Development of an ultrafast fluorescent probe for specific recognition of hypochlorous acid and its application in live cells

Article abstract of DOI:10.1039/d1ra04082k

Hypochlorous acid (HOCl), a highly potent oxidant of reactive oxygen species, plays critical roles in many physiological and pathological processes. In this work, a novel coumarin-based fluorescent probe, Cou-HOCl, was prepared for the detection of HOCl. The probe exhibited good selectivity over other analytes, excellent sensitivity with a detection limit of 16 nM, and fast response within 5 s. And further study demonstrated that the probe could be used not only to image exogenous HOCl in various cells, but also to determine the fluctuating levels of HOCl in macrophage cells during inflammation.

Full text of DOI:10.1039/d1ra04082k

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Development of an ultrafast fluorescent probe for
specific recognition of hypochlorous acid and its
application in live cells†
Cite this: RSC Adv., 2021, 11, 24669
Zhencai Xu,‡ab Xiaofeng Wang,‡a Tingting Duan,a Rong He,a Fangwu Wanga
a
*
and Xuejun Zhou
Hypochlorous acid (HOCl), a highly potent oxidant of reactive oxygen species, plays critical roles in many
physiological and pathological processes. In this work, a novel coumarin-based fluorescent probe, Cou–
HOCl, was prepared for the detection of HOCl. The probe exhibited good selectivity over other analytes,
excellent sensitivity with a detection limit of 16 nM, and fast response within 5 s. And further study
demonstrated that the probe could be used not only to image exogenous HOCl in various cells, but also
to determine the fluctuating levels of HOCl in macrophage cells during inflammation.
Received 25th May 2021
Accepted 2nd July 2021
DOI: 10.1039/d1ra04082k
rsc.li/rsc-advances
made to develop uorescent probes of HOCl on the basis of
reactions with various sensing moieties including oxime
oxidation,15,16 selenium oxidation,17,18 p-aminophenol oxida-
Introduction
Hypochlorous acid (HOCl) is an indispensable reactive oxygen
species (ROS) in organisms, which is intimately involved in
various pathophysiological processes and has a signicant
impact on organisms.1 HOCl is produced in the body by
inammatory cells such as macrophages and neutrophils via
the reaction of hydrogen peroxide (H2O2) and chloride ions
(Clꢀ) catalyzed by myeloperoxidase (MPO).2,3 HOCl can effec-
tively kill bacteria in cells, which allows HOCl to be used as
a powerful tool to resist pathogens that invade the immune
system.4 However, abnormal production of HOCl may cause
oxidative stress, leading to the occurrence of various diseases
including inammation, cardiovascular disease, nervous
system disease, and even cancer.5–7 In view of the important role
of HOCl in living systems, there is an urgent need to develop
tools to study the dynamic changes of HOCl in vivo.
Various methods such as electrochemistry, iodometry and
colorimetry are available to detect HOCl, but they are very
complicated to execute.8,9 In recent years, uorescence imaging
technology has developed rapidly and attracted increasing
attention because it can be used to study changes of molecules
at the cellular level in real time, and do so non-invasively and
with high selectivity and sensitivity. Fluorescent probes are the
ideal tools for use with uorescence imaging technology to
investigate molecular life activities.10–14 Many efforts have been
tion,19,20 lactam oxidation,21–23 double bond oxidation,24–26 thio-
ester chlorination,27 and others.28 However, some of these HOCl
probes are limited by poor sensitivity, poor photostability, and
slow response.29,30 Therefore, developing HOCl uorescent
probes featuring advantages such as high sensitivity, high
photostability, and rapid response is still very important.
Herein, we describe a simple coumarin-based uorescent
probe, Cou–HOCl, for detecting HOCl based on an HOCl-
mediated oxidation deprotection mechanism. Cou–HOCl was
obtained by simple condensation of coumarin derivatives and
mercaptoethanol (Scheme 1). In the absence of HOCl, Cou–
HOCl itself exhibited a strong green uorescence. Aer reacting
with HOCl, Cou–HOCl showed a dramatic decrease in uores-
cence. It was worth noting that Cou–HOCl displayed excellent
sensitivity and selectivity. The biological evaluation also
demonstrated that Cou–HOCl could be employed to visualize
the uctuating levels of HOCl in cells.
aDepartment of Otolaryngology, Head and Neck Surgery, The First Affiliated Hospital
of Hainan Medical University, Haikou 570102, China. E-mail: xuejunzhouent@
hainmc.edu.cn
bGuanyun People's Hospital, Lianyungang 222000, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ra04082k
Scheme 1 (A) Synthesis of Cou–HOCl. (B) Reaction of Cou–HOCl
‡ These two authors contributed equally to this work.
with HOCl.
