Fast detecting hypochlorous acid based on electron-withdrawing group promoted oxidation and its biological applications in cells and root tips of plants

Article abstract of DOI:10.1016/j.saa.2019.118001

Hypochlorous acid, a type of reactive oxygen species, has been shown to play an important role in organisms. Nowadays, there are many kinds of fluorescence detecting mechanisms to detect hypochlorous acid in vivo. Due to the high selectivity, the mechanism of using the strong oxidation of hypochlorous acid to break carbon?carbon double bonds has been favored by many scientists. However, the reported probes of breaking carbon?carbon double bonds still had drawback such as slow response. Based on this, we introduced electron-withdrawing group malonitrile to accelerate the oxidation of hypochlorous acid, resulting in reaction time less than 150 s. Meanwhile, the probe exhibited excellent selectivity, optical stability, high sensitivity and the detection limit as low as 0.19 μM. More importantly, we also successfully proved the potential application of the probe for the detection of intracellular ClO^? living cells and Arabidopsis root tip by fluorescence imaging.

Full text of DOI:10.1016/j.saa.2019.118001

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Fast detecting hypochlorous acid based on electron-withdrawing
group promoted oxidation and its biological applications in cells
and root tips of plants
Ming Li, Jianbin Chao, Yaoming Liu, Miao Xu, Yongbin Zhang,
Fangjun Huo, Juanjuan Wang, Caixia Yin
PII:
S1386-1425(19)31400-3
https://doi.org/10.1016/j.saa.2019.118001
SAA 118001
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 9 September 2019
Revised date:
23 December 2019
5 December 2019
Accepted
date:
2
Please cite this article as: M. Li, J. Chao, Y. Liu, et al., Fast detecting hypochlorous acid
based on electron-withdrawing group promoted oxidation and its biological applications
in cells and root tips of plants, Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy(2018), https://doi.org/10.1016/j.saa.2019.118001
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2018 Published by Elsevier.

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Fast
detecting
hypochlorous
acid
based
on
electron-withdrawing group promoted oxidation and its
biological applications in cells and root tips of plants
ab
a
a
ab
d
d
Ming Li, Jianbin Chao, Yaoming Liu, Miao Xu, Yongbin Zhang, Fangjun Huo,
a
c*
Juanjuan Wang and Caixia Yin
a
Scientific Instrument Center, Shanxi University, Taiyuan 030006, China
b
School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
China
c
Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China
d
Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China
*
Corresponding author: C.X. Yin, E-mail: yincx@sxu.edu.cn,
Tel/Fax: +86-351-7011022.
Abstract: Hypochlorous acid, a type of reactive oxygen species, has been shown to
play an important role in organisms. Nowadays, there are many kinds of fluorescence
detecting mechanisms to detect hypochlorous acid in vivo. Due to the high selectivity,
the mechanism of using the strong oxidation of hypochlorous acid to break
carbon-carbon double bonds has been favored by many scientists. However, the
reported probes of breaking carbon-carbon double bonds still had drawback such as
slow response. Based on this, we introduced electron-withdrawing group malonitrile
to accelerate the oxidation of hypochlorous acid, resulting in reaction time less than
1
50 s. Meanwhile, the probe exhibited excellent selectivity, optical stability, high
sensitivity and the detection limit as low as 0.19 μM. More importantly, we also
successfully proved the potential application of the probe for the detection of
-
intracellular ClO living cells and Arabidopsis root tip by fluorescence imaging.
-
Key words: Electron-withdrawing group, Fluorescent probe, ClO , Imaging
1

