A fluorescent sensor for selective detection of hypochlorite and its application in Arabidopsis thaliana

Article abstract of DOI:10.1016/j.saa.2020.118830

Hypochlorite, as one of reactive oxygen species, has drawn much attention due to its essential roles in special biological events and disorders. The exogenous hypochlorite remains a risk for human, animals and plants. In this work, a novel water soluble quinolin-containing nitrone derivative T has been developed for fluorometric sensing hypochlorite. The response mechanism of T towards ClO^? was reported for the first time. In comparison with the reported sensors for ClO^?, the sensor T in this work exhibited advantages including high selectivity (80 fold over other analytes), rapid response (within 5 s) and lipid-water distribution transformation (LogP from 2.979 to 6.131). Further biological applications suggested that T was capable of monitoring both exogenous and endogenous ClO^? in living cells. The imaging in Arabidopsis thaliana indicated that the absorption and transmission of ClO^? in plant could be monitored by this sensor through the chlorine-related mechanism. This work might raise referable information for further investigations in the physiological and pathological events in both tumor and plants.

Full text of DOI:10.1016/j.saa.2020.118830

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118830
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A fluorescent sensor for selective detection of hypochlorite and its
application in Arabidopsis thaliana
a,
a,
Zi-Xuan Zenga,1, Jin Gu a,1, Ya-Ni Liu a, Dong-Dong Lib, Yu-Shun Yanga, , Bao-Zhong Wang , Hai-Liang Zhu
⁎
⁎
⁎
a
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, China
b
College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2020
Received in revised form 4 August 2020
Accepted 9 August 2020
Available online 15 August 2020
Hypochlorite, as one of reactive oxygen species, has drawn much attention due to its essential roles in special bi-
ological events and disorders. The exogenous hypochlorite remains a risk for human, animals and plants. In this
work, a novel water soluble quinolin-containing nitrone derivative T has been developed for fluorometric sensing
hypochlorite. The response mechanism of T towards ClO− was reported for the first time. In comparison with the
reported sensors for ClO−, the sensor T in this work exhibited advantages including high selectivity (80 fold over
other analytes), rapid response (within 5 s) and lipid-water distribution transformation (LogP from 2.979 to
6.131). Further biological applications suggested that T was capable of monitoring both exogenous and endoge-
nous ClO− in living cells. The imaging in Arabidopsis thaliana indicated that the absorption and transmission of
ClO− in plant could be monitored by this sensor through the chlorine-related mechanism. This work might
raise referable information for further investigations in the physiological and pathological events in both
tumor and plants.
Keywords:
Hypochlorite detection
Fluorescent sensor
Biological imaging
Plant tissue
Nitrone-involved mechanism
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
and chloride ions in a biosystem catalyzed by myeloperoxidase (MPO)
[17]. It can cause the damage of DNA, RNA, fatty acids, cholesterol, and
Since the fluorescent sensors guided the prosperity in chemical biol-
ogy in the recent decade [1,2], the concomitant sensing approaches
have started to challenge the traditional analytical methods including
electrochemistry [3], Raman spectroscopy [4], colorimetry [5],
titrimetry [6], chromatography [7], and so on. Generally, the advantages
of the fluorescent implements include simple preparation of samples,
convenient technical procedures, and of course the most important
properties such as high sensitivity, high selectivity and good biocompat-
ibility [8,9]. To date, many practical fluorescent sensors have been
exploited for the detection of various chemical species via lots of mech-
anisms [10].
Among the chemical species, reactive oxygen species (ROS) act as the
significant indicators of physiological and pathological procedures
[11,12], thus have accordingly been utilized in diagnostic and therapeutic
applications [13–15]. In particular, as one of the ROS, hypochlorite (ClO−)
has drawn much attention due to its essential roles in special biological
events such as the prevention of microorganism invasion [16]. In organ-
isms, hypochlorite is commonly synthesized from hydrogen peroxide
proteins [18]. Therefore, the hypochlorite level is tightly associated with
disorders and diseases such as atherosclerosis, arthritis, and cancers
[19]. The exogenous hypochlorite mainly came from the disinfection
agent in drinking water treatment, which remains a risk for human, ani-
mals and plants [20,21]. Thus the detection of hypochlorite could not
only reveal the indistinct mechanism of disease occurrence and develop-
ment, but also hint new therapeutic schedules via ROS production [22].
