Two coumarin-based turn-on fluorescent probes based on for hypochlorous acid detection and imaging in living cells

Article abstract of DOI:10.1016/j.saa.2018.12.019

This work, two turn-on fluorescent probes (3-acetyl-2H-chromen-2-one (ACO) & (1E)-1-(1-(2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (CETC)) based on coumarin have been designed and synthesized, which could selectively and sensitively recognize ClO^? with fast response time. ACO & CETC were almost non fluorescent possibly due to both the lacton form of coumarin and unbridged C[dbnd]N bonds which can undergo a nonradiative decay process in the excited state. Upon the addition of ClO^?, ACO & CETC were oxidized to ring - opened by cleavage the C–O and C[dbnd]N and the fluorescence intensity were increased considerably. Fluorescence titration experiments showed that the detection limit ACO & CETC is as low as 22 nm and 51 nm respectively. In particular, some relevant reactive species, including [rad]OH, ¹O₂, H₂O₂, KO₂, some anions and cations cannot be interference with the test. In live cell experiments, ACO & CETC were successfully applied to image exogenous ClO^? in HepG2 cells. Therefore, ACO & CETC not only could image ClO^? in living cells but also proved that C–O and C[dbnd]N can be cleavage by ClO^?.

Full text of DOI:10.1016/j.saa.2018.12.019

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 211 (2019) 239–245
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Two coumarin-based turn-on fluorescent probes based on for
hypochlorous acid detection and imaging in living cells
Qingming Wang a, , Lei Jin a,b, Wenling Wang a, Lihui Dai a, Xiaoxue Tan a, Cong Zhao a
⁎
a
School of Pharmacy, Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers' University, Yancheng, Jiangsu 224051,
People's Republic of China
b
College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, 210009, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
This work, two turn-on fluorescent probes (3-acetyl-2H-chromen-2-one (ACO) & (1E)-1-(1-(2-oxo-2H-chro-
men-3-yl)ethylidene)thiosemicarbazide (CETC)) based on coumarin have been designed and synthesized,
which could selectively and sensitively recognize ClO− with fast response time. ACO & CETC were almost non
fluorescent possibly due to both the lacton form of coumarin and unbridged C_N bonds which can undergo a
nonradiative decay process in the excited state. Upon the addition of ClO−, ACO & CETC were oxidized to ring -
opened by cleavage the C\\O and C_N and the fluorescence intensity were increased considerably. Fluorescence
titration experiments showed that the detection limit ACO & CETC is as low as 22 nm and 51 nm respectively. In
particular, some relevant reactive species, including •OH, 1O2, H2O2, KO2, some anions and cations cannot be inter-
ference with the test. In live cell experiments, ACO & CETC were successfully applied to image exogenous ClO− in
HepG2 cells. Therefore, ACO & CETC not only could image ClO− in living cells but also proved that C\\O and C_N
Received 25 September 2018
Received in revised form 19 November 2018
Accepted 9 December 2018
Available online 10 December 2018
Keywords:
Coumarin-based fluorescence probe
Hypochlorous acid
Reactive species
Mechanism
can be cleavage by ClO−
.
Cell imaging
© 2018 Elsevier B.V. All rights reserved.
1. Introduction
of water and organic solvents, this may be a limiting factor for applica-
tions of these probes. So it is still significative to extend a new fluores-
cent probe for the recognition moiety of ClO−.
Reactive oxygen species (ROS), such as •OH, 1O2, H2O2, O2−, ClO− and
so on, play important roles in cell signaling and maintaining redox ho-
meostasis [1–5]. Among of the broad range of biologically reactive spe-
cies mentioned above [6], hypochlorite acid (HClO) is a weakly acidic
(pKa = 7.53) and partially exists in the form of hypochlorite ion
(ClO−) in physiological conditions [7–9]. It is produced by the peroxida-
tion reaction between hydrogen peroxide and chloride ions which was
catalyzed by myeloperoxidase (MPO) [10–13]. In view of the highly ox-
idant property in immune systems, the normal pathology concentration
of HClO is b200 μM [14]. The abnormal production of hypochlorite can
lead to tissue damage and various of human diseases, including arthritis,
hepatic ischemia-reperfusion damage, cardiovascular diseases, rheuma-
toid, lung injury, cancer, atherosclerosis [15–23]. Due to the importance
of HClO, more and more scientists have attracted a great deal of interest
to develop new suitable chemical methods for real-time monitoring of
HClO or rapid imaging in living systems [24–26].