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1
. Introduction
-
Hypochlorous acid (HClO)/Hypochlorite (ClO ), as an important reactive oxygen
species (ROS), is widely used in daily life and industry, such as drinking water
disinfection, household bleach, cyanide treatment and cooling water treatment of
coastal power stations [1-3]. At the same time, more and more scientific experiments
-
have proved that moderate amount of HClO/ClO has a certain protective effect on
human health [4-5], but the abnormal concentration of HClO in the body's cells will
cause tissue destruction and cell damage [6-8]. Based on this, it is particularly
important to study the physiological process of hypochlorous acid in the body [9].
Compared to traditional techniques, chemical probes based on absorption or
fluorescence changes are more feasible for detecting analytes owing to their many
appealing advantages such as low detection limit, high selectivity, real-time
monitoring and its potential for in vivo imaging of living cells [10-11]. Therefore, it is
a wise choice to use fluorescence probe strategy to detect HClO/ClO in vivo. At
-
present, the detection of HClO/ClO by fluorescent probes is mainly based on the
following mechanisms: oxidation of p-methoxyphenol to benzoquinone [12], oxime
isomerization [13], chlorination of thioester and amide oxidation of p-aminophenol
analogs [14,15], oxidation of thioethers into sulfonates [16,17], and cleavage of
carbon-carbon double bonds [18]. Compared with among detection mechanisms, the
mechanism of cleavage of carbon-carbon double bonds shows better selectivity and
can effectively avoid interference of other reactive oxygen species [19]. However, the
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cleavage of carbon-carbon double bonds generally requires a long reaction time, so it
is a challenge to design a hypochlorous acid fluorescence probe with fast response
speed and high selectivity.
Based on the above situation, we reasonably designed a probe 1 (2-((2E) -
1
-(7-(diethylamino)
-
2-oxo-2H-chromen-3-yl) - 3-(9-ethyl-9H-carbazol-3-yl)
-
.
allylidene) malononitrile) to be used to detect ClO In our design strategy,
malononitrile is not only a linker between the coumarin and carbazole, but also can
-
accelerate the oxidation of ClO as an electron-withdrawing group. After a series of
-
spectral tests, the response time of the probe with HClO/ClO was less than 150 s.
Besides, the probe 1 had the advantages of good stability, low detection limit, high
selectivity and sensitivity for the detection of hypochlorite. Furthermore, probe 1 also
-
applied to monitor the fluctuation of ClO in HepG2 cells and Arabidopsis root tip
successfully.
2
. Materials and methods
2
.1. Materials and measurement
All chemicals and reagents were purchased from Aladdin Reagent Shanghai Co.,
Ltd., all of which were of analytical grade. Distilled water was used throughout all
1
13
experiments. H NMR and C NMR spectra were performed with Bruker instrument
with TMS (Tetramethylsilane) as the internal standard for 600 MHz and 150 MHz
spectrometer (AVAVCE ⅢHD), respectively. Shimadzu UV-2450 spectrophotometer
and F-7000 fluorescence spectrophotometer (Hitachi, Japan) was employed to
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measure absorption spectra and fluorescence spectra, respectively. Mass spectra were
obtained with a Thermo Scientific Q Exactive LC-MS/MS system. The fluorescence
images of probe were obtained using a ZEISS LSM 880 confocal laser scanning
microscope.
2
.2. Synthesis of compound 1
A mixture of 3-acetyl-7-diethylaminocoumarin (2.60 g, 10 mmol), malononitrile
(
1.25 mL, 20 mmol), acetic acid solution (0.45 mL, 8 mmol) and ammonium acetate
0.16 g, 2 mmol) was dissolved in 20 mL of benzene and heated to reflux with the aid
(
of a water separator for 18h. After completion of the reaction, the solvent was
evaporated, and a yellow oily substance was precipitated by adding 5 ml of ethanol,
and then filtered under vacuum to give a crude product. Finally, the crude product was
recrystallized from ethanol to give a yellow needle product (compound 1) (2.61g,
1
yield 85 %). H NMR (600 MHz, CDCl ): δ 7.91 (s, 1H), 7.38 (d, J = 9.0 Hz, 1H),
3
6
3
2
.66 (d, J = 8.9 Hz, 1H), 6.51 (s, 1H), 3.49 (q, J = 7.0 Hz, 4H), 2.69 (d, J = 12.8 Hz,
H), 1.27 (t, J = 7.1 Hz, 6H).
.3 Synthesis of probe 1
Compound 1 (0.307 g, 1 mmol), N-ethylcarbazole-3-carbaldehyde (0.223 g, 1
mmol) and 2 mL DES (deep eutectic solvent) were added into 10 mL round bottom
flasks in turn and stirred for 1-2 hours at 30-40℃. The reaction was followed by TLC.
After completion of the reaction, the mixture was cooled to room temperature, washed
with distilled water, and the precipitate was filtered and dried to give an orange solid
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1
(
0.38 g， yield 75 %). H NMR (600 MHz, DMSO-d ): δ 8.64 (s, 1H), 8.20 (d, J = 7.6
6
Hz, 1H), 8.15 (s, 1H), 7.94 (d, J = 8.6 Hz, 1H), 7.
76–7.64 (m, 3H), 7.58 (d, J = 8.9
Hz, 1H), 7.52 (d, J = 13.9 Hz, 2H), 7.29–7.23 (m, 1H), 6.83 (d, J = 8.8 Hz, 1H), 6.69
(s, 1H), 4.49 (d, J = 7.3 Hz, 2H), 3.52 (d, J = 7.2 Hz, 4H), 1.33 (t, J = 6.8 Hz, 3H),
1
3
1
1
1
4
5
2
.17 (t, J = 6.3 Hz, 6H). C NMR (151 MHz, DMSO-d ): δ 167.4, 158.8, 157.7,
6
52.5, 151.1, 147.1, 142.2, 140.7, 131.3, 127.5, 127.1, 126.0, 124.1, 123.4, 122.7,
21.3, 120.5, 120.4, 114.9, 114.1, 112.7, 110.6, 110.3, 110.2, 107.8, 96.9, 78.7,60.2,
+
4.9, 37.8,21.2, 14.3, 12.8. HRMS: m/z calculated for C H N O [M+H] :
3
3
29
4
2
13.22458, obtained: 513.22891. (Fig.S1, S2, S3)
.4 Measurement procedure
0
.001 g of probe 1 was accurately weighed and dissolved in 2 mL of DMSO
solution to obtain a 1 mM stock solution. The absorption and fluorescence spectra
experiments were measured in C H OH/PBS (v/v, 1/1, pH 7.4). In the fluorescence
2
5
spectroscopy experiment, the selected excitation wavelengths were 420 nm and the
slits were 5/2.5 nm respectively. All substances in the selective experiment were
-
-
dissolved or diluted with distilled water to make a stock solution of 0.1 M. CN , SCN ,
-
-
Br , MnO were obtained by dissolving its potassium salt. The remaining ions were
4
obtained by dissolving the sodium salt.
2
.5 Cell culture and fluorescence imaging
HepG2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)
supplemented with 12 % (v/v) fetal bovine serum (FBS) and 1 % antibiotics at 37 ℃
5

