The detection for hypochlorite by UV-Vis and fluorescent spectra based on oxidized ring opening and successive hydrolysis reaction

Article abstract of DOI:10.1016/j.saa.2016.05.016

In this work, two high selective and sensitive fluorescent probes for ClO^-, 7-Hydroxycoumarin and 4-Hydroxycoumarin were designed. The reaction mechanism that we speculated was the oxidized ring opening reaction and hydrolysis. The detection could be realized in quasi-aqueous phase and the detection limits of probe [7] and probe [4] for ClO^- were found to be 56.8 nM and 70.5 nM. Furthermore, the probes can be used to cell imagings.

Full text of DOI:10.1016/j.saa.2016.05.016

The detection for hypochlorite by UV–Vis and fluorescent spectra based on
oxidized ring opening and successive hydrolysis reaction
Kangming Xiong, Caixia Yin, Jianbin Chao, Yongbin Zhang, Fangjun
Huo
PII:
DOI:
Reference:
S1386-1425(16)30268-2
doi: 10.1016/j.saa.2016.05.016
SAA 14437
To appear in:
Received date:
Revised date:
Accepted date:
9 December 2015
26 April 2016
13 May 2016
Please cite this article as: Kangming Xiong, Caixia Yin, Jianbin Chao, Yongbin
Zhang, Fangjun Huo, The detection for hypochlorite by UV–Vis and fluorescent spec-
tra based on oxidized ring opening and successive hydrolysis reaction, (2016), doi:
1
0.1016/j.saa.2016.05.016
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The detection for hypochlorite by UV-Vis and fluorescent spectra based on
oxidized ring opening and successive hydrolysis reaction
a
a
b
b
b
Kangming Xiong, Caixia Yin,* Jianbin Chao, Yongbin Zhang, Fangjun Huo*
a
Institute of Molecular Science, Key Laboratory of Materials for Energy Conversion
and Storage of Shanxi Province, Shanxi University, Taiyuan, 030006, China.
b
Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China.
*
Corresponding author:
E-mail: yincx@sxu.edu.cn; huofj@sxu.edu.cn
−
Abstract: In this work, two high selective and sensitive fluorescent probes for ClO ,
7
-Hydroxycoumarin and 4-Hydroxycoumarin were designed. The reaction mechanism
that we speculated was the oxidized ring opening reaction and hydrolysis. The
detection could be realized in quasi-aqueous phase and the detection limits of probe (7)
−
and probe (4) for ClO were found to be 56.8 nM and 70.5 nM. Furthermore, the
probes can be used to cell imagings.
Keywords: UV-Vis and fluorescent spectra, Probes; 7-Hydroxycoumarin;
4
-Hydroxycoumarin; Cell imagings.

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1
. Introduction
The detection and quantification of inorganic anions have been invigorated over
the past years from environmental, biological, and medical aspects. And a variety of
1
-6
probes that are able to selectively recognize anions were designed. As one of the
reactive oxygen species (ROS), hypochlorite is generated from the reaction of H O
2
2
7
-8
and chloride ion catalyzed by myeloperoxidase (MPO) enzyme in organisms. In the
−
physiological pH solution HOCl is partially dissociated into ClO , and it plays a
9
-10
crucial role in vivo due to its antibacterial properties.
However, the excessive
generation of the potent oxidant also poses a risk to the host, and there is evidence
11
that HOCl contributes to the tissue injury associated with inflammation. Neutrophil
1
2-14
derived HOCl has been considered to be related to lung injury,
rheumatoid
1
5-16
17
18-19
arthritis,
hepatic ischemia-reperfusion injury, and renal disease.
Current
chemo/biosensors for hypochlorous acid or hypochlorite detections are usually limited
to the submicromolar level because of their insufficient sensitivity, which is a problem
because the concentrations in biological matrices is generally on the nanomolar scale
or even lower. The intracellular concentrations of hypochlorite are generally on the
submicromolar level. It has been reported that the average production concentration of
−
1
6
20
HOCl in neutrophils is about 0.47 nmol min per 10 cells. So it is essential to
design some chemosensors with high enough sensitivity for the detection of
−
HOCl/ClO . There have been some successful examples reported very recently such
2
1-33
as colorimetric, luminescent, electrochemical and chromatographic methods.
Even
so, fluorescent probe detection is a promising method for detection of hypochlorous

