft
Yu Miao: Data curation;
Zhixue Liu: Data curation;
Xue Wu: Funding acquisition; Supervision;
Chunxiang Piao: Data curation; Investigation; Writing – review & editing.
Xin Zhou: Conceptualization; Project administration; Funding acquisition; Writing –
review & editing.

Graphical abstract
A new BODIPY-based fluorescent probe for ClO- was developed successfully. This
probe can be used in visualizing the changes of exogenous ClO- in the mitochondria.

A mitochondria-targeted fluorescent probe for fast detecting
hypochlorite in living cells
Hongfang Li,a Yu Miao,a Zhixue Liu,a Xue Wu,a Chunxiang Piaob* and Xin Zhoua, c*
aResearch Centre for Chemical Biology, Department of Chemistry, Yanbian
University, Yanji 133002, China
bFood Research Centre, Colleget of Agriculture, Yanbian University, Yanji 133002,
China
cDepartment of Chemistry, Qingdao University, Qingdao 266071, China
Abstract
A fast-responsive fluorescent probe PI-Py was designed and synthesized for
sensitive and selective detecting hypochlorite (ClO-). The probe PI-Py was
intrinsically non-fluorescent due to the PET effect of the phenothiazine moiety. After
reacting with ClO-, the PET effect was blocked, and a strong green emission of the
BODIPY unit was recovered. The probe PI-Py showed an excellent selectivity
towards ClO- in comparison of other ROS. The limit of detection for ClO- was
calculated to be 1.7 nM, proving the probe's high sensitivity. Moreover, the probe
PI-Py has been proved to be effective in visualizing the variations of exogenous ClO-
in living HeLa cells. A co-localization study employing Mito Tracker red reveals that
the PI-Py is specifically located in the mitochondria.
Keywords: Fluorescent probe, Hypochlorite, Cell imaging, PET
*Corresponding authors.
E-mail address: cxpiao@ybu.edu.cn, zhouxin@qdu.edu.cn
Introduction
As a typical reactive oxygen species (ROS), hypochlorite (ClO-) has been
reported to be associated with many biological processes in living organisms, such as

innate host immune reaction, defenses against invasive bacteria and killing a wide
range of pathogens.[1-7] Hypochlorite is mainly generated from hydrogen peroxide
and chloride ions through a heme enzyme myeloperoxidase (MPO) catalyzed
reaction.[8, 9] The maintenance of the appropriate level of ClO- is fairly crucial for
numerous cellular functions. However, abnormal overproduction of ClO- is
considered to induce some diseases, such as atherosclerosis, osteoarthritis, rheumatoid
arthritis and even cancers.[10-14] Therefore, detecting ClO- has attracted extensive
interests.
Recently, due to their advantages of high sensitivity, selectivity, rapid response
and ease synthesis,[15-19] fluorescence probe-based imaging technique has been
widely developed for detecting ClO- in vitro and in vivo.[20-32] However, only a few
mitochondria-targeted fluorescent probes have been reported to date.[33-36]
Therefore, there is an urgent need to explore a new effective mitochondria-targeted
fluorescent probe for ClO- with high selectivity and sensitivity.[37-39] Herein, we
designed and synthesized a BODIPY based fluorescent probe PI-Py by employing a
phenothiazine group as a ClO- reactive site. The probe PI-Py showed an OFF-ON type
fluorescent response towards ClO-. The PET effect of the phenothiazine moiety
intrinsically quenched the emission of the probe. After reacting with ClO-, a strong
green emission of the BODIPY unit was recovered. The probe PI-Py showed an
excellent sensitivity towards ClO- with a limit of detection as low as 1.7 nM.
Moreover, we proved the successful usage of the probe in visualizing the variations of
exogenous ClO- in mitochondria in living HeLa cells.
Experimental
1. Materials and apparatus
1
All the reagents and solvents were used without further purification. H NMR
and 13C NMR spectra were recorded on a Bruker Ultrashield 300 MHz NMR
spectrometer. Chemical shifts were expressed in ppm (in CDCl3: TMS as an internal