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Cou–HOCl could be deployed for the detection of HOCl in the
physiological pH range of living systems. Various competing
analytes such as reactive sulfur species, metal ions, reactive
nitrogen species and reactive oxygen species were applied
together with uorescence spectroscopy to investigate the
selectivity of Cou–HOCl toward HOCl. As shown in Fig. S2,† the
presence of HOCl triggered an apparent uorescence decrease,
while other analytes such as GSH, Cys, Hcy, Fe2+, Fe3+, Zn2+,
Cu2+, NO, KO2, H2O2, 1O2, HOc, and t-BuOOH did not cause
such uorescence changes. All of these results showed the
excellent sensing performance of Cou–HOCl toward HOCl over
other competing analytes. To verify the reaction mechanism, we
acquired 1H NMR spectra of Cou–HOCl to which HOCl was
added in CD3CN/H2O (v/v ¼ 9/1). As depicted in Fig. S3,† Cou–
HOCl was observed to convert to Cou–Ac.
Results and discussion
Spectral properties of Cou–HOCl
With the probe in hand, UV-Vis and uorescence spectra were
rst acquired. As shown in Fig. 1A, upon being excited with light
having a wavelength of 410 nm, Cou–HOCl displayed an emis-
sion peak at 510 nm. As the HOCl concentration was increased
from 0 to 50 mM, the uorescence emission showed a prompt
decrease in intensity and a slight red shi. The uorescence
intensity of Cou–HOCl at about 510 nm and the concentration
of HOCl (0–50 mM) showed a predominantly linear relationship
(F510 nm ¼ ꢀ17.99 ꢁ [HOCl]/mM + 979.83, R2 ¼ 0.9987). Based on
the standard method of 3s/k, the detection limit of HOCl was
calculated to be 16 nM (Fig. S1†). Free Cou–HOCl yielded
a maximum absorption peak at 410 nm, as shown in Fig. 1B.
Upon increasing the concentration of HOCl from 0 to 50 mM,
a new absorption peak at 470 nm emerged and increased in
intensity with a well-dened isobestic point at 430 nm. The
uorescence intensity changes at 510 nm over time are shown
in Fig. 1C. Aer adding 50 mM HOCl to the solution of Cou–
HOCl, the reaction was basically completed within 5 s, and the
uorescence intensity remained stable over the course of 30 s.
The time-dependent changes in uorescence indicated the
ability of the probe to quickly respond to HOCl, and in fact do so
more quickly than most of the previously reported HOCl
probes.30 Next, we investigated the ability of Cou–HOCl to
recognize HOCl in conditions with different pH values from 2.5
to 10.0. As shown in Fig. 1D, the uorescence intensity at
510 nm remained unchanged in the pH range 6.0–10.0, showing
the excellent stability of Cou–HOCl. Throughout the pH range
2.5–10.0, the uorescence intensity of Cou–HOCl was less in the
presence of HOCl than in its absence. The results implied that
Imaging application of Cou–HOCl
The excellent spectral properties of Cou–HOCl motivated us to
investigate its potential applications in live cells. Prior to the
cell imaging, the cytotoxic activity of Cou–HOCl was rst eval-
uated by performing Cell Counting Kit-8 (CCK-8) assays. As can
be seen from Fig. S4,† Cou–HOCl showed inconspicuous cyto-
toxicity even at a concentration of 100 mM in various cells, which
indicated that the probe could be applied for cell imaging. Cou–
HOCl was incubated with different types of cells including A549,
HepG 2 and HeLa cells for 30 minutes and in each case
a remarkable green uorescence was seen (Fig. 2). However,
following incubation with 50 mM of HOCl, the intensity of the
green uorescence decreased rapidly. The results conrmed
that Cou–HOCl could sensitively detect HOCl in biological
systems.