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acid because of the low cytotoxicity of fluorescent probe which can realize detection
3
4-37
in living cells.
Coumarin and its derivatives are one of the important classes of heterocyclic
compounds and are known to possess a wide range of biological activities including
anti-HIV, anti-biotic, anti-fungal, anti-bacterial, anti-viral, anti-cancer, anti-clotting
3
8-45
activity, and especially as anti-coagulants.
Meanwhile, 7-Hydroxyl coumarin as a
commercial available fluorescent dye can be used for fluorescent indicator and pH
indicator; 4-Hydroxyl coumarin as a commercial available dye can be used for a
solvent dyes, fluorescent whitening agent and laser dye. In the process of studying
their fluorescence properties, we found that they can selective recognition
hypochlorite ion.
In this work, 7-hydroxyl coumarin and 4-hydroxy coumarin were developed as
two high selective fluorescent probes for hypochlorite ion over other anions. These
−
probes worked well at physiological pH and had low detection limits for ClO .
2
. Experimental
2
.1 Materials
4
-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased
from Sigma-Aldrich (St. Louis, MO). 7-hydroxyl coumarin and 4-hydroxy coumarin
were purchased from Aladdin Industrial Corporation (Shanghai, China). Amino acids
were purchased from Shanghai Experiment Reagent Co., Ltd (Shanhai, China). All
other chemicals used were of analytical grade.
2
.1. Physical measurements

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A pH meter (Mettler Toledo, Switzerland) was used to determine the pH.
Ultraviolet-visible (UV-Vis) spectra were recorded on a Cary 50 Bio UV-Visible
spectrophotometer. Fluorescence spectra were measured on Cary Eclipse fluorescence
spectrophotometer. A PO-120 quartz cuvette (10 mm) was purchased from Shanhai
1
13
Huamei Experiment Instrrument Plants, China. H NMR, C NMR spectra were
recorded on a Bruker AVANCE-300 MHz and 75 MHz NMR spectrometer,
respectively (Bruker, Billerica, MA). ESI-MS was measured with an LTQ-MS
-
(
Thermo) instrument. The ability of probe (7) reacting to OCl in the living cells was
also evaluated by laser confocal fluorescence imaging using an Olympus FV1000
laser scanning microscope.
3
. Results and Discussions
−
3
.1. UV–Vis and fluorescence spectra of detecting ClO
−
The detections of 7-hydroxyl coumarin and 4-hydroxy coumarin for ClO were
investigated by the UV–Vis and fluorescence spectra. Detailed UV–Vis spectral
−
titrations for ClO were carried out (Fig. 1). Fig. 1a showed the change in the
UV-Visible spectra upon addition of hypochlorite solution to a solution of the probe
−
(7) (50 µM) in HEPES (10 mM, pH 7.4). With increasing concentration of ClO
(0–33.5 µM), the absorption peak of the probe at 323 nm was gradually decreased and
two new absorption peaks appeared at 263 nm and 369 nm. Two isosbestic points
appeared at 292 nm and 343 nm, indicating the formation of a new compound. Fig. 1b
−
showed the change in the UV-Visible spectra upon addition of ClO to the probe (4)
−
(
50 µM) in HEPES (10 mM, pH 7.4). With increasing concentration of ClO (0–22.8