standard) and coupling constants (J) in Hz. Mass spectra were recorded using
SHIMADZU LCMS-2020 and Agilent Technologies 6410 UHD Accurate-Mass
Q-TOF LC/MS instruments. UV/vis spectra were recorded on imadzu UV-2550
spectrophotometer with a 1.0 cm quartz cell. Fluorescence spectra were conducted on
Shimadzu RF-5301PC fluorescence spectrophotometer. The probe PI-Py dissolved
into THF to prepare the stock solutions with a concentration of 1 mM.
2. Methods for cell culture and fluorescent imaging
Hela cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco,
USA), supplemented with 10% FBS (ScienCell, USA) and (1000 Units/ml) penicillin
and (1000µg/ml) streptomycin (Gibco, USA). The cells were incubated at 37℃ in a
humidified atmosphere of 5% CO2. They were seeded in 6-well culture plate and
allowed to adhere for overnight. Stock solutions of probe PI-Py (5 µM) in THF,
NaClO solution (10 mM, pH 7.4) were prepared in experiment. For the experiments,
cells were washed with PBS as least three times and incubated with PI-Py (5 µM) in
PBS-EtOH (pH 7.4, 1 : 1，v/v) for 10 min at 37 °C. Then, 50 µM NaClO solution is
added for 10 min at 37°C. The cell was used a Fluorescence Microscope (Confocal
laser microscope A1) with a 40×oil-immersion objective lens. The cells were washed
with physiological saline after having detected the imaging. The Fluorescence
Emission was collected at 500∼550 nm (excited at 488 nm) for green channel.
3. Synthesis
Synthesis of compound 2 In a 100 mL Schlenk flask, phenothiazine (1.0 g, 5
mmol) was dissolved in DMF (20 mL). To this solution, NaH (360 mg, 15 mmol) was
slowly added. The mixture was stirred at room temperature under N2 for 30 min,
Iodoethane (0.48 mL, 6 mmol) was slowly added. Then, the reaction mixture was
heated at 70 ℃ under N2 atmosphere for 3 hours. After cooling to room temperature,
the mixture was extracted with ethyl acetate and saturated brine. The extractions were
combined and dried over anhydrous sodium sulfate. After evaporation of solvent, the

crude compound was purified over a silica gel column and the desired compound was
eluted using PE: DCM (20: 1 v/v) to obtain the 0.996 g pure product as a white solid.
Yield: 88%.
Synthesis of compound 3. In a 50 mL two-necked round-bottom flask，POCl3 (3
mL, 30 mmol) was added to DMF (10 mL). After stirring the reaction mixture under
N2 for 30 minutes, 10-ethyl-10H-phenothiazine (500 mg, 2.2 mmol) was added. The
reaction mixture was heated at 70 ℃ for 6 h. After cooling to room temperature, the
mixture was poured into ice water slowly and then neutralized with 2 M sodium
hydroxide solution. Afterwards, the mixture was extracted with dichloromethane
(3*50 ml). The extractions were combined, washed with distilled water and saturated
brine, and then dried over anhydrous sodium sulfate. After evaporation of solvent, the
crude compound was purified over a silica gel column and the desired compound was
eluted using PE: EA (5: 1 v/v) to obtain 0.33 g pure product as a yellow solid. Yield:
58%. 1H NMR (300 MHz, CDCl3) δ 9.81 (s, 1H), 7.65 (dd, J = 8.4, 1.9 Hz, 1H), 7.59
(d, J = 1.9 Hz, 1H), 7.23 – 7.15 (m, 1H), 7.12 (d, J = 1.8 Hz, 1H), 6.99 (dd, J = 8.4,
1.8 Hz, 1H), 6.92 (d, J = 8.3 Hz, 2H), 4.00 (q, J = 7.0 Hz, 2H), 1.47 (t, J = 7.0 Hz,
3H).
Synthesis of the PI-Py probe In a 100 mL Schlenk flask, Compound 2 (200 mg,
0.78 mmol) and 2, 4-dimethyl pyrrole (0.16 ml, 1.56 mmol) were dissolved in
absolute CH2Cl2 (30 ml) under N2 atmosphere, 10 drops of TFA were added and the
solution was stirred at room temperature until TLC analysis showed complete
consumption of the aldehyde. At this time of DDQ (178 mg, 0.78 mmol) were added
and stirring continued for 2 h. Then, Et3N (1.5 mL) and BF3·Et2O (1.5 mL) were
added. The mixture was stirred for 2 h and the organic layer containing the crude
product was subsequently washed three times with water; the organic solution was
dried over Na2SO4, and evaporated to dryness. The raw material was purified over a
silica gel column and the desired compound was eluted using PE: EA (20: 1, 10: 1v/v)
to obtain 69 mg pure product as a red solid. Yield: 20%. 1H NMR (300 MHz, CDCl3)