A certain number of fluorescent sensors have been developed for
monitoring hypochlorite. Begining at about 2010 [23] and booming
from approximate 2016 [24], the researches on sensing hypochlorite
have involved lots of chromophores such as Nile Blue, phenothiazines,
fluorescein, benzothiazole, 1,8-naphthalimide, BODIPY and Iridium
complexes as well as various mechanisms including oxidation,
deprotection, breaking double bond and logical gate [25–44]. To over-
come the reported limitations such as narrow practical pH range, com-
plicated synthesis, poor water-solubility and low selectivity, novel
fluorescent sensors with new mechanisms are still in urgent need.
In this work, a novel water soluble quinolin-containing nitrone de-
rivative T has been developed for fluorometric sensing hypochlorite
(Fig. 1). The response mechanism of T towards ClO− was reported for
the first time. These fluorescent sensors seemed unique among the re-
cent hypochlorite sensing reports [25–44]. With practical optical and
recognizing performances, the sensor was further applied to monitor
the ClO− level in cancer cells and Arabidopsis thaliana.
⁎
Corresponding authors.
E-mail addresses: ys_yang@nju.edu.cn (Y.-S. Yang), wbz@nju.edu.cn (B.-Z. Wang),
zhuhl@nju.edu.cn (H.-L. Zhu).
1Both authors contributed equally to the work.
https://doi.org/10.1016/j.saa.2020.118830
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
Z.-X. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118830
Fig. 1. Illustration of the fluorescent sensor T for monitoring hypochlorite.
2. Experimental
and the reaction was kept refluxing at 115 °C overnight. After cooling
down to room temperature, the toluene layer was removed. The satu-
rated sodium hydroxide solution was added to neutralize the aqueous
layer. The solution was extracted with CH2Cl2, washed with saturated
sodium chloride solution for three times, dried over anhydrous sodium
sulfate, filtered and concentrated by evaporation under reduced pres-
sure. The crude product was purified by column chromatography on sil-
ica gel (PE: EA = 4:1 v/v) to gain intermediate 2 as a sand-brown solid
(3.6 g, 70%). Subsequently, selenium dioxide (2.8 g, 25.1 mmol, 1.3
equiv) was dissolved. The solution was heated at 80 °C for 30 min. Inter-
mediate 2 (3.5 g, 19.2 mmol, 1 equiv) was then added and the mixture
was refluxed at 80 °C for 4 h. After being cooled down to room temper-
ature, the mixture was filtered and the residue was washed three times
by CH2Cl2. The filtrate was concentrated by evaporation under reduced
pressure to obtain crude product which was purified by column chro-
matography on silica gel (PE: EA = 6:1 v/v). The acquired quinoline al-
dehyde T-Prod (also the detecting product) was yellow solid
(1.52 g, 40%).
The intermediate T-Prod (200 mg, 1.0 mmol) and pyrrolidine
(85.44 mg, 1.2 mmol) were dissolved in CH2Cl2 (10 mL) for 10 min. N-
(tert-Butyl) hydroxylamine hydrochloride (125.6 mg, 1 mmol) was
added into the solution. The mixture was stirred at room temperature
for 4 h. After the completion of the reaction confirmed by TLC, the
solvent was condensed by evaporation and was then purified by thin-
layer chromatography (PE: EA = 1:2, v/v) to gain the sensor T, (Z)-N-
tert-butyl-1-(6-(dimethylamino)quinolin-2-yl)methanimine oxide as
tawny solid (35 mg). Yield: 17.5%. 1H NMR (600 MHz, Chloroform‑d) δ
9.15 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 9.2 Hz,
1H), 7.37 (d, J = 12.0 Hz, 1H), 6.79 (s, 1H), 3.11 (s, 6H), 1.66 (s, 9H).
13C NMR (151 MHz, DMSO‑d6) δ 152.02, 146.63, 141.01, 132.95,
127.98, 127.45, 121.21, 120.52, 100.21, 29.16, 23.69.