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µM), the absorption peak of the probe (4) at 285 nm was gradually decreased and a
new absorption peak appeared at 252 nm. An isosbestic point formed at 270 nm,
indicating the formation of a new compound. Detailed fluorescence spectral titrations
−
for ClO were carried out (Fig. 2). Fig. 2a displayed the fluorescence response of the
−
probe (7) to ClO in HEPES (10 mM, pH 7.4). Initially, the probe (0.1 μM) had strong
−
fluorescence intensity (λ = 323 nm, slit width: 5 nm/5 nm), but the addition of ClO
ex
(0–241.2 µM) caused a remarkable reduction emission at 463 nm. Fig. 2b showed the
−
change in the fluorescence spectra when the ClO (0–4.7 µM) was gradually added to
−
the probe (4) (15 μM) in HEPES (10 mM, pH 7.4). Upon increasing ClO
concentration, the initial fluorescence intensity of 377 nm gradually decreased (λ =
ex
2
87 nm, slit width: 5 nm/5 nm).
(a)
(b)
Fig. 1. (a) The UV–Vis absorption spectra of probe (7) (50 μM) in the presence of various
−
concentrations of ClO (0–33.5 μM) in HEPES (10 mM, pH 7.4). (b) The UV–Vis absorption
−
spectra of probe (4) (50 μM) in the presence of various concentrations of ClO (0–22.8 μM) in
HEPES (10 mM, pH 7.4).

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(a)
(b)
Fig. 2. (a) Fluorescence spectra of probe (7) (0.1 μM) in the presence of various concentrations of
−
ClO (0–241.2 μM) in HEPES (10 mM, pH 7.4) (λ = 323 nm, slit width: 5 nm/5 nm). Inset: a
ex
−
color change photograph for ClO . (b) Fluorescence spectra of probe (4) (15 μM) in the presence
−
of various concentrations of ClO (0–4.7 μM) in HEPES (10 mM, pH 7.4) (λ = 287 nm, slit
ex
−
width: 5 nm/5 nm). Inset: a color change photograph for ClO .
−
3
.2. The Selective response of probes to ClO
An important feature of probe is that it has special selectivity for a kind of
analyte over other substances. In order to study its special recognition ability, we

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carried out the experiment by UV-Vis and fluorescence spectrometer. Detailed
−
UV–Vis spectra of probes for ClO and other analytes were carried out (Fig. 3). Fig.
3
(a) showed the absorbance curve changes that probe (7) (50 µM) undergowent upon
the addition of various analytes in HEPES (10 mM, pH 7.4) (2 mL). And they all
−
−
triggered minor changes except ClO . Upon the addition of ClO , the absorption peak
of the probe (7) at 323 nm was decreased and two new absorption peaks appeared at
2
63 nm and 369 nm. Fig. 3 (b) showed the absorbance curve changes that probe (4)
(50 µM) undergowent upon the addition of various analytes in HEPES (10 mM, pH
−
7
.4) (2 mL). And they all triggered minor changes except ClO . Upon the addition of
−
ClO , the absorption peak of the probe (4) at 285 nm was decreased. Detailed
−
fluorescence spectra of probes for ClO and other analytes were carried out (Fig. 4
and Fig. S1). Fig. 4(a) showed the probe (7) (0.1 μM) displayed a remarkable
fluorescence intensity reduction (λ = 323 nm, λ = 463 nm, slit width: 5 nm/5 nm)
ex
em
−
in the presence of the ClO (241.2 μM). And other analytes only triggered minor
changes. Besides, from the Fig. 4a, we could see that the other analytes induced no
−
changes in the solution color, but ClO induced obvious changes in the solution color
(from strong blue fluorescent to colorless) under illumination with a 365 nm UV lamp.
Fig. 4b showed the probe (4) (15 μM) displayed a remarkable fluorescence intensity
reduction (λ = 287 nm, λ = 377 nm, slit width: 5 nm/5 nm) in the presence of the
ex
em
−
ClO (4.7 μM). Also from Fig. 4b, we could see that the other analytes induced no
−
changes in the fluorescence spectra and solution color, but ClO induced obvious
changes in fluorescence intensity reduction and solution color (from weak blue