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in a 5 % CO incubator, and replaced culture media with fresh media every day. The
2
cells were plated into 6 well culture-plates and incubated for 24 h to allow the cells
adhere. The fluorescence images of probe were obtained using a ZEISS LSM 880
confocal laser scanning microscope. And fluorescence imaging at blue channel
(410-500nm), the excitation wavelength was 405 nm.
2
.6 Arabidopsis thaliana culture and fluorescence imaging
The culture of Arabidopsis thaliana was cultured according to the methods in the
literature [20]. The root tip of Arabidopsis thaliana was selected as the experimental
object. The fluorescence images of probe were obtained using a ZEISS LSM 880
confocal laser scanning microscope. And fluorescence imaging at blue channel
(410-500nm), the excitation wavelength was 405 nm.
3
. Results and discussion
3
.1 Time response and optical stability study of probe 1
Before spectral titration, we first determined the stability of 1 and the reaction time
-
with ClO by time-dependent study in C H OH/PBS (v/v, 1/1, pH 7.4). Fig. 1
2
5
displayed the change in fluorescence intensity at 490 nm of the 1 with an excitation
-
4
20 nm in 10 minutes. The probe 1 also exhibited good stability. When ClO was
added, the fluorescence intensity reached a maximum around 3 minutes and remained
stable.
Insert Fig.1
3
.2 Spectroscopic characteristics of probe 1
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-
The spectroscopic experiment of 1 (10 μM) for detecting ClO was carried out in
C H OH/PBS (v/v, 1/1, pH 7.4). As shown in Fig. 2a, 1 itself had a weak absorption
2
5
-
peak at 410 nm. With the addition of ClO , the absorption at 475 nm gradually
decreased up to disappear, and new absorption appeared at 410 nm, at the same time
the color of the solution changed from orange to colorless. Subsequently, we found
-
that 1 exhibited a good linear relationship with ClO in the concentration range of
-
2
1
47.4-522.6 μM. The regression equation was A=0.271×[ClO ]-4.448，R =0.99626
(Fig. 2b).
Inset Fig.2
The changes in the fluorescence spectra of 1 (10 μM) in C H OH/PBS (v/v, 1/1, pH
2
5
-
7
.4) system were shown in Fig. 3a. As we can see, with the addition of ClO (0 – 469
μM), 1 produced a distinct fluorescent signal at 490 nm. It is speculated that the
changes in the ultraviolet and fluorescence spectra resulting from the strong oxidative
-
nature of ClO cleaving double bond to form
a
new compound,
N-ethylcarbazole-3-carbaldehyde. Therefore, we dissolved the molecular in DMSO
and performed fluorescence detection in the test system. The emission wavelength of
the molecular is almost same as the emission wavelength of the 1 (Fig. S4). The
−
fluorescence intensity enhanced linearly with the ClO concentration from 93.8-361.8
-
μM. The linear equation could be represented by F=11.972×[ClO ]-1100.071 with the
2
correlation coefficient of R = 0.9942 (Fig.3b). The detection limit was calculated to
be 0.19 μM according to the IUPAC standard method LOD=3δ/k. These data
7