Herein, we reported two coumarin-based probes for ClO−. The syn-
thesized coumarin derivative ACO & CETC were followed early reported
method with some modify. The ACO & CETC shown almost non-
fluorescent emission which is due to the donor-excited photo induced
electron transfer(D-PET)process [38]. However, ACO & CETC could con-
vert to (Z)-2-(2-hydroxybenzylidene)-3-oxobutanoic acid (HOA) in
presence of ClO− (Scheme 1) by breaking the C\\O and C_N.
2. Experimental Section
2.1. Materials and Instruments
We purchase all of the reagents from commercial providers and use
them without further purification. The anions like Cl−, Br−, I−, SO42−
,
Due to higher selectivity, better sensitivity, readily operation and po-
tentially of real time monitoring, there are many fluorescence probes
developed for the detection of ClO− [7,27–37]. Unfortunately, most of
the reported fluorescence probes can only be operated in the mixture
PO43−, and so on, came from their sodium salts or potassium salts. The
cations, for example, Na+, K+, Mg2+, Ca2+, Ba2+, Cr3+, Co2+, and so
on, came from their chloride or nitrate, both anions and cations were
purchased from Shanghai Experiment Reagent Co., Ltd. (Shanghai,
China). The probes 3-acetyl-2H-chromen-2-one (ACO) & (1E)-1-(1-
(2-oxo-2H-chromen-3-yl)ethylidene)thiosemicarbazide (CETC) were
prepared according to the literature method with some modify [39–41].
⁎
Corresponding author.
E-mail address: wangqm@yctu.edu.cn (Q. Wang).
https://doi.org/10.1016/j.saa.2018.12.019
1386-1425/© 2018 Elsevier B.V. All rights reserved.

240
Q. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 211 (2019) 239–245
Scheme 1. Mechanism of ClO− sensing by probe ACO & CETC.
1H NMR and 13C NMR spectra were recorded on a Bruker DRX 400
was collected and washed with ethanol for three times and dried in
vacuo. Yield, 57.2%. FT-IR (KBr, cm−1): 3030 (Ar\\H), 2927 (C\\H),
1740 (C\\O), 1614, 1558, 1454 (Ar, C_C), 1211, 1122 (C\\O\\C).
spectrometer with TMS as the internal standard. FT-IR spectrum
(4000–400 cm−1) were obtained on NICOLET380 FT-IR spectrometer
in KBr disks. Elemental analyses (EAs) were carried out with a VARI-
EL elemental analyzer. Electrospray ionization mass spectra (ESI - MS)
were measured on a Triple TOF TM 5600+ system. Fluorescence spectral
data were calculated by a RF-5301 fluorescence spectrophotometer. Ul-
traviolet spectrum was performed on UV-1800 ENG 240 V. Fluorescence
images of HepG2 cells were performed using a confocal laser scanning
microscope with model LSM-880.
Exact mass for ACO: 188.0473, ESI - MS (positive mode) [ACO + H+
]
+ (m/z,189.0638), [ACO + Na+] + (m/z,211.0486). Elemental analysis
(calcd. %) for C11H8O3·0.2H2O: C, 68.89; H, 4.41; Found: C, 68.76; H,
4.68. 1H NMR (400 MHz, DMSO) δ 11.22 (s, 1H), 8.67 (s, 1H), 7.96 (dd,
J = 7.7, 1.4 Hz, 1H), 7.78–7.73 (m, 1H), 7.49–7.40 (m, 2H), 3.34 (s,
1H). 13C NMR (101 MHz, DMSO) δ 195.59 (s), 158.92 (s), 155.08 (s),
147.53 (s), 134.96 (s), 131.26 (s), 125.42 (s), 124.90 (s), 118.65 (s),
116.59 (s), 40.61 (s), 40.40 (s), 40.19 (s), 39.98 (s), 39.78 (s), 39.57
(s), 39.36 (s), 30.54 (s).
2.2. Absorption and Fluorescence Spectroscopy
ACO & CETC were dissolved in DMSO for a stock solution (0.01 M).