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fluorescent to colorless) under illumination with a 365 nm UV lamp.
(a)
(b)
Fig. 3. (a) UV-Vis absorption spectra of probe (7) (50 μM) in HEPES (10 mM, pH 7.4) in the
−
−
−
−
−
−
2−
−
2−
2−
−
−
−
presence of F , Cl , Br , NO , SCN , CN , SO , HSO , S O , CO3 , HCO , AcO , H PO ,
3
2−
4
3
2
3
3
2
4
−
−
2−
−
4−
−
−
Cys, H O , I , ClO , C O
, S2O4 , ClO , P O , H O , HS , GSH, Hcy (600 µM) and ClO
4 2 7 2 2
2
2
2
2
4
(
33.5 µM). (b) UV-Vis absorption spectra of probe (4) (50 μM) in HEPES (10 mM, pH 7.4) in the
−
−
−
−
−
−
2−
−
2−
2−
−
−
−
presence of F , Cl , Br , NO , SCN , CN , SO , HSO , S O , CO , HCO , AcO , H PO ,
3
4
3
2
3
3
3
2
4
−
−
2−
4
2−
−
4−
−
Cys, H O , I , ClO , C O
, S2O4 , ClO , P O , H O , HS , GSH, Hcy (410.0 µM) and
4 2 7 2 2
2
2
2
2
−
ClO (22.78 µM).
(a)

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(
b)
−
Fig. 4. (a) Fluorescence spectra of probe (7) (0.1 μM) with various analytes and ClO in HEPES
(
10 mM, pH 7.4) (λ = 323 nm, slit: 5 nm/5 nm). Inset: a visual fluorescence color change
ex
−
photograph for ClO and other analytes under illumination with a 365 nm UV lamp. (b)
−
Fluorescence spectra of probe (4) (15 μM) with various analytes and ClO in HEPES (10 mM, pH
−
7
.4) (λ = 287 nm, slit: 5 nm/5 nm). Inset: a visual fluorescence color change photograph for ClO
ex
and other analytes under illumination with a 365 nm UV lamp.
3
.3. pH dependent of the probes
−
The pH range for the determination of ClO was also studied. Fig. S2 showed the
−
fluorescence intensity obtained for the free probes and probes-ClO in different pH
values. From Fig. 2S, we could find that both probe (7) and probe (4) in HEPES (10
mM, pH 7.4) were more effective for spectral detections.
−
3
.4. The kinetic study in the detection process of ClO
−
We investigated the reaction time between probes and ClO (Fig. 5). From Fig. 5,
−
we could figure out that the minimum fluorescence intensities of probe (7)-ClO and
−
probe (4)-ClO were achieved after 9 s and 17 s, respectively.

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(a)
(b)
Fig.5. (a) Reaction time profile of fluorescence spectra of probe (7) (0.1 μM) upon addition of
−
ClO (241.2 μM) at 463 nm in HEPES (10 mM, pH 7.4) (λ = 323 nm, slit width: 5 nm/5 nm). (b)
ex
−
Reaction time profile of fluorescence spectra of probe (4) (15 μM) upon addition of ClO (4.7 μM)
at 377 nm in HEPES (10 mM, pH 7.4) (λ = 287 nm, slit width: 5 nm/5 nm).
ex
−
3
.5. The detection limit of probes for ClO
−
To investigate the detection limits of the probes for ClO , probe (7) (0.1 μM) was
−
treated with various concentrations of ClO (0–201 μM) and the fluorescence
−
intensity at 463 nm was plotted as a function of ClO concentration, probe (4) (15 μM)
−
was treated with various concentrations of ClO (0–4.7 μM) and the fluorescence
−
intensity at 377 nm was plotted as a function of ClO concentration (Fig. S3). The
fluorescence intensities of probe (7) and probe (4) were linearly proportional to the
−
ClO concentrations, and the detection limits were 56.8 nM and 70.5 nM, respectively,
4
6
based on the definition by IUPAC (C = 3 Sb/m). The detection limits indicated that
DL
commercially available fluorescence probes: 7-hydroxyl coumarin and 4-hydroxy
−
coumarin showed certain sensitivities towards ClO and their detection limits were
4
7-54
compared to the other reported hypochlorite probes (Fig. S4).
.6. Proposed mechanism
3

