A novel ratiometric fluorescent probe for the selective determination of HClO based on the ESIPT mechanism and its application in real samples

Article abstract of DOI:10.1039/c9ra04569d

Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed and synthesized for the detection of HClO. The reaction site of diaminomaleonitrile at the ortho-position of the phenolic hydroxyl group made the probe e

Full text of DOI:10.1039/c9ra04569d

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A novel ratiometric fluorescent probe for the
selective determination of HClO based on the ESIPT
Cite this: RSC Adv., 2019, 9, 30943
mechanism and its application in real samples†
ab
Jingrui Li,ab Aijun Gong,
Guoqing Shia and Chengwen Chaia
*
Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed
and synthesized for the detection of HClO. The reaction site of diaminomaleonitrile at the ortho-position of
the phenolic hydroxyl group made the probe exhibit a ratiometric fluorescence response towards
hypochlorous acid (HClO). The specific sensing mechanism was verified via MS, HPLC and 1H NMR
spectroscopy. Moreover, the probe showed excellent performance with high sensitivity and good
selectivity towards HClO in the presence of other reactive oxygen species. In addition, the probe was
successfully applied to detect HClO spiked in tap water, river water and diluted human serum with good
recoveries.
Received 18th June 2019
Accepted 15th September 2019
DOI: 10.1039/c9ra04569d
rsc.li/rsc-advances
HClO in complicated and living systems.11–14 Therefore, many
uorescent probes have been designed and developed in recent
years. Wang et al.15 developed an effective method for the imaging of
1. Introduction
In our modern daily life, for clean drinking water, hypochlorous
acid (HClO) is widely used as a common disinfectant, especially
in water treatment and distribution; in addition, HClO is used
on a large scale in the food industry. As one of the natural
oxidants, HClO can be dissociated into hypochlorite anion
(OClꢀ) under physiological conditions (pKa ¼ 7.46).1 In biology,
its oxidation property make it react with many biomolecules
such as DNA, RNA, fatty acid groups, cholesterol and proteins.
HClO as a typical reactive oxygen species (ROS) plays an
important role in some pathological and biological events;2 for
instance, HClO mediates the peroxidation of chloride ions in
activated leukocytes3 and acts as a critical microbicidal agent in
response to inammatory stimuli; moreover, the abnormal
generation of HClO from phagocytes can cause many
inammation-related diseases including cardiovascular and
cancer,4 neurodegeneration,5 renal disease6 and osteoarthritis.7
Compared to traditional analytical techniques, such as electro-
analysis, chemiluminescence, or chromatography, used for the
determination of HClO, uorescence detection8–10 has been found
to be superior in some aspects such selectivity and sensitivity. The
uorescence method also meets the requirement of a fast response
and real-time detection, which is appropriate for the investigation of
lysosomal HClO to better understand its role in HClO-related
diseases. Xue et al.16 designed a simple and efficient ratiometric
uorescent nanoprobe for the imaging of exogenous/endogenous
HClO in lysosomes. Zhang et al.17 developed a lysosome-targetable
uorescent probe for HClO detection by combining a 4-(2-
aminoethyl)-morpholine moiety and a HClO-capturing group
phenyl-thiourea together. Yang et al.18 synthesized a uorescent
probe for the quantitative detection of HClO and live cell imaging,
which provided a powerful tool for the investigation of HClO. Peng
et al.19 reported a BODIPY-based HClO probe with high sensitivity
and fast response that was applied to image basal HClO in cancer
cells. In addition to the abovementioned uorophores, the other
uorophores used in probe designs include coumarin,20 rhoda-
mine,21 heptamine cyanine22 and iridium complexes.23
2-(20-Hydroxyphenyl)benzimidazole (HBI) is a typical uores-
cent compound, which emits intense uorescence via an excited-
state intramolecular proton transfer (ESIPT) reaction. Its unique
characteristic of a large Stokes shi (>100 nm) with a good
quantum yield (40–65%, in aqueous solution) makes it suitable for
wide applications in laser dyes, high-energy radiation detectors
and uorescence imaging. Most of the probes synthesized on the
basis of HBI or its derivatives exhibit uorescence emission of
a typical “turn-on” type. He et al.24 reported a highly selective and
sensitive probe for HClO detection by choosing HBI as a uo-
rophore. Compared to “turn-on” probes, the ratiometric uores-
cent probes are more reliable because these probes can reduce the
detection error by self-calibration based on two uorescence
emission bands; however, to date, only few ratiometric uorescent
probes have been designed based on HBI or specically used for
HClO detection.