2.1. The experimental materials and equipment
All chemicals purchased were used as received without further puri-
fication. The products were purified by 300–400 mesh chromatography
silica gel. All NMR data of the compounds were measured by Bruker
(Rhenistetten-Forchheim, Germany) AM 600 MHz, collected in
Chloroform‑d or DMSO‑d6 and dealt with MestReNova software
(Mestrelab Research, Santiago de Compostela, Spain). Mass spectra
were acquired from an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS
(Agilent Technologies Inc., California, USA). UV–vis spectra were de-
tected on Shimadzu UV-2550 spectrometer (Shimadzu Corp., Tokyo,
Japan). The fluorescence intensity detection was accomplished on
Hitachi Fluorescence Spectrophotometer F-7000 (Hitachi Ltd., Tokyo,
Japan). pH calibration was conducted by pH meter of PHS-25. Cell viabil-
ity experiments were carried out using MTT method. Bioimaging exper-
iment was presented on Leica TCS SP8-MaiTai M double-photon
confocal fluorescent microscope (Leica Camera AG, Weztlar, Germany).
2.2. Determination of LogP values
Aqueous sodium chloride (0.9% w/v) and organic (n-octanol) phases
were saturated for 1 week. The tested compounds were dissolved at a
final concentration of 300 μM in the aqueous phase. An equal volume
of saturated n-octanol was added and the solutions mixed for 30 min
under 45 rpm. Samples were centrifuged (1500g, 10 min) and the tested
compound content of the organic and aqueous phases was determined
by UV absorbance at 280 nm. The LogP values were then defined as the
logarithmic ratio of test compound concentrations in the organic and
aqueous phases.
2.3. Synthesis of the sensors
3. Results and discussion
The general synthesis route of the sensor T and the intermediate T-
Prod (also the detecting product) was illustrated in Fig. 2. 4-N,N-
Dimethylamino aniline (Compound 1, 5 g, 36.7 mmol, 1.0 equiv) was
added into a solution of HCl (6 M, 66 mL). Then crotonaldehyde
(6.0 mL, 73.5 mmol, 2 equiv) was added into the mixture and stirred
at room temperature for 1 h. Afterwards, toluene (35 mL) was added
3.1. Synthesis of the sensor
The sensor T and the intermediates were synthesized as shown in
Fig. 2 and confirmed by satisfactory spectroscopic data (1H NMR, 13
NMR, HRMS, Figs. S5–10, SI).
C
Fig. 2. General synthesis route of the sensor T and the intermediates.

Z.-X. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118830
3
Fig. 3. (A) The fluorescence spectra of the sensor T (10 μM) in PBS buffer (10 mM, pH 7.4, 1% DMSO) after treatment with hypochlorite (0–20 equiv.) for 5 min; (B) The linear relationship
between the fluorescence intensity and the concentration of hypochlorite (0–20 equiv.) in PBS buffer (10 mM, pH 7.4, 1% DMSO) for 5 min.
3.2. Fluorescent analytical performance of sensor T
3.3. Selectivity and anti-interference ability
Initially, we evaluated the fluorescent properties of the sensor T in
buffered aqueous media (physiological pH 7.4, 10 mM PBS, 1% DMSO
as a co-solvent). With the addition of ClO−, the absorption peak exhib-
ited a blue-shift and the color changed to transparency under naked-eye
observation instantaneously (Fig. S1, SI). As we expected, under the ex-
citation wavelength of 460 nm, the free sensor T presented an obvious
luminous chartreuse fluorescence emission peak at 561 nm, whereas
with the addition of hypochlorite, the fluorescence was quenched im-
mediately (Fig. 3). With the increasing hypochlorite concentration, the
fluorescence intensity of the systems decreased with a high ratio up to
80-fold reduction. The fluorescent response of T towards ClO− was cal-
culated to cover a linear range from 1 × 10−7 to 2 × 10−4 M, according
to linear formula: y = 3986–205.4 × x. The detection limit was calcu-
lated as 0.47 μM.
The selectivity and anti-interference ability of the sensor T were both
evaluated (Fig. 4). A variety of competing analytes including ROS, anions
and cations were used independently or together with ClO−. The con-
centration of the sensor was 10 μM, and the analytes were all set as
200 μM. The data were presented as the ratios of the fluorescence inten-
sity of T before (F0) and after (F1) the addition of ClO−. As shown in
Fig. 4A, no remarkable fluorescence variation was found in the presence
of the other analytes including ions, amino acids and macromolecules
(HSA: Human Serum Albumin; BSA: Bovine Serum Albumin). Only
ClO− could quench the fluorescence. Meanwhile, as shown in Fig. 4B,
during the incubation of the involved analytes together with ClO−, the
interference on the detecting system was not obvious. Therefore, in
the complex intracellular environment, T might be a selective sensor
with a reliable detecting performance.