2.4.2. Synthesis of (1E)-1-(1-(2-oxo-2H-chromen-3-yl)ethylidene)
thiosemicarbazide (CETC)
5% of H2O2 was by dilution of 30% solution in water. KO2 was diluted
in anhydrous DMSO to give ·O2− 1O2 was obtained by mixing H2O2
.
CETC was prepared by mixing of 3-acetyl-2H-chromen-2-one with
thiosemicarbazide in a 1:1 ratio in 30 mL ethanol solution. Then a few
drops of acetic acid were added drop-wise into the mentioned solution
above. The solution was reflux for 12 h. The dark brown color product
was filtered off and recrystallized from ethanol solution. The compound
was dried in vacuum desiccator. Yield, 36.4%. FT-IR (KBr, cm−1): 3389
(N\\H), 3030(Ar\\H), 2927 (C\\H), 1719 (C_O), 1605, 1497, 1454
(Ar, C_C), 1240, 1116 (C\\O\\C).Exact mass for CETC: 261.0572, ESI -
MS (positive mode) [CETC + H+] + (m/z, 262.0804), [CETC + H+] +
(m/z, 284.0624). Elemental analysis (calcd. %) for C12H11N3O2S: C,
55.16; H, 4.24; N, 16.08; Found: C, 55.20; H, 5.18, N, 4.24. 1H NMR
(400 MHz, DMSO) δ 10.63 (s, 1H), 10.46 (s, 2H), 8.48 (s, 2H), 8.43 (s,
2H), 8.02 (s, 1H), 7.97 (s, 2H), 7.77 (d, J = 8.9 Hz, 2H), 7.68–7.62 (m,
2H), 7.45–7.37 (m, 4H), 3.44 (dd, J = 14.0, 7.0 Hz, 1H), 2.26 (s, 5H),
2.18 (s, 1H), 1.07 (s, 1H), 1.06 (s, 1H), 1.04 (s, 1H). 13C NMR
(101 MHz, DMSO) δ 179.70 (s), 159.61 (s), 153.84 (s), 146.43 (s),
142.47 (s), 132.86 (s), 129.59 (s), 126.27 (s), 125.24 (s), 119.42 (s),
116.45 (s), 40.61 (s), 40.40 (s), 40.19 (s), 39.98 (s), 39.77 (s), 39.59
(s), 39.46 (d, J = 21.0 Hz), 16.53 (s).
and 100-exceed of NaClO.·OH was produced by Fenton reaction, mixing
H2O2 and 10-exceed (NH4)2Fe(SO4)2 [42,43]. Absorption and fluores-
cence spectrum were recorded in a quartz optical of 1.0 cm optical
path length at room temperature. All of the test solutions were prepared
by displacing 2 μL of the stock solution into a 2 mL of 20 mM Tris - HCl at
pH = 7.2.
2.3. Cell Culture and Cell Imaging
HepG2 cells were cultured in Dulbecco's Modified EagleMedium
(DMEM) medium supplemented with 10% fetal bovine serum (FBS)
and 0.1% antibiotic-antimycoticmix antibiotic and grown at 37 °C in a
5% CO2. Cells were treated with ACO & CETC (10 μM) in PBS buffer (2%
DMSO, pH = 7.2) for 30 min. After, the cells were then incubated with
ClO− (40 μM) for another 40 min. Then the cells were washed with
PBS buffer for three times. And the living cell images were captured
through a confocal laser scanning microscope. The survivance of
HepG2 cells incubated with ACO & CETC respectively, was determined
by the 3-[4,5-Dimethylthiazo-2 -yl]-2,5-diphenpyltetra-zolium bro-
mide (MTT) assay.
3. Results and Discussion
2.4. Synthesis of Probe ACO & CETC
3.1. Synthesis and Characterization
2.4.1. Synthesis of 3-acetyl-2H-chromen-2-one (ACO)
Following the reported method [44,45] to synthesize probe ACO, as
showed in Scheme S1. 0.6106 g (5 mmol) 2-hydroxybenzaldehyde
and 0.646 g (5 mmol) ethyl 3-oxobutanoate were added to 30.00 mL
ethanol. Then piperidine was added drop-wise into the mentioned solu-
tion above. After the resulting solution was reflexed for 16 h with a con-
stant stirring, the color of the reaction was dark green and cooled to
room temperature, yellow solid was obtained and the precipitation
The synthesis of ACO began with 2-hydroxybenzaldehyde and ethyl
3-oxobutanoate which were catalyzed by piperidine. The compound
CETC was synthesized by using the intermediate ACO as reaction mate-
rial undergoing condensation reaction with thiosemicarbazide. Both
ACO and CETC were recrystalled by CH2CH3OH/H2O to give pure prod-
ucts. Detail characterization data and experimental procedure were
shown in the Scheme 2. The structures of ACO & CETC were confirmed
Scheme 2. Design and synthesis of the probe ACO & CETC.