aSchool of Chemistry and Biological Engineering, University of Science and Technology
Beijing, Beijing 100083, China. E-mail: Gongaijun5661@ustb.edu.cn; Fax: +86-010-
62334071; Tel: +86-010-82375661
bBeijing Key Laboratory for Science and Application of Functional Molecular and
Crystalline Materials, University of Science and Technology Beijing, Beijing 100083,
China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra04569d
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Therefore, in this study also, HBI was chosen as a uo- hydroxide. A yellow solid precipitated from the solution. The
rophore for the synthesis of a novel ratiometric probe. Dia- product was obtained through ltration and puried with silica
minomaleonitrile was taken as the reaction site for the sensor chromatography using hexane–ethyl acetate (10 : 1) as the
design. Compared to the former ESIPT uorophore HBO (2-(20- eluent to obtain a pure product (70 mg, 56% yield). 1H NMR
hydroxyphenyl)-benzoxazole),25 the uorescent response group (300 MHz, DMSO-d6) d 13.84 (s, 3H), 10.47 (s, 1H), 8.19 (dd, J ¼
diaminomaleonitrile led to a ratiometric probe that was uo- 2.2, 0.8 Hz, 1H), 7.68 (dt, J ¼ 8.0, 4.0 Hz, 2H), 7.60 (dd, J ¼ 2.2,
rescence emission type instead of intensity-based type. Upon 0.9 Hz, 1H), 7.37–7.25 (m, 2H), 2.36 (t, J ¼ 0.7 Hz, 3H). MS (ESI):
the addition of HClO, the probe subsequently reacted with it to calcd for C15H12N2O2: 252.09; found: 253.62.
realize hypochlorous acid-initiated oxidative intramolecular
Compound 2 was reacted with diaminomaleonitrile to
cyclization, thus providing a rapid and sensitive uorescence obtain the probe. Compound 2 (51 mg, 0.2 mmol) and dia-
response towards HClO. Due to its improved performance, the minomaleonitrile (22 mg, 0.2 mmol) were dissolved in EtOH (15
probe could be applied to detect HClO in tap water, river water mL). Then, 3 drops of acetic acid were added to the mixture. The
and diluted human serum samples.
solution was further reuxed for 5 hours under N2 gas protec-
tion. The nal yellow solution was concentrated to obtain
precipitates, which were ltered off and washed with EtOH. The
product was recrystallized in EtOH to obtain a yellow solid
(41 mg, 60% yield). 1H NMR (300 MHz, DMSO-d6, TMS): d 13.37
(s, 1H), 8.71 (s, 1H), 8.20 (d, J ¼ 2.1 Hz, 1H), 8.02 (d, J ¼ 2.2 Hz,
1H), 7.95 (s, 2H), 7.67 (s, 1H), 7.30 (d, J ¼ 7.4 Hz, 1H), 2.37 (s,
3H). MS (ESI): calcd for C19H14N6O: 342.12; found: 343.36.
2. Experimental
2.1 Reagents and instruments
All the solvents and reagents employed herein were obtained from
commercial suppliers and used directly without any further puri-
cation. Herein, the UV-vis spectra were obtained using the UV-vis
spectrophotometer UV-8000S (Shanghai Metash Instruments Co.,
Ltd.). The uorescence spectra in this study were obtained using
the uorescence spectrophotometer FS5 (Edinburgh Instruments).
Quantum yield measurements were carried out using a spectro-
2.3 Fluorescence detection procedure
The stock solution of the probe was prepared by dissolving the
probe in methanol. All the stock solutions of the other analytes
were obtained by dissolving them in ultra-pure water (see ESI†).
The uorescence properties of the probe were investigated in
PBS/MeOH (v/v, 4 : 1, pH ¼ 7.4, 10 mM). Each spectrum was
obtained aer 1 min. The detection limit (LOD) was calculated
based on the uorescence titration reported in the literature.
The uorescence intensity of ten blank samples was measured.
Then, the corresponding mean value and the standard devia-
tion were calculated. Moreover, LOD was calculated based on
the formula LOD ¼ 3s/k, where s is the standard deviation of
the blank samples measured 11 times and k is the slope ob-
tained from the calibration curve.
The quantum yields (QY,h) of the probe and the probe–HClO
system were determined according to the literature.25,26 All the
data were acquired in PBS/MeOH (v/v, 4 : 1, pH ¼ 7.4, 10 mM).