Besides, we checked two important parameters for ROS sensors, pH
and response time. In the pH range of 3–12, the sensor T showed a con-
stant fluorescence intensity within pH 7–12 while the detecting system
of T and ClO− exhibited no fluorescence within pH 3–10 (Fig. S2, SI).
Therefore the workable range of T was set as pH 7–10, which could
cover most of the physiological environments for ROS generation. On
the other hand, a time scan (Fig. S3, SI) suggested that the detecting pro-
cess completed in a very short moment (<5 s), which was extremely
suitable for monitoring the rapidly changing ROS level.
3.4. The reaction mechanism and characteristic
As we claimed in the Introduction, the response mechanism of T was
achieved via the departure of the new reported nitrone recognizing
group for hypochlorite sensing (Fig. 5). After comparing the HRMS
and 1H NMR spectra of T and T-Prod, we confirmed the detecting prod-
uct of this system (Figs. S5, S7, S8, S10). Being incubated with ClO−, the
nitrone moiety was transferred into the aldehyde, which led to the
Fig. 4. The ratios of the fluorescence intensity at 561 nm of T before (F0) and after (F1) the addition of ClO− in PBS buffer (10 mM, pH 7.4, 1% DMSO). (A) Independent analytes and (B) the
coexistence systems with ClO−: (1) Mn2+; (2) Br−; (3) K+; (4) Ni2+; (5) Mg2+; (6) Al3+; (7) I−; (8) Zn2+; (9) Co2+; (10) Fe3+; (11) SCN−; (12) HCO−3 ; (13) HSO−4 ; (14) S2O8−; (15) H2O2;
(16) KO2; (17) Leu; (18) Hcy; (19) Arg; (20) His; (21) Met; (22) Gln; (23) Gly; (24) Ala; (25)Asp; (26) Thr; (27) Ile; (28) Trp; (29) Ser; (30) Glu; (31) Cys; (32) Hsp; (33) GSH; (34) GSH;
(35) Val; (36) HSA; (37) BSA; (38) ClO−; (39) Sensor T only.

4
Z.-X. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118830
H
H
ClO-
O
Cl
O
+ ClO-
+ H2O
H+
N
N
N
N+
O-
N+
O-
N
O-
N
N
N
T
Possible
Industrial
Reproduction
Cl
O
N
O
N
O
H
HN
OH
N
Redox
N
T-Prod
ClO-
Fig. 5. The illustration of the sensing mechanism of the sensor T towards hypochlorite.
change of the chartreuse fluorescence into a faint orange one (even no
obvious signal if the excitation of 460 nm was not adjusted). It was a
pity that it seemed difficult for us to capture the intermediate during
the response due to the transitory process, but we could still be hinted
from the rare report on this reaction that the chlorine of hypochlorite
participated in the sensing procedure [45]. Instead of experiencing a
catalysis-like procedure, the redox pair of hydroxylamine and ClO− lim-
ited the regeneration of ClO−, thus promoted the equilibrium of the de-
tecting reaction. Moreover, we believed that the sensor T experienced a
transformation in the preference of lipid-water distribution during the
detection. The logP (a model for evaluating oil-water partition coeffi-
cient) values of T and T-Prod were calculated as 2.979 and 6.131
(both around the 2–5 range of Lipinski's “Rule of Five” for drugs), re-
spectively. This result suggested that the sensor itself, as a salt-like com-
pound, was more suitable for delivery, while the detecting product, as
an amphiphilic molecule, was more favorable for the intracellular
imaging.
3.5. Biological imaging
Before the biological imaging, the cytotoxicity of the sensor T was
evaluated upon MCF-7 (human breast adenocarcinoma cell line) and
HEK293T (human embryonic kidney epithelial cell line) cells by using
MTT method. As shown in Fig. S4 in SI, after being incubated with the
Fig. 6. The confocol images of MCF-7 cells incubated with T in different conditions. The concentrations of ClO- were set as 0, 100, 200, 500 μM, respectively. After the incubation with ClO−
for 10 min, a further incubation with 10 μM T for 10 min was conducted for each group. The temperature was set as 37 °C. Green channel: collected at 500–600 nm, λex = 460 nm. Scale bar:
75 μm.

Z.-X. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118830
5
sensor T (0.1–100 μM) for 24 h, the studied cells maintained a high sur-
vival rate over 75%.