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The reaction mechanism that we speculated was the oxidized ring opening
reaction and hydrolysis. As we known, hypochlorite is a strong oxidant, it maybe can
oxidize ethylenic bond of coumarin to form carboxyl product. Furthermore, the C-O
bond of ester group may be disconnected by hydrolysis of ester bond (Scheme 1). To
1
13
elucidate the detailed signal mechanism, H NMR、 C NMR and ESI-MS analyses of
−
probes-ClO products were carried out (Fig. S5, ESI†). The ESI-MS of probe
−
-
−
-
(
7)-ClO , m/z: 188.67, [M+Cl] ; the ESI-MS of probe (4)-ClO , m/z: 138.83, [M] .
Scheme 1. The reaction mechanism of probes.
3
.7. Cellular Imaging
We further investigated the utility of the probe (7) in tissue imaging. The ability
–
of probe (7) to detect ClO within living cells was also evaluated by laser confocal
fluorescence imaging using an Olympus FV1000 laser scanning microscope. The
optical window at the channel (400 – 550 nm) was chosen as a signal output. Under
selective excitation at 323nm, HepG2 cells incubated with probe (7) (0.3 μM) for 30
min at 37 °C showed light blue fluorescence (Fig. 6a). In a further experiment it was
found that HepG2 cells displayed no fluorescence when the cells were first incubated
–
with 0.3 μM of probe (7) for 30 min at 37 ℃ and then incubated with 30 μM ClO
(Fig. 6b). These cell experiments showed the good cell-membrane permeability of
–
probe (7), thus it could be used to mark ClO within living cells.

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Fig. 6 Confocal fluorescence images in HepG2 cells: (a) Fluorescence image of HepG2 cells with
adding probe (7) (0.3 μM) and its bright field image (c); (b) Fluorescence image of HepG2 cells
incubated with 0.3 μM probe (7) for 30 mins at 37 °C, then incubated with NaClO (30 μM) for 30
mins at 37 °C and its bright field image (d).
4
. Conclusion
In summary, we have developed two effective fluorescent probes for ClO in
−
quasi-aqueous phase. The probes are based on commercially available coumarin
−
compounds which have high selectivities and sensitivities for ClO over other
analytes in HEPES (10 mM, pH 7.4). Moreover, the probes are sensitive enough to
−
−
monitor ClO comparable to that of other reported ClO chemosensors (Fig. S4).
Interestingly, the practical appilication was realized by cell imagings.
Acknowledgments
National Natural Science Foundation of China (NO. 21472118), the Shanxi
Province Science Foundation for Youths (2013011011-1), the Program for the Top
Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi
(2013802), the Program for the Top Young and Middle-aged Innovative Talents of
Higher Learning Institutions of Shanxi (No. 2013802), talents Support Program of
Shanxi Province (No. 2014401), Shanxi Province Outstanding Youth Fund (No.

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014021002), and Shanxi University funds for study aboard 2014.

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Graphical abstract

ACCEPTED MANUSCRIPT
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Highlights
1
2
3
. Two new fluorescent probes for hypochlorite based on a novel
recognition mechanism: oxidized ring opening reaction and hydrolysis
were developed.
. The detection could be realized in quasi-aqueous phase and the
detection limits were found to be as low as 56.8 nM and 70.5 nM,
respectively.
−
. The probe could be used to monitor intracellular ClO in HepG2 cells.