The QY, dened as the ratio of the emitted photons to the
absorbed photons, was determined according to the following
expression:
1
uorometer (FS5) equipped with an integrating sphere. The H
NMR and 13C NMR spectra were obtained using the Bruker AV-300
spectrometer (Bruker), with chemical shis reported as ppm (in
CDCl3 and DMSO-d6, with TMS as the internal standard). Mass
spectra analysis was carried out using the LCQ Fleet mass spec-
trometer (Thermo Fisher).
2.2 Synthesis of the probe
In a round-bottom ask equipped with a magnetic stirring bar,
5-methylsalicyladehyde (136 mg, 1 mmol) and sodium bisulte
(104 mg, 1 mmol) were dissolved in ethanol (15 mL). The
solution was stirred at room temperature for 4 h. Then, the
solution of o-diaminobenzene (108 mg, 1 mmol) dissolved in
ethanol (5 mL) was added to the ask and heated to reux for
4 h. The reaction was quenched by pouring the solution into ice
water. Aer a few minutes, a white solid precipitated from the
aqueous phase. The crude product, i.e. compound 1, was ob-
tained by ltration under diminished pressure, and then, it was
applied directly in the next step for the synthesis of the uo-
rescent probe without any purication (168 mg, 75% yield). 1H
NMR (300 MHz, DMSO-d6) d 13.13 (s, 1H), 12.86 (s, 1H), 7.90–
7.83 (m, 1H), 7.69 (d, J ¼ 7.5 Hz, 1H), 7.58 (d, J ¼ 7.5 Hz, 1H),
7.27 (p, J ¼ 5.4 Hz, 2H), 7.18 (ddd, J ¼ 8.4, 2.2, 0.7 Hz, 1H), 6.92
(d, J ¼ 8.3 Hz, 1H), 2.31 (s, 3H). MS (ESI): calcd for C14H12N2O:
224.09; found: 225.39.
number of photons emitted
number of photons absorbed
Lsample
h ¼
¼
Ereference ꢀ Esample
where h represents QY, Lsample is the emission intensity, and
Ereference and Esample are the intensities of the excitation light not
absorbed by the reference and the sample, respectively.
3. Results and discussion
3.1 Probe design and synthesis
The compound 2 was synthesized according to the Duff
reaction. Typically, compound 1 (112 mg, 0.5 mmol) and
hexamethylenetetramine (HMTA, 210 mg, 1.5 mmol) were dis- In the probe proposed herein, HBI was used as a uorophore,
solved in triuoroacetic acid (15 mL). The mixture was heated which generated uorescence under ultraviolet excitation; its
and reuxed for 8 h under the protection of nitrogen gas. The unique ESIPT process endowed the probe with excellent uo-
reaction was quenched by pouring the solution into ice water. rescence properties such as a large Stokes shi (>100 nm) and
The obtained solution was adjusted to neutral with sodium fast uorescence response towards HClO. As
a typical
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uorophore based on the ESIPT mechanism, HBI emits intense
uorescence via an excited-state intramolecular proton-transfer
reaction, which guarantees uorescence emission during the
detection process. On the other hand, the reaction site on the
probe could change the uorescence emission type to realize
the detection of HClO.
In this study, HBI-CH3 was easily obtained under mild
conditions. Without any purication, it could be directly used in
the following reaction. The introduction of an aldehyde group
was realized through the reaction of HBI-CH3 with hexamethy-
lenetetramine (HMTA) in an acid solution; the nal reaction to
synthesize the probe involved the stirring of HBI-CHO and
diaminomaleonitrile in methanol under reux conditions. The
introduction of a reaction site at the ortho-position of the
phenolic hydroxyl group caused the corresponding probe to
exhibit different uorescence emissions. Its uorescence
property and detection mechanism were investigated via the
following experiments (Scheme 1).
Fig. 1 Fluorescence spectral changes of the probe (10 mM) in PBS/
MeOH (v/v, 4 : 1, pH ¼ 7.4, and 10 mM) upon the addition of different
amounts of HClO (2.0–80.0 mM). Fluorescence intensity was deter-
mined 1 min after the addition of HClO at room temperature (slit width:
2/8 nm, lex ¼ 340 nm).