Q. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 211 (2019) 239–245
241
that the addition of these anions to the solution of ACO & CETC, there
was almost no effect on the fluorescence emission enhancement was
found. But when 5.0 equiv. ClO− was added, the fluorescence emission
of ACO & CETC was increased rapidly. Thus, ACO & CETC exhibited ex-
cellent selectivity for ClO− over the tested anions. The competitive stud-
ies of ACO & CETC were conducted with the addition of 5.0 equiv. ClO−
to induced fluorescence enhancement before mixed 20 equiv. of Cl−
,
,
Br−, I−, SO42−, PO43−, H2PO4−, OH−, HCO3−, NO3−, C2O42−, F−, HPO42−
S2O32−, NO2−, Ac−, P2O74−, B4O72−, CO3−, HSO3−, SO32−, respectively. The
fluorescence emission of the mixed system at 478 nm was showed in
Fig. 1, it was demonstrated that the co-existent anions impacted a neg-
ligible effect on the recognition of the ACO & CETC with ClO−. In the
same way, some selected cations, such as Na+, K+, Mg2+, Ca2+, Ba2+
Cr3+, Co2+, Mn2+, Fe3+, Ni2+, Cu2+, Cd2+, Zn2+, Al3+, Pb2+, Bi3+
,
,
Ag+, Sn2+, Sr+, NH4+, Hg2+, were also tested. The results revealed that
all the tested cations could not interfere the ACO & CETC to recognize
the ClO−(as showed in Fig. S9–Fig. S14). Thus, the dramatic response
suggested that ACO & CETC were highly selective and selective fluores-
cence probe for ClO− in our prospective.
3.3. UV–vis absorption spectra of ACO & CETC towards ClO−
The Uv-vis spectras of ACO & CETC was examined in Tris - HCl buffer/
DMSO (v:v = 9:1, 20 mM, pH = 7.0). Uv-vis spectra of ACO (10 μM) ex-
hibited nearly no absorption. After the treatment of ClO− at room tem-
perature, the absorption peak at 407 nm obviously increased gradually,
a new absorption peak was present at 407 nm, with the increasing of
ClO−, the absorbance at 407 nm was gradually increased (Fig. 2A). Be-
sides, a color change from colorless to yellow can be visualized clearly
by naked eye. Fig. 2B showed the Uv-vis spectra of CETC (10 μM),
which exhibited an absorption maximum at λ = 334 nm and λ =
257 nm. When the solution of ClO− was titrated into Tris - HCl buffer/
DMSO (v:v = 9:1, 20 mM, pH = 7.0) containing CETC, the maximum
absorption peak at 354 nm was declined gradually, and another absorp-
tion peak at 257 nm was increased gradually.
Fig. 1. Examination of selectivity of ACO & CETC towards ClO− (1.0 μM) in the presence of
various anions (20 equiv.) in Tris - HCl buffer/DMSO (v:v = 9:1, 20 mM, pH = 7.0).
A) ACO; B) CETC.
by Elemental analyses (EAs), FT - IR spectra (IR) analysis, Electrospray
3.4. Fluorescence Spectra of ACO & CETC towards ClO−
ionization mass spectra (ESI - MS), 1H NMR and 13C NMR, (Fig. S1–S8).