Subsequently, we set out to investigate the ability of using
Cou–HOCl to detect HOCl in inammatory cells. Different
samples of live RAW264.7 cells were rst pretreated with
different concentrations of HOCl (0, 5, 10, 15, 20, 30, and 50 mM,
respectively), and then each incubated with Cou–HOCl. As
shown in Fig. 3A, in the absence of HOCl, the cells loaded with
Cou–HOCl showed a strong green uorescence signal. As the
concentration of the exogenous HOCl was increased, the
intracellular green uorescence gradually weakened in
a concentration-dependent manner. Compared with the control
group, the addition of 50 mM HOCl reduced the uorescence
intensity at least ve fold (Fig. 3B), which indicated that Cou–
HOCl could effectively penetrate the cell membrane and be used
to detect changes in the level of exogenous HOCl in RAW264.7
cells. To investigate the feasibility of using Cou–HOCl to image
endogenous HOCl in inammatory cells, we chose LPS (an
inammation activator) to stimulate RAW264.7 cells to produce
endogenous HOCl. As displayed in Fig. 3C and D, the green
uorescence of the LPS-treated group was weaker than that of
the control group. Myeloperoxidase (MPO) is a well-known
peroxidase that could catalyze the conversion of hydrogen
peroxide and chloride ions to HOCl. To investigate the mecha-
nism of the catalysis of the production of HOCl by MPO,
different samples of the cells were treated with MPO, H2O2 +
Clꢀ, and MPO + H2O2 + Clꢀ, respectively. For the MPO and H2O2
Fig. 1 (A) Fluorescence response of Cou–HOCl (10 mM) toward HOCl
(0–50 mM). (B) Absorption spectra of Cou–HOCl (10 mM) with HOCl
(0–50 mM). (C) Time course of the fluorescence intensity of Cou–
HOCl before and after adding 50 mM HOCl. (D) Fluorescence
responses of Cou–HOCl toward HOCl at various pH levels. All of the
data were collected from the samples in CH3CN/PBS solutions
(10 mM, v/v ¼ 3/7, pH ¼ 7.4). lex ¼ 410 nm.
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Fig. 2 Fluorescence images of exogenous HOCl in A549, HeLa and HepG 2 cells. (A) A549, HeLa and HepG 2 cells were pretreated with or
without HOCl for 30 minutes, and then treated with Cou–HOCl (10 mM) for 5 minutes. After being washed with PBS, the cells were imaged. (B–D)
Quantification of the relative fluorescence intensities of the A549, HepG2 and HeLa cells. The data are shown as mean (ꢂSD). Scale bar ¼ 30 mm.
+ Clꢀ treatment groups, the green uorescence intensities were salicyl hydroxamic acid (SHA) (two well-known MPO inhibitors),
slightly reduced compared to the control, while the intensity for and Cou–HOCl, respectively. The green uorescence signals of
the MPO + H2O2 + Clꢀ treatment group was signicantly the cells were well maintained in the presence of ABH or SHA.
reduced. To further verify the intracellular uorescence changes Overall, these results demonstrated that Cou–HOCl was
caused by endogenous HOCl, different samples of the cells were a simple tool capable to detect and image exogenous and
treated with LPS, 4-aminobenzoic acid hydrazide (ABH) or endogenous HOCl levels in inammatory cells.
Fig. 3 Fluorescence images of HOCl flux in RAW264.7 cells. (A) Different samples of RAW264.7 cells were pretreated with different concen-
trations of HOCl (0, 5, 10, 15, 20, 30 and 50 mM, respectively) for 30 minutes, and then each incubated with Cou–HOCl (10 mM) for 30 minutes
before being subjected to fluorescence imaging. (B) Quantitative plot of the relative proportion of the fluorescence intensity of the cells in A. (C)
Different samples of cells were preincubated with or without LPS (1 mg mLꢀ1, 12 h), MPO (100 ng mLꢀ1, 1 h), H2O2 (100 mM, 1 h), Clꢀ (1 mM, 1 h), 4-
ABAH (500 mM, 1 h), and SHA (500 mM, 1 h), respectively, and then each treated with Cou–HOCl (10 mM) for another 30 minutes. Then, after being
washed with PBS, the cells were imaged. (D) Quantitative plot of the relative proportion of the fluorescence intensity of the cells in C. The data are
shown as mean (ꢂSD). Scale bar ¼ 30 mm.
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