1H NMR (300 MHz, CDCl3) δ 7.20 (td, J = 7.7, 1.4 Hz, 1H), 7.12 (dd, J = 7.6, 1.4 Hz,
1H), 7.05 – 6.99 (dd, J = 7.7, 1.4 Hz, 2H), 6.96 (d, J = 7.0 Hz, 2H), 6.91 (s, 1H), 5.99
(s, 2H), 3.98 (q, J = 6.9 Hz, 2H), 2.56 (s, 6H), 1.53 (s, 6H), 1.47 (t, J = 7.0 Hz,
3H).13C NMR (75 MHz, CDCl3) δ 155.49, 145.53, 144.72 (s), 142.76 (s), 135.09,
133.74, 133.60, 130.57, 130.41, 128.89, 128.03, 127.28 (s), 126.40 (s), 124.28 (s),
122.84 (s), 121.05 (s), 118.38 (s), 117.37 (s), 116.67 (s), 44.99 (s), 25.84 (s), 22.33 (s),
19.07 (s), 14.76 (s), 14.48 (s). A prominent peak at m/z 473.4 (calculated to be 473.19)
was found in MS spectra.
Scheme 1 Synthesis routs of the probe PI-Py.
Results and discussion
Rational Design of the Probe
As shown in Scheme 1, the probe was synthesized easily (Scheme 1) and
1
characterized unambiguously by H NMR, 13C NMR and ESI-MS spectra (see
ESI†)[40]. Here, BODIPY group was employed as the fluorophore of the probe due to
its relatively long emission wavelength, high quantum yield, and good
photo-stability.[41, 42] Meanwhile, electron-rich phenothiazine group was selected as
an effective PET donor, which can effectively quench the emission of the excited state
of BODIPY moiety.[43] After reacting with ClO-, the phenothiazine group can be
oxidized into its sulfoxide form, resulting in blocking the PET process and recovering

the emission of the BODIPY unit.
Fluorescence response of the probe PI-Py to different ROS
The selectivity of the probe PI-Py towards ClO- was firstly investigated. As
shown in Fig. 1a, the probe is almost nonfluorescent, upon excitation at 500 nm,
owing to quenching of this excited state by the well-known PET effect from the
phenothiazine moiety. This indicated that the phenothiazine group can be regarded as
an effective PET donor in our design strategy. After addition of 9 µM NaClO (a
common ClO- source), a noticeable fluorescent enhancement at 515 nm was observed.
1
-
Nevertheless, other ROS including H2O2, O2, O2 , ·OH, NO, ROO· and ONOO-,
induced nonsignificant fluorescent change even with a high concentration (100 µM).
Under excitation at 365 nm (hand-held UV lamp), strong green fluorescence only
could be observed upon the addition of ClO- (inset of Fig.1b). These data indicate that
the probe shows competitive selectivity towards ClO- rather than other ROS.
Fig. 1 (a) Fluorescence spectra and (b) fluorescence intensity of PI-Py (1 µM) at 515
nm with addition of various ROS (9 µM ClO-, and 100 µM other ROS) in PBS (pH
7.4,10 mM, containing 50% EtOH as a cosolvent) (Ex: 500 nm, slit: 1.5/1.5 nm). Inset:
fluorescent picture of probe with varous ROS (1: blank, 2: ClO-, 3: H2O2, 4: 1O2, 5:
-
O2 , 6: ·OH, 7: NO, 8: ROO·, 9: ONOO-.).
Fluorescence titration experiment
The fluorescent titrations of probe PI-Py (1 µM) towards ClO- were evaluated in
PBS buffer pH 7.4 (containing 50% EtOH as a cosolvent) at room temperature. As