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-
demonstrated that 1 could achieve quantitative detection of ClO .
Inset Fig.3
3
.3 The selective response of probe 1
Excellent selectivity and anti-interference performance are key parameters for
evaluating the suitability of a probe for biological applications. Therefore, we
-
evaluated the selectivity of 1 for ClO by adding an excess of different anions, strong
oxidizing substances or some common nucleophilic thiols. As could be seen in Fig. 4,
the addition of other analytes each did not cause significant fluorescence changes in
the probe 1 system. We further added different analytes to 1, and then added 469 μM
-
ClO to detect the anti-interference ability of 1. Those results showed that 1 has
-
excellent selectivity and anti-interference ability for ClO detection.
Inset Fig.4
3
.4 The effect of pH for probe 1
Next, we tested the fluorescence response of 1 to ClO in different pH environments
-
(pH 3-9). As observed in Fig. 5, 1 had no significant difference in fluorescence
-
intensity between pH 3-9. When 469 μM ClO was added, 1 showed the largest
-
response signal to ClO only in the pH 7.4 environment. Therefore, 1 could be used to
-
monitor ClO without being affected by physiological pH.
Inset Fig.5
3
.5 Proposed mechanism
-
We further explored the recognition mechanism of probe 1 for ClO by NMR
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-
titration. The olefin in the 1 structure was easily oxidized by ClO to an epoxy
compound and then formed an aldehyde (Scheme 2). As Fig. S5 shown, after the
-
addition of ClO , a new peak appeared at a chemical shift of 10.04 ppm, which was
attributed to the aldehyde group peak. The other peaks shifted to higher field. The
suggested mechanism cleavage is accordance with the absorption and fluorescence
spectra.
Inset Scheme 2
3
.6 Cellular imaging
Based on the excellent characteristics of the above probe 1 in vitro, we applied 1 to
-
HepG2 intracellular imaging to detect exogenous ClO . HepG2 cells were incubated
with 1 (5 μM) for 15 min and then washed three times with PBS buffer. Fig. 6 A1 and
A2 showed that there is almost no fluorescence change in the cells under the blue
-
channel. However, after treated with 154.1 μM ClO for 30 min and then washed three
times with PBS buffer in which an obvious fluorescence signal was appeared in blue
-
channel (B1 and B2). All the results illustrated that 1 could be used to monitor ClO in
living cells.
Inset Fig.6
3
.7 Arabidopsis thaliana imaging
In order to verify that the probe 1 has better cell permeability, we also performed
Arabidopsis root tip imaging. Arabidopsis root tips were firstly incubated with 1 for
3
0 min and washed three times. After co-incubation of 1 and Arabidopsis root tips,
9