(slightlt behind the tip because the tip was relatively dense) caused
an initial enrichment of ClO−, which could be monitored by the sensor
T through a decrease of the green fluorescence. The absorbed ClO−
came up along the catheter, therefore the decrease of fluorescence
could be observed also in the central catheter region. When it came to
the catheter top, the decrease of fluorescence seemed less obvious,
which indicated the transformation of ClO− into other species. How-
ever, in the CHL group, the absorption of chlorine was blocked from
the very beginning, thus the green fluorescence was not affected in all
the investigated regions. Since the major difference of the two groups
was the absorption of chlorine, we could infer that the response mech-
anism was associated with chlorine. Moreover, T could be used to mon-
itor ClO− during specific events in plant physiology.
Based on the above results, we used T to monitor exogenous and en-
dogenous ClO− in living MCF-7 cells under the fluorescent confocal mi-
croscope. As shown in Fig. 6, with the addition of gradient
concentrations (0, 100, 200, 500 μM) of ClO−, the fluorescence of the
system remarkably decreased along with the increase of ClO−
concentration.
Furthermore, we studied whether
T could monitor the
endogenously-produced ClO− in living cells. For the endogenous
ClO−, a typical pair of 1.0 μg/mL lipopolysaccharide (LPS) and
1.0 μg/mL phorbol myristate acetate (PMA) was used as the stimulator,
whereas 200 μM 4-azidobenzohydrazide (ABH, a well-known inhibitor
of MPO) was used as the blocker [19]. As shown in Fig. 7, the addition of
ABH could maintain the fluorescence, whereas the incubation of the
LPS/PMA pair could cause an obvious fluorescence decrease in the
green channel. A further group containing the LPS/PMA pair together
with ABH exhibited no obvious decrease of the fluorescence, which
was more close to the control group. The results above indicated that
the sensor T was capable of monitoring both exogenous and endoge-
nous ClO− in living cells.
4. Conclusion
In summary, a novel water soluble quinolin-containing nitrone de-
rivative T has been developed for fluorometric sensing hypochlorite.
The response mechanism of T towards ClO− was reported for the first
time. In comparison with the reported sensors for ClO− (Table S1, SI),
the sensor T in this work exhibited advantages including high selectivity
(80 fold over other analytes), rapid response (within 5 s) and lipid-
water distribution transformation (LogP from 2.979 to 6.131). The bio-
logical application of the sensor T was further performed in cancer
cells and Arabidopsis thaliana. The results indicated that T was capable
of monitoring both exogenous and endogenous ClO− in living cells.
Moreover, the absorption and transmission of ClO− in plant could be
monitored by this sensor through the chlorine-related mechanism.
We have demonstrated in the introduction that ClO− could also af-
fect the plant physiology. In this work, we applied the sensor T into
the ClO− detection in both the wild type (WT) and chlorate resistant
1-mutant (CHL, a mutant strain which cannot absorb chlorate)
Arabidopsis thaliana. Thus we could track the absorption and transmis-
sion of ClO− in this model plant, as well as conversely evidence the par-
ticipation of chlorine in the response mechanism. As shown in Fig. 8, in
the WT group, the absorption of ClO− within the region near root tip
Fig. 7. The confocol images of MCF-7 cells incubated with T in different conditions. The concentration of the LPS/PMA pair was set as 1.0 μg/mL each, and that of ABH was set as 200 μM.
After the incubation with corresponding agents above for 30 min, a further incubation with 10 μM T for 10 min was conducted for each group. The temperature was set as 37 °C. Green
channel: collected at 500–600 nm, λex = 460 nm. Scale bar: 75 μm.

6
Z.-X. Zeng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 244 (2021) 118830
Fig. 8. The confocol images of Arabidopsis thaliana incubated with T at 25 °C (room temperature). Both the wild type and the chlorate resistant 1-mutant one were involved with the
catheter top, central catheter and root tip regions studied. The incubation condition was set as 100 μM ClO− (30 min, room temperature) and 10 μM T (10 min, room temperature).
λex = 460 nm. Scale bar: 100 μm.
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Zi-Xuan Zeng: Methodology, Formal analysis. Jin Gu: Validation, Vi-
sualization. Ya-Ni Liu: Writing - original draft, Validation. Dong-Dong
Li: Writing - review & editing. Yu-Shun Yang: Conceptualization, Writ-
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pervision. Hai-Liang Zhu: Funding acquisition.
Declaration of competing interest
All authors declare that there are no conflicts of interest.
Acknowledgments
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This work is supported by the Natural Science Research of Jiangsu
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
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