3.2 Fluorescence response of the probe toward HClO
The uorescence property of the probe was investigated in PBS/
MeOH (v/v, 4 : 1, pH ¼ 7.4, and 10 mM) at room temperature. As
shown in Fig. 1, the probe displayed a uorescence emission
band at 600 nm. Upon the addition of HClO, a new uorescence
emission band appeared at 485 nm. Its uorescence intensity at
485 nm gradually increased with an increase in the amount of
HClO. However, the uorescence intensity at 600 nm did not
change and maintained its original level. Therefore, the probe
exhibited a ratiometric characteristic towards HClO. By plotting
the uorescence intensity ratio (FI485/FI600) versus the concen-
tration of HClO, a good linear relationship was obtained in the
concentration range from 2.0 mM to 45.0 mM. The detection
limit was calculated to be 14.6 nM based on 3s/k. The result
proved that the probe could quantitatively and qualitatively
determine HClO with a low detection limit. Based on the
abovementioned experimental results, it can be concluded that
the proposed probe shows some advantages over the other
probes listed in Table 1 in terms of the detection solvents,
Stokes shi, detection limit and response time; this indicates
that the probe proposed herein has great potential in HClO
detection (Fig. 2).
Moreover, the UV-vis absorption of the probe was investi-
gated. As shown in Fig. 3, the probe exhibited an absorption
band between 350 nm and 425 nm with a peak centered at
400 nm. Upon the addition of HClO, the absorption band at
400 nm went down to the lower absorption value. However,
a new absorption band at 335 nm appeared in the spectrum.
HClO was added to the detection solution to react with the
probe, leading to the uorescence and UV-vis response. The
specic detection mechanism was investigated to explain the
internal changing processes.
From the initial investigation of the uorescence properties,
it was clear that the introduction of diaminomaleonitrile did
not quench the uorescence. Thus, the proposed probe is
uorescence intensity ratio type.
3.3 Time-dependent uorescence response
In addition, the time-course uorescence response of the probe
towards HClO was tested. As shown in Fig. 4, upon the addition
of 26 and 60 mM HClO, the uorescence ratio (FI485/FI600
)
increased signicantly and reached a maximum value within 30
seconds. This result showed that the probe exhibited a kind of
‘fast response’ characteristic for HClO and could be appropriate
for the real-time detection of HClO.
3.4 Selectivity of the probe
The selectivity of the probe towards HClO was also investigated.
The interfering components could be divided into three cate-
gories: anions, metal cations and ROS. Other reactive oxygen
species (ROS) were chosen to verify the probe's uorescence
response towards them. The preparation details of ROS, which
t
include 1O2, cOH, H2O2, Fe3+, ONOOꢀ, NO, and BuOOH, are
Scheme 1 Synthesis of the probe.
provided in the ESI.†
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Table 1 Typical ratiometric fluorescent probes for the determination of HClO
Detection
limit
Response
time
Probe
lex (nm), lem (nm)
Detection solvent
PBS
Ref.
27
530, 605/760
100 nM
89 nM
4.6 nM
10 min
1 min
200 s
360, 492/562
475, 495/618
PBS, pH7.4, 5% DMF
PBS, pH7.4, 1% ACN
28
29
350, 425/600
475, 512/653
PBS, pH7.4, 10% DMSO
PBS, pH6.0, 50% ACN
15.2 nM
56 nM
50 s
30
31
13 min
488, 511/713
340, 485/600
PBS, pH7.4, 50% EtOH
PBS/MeOH (v/v, 4 : 1)
10.6 nM
14.6 nM
10 s
30 s
32
This work
As shown in Fig. 5, the commonly used anions did not affect quench the uorescence of HBI.33 Most importantly, other ROS
the detection. All the chosen metal cations did not produce new also did not trigger an increase in the uorescence of the probe.
interference, except for the copper ions. The copper ions could Especially, the probe exhibited about an 8-fold increase in the
Fig. 2 A linear relationship between the fluorescence ratio for the
probe (10 mM) and the HClO concentration (2.0–45.0 mM). Fluores-
cence intensity was determined 1 min after the addition of HClO at
room temperature (slit width: 2/8 nm, lex ¼ 340 nm).
Fig. 3 UV-vis absorption spectra of the probe in PBS/MeOH (v/v, 4 : 1,
pH ¼ 7.4, and 10 mM) upon the addition of different amounts of HClO
(4.0, 20.0, 40.0, and 60.0 mM).
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the probe at 600 nm over the range from pH 4.0 to 9.0. Upon the
addition of HClO, the uorescence intensity of the probe at
600 nm did not change, and a new uorescence emission at
485 nm appeared but did not show a signicant difference.