The fluorescent emission spectrum was monitored by titration ex-
periment with various concentrations of ClO−. As showed in Fig. 3A,
for free ACO (1.0 μM), no obvious emission was observed with the exci-
tation at 420 nm in the detection system. It was interesting to us that
with the incremental changes of ClO− (0–8.0 μM) brought about a sig-
nificant fluorescence turn-on response at 478 nm with a visual fluores-
cence change from colorless to blue. There was an approximately 36-
fold increasing could be observed. From Fig. 3B we could find that
CETC (1.0 μM) display very weak fluorescence at 495 nm when it was
3.2. Spectral Characteristics of ACO & CETC for Various Anions and Cations
To evaluate the anion-binding properties of ACO & CETC, a wide
range of environmentally and physiologically anions and cations were
investigated using the fluorescence spectra. The solution of ACO &
CETC (1.0 μM) and added separately 20 equiv. selected anions, such as
Cl−, Br−, I−, SO42−, PO43−, H2PO4−, OH−, HCO3−, NO3−, C2O42−, F−
,
HPO42−, S2O32−, NO2−, Ac−, P2O74−, B4O72−, CO3−, HSO3−, SO32−, it is evident
Fig. 2. Changes in the absorption spectra of ACO & CETC in the presence of ClO−
.

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Fig. 3. Fluorescence titration of ACO & CETC (1.0 μM) with different concentration of ClO− in Tris - HCl buffer/DMSO (v:v = 9:1, 20 mM, pH = 7.0) solution. Inset: the change of
fluorescence depending on the concentrations of ClO− by illumination with a 365 nm UV lamp.(A) for ACO; (B) for CETC.
excited at 380 nm, the fluorescence emission was enhanced gradually
with the various concentrations of ClO− was titrated. It is about 3-fold
increasing could be found. Compared with ACO & CETC, we could find
that probe ACO caused more fluorescence enhancement than CETC.
The quantum yield was 82.05% and 14.25%, respectively (Fig. S15).
The fluorescence variation experiment indicated that ACO & CETC
were enough to detect the concentration of ClO−.
By the fluorescence titration method, the sensitivity of ACO & CETC
to ClO− was well examined by using the working curve. As showed in
Fig. 4A & B, 1.0 μM of ACO & CETC were treated with various concentra-
tions of ClO− (0–8.0 μM) in Tris - HCl buffer/DMSO (v:v = 9:1, 20 mM,
pH = 7.0). The correlation coefficient of R2 is 0.998 and 0.996, respec-
tively. The detection limit (LOD) for ClO− by ACO & CETC based on the
definition by IUPAC (CDL = 3 Sb/m) were calculated to be 22 nM and
51 nM, respectively. The lower LOD is similar to the early results
[43,46,47] and better than other result [48] (as showed in Table S1).
The results meant that ACO & CETC could be potentially applied to de-
tect ClO− quantitatively. Moreover, there was also demonstrated that
ACO & CETC to be very highly sensitive probe for ClO−.
3.5. Selectivity Studies over some Oxidative Species
Next, parallel experiments for specificity of ACO & CETC towards
ClO− over other ROS (·OH, 1O2, H2O2, KO2) were carried out. The ACO
& CETC barely showed any fluorescence enhancement in response to
hydroxylradical(·OH), singlet oxygen (1O2), H2O2, potassium superox-
ide (KO2−). While the ClO− was added to the solution of ACO & CETC
in Tris - HCl buffer/DMSO (v:v = 9:1, 20 mM, pH = 7.0), an obviously
enhanced fluorescence at 478 nm (for ACO, 498 nm for CETC) was ob-
tained. The results indicated that the reactive oxygen species ·OH,
ClO− 1O2, H2O2, KO2 cannot interference the test results. As showed
,
in Fig. 5, the relative enhancement of fluorescence emission caused by
ClO− was 24-fold (for ACO, 2-fold for CETC), which was much higher
than other oxidative species. These results manifested that ACO &
CETC could detect ClO− not only qualitatively but also quantitatively
in complex biological systems.
3.6. Cell Imaging
Furthermore, the response time of ACO to ClO− was also performed.
A plot of fluorescence intensity vs time was showed when ACO is ti-
trated with 0, 1.0 μM, 2.0 μM of ClO−, respectively. Within the time of
0–30 s, the fluorescence intensity increased with the time and when
the time reached 30 s, the fluorescence intensity was reached to the
maximum. The maximum could be maintained for 5 min (Fig. S16).
Inspired by the favorable fluorescence response of ACO & CETC to
ClO−, fluorescent imaging of ClO− using ACO & CETC in living HepG2
were determinated. Initially, the cytotoxicity of ACO & CETC were re-
corded by using the MTT assay to evaluate the possibility of probes to
be applied to living HepG2 cells. As showed in Fig. S17, both of the sur-
vival rates of cell remained N90% after incubating with ACO or CETC in
Fig. 4. Fluorescent emissions spectra of ACO & CETC (1.0 μM) in the presence of ClO−.(C) for ACO; (D) for CETC.