shown in Fig. 2a, the fluorescence intensity of probe PI-Py at 515 nm increased
gradually upon addition of ClO-, resulting in a OFF-ON type fluorescent response to
ClO-. Meanwhile, the addition of the ClO- had little effect on the absorption spectra
(Fig. 2b), which indicated that the phenothiazine group may adopt an almost vertical
molecular configuration with BODIPY moiety,[44, 45] resulting in a more effective
PET OFF-ON process.
Time-dependent study of the probe PI-Py to ClO- was further conducted by using
fluorescence changes. As showed in Fig. 3a, the probe PI-Py revealed a fast response
within just a few seconds. This fast response time is considered beneficial for our
probe to monitor ClO- variations in real time. Moreover, the fluorescence intensity
was found to be linearly proportional to the concentration of NaClO in the
concentration range of 0-9 µM (Fig. 3b). On the basis of a signal-to-noise (S N1-) ratio
of 3, a detection limit of 1.7 nM was calculated, demonstrating the very high
sensitivity of PI-Py to ClO-.
Fig. 2 Fluorescence (a) and absorbance (b) titrations spectra of PI-Py (1 µM) upon
addition of NaClO (0-9 µM) in PBS (pH 7.4, 10 mM, containing 50% EtOH as a
cosolvent) (Ex: 500 nm, slit: 1.5/1.5 nm). Inset: the fluorecent imaging change of
PI–Py (1 µM) with ClO-.

Fig.3 (a) Time-dependent change of fluorescence intensity of PI-Py (1 µM) upon
addition of ClO– (9 µM). λex = 500 nm; λem = 515 nm. (b) The titration curve plotted
with the fluorescence intensity of PI-Py (1 µM) at 515 nm as a function of ClO–
concentration in range of 0-9 µM.
Investigation of the sensing mechanism
The phenothiazine group has been reported as an effective PET donor for many
fluorophores. In our case, we also assumed that the divalent Sulphur atom of
phenothiazine group of our probe could be oxidized in to its sulfoxide form by NaClO.
Internally, the probe has no fluorescence due to the PET effect coming from the lone
pair electrons of S atom. After adding the hypochlorous acid, the PET effect was
suppressed due to the oxidation reaction of the phenothiazine group, in which the
Sulphur atom was oxidized into sulfoxide, resulting in the restoration of the strong
green emission of BODIPY. To further verify the assumed mechanism of PI-Py
towards ClO-, the product of PI-Py reacted with ClO- was analyzed by ESI-MS (Fig.
S10). A prominent peak at m/z 489.3 (calculated to be 489.39) was found in MS
spectra, confirming the proposed sulfoxide product as depicted in Scheme 2.

Scheme 2 The proposed mechanism of PI-Py with ClO-.
Effect of pH
There were significant differences in the pH values in different cell organelles.
To monitor ClO- in living cells, the probe should remain stable in different pH
conditions. Thus, before testing the real application of the PI-Py as a ClO- probe in
living cell imaging, we evaluated the fluorescence responses of the probe to ClO- in a
series of buffers with different pH values ranging from 4 to 9. As show in Fig. S9, in
this pH range, the responses of the probe to ClO- was pH-independent, indicating that
the probe is compatible with most biological environment, and suitable to detect ClO-
in living cells.
Cell experimental
The desirable fluorescence properties of PI-Py for ClO- prompted us to utilize it
for the detection ClO- in living cells. The ability to detect exogenous ClO- was firstly
examined. As shown in Fig. 4a, it was found that HeLa cells stained with PI-Py (5 µM)
showed almost no fluorescence in the green channel. On this basis, it is concluded that
negligible amount of ClO- is present in living HeLa cells. After the addition of 50 µM
NaClO, the fluorescence intensity increased in the green channel (Fig. 4d). The
subsequent addition of NaClO led to an increase in the fluorescence. In the presence
of 100 µM exogenous NaClO, the fluorescence in the green channel became
commensurately brighter (Fig. 4g). The merged picture, as shown in Fig. 4f and 4i,
confirmed that the fluorescence signals are only located within cells. The above