When the added concentration of HClO was 60 mM, the uo-
rescence intensity ratio (FI485/FI600) was maintained at 4.2 from
pH 4.0 to 9.0. This result indicated that the probe could
potentially be applied for determining HClO in real samples.
3.6 Detection mechanism
According to the literature,34,35 there are two kinds of typical
detection mechanisms (Scheme 2): hypochlorous acid-initiated
oxidative intramolecular cyclization and removal of the
unbridged imine C]N bond. The designed probes based on the
abovementioned two chemical reactions were uorescent “turn-
on” types. Although the recognition functional group was the
same as that in the former probes, the uorescence emission
type of the probe in this study was different. Therefore, it was
urgent to investigate the mechanism involved in the uores-
cence detection.
Fig. 4 Time-dependent fluorescence ratio (FI485/FI600) of the probe
(10 mM) towards HClO (26 mM and 60 mM) in PBS/MeOH (v/v, 4 : 1, pH
¼ 7.4, and 10 mM). Fluorescence intensity was determined 1 min after
the addition of HClO at room temperature (slit width: 2/8 nm, lex
¼
340 nm).
To verify the detection mechanism of the probe for HClO in
a detection solution, related experiments were performed. At
rst, the detection solution was directly injected into the mass
spectrometer to mainly analyze its composition. Based on the
obtained MS spectrum (Fig. 7), it was preliminarily concluded
that the probe reacted with HClO in solution and converted to
a new species, which was assigned to the new peak at m/z
339.18. Moreover, when the probe reacted with a 3 equivalent
amount of HClO, the probe was completely converted to a new
species (inner table in Fig. 7). This reaction was not affected by
the pH of the detection solution, which was in agreement with
the effect of pH on the uorescence emission of the probe. The
new species at m/z 339.18 was the unique product of the
detection reaction.
uorescence intensity ratio towards HClO over other ROS. The
competition experiment results were in agreement with the
abovementioned results. These results demonstrate the selec-
tivity of the probe for HClO.
3.5 Effect of pH on the uorescence emission of the probe
The inuence of pH on the detection assay was also investi-
gated. In real sample detection, pH is important for the appli-
cation of a probe. Therefore, the effect of pH on the emission
spectra of the probe was investigated. As shown in Fig. 6, pH did
not exert an obvious inuence on the uorescence intensity of
Fig. 5 Fluorescence response of the probe (10 mM) toward anions (50
mM), metal cations (50 mM), and ROS in PBS/MeOH (v/v, 4 : 1, pH ¼ 7.4,
10 mM). (1) 1O2, (2) cOH, (3) H2O2, (4) Fe3+, (5) ONOOꢀ, (6) NO, (7)
tBuOOH, (8) Cu2+, (9) Zn2+, (10) Ca2+, (11) Cd2+, (12) Fe2+, (13) Co2+, (14) Fig. 6 Effect of pH on the fluorescence ratio (FI485/FI600) of the probe
Al3+, (15) CH3COOꢀ, (16) Hg2+, (17) Mg2+, (18) Na+, (19) K+, and (20) (15 mM) in the absence/presence of HClO (15 mM and 60 mM). Fluo-
HClO. Fluorescence intensity was determined 1 min after the addition rescence intensity was determined 1 min after the addition of HClO at
of HClO at room temperature (slit width: 2/8 nm, lex ¼ 340 nm).
room temperature (slit width: 2/8 nm, lex ¼ 340 nm).
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Scheme 2 Detection mechanism of the probe towards HClO.
Subsequently, we tried to separate the product generated in As shown in Fig. S3,† compared to the chromatogram of the
the detection reaction through HPLC. The concentration of the probe with the detection solution, a new peak at the retention
probe in the detection solution was increased to meet the test time of 7.25 min appeared, which could be attributed to the new
requirement. Aer optimizing the instrumental conditions, the species. The mobile phase effluent at this retention time was
components of the detection solution were efficiently separated. obtained, concentrated and directly injected into the mass
Fig. 7 Mass spectrum of the probe (10 mM) treated with HClO (inner table: 0, 10, and 30 mM).
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Fig. 8 Molecular orbital surfaces and energy levels obtained using the DFT/B3LYP method for the probe and its corresponding product.
Table 2 Determination of HClO in real samples
spectrometer to conrm its exact molecular weight. The result
showed that the molecular ion peak was at m/z 339.18, which
Sample
Spiked/mM
Found/mM
Recovery/%
RSD/%
was identical to the new peak shown in Fig. 7. It was concluded
that the detection mechanism was as follows: the probe reacted
with HClO in the detection solution and was converted to an
intramolecular cyclization product. This proposed sensing
mechanism is in agreement with the mechanism reported in
the literature (Scheme 2, path 2).