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-
incubation with ClO continuously for 40 min. As shown in Fig.7, there was a
significant fluorescence change from A1 to B1 under blue channel. This was
consistent with the phenomenon of fluorescence imaging in cells. Those data showed
that 1 had good cell permeability and could be successfully applied to the root tip of
plants.
Inset Fig.7
4
. Conclusions
In summary, we designed and synthesized a fluorescent probe 1 with
-
electron-withdrawing group as an accelerator for the detection of ClO . The addition
-
of ClO caused 1 to produce a strong fluorescence emission at a wavelength of 490
nm and exhibited a good linear relationship in the concentration range of 147.4-522.6
-
μM. 1 showed excellent time response to ClO less than 150s. Simultaneously, the
probe exhibited good selectivity, optical stability, high sensitivity and low detection.
Furthermore, the results of fluorescence imaging confirmed that 1 can be applied to
-
HepG2 cells and Arabidopsis root tip to identify exogenous ClO . Most importantly, it
will provide a new idea to improve the reaction speed between the probe and HClO to
realize in situ detection of HClO in vivo.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
2
1775096, 21672131), Natural Science Foundation of Shanxi Province of China (No.
1
0

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2
01701D121018), One hundred people plan of Shanxi Province, Shanxi Province
"1331 project" key innovation team construction plan cultivation team (2018-CT-1),
Shanxi Province Foundation for Returness (2017-026), Shanxi Collaborative
Innovation Center of High Value-added Utilization of Coal-related Wastes, 2018
xiangyuan county solid waste comprehensive utilization Science and technology
project (2018XYSDJS-05), the Shanxi Province Science Foundation for Youths (No.
2
01901D111015), Key R&D Program of Shanxi Province (201903D421069), and
Scientific Instrument Center of Shanxi University (201512).
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Figure captions
Scheme 1. The synthetic route of probe 1.
-
Scheme 2. Response mechanism of probe 1 to ClO .
Fig.1 Time-dependent fluorescence intensity of probe 1 before and after the addition
-
of ClO in C H OH/PBS (v/v, 1/1, pH 7.4). λ = 420 nm, slit: 5nm/2.5nm.
2
5
ex
-
Fig.2 (a) The absorption spectra of probe 1 (10 μM) vary with increasing ClO (469
μM) concentration in C H OH/PBS (v/v, 1/1, pH 7.4); inset: the color changes from
2
5
-
orange to colorless. (b) Linear fit between absorbance (475 nm) and ClO
concentration (147.4-522.6 μM); inset: the absorbance of probe 1 at 475 nm.
Fig.3 (a) The fluorescence spectrum of probe 1 (10 μM) in the presence of different
-
concentration of ClO (0-469 μM) in C H OH/PBS (v/v, 1/1, pH 7.4). (b) Linear fit
2
5
-
between the fluorescence intensity (490 nm) and ClO concentration (93.8-361.8 μM).
λex=420nm, slit: 5 nm/2.5 nm.
-
Fig.4 Fluorescence intensity of probe 1 (10 μM) towards ClO in C H OH/PBS (v/v,
2
5
-
-
-
1
/1, pH 7.4) in the absence and presence of 200 μM various analytes (F , Cl , Br ,
-
2-
4
2-
3
2-
2-
-
-
-
-
-
HCO , SO , SO , S , CO , CNS , AC , NO , HSO , CN , Cys, GSH, Hcy, H O ,
3
3
3
3
2
2
2
-
MnO ). λ =420nm, slit: 5 nm/2.5 nm.
4
ex
Fig.5 Fluorescence intensity of the probe 1 (10 μM) at different pH values in the
-
absence or presence of ClO (469 μM) in C H OH/PBS (v/v, 1/1, pH 7.4). λ =420nm,
2
5
ex
slit: 5 nm/2.5 nm.
Fig.6 (A1-A2) Confocal fluorescence images of HepG2 cells incubated with probe 1
1
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(5 μM) for 15 min (A1, blue channel); (B1-B2) Confocal fluorescence images of
-
HepG2 cells were further cultured with ClO (154.1 μM) for 10 min (B1, blue
channel). Fluorescence images of HepG2 cells were indicated by blue channel (λ =
ex
4
05nm, λ = 410-500 nm).
em
Fig.7 (A1-A2) Confocal fluorescence images of Arabidopsis root tip with probe 1 (10
μM) for 30 min (A1, blue channel); (B1-B2) Confocal fluorescence images of
-
co-incubation of 1 and Arabidopsis root tips towards addition of ClO (154.1 μM) for
4
0 min (B1, blue channel). Blue channel: λ = 405nm, λ = 410-500 nm.
ex em
1
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Figures
Scheme 1. The synthetic route of probe 1.
-
Scheme 2. Response mechanism of probe 1 to ClO .
Fig.1 Time-dependent fluorescence intensity of probe 1 before and after the addition
-
of ClO in C H OH/PBS (v/v, 1/1, pH 7.4). λ = 420 nm, slit: 5nm/2.5nm.
2
5
ex
1
7