experiments proved that the probe PI-Py was well cell-permeated, and can be used to
detect exogenous ClO- in living HeLa cells.
Next, to assess the mitochondrial staining ability of PI-Py, colocalization
experiments were carried out using Mito Tracker red, a commercially available
mitochondrial tracker (Fig. 5). As depicted in Fig. 5d, the fluorescence signals of
PI-Py responded to ClO- overlaid well with the fluorescence of Mito Tracker red.
Moreover, colocalization was qualified using Pearson’s sample correlation factors
(Rr). The PI-Py response well as reflected in a Pearson’s colocalization coefficient of
0.9664. Considered in concert, these results provide support for the notion that PI-Py
acts as organelle-specific probes that target the mitochondria of the living HeLa cells.
Fig. 4 Confocal fluorescence images of HeLa cells (λex=488 nm, λem: 500-550 nm)
(a-c) Fluorescent image of HeLa cells stained with probe PI-Py (5 µM) for 10 min. (a)
fluorescent image; (b) bright field image; and (c) merged image. (d–f) HeLa cells
were then incubated with NaClO (50 µM) for another 15 min. (d) fluorescent image;
(e) bright field image; and (f) merged image. (g–i) HeLa cells were then incubated
with NaClO (100 µM) for another 15 min. (g) fluorescent image; (h) bright field
image; and (i) merged image.

Fig. 5 (a–d) Confocal fluorescence images of HeLa cells. The cells were incubated
with PI-Py (5 µM) for 10 min, further incubated with NaClO (100 µM) for 15 min,
and finally incubated with Mito Tracker red (100 nM) for 20 min. (a) emission from
the green channel, λex=488 nm, λem: 500-550 nm; (b) emission from the red channel
(mitochondrial staining, λex=561 nm, λem: 570-1000 nm); (c) the merged bright-field
image; (d) the merged image.
Conclusion
In summary, we developed here a new BODIPY-based fluorescent probe (PI-Py)
for selective and sensitive detecting ClO-. The probe PI-Py was initially no
fluorescence due to the PET effect of the electron-rich phenothiazine moiety, and gave
a strong green fluorescent response after addition of ClO-. The fluorescent titration
experiments indicated that probe PI-Py showed an excellent selectivity and sensitivity
towards ClO- (LOD as low as 1.7 nM). Moreover, the probe was further used in
visualizing the changes of exogenous ClO- in living HeLa cells. The result indicated
that the probe can trace the change of ClO- content in mitochondria.
Acknowledgement
Dr. Xin Zhou acknowledge the Natural Science Foundation of China (NSFC.
21662037, 21762045, 21911540466), Shandong Provincial Natural Science
Foundation (ZR2019YQ12), China Postdoctoral Science Foundation (219M652306),
and Taishan Scholar Project (tsqn201812049) for supporting this work.
Conflict of interest
The authors declared that they have no conflicts of interest to this work.
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Highlights
ò A novel BODIPY-based fluorescent probe PI-Py was synthesized.
ò The probe PI-Py showed an excellent selectivity and sensitivity towards ClO-.
ò Fluorescent titrations, MS spectra support a PET OFF-ON mechanism.
ò Living cells imaging for exogenous ClO- located in the mitochondria was
observed.

The authors declared that they have no conflicts of interest to this work.