Tap water
River water
Human serum
0
2.0
5.1
4.9
10.2
4.9
9.2
—
—
3.0
5.0
10.0
5.0
10.0
104.0
98.8
102.0
98.0
92.1
1.5
2.1
1.4
3.0
1.6
Finally, the uorescent product in the detection solution was
isolated and characterized by 1H NMR to clarify the actual
reaction between the probe and HClO. As shown in Fig. S6,†
a signal of the Schiff base proton at d 8.73 ppm appeared in the
spectrum of the probe but disappeared in the product; this
indicated that the substituted imidazole structure was gener-
ated in the product.34
3.8 Detection of HClO in real samples
Briey, tap water was obtained from our laboratory and directly
used for detection without any pretreatment. The river water
was obtained from the Xiaoqing River near Beijing North Fih
Ring Road. Before detecting HClO, the river water was ltered to
remove the solid impurities. Finally, pretreatment for human
serum was conducted as follows: 1.0 mL acetonitrile was added
to 5.0 mL human serum and mixed thoroughly to precipitate
the proteins. Then, the mixture was centrifuged to obtain the
supernatant. The supernatant was diluted 50 times with PBS
buffer (containing 20% ethanol, v/v, and pH 7.0). These real
samples were spiked with the NaClO stock solution at the
concentration of 100 mmol Lꢀ1 and investigated by the above-
mentioned established analytical method.
The detection results are summarized in Table 2. When the
real samples were spiked with the HClO solution of known
concentration, the total concentrations in the samples were
determined through the established method. Good recoveries
from 92.1% to 104.0% with the RSDs from 1.4% to 3.0% were
obtained. The abovementioned results indicated that the probe
could quantify HClO in real samples.
3.7 Theoretical computation of the probe
To better understand the geometry and electron density of the
probe and its product, the Material Studio soware was used to
conduct a basic computational investigation; density functional
theory (DFT) calculations with the Becke-3-Lee-Yang-Parr
(B3LYP) exchange functional were performed using the DMol3
package of the soware.
Via the analysis of the product generated in the detection
solution, the molecular orbitals of the probe and its product
were obtained (Fig. 8). In Fig. 8, the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular orbital
(LUMO) plots of the probe and its product are plotted. The 3D
isosurface HOMO of the probe was mainly located on the u-
orophore. The electronic spatial distributions indicated that the
ESIPT process of the probe was not broken, which guaranteed
uorescence emission of the probe; on the other hand, the
LUMO of the probe showed that the reaction with HClO most
probably occurred on the C]N bond. When the HClO-initiated
oxidative intramolecular cyclizing reaction was completed, the
energy difference of the product was much larger than that of
4. Conclusion
the probe, suggesting that the product was more chemically In summary, a novel ratiometric uorescent probe for the fast
stable than the probe.
determination of HClO was designed and synthesized based on
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the ESIPT uorophore HBI. The probe exhibited high selectivity 11 Y. R. Zhang, Z. M. Zhao, J.-Y. Miao and B.-X. Zhao, A
for the detection of HClO over a wide pH range. Through MS
and HPLC, the specic sensing mechanism was determined.
Finally, the probe was applied to determine HClO spiked in real
ratiometric uorescence probe based on a novel FRET
platform for imaging endogenous HOCl in the living cells,
Sens. Actuators, B, 2016, 229, 408–413.
samples, and the results indicated high potential of the probe in 12 Y. R. Zhang, N. Meng, J. Y. Miao and B. X. Zhao, A ratiometric
analytical and bioanalytical applications.
uorescent probe based on a through-bond energy transfer
system for imaging HClO in living cells, Chem.–Eur. J.,
2015, 21, 19058–19063.
Conflicts of interest
13 H. D. Xiao, J. H. Li, J. Zhao, G. Yin, Y. W. Quan, J. Wang and
R. Y. Wang, A colorimetric and ratiometric uorescent probe
for ClO- targeting in mitochondria and its application in
vivo, J. Mater. Chem. B, 2015, 3, 1633–1638.
The authors declare no competing nancial interest.
Acknowledgements
14 P. Zhang, H. Wang, Y. Hong, M. Yu, R. Zeng, Y. Long and
This work was nancially supported by National Key R&D
Program of China (2018YFF0212503) and National Key R&D
Program of China No. 2017YFF0106006. We thank Dr Wang
from school of chemistry and biological engineering for
providing the theoretical computation of the probe and its
product.