Q. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 211 (2019) 239–245
243
product HOA is formed after the reaction between ACO and ClO−. From
Fig. 8 we could find that a peak at m/z = 239.1753, corresponding to
[HOA + H + CH3OH]+ (cal. M/z = 239.1519), a peak at m/z =
261.0678, corresponding to [HOA + Na + CH3OH] + (cal. M/z =
261.0739), a peak at m/z = 301.1594, corresponding to [HOA + Na
+ 4H2O] + (cal. M/z = 301.1556), and a peak at m/z = 360.3465, corre-
sponding to [HOA + 5H2O + 2CH3OH] (cal. M/z = 360.3602) are clearly
observed, which suggested that the C\\O of ACO could be cleaved by the
oxidation of ClO−. For the mechanism between CETC and ClO− is also in-
vestigated by mass spectrometry in CH3OH/H2O(v:v = 1: 9). From
Fig. S18, a peak at m/z = 239.1750 corresponds to [HOA + H
+ CH3OH] + (cal. M/z = 239.1519), a peak at m/z = 301.1594, corre-
sponding to [HOA + Na + 4H2O] + (cal. M/z = 301.1556), and a peak
at m/z = 360.3456, corresponding to [HOA + 5H2O + 2CH3OH] (cal.
M/z = 360.3602) are clearly observed, which indicated that the C\\O
and C_N of CETC could also be cleaved by the oxidation of ClO−. These
results demonstrated that ClO− could cleave the C\\O and C_N by
oxidation.
Fig. 5. Fluorescence response of ACO & CETC (1.0 μM) to NaClO (5 μM) with the
competition analytes.
range of 0–60 μM for 24 h, revealing that ACO & CETC have almost no
remarkable cytotoxicity to HepG2 cells, and can be used to biological
body. Then the ability of ACO or CETC to image ClO− in HepG2 cells
was evaluated. From Fig. 6 and Fig. 7, HepG2 cells were incubated
with 10 μM of ACO or CETC for 30 min at 37 °C displayed no obvious
fluorescence for ACO or weak blue fluorescence for CETC. After incuba-
tion of the probes treated HepG2 cells with 40 μM ClO− for another
40 min, strong blue fluorescence image of ACO and CETC was found
through the fluorescent micro-scope. Therefore, ACO & CETC were cell
membrane permeable and have potential applicability for detecting
ClO− in living cells.
4. Conclusions
In conclusion, we have successfully synthesized two turn-on fluores-
cent probes (ACO & CETC) for highly sensitive detection of ClO− with
fast response time by oxidative cleavage of C\\O and C_N. The ACO &
CETC displayed high selectivity for ClO− over other anions and cations
with a detection limit of 22 nM and 51 nM, respectively. What is
more, some relevant reactive species, including ·OH, 1O2, H2O2, KO2
could not interference the test results. More importantly, these probes
can sense intracellular ClO− levels.
Acknowledgments
3.7. Proposed Mechanism
This work are financially supported by the National Natural Science
Foundation for Young Scientists of China (21301150), the Practice Inno-
vation Training Program Projects for the Jiangsu College Students
The mass spectrometry is widely used to analyze the product between
ACO & CETC and ClO− in CH3OH/H2O(v:v = 1: 9). Scheme 1 shows the
Fig. 6. Fluorescence microscopic images of HepG2 cells: (a) Untreated Cell control; (b) Cells treated with HL; (c) overlap image of (a) and (b); (d) Untreated Cell control; (e) Cells treated
with ACO and ClO−; (f) overlap image of (d) and (e).

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Q. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 211 (2019) 239–245
Fig. 7. Fluorescence microscopic images of HepG2 cells: (a) Untreated Cell control; (b) Cells treated with HL; (c) overlap image of (a) and (b); (d) Untreated Cell control; (e) Cells treated
with CETC and ClO−; (f) overlap image of (d) and (e).
Fig. 8. ESI mass spectra of ACO upon addition of excess ClO−
.
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Appendix A. Supplementary data
Bai,
A coumarin-based two-photon probe for hydrogen peroxide, Biosens.
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