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-
Fig.2 (a) The absorption spectrum probe 1 (10 μM) vary with increasing ClO (469
μM ) concentration in C H OH/PBS (v/v, 1/1, pH 7.4); inset: the color changes from
2
5
-
orange to colorless. (b) Linear fit between absorbance (475nm) and ClO
concentration (147.4-522.6 μM); inset: the absorbance of probe 1 at 475 nm.
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Fig.3 (a) The fluorescence spectrum of probe 1 (10 μM) in the presence of different
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concentration of ClO (0-469 μM) in C H OH/PBS (v/v, 1/1, pH 7.4). (b) Linear fit
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between the fluorescence intensity (490 nm) and ClO concentration (93.8-361.8 μM).
λex=420nm, slit: 5 nm/2.5 nm.
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Fig.4 Fluorescence intensity of probe 1 (10 μM) towards ClO in C H OH/PBS (v/v,
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1
/1, pH 7.4) in the absence and presence of 200 μM various analytes (F , Cl , Br ,
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3
2-
2-
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-
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HCO , SO , SO , S , CO , CNS , AC , NO , HSO , CN , Cys, GSH, Hcy, H O ,
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3
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MnO ). λ =420nm, slit: 5 nm/2.5 nm.
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ex
Fig.5 Fluorescence intensity of the probe 1 (10 μM) at different pH values in the
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absence or presence of ClO (469 μM) in C H OH/PBS (v/v, 1/1, pH 7.4). λ =420nm,
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ex
slit: 5 nm/2.5 nm.
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Fig.6 (A1-A2) Confocal fluorescence images of HepG2 cells incubated with probe 1
(5 μM) for 15 min (A1, blue channel); (B1-B2) Confocal fluorescence images of
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HepG2 cells were further cultured with ClO (154.1 μM) for 10 min (B1, blue
channel). Fluorescence images of HepG2 cells were indicated by blue channel (λ =
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05nm, λ = 410-500 nm).
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Fig.7 (A1-A2) Confocal fluorescence images of Arabidopsis root tip with probe 1 (10
μM) for 30 min (A1, blue channel); (B1-B2) Confocal fluorescence images of
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co-incubation of 1 and Arabidopsis root tips towards addition of ClO (154.1 μM) for
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0 min (B1, blue channel). Blue channel: λ = 405nm, λ = 410-500 nm.
ex em
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Author contributions
Ming Li and Miao Xu carried out synthesis and spectrometry
Juanjuan Wang’s cell imaging
Caixia yin and Fangjun huo’s idea and writing
Jianbin Chao and Yaoming Liu’s NMR
Yongbin Zhang’s sample detection.
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Declaration of interest statement
We have no any interest conflict.
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Graphic
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Highlights
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. A novel fluorescent probe with electron-withdrawing group as an accelerator for
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the detection of ClO .
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The probe exhibited good selectivity, optical stability, high sensitivity and low
detection.
. The probe can be applied to HepG2 cells and Arabidopsis root tip to identify
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exogenous ClO .
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