J.
Chen,
Selective
visualization
of
endogenous
hypochlorous acid in zebrash during lipopolysaccharide-
induced acute liver injury using a polymer micelles-based
ratiometric uorescent probe, Biosens. Bioelectron., 2018,
99, 318–324.
15 H. Wang, P. Zhang, Y. Hong, B. Zhao, P. Yi and J. Chen,
Ratiometric imaging of lysosomal hypochlorous acid
enabled by FRET-based polymer dots, Polym. Chem., 2017,
8, 5795–5802.
References
1 D. C. Harris, Exploring Chemical Analysis, 4th edn, 2009, p. 16 M. Xue, H. Wang, J. Chen, J. Ren, S. Chen, H. Yang, R. Zeng,
538.
Y. Long and P. Zhan, Ratiometric uorescent sensing of
endogenous hypochlorous acid in lysosomes using AIE-
based polymeric nanoprobe, Sens. Actuators, B, 2019, 282,
1–8.
2 M. K. Shigenaga, T. M. Hagen and B. N. Ames, Oxidative
damage and mitochondrial decay in aging, Proc. Natl. Acad.
Sci. U. S. A., 1994, 91, 10771–10778.
3 X. Q. Chen, X. Z. Tian, I. Shin and J. Yoon, Fluorescent and 17 P. Zhang, H. Wang, D. Zhang, X. Zeng, R. Zeng, L. Xiao,
luminescent probes for detection of reactive oxygen and
nitrogen species, Chem. Soc. Rev., 2011, 40, 4783–4804.
4 D. I. Pattison and M. J. Davies, Evidence for rapid inter- and
H. Tao, Y. Long, P. Yi and J. Chen, Two-photon uorescent
probe for lysosome-targetable hypochlorous acid detection
within living cells, Sens. Actuators, B, 2018, 255, 2223–2231.
intramolecular chlorine transfer reactions of histamine and 18 J. Hu, N. Wong, M. Lu, X. Chen, S. Ye, A. Zhao, P. Zhao,
carnosine chloramines: implications for the prevention of
hypochlorous-acid-mediated damage, Biochemistry, 2006,
45, 8152–8162.
R. Kao, J. Shen and D. Yang, HKOCl-3: a uorescent
hypochlorous acid probe for live-cell and in vivo imaging
and quantitative application in ow cytometry and a 96-
well microplate assay, Chem. Sci., 2016, 7, 2094–2099.
5 Y. W. Yap, M. Whiteman and N. S. Cheung, Chlorinative
stress:
An
underappreciated
mediator
of 19 H. Zhu, J. Fan, J. Wang, H. Mu and X. Peng, An “enhanced
PET”-based uorescent probe with ultrasensitivity for
imaging basal and elesclomol-induced HClO in cancer
cells, J. Am. Chem. Soc., 2014, 136, 12820–12823.
neurodegeneration signaling?, Cell, 2007, 19, 219–228.
6 S. M. Wu and S. V. Pizzo, a2-Macroglobulin from rheumatoid
arthritis synovial uid: functional analysis denes a role for
oxidation in inammation, Arch. Biochem. Biophys., 2001, 20 J. Zha, B. Fu, C. Qin, L. Zeng and X. Hu, A ratiometric
391, 119–126.
uorescent probe for rapid and sensitive visualization of
hypochlorite in living cells, RSC Adv., 2014, 4, 43110–43113.
in 21 J. T. Hou, M. Y. Wu, K. Li, J. Yang, K. K. Yu, Y. M. Xie and
X. Q. Yu, Mitochondria-targeted colorimetric and
uorescent probes for hypochlorite and their applications
for in vivo imaging, Chem. Commun., 2014, 50, 8640–8643.
7 M. J. Steinbeck, L. J. Nesti, P. F. Sharkey and J. Parvizi,
Myeloperoxidase
osteoarthritis: potential biomarkers of the disease, J.
Orthop. Res., 2007, 25, 1128–1135.
8 J. T. Hou, M. Y. Wu, K. Li, J. Yang, K. K. Yu, Y. M. Xie and
and
chlorinated
peptides
X. Q. Yu, Mitochondriatargeted colorimetric and 22 G. Cheng, J. Fan, W. Sun, J. Cao, C. Hu and X. Peng, A near-
uorescent probes for hypochlorite and their applications
for in vivo imaging, Chem. Commun., 2014, 50, 8640–8643.
9 F. Ma, M. T. Sun, K. Zhang, Y. J. Zhang, H. J. Zhu, L. J. Wu,
infrared uorescent probe for selective detection of HClO
based on Se-sensitized aggregation of heptamethine
cyanine dye, Chem. Commun., 2014, 50, 1018–1020.
D. J. Huang and S. H. Wang, An oxidative cleavage-based 23 F. Lu and T. Nabeshima, A highly selective and sensitive
ratiometric uorescent probe for hypochlorous acid
detection and imaging, RSC Adv., 2014, 4, 59961–59964.
10 W. Lin, L. Long, B. Chen and W. Tan, A ratiometric
turn-on chemodosimeter for hypochlorous acid based on
an iridium(III) complex and its application to bioimaging,
Dalton Trans., 2014, 43, 9529–9536.
uorescent probe for hypochlorite based on a deoximation 24 Y. He, Y. Xu, Y. Shang, S. Zheng, W. Chen and Y. Pang, An
reaction, Chem.–Eur. J., 2009, 15, 2305–2309.
ESIPT-based uorescent probe for the determination of
30950 | RSC Adv., 2019, 9, 30943–30951
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
hypochlorous acid (HClO): mechanism study and its
RSC Advances
based on
a
selenide oxidation/elimination tandem
application in cell imaging, Anal. Bioanal. Chem., 2018,
410, 7007–7017.
25 M. Z. K. Baig, D. Majhi, R. N. P. Tulichala, M. Sarkarb and
M. Chakravarty, Easy access to new anthracenyl p-
conjugates: generation of distinct AIE-active materials, J.
Mater. Chem. C, 2017, 5, 2380–2387.
reaction, Chem. Commun., 2018, 54, 11965.
30 K. Dou, G. Chen, F. Yu, Z. Sun, G. Li, X. Zhao, L. Chen and
J. You, A two-photon ratiometric uorescent probe for the
synergistic detection of the mitochondrial SO2/HClO
crosstalk in cells and in vivo, J. Mater. Chem. B, 2017, 5, 8389.
31 L. Zhang, J. Ning, J. Miao, J. Liu and B. Zhao, A new
ratiometric uorescent probe for detecting endogenous
HClO in living cells, New J. Chem., 2018, 42, 2989.
26 D. Chen, W. Wu, Y. Yuan, Y. Zhou, Z. Wana and P. Huang,
Intense multi-state visible absorption and full-color
luminescence of nitrogen-doped carbon quantum dots for 32 Z. Zhang, J. Fan, G. Cheng, S. Ghazali, J. Du and X. Peng,
blue-light-excitable solid-state-lighting, J. Mater. Chem. C,
2016, 4, 9027–9035.
Fluorescence completely separated ratiometric probe for
HClO in lysosomes, Sens. Actuators, B, 2017, 246, 293–299.
27 X. Zhang, W. Zhao, B. Li, W. Li, C. Zhang, X. Hou, J. Jiang and 33 M. Qin, D. Jin, W. Che, Y. Jiang, L. Zhang, D. Zhu and Z. Su,
Y. Dong, Ratiometric uorescent probes for capturing
endogenous hypochlorous acid in the lungs of mice, Chem.
Sci., 2018, 9, 8207.
Fluorescence response and detection of Cu2+ with 2-(2-
hydroxyphenyl)benzimidazole in aqueous medium, Inorg.
Chem. Commun., 2017, 75, 25–28.
28 Z. Mao, M. Ye, W. Hu, X. Ye, Y. Wang, H. Zhang, C. Li and 34 B. Chen, H. Fu, Y. Lv, X. Li and Y. Han, An oxidative
Z. Liu, Design of a ratiometric two-photon probe for
imaging of hypochlorous acid (HClO) in wounded tissues,
Chem. Sci., 2018, 9, 6035.
cyclization reaction based uorescent “Turn-On” probe for
highly selective and rapid detection of hypochlorous acid,
Tetrahedron Lett., 2018, 59, 1116–1120.
29 X. Xie, T. Wu, X. Wang, Y. Li, K. Wang, Z. Zhao, X. Jiao and 35 L. Chen, S. Park, D. Wu, H. Kim and J. Yoon, A two-photon
B. Tang, A two-photon uorescent probe for ratiometric
visualization of hypochlorous acid in live cells and animals
ESIPT based uorescence probe for specic detection of
hypochlorite, Dyes Pigm., 2018, 158, 526–532.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 30943–30951 | 30951