A “turn-on” fluorescent probe with high selectivity and large stokes shift for the detection of hydrogen peroxide and its bioimaging applications

Article abstract of DOI:10.1016/j.saa.2021.120320

Hydrogen peroxide (H₂O₂) plays pivotal roles in various biological functions and pharmacological activities. High selectivity and sensitivity remain challenges for fluorescent probes to detection of H₂O₂ with a large stokes shift. Herein, a new “turn-on” fluorescent probe (DCM-C) was designed based on the mechanism of intramolecular charge transfer (ICT). In PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v), DCM-C exhibited high selectivity and sensitivity for H₂O₂ over other interfering analytes with a large stokes shift (187 nm), and the detection limit was as low as 35.5 nM. In addition, the probe was effective for detecting exogenous and endogenous H₂O₂ in living cells, and identifying stained in cytoplasm. Moreover, the probe has been used successfully for determining H₂O₂ in zebrafish by fluorescence imaging.

Full text of DOI:10.1016/j.saa.2021.120320

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A ‘‘turn-on” fluorescent probe with high selectivity and large stokes shift
for the detection of hydrogen peroxide and its bioimaging applications
b
a
b,
Yun Hua a, Yajing Shang a, , Mengjiao Gao , Jin Li , Yanfei Kang
⇑
⇑
a College of Chemical and Biological Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China
b Zhang Jiakou Key Laboratory of Organic Light Functional Materials, College of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
A turn-on fluorescent probe (CDM-C)
was developed for H2O2 detection.
The probe CDM-C exhibited a large
Stokes shift of 187 nm.
The probe CDM-C possessed favorable
cell membrane permeability and low
cytotoxicity.
ꢀ
ꢀ
The probe CDM-C was successfully
used for imaging exogenous and
endogenous H2O2 in living cells.
The probe CDM-C was applied for
sensing H2O2 in the zebrafish.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2021
Received in revised form 1 August 2021
Accepted 23 August 2021
Available online 26 August 2021
Hydrogen peroxide (H2O2) plays pivotal roles in various biological functions and pharmacological activ-
ities. High selectivity and sensitivity remain challenges for fluorescent probes to detection of H2O2 with a
large stokes shift. Herein, a new ‘‘turn-on” fluorescent probe (DCM-C) was designed based on the mech-
anism of intramolecular charge transfer (ICT). In PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v), DCM-C
exhibited high selectivity and sensitivity for H2O2 over other interfering analytes with a large stokes shift
(187 nm), and the detection limit was as low as 35.5 nM. In addition, the probe was effective for detecting
exogenous and endogenous H2O2 in living cells, and identifying stained in cytoplasm. Moreover, the
probe has been used successfully for determining H2O2 in zebrafish by fluorescence imaging.
Ó 2021 Elsevier B.V. All rights reserved.
Keywords:
Fluorescence probe
Hydrogen peroxide
Large stokes shift
Fluorescence imaging
1. Introduction
such as glucose oxidase, uric acid oxidation, amino acid oxidation,
glutamate and lysine oxidation and so on, which are closely corre-
Hydrogen peroxide (H2O2) is an irreplaceable small molecules
that plays a pivotal role in organisms: (1) H2O2 is an important sig-
naling molecules as it can penetrate through the membrane
directly and has better stability than other ROS, which plays a
physiological role in cellular increment, lesion, apoptosis, immune
cell activation, vascular remodeling, stomatal closur [1-5]. (2) H2O2
is one of the byproducts of many classical enzymatic reactions,
lated with human health [6-10]. For example, an abnormal level of
H2O2 is associated with Parkinson’s disease, cancer, stroke and
arteriosclerosis [11,12]. Thus, there is a great pressing need to
develop effective methods to detect H2O2 in order to understand
both a range of cellular signaling processes and the mechanisms
of the pathological formation.
Taking advantages of timely reporting, high sensitivity, and
non-invasive visualizing, fluorescent probes have been widespread
applications in in vitro studies and in vivo bioimaging [7,12-16]. Up
to now, many well-designed fluorescent probes for H2O2 in biolog-
ical systems mainly based on the chemical reactivity, such as bor-
⇑
Corresponding authors.
E-mail addresses: shangyaj@mail.lzjtu.cn (Y. Shang), kangyanfei172@163.com
(Y. Kang).
https://doi.org/10.1016/j.saa.2021.120320
1386-1425/Ó 2021 Elsevier B.V. All rights reserved.

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
2. Experimental
onate oxidation reactions[12,15,16], tandem Payne/Dakin reaction
[12,18,19], and Baeyer-Villiger oxidation reactions [20-23], have
been reported. However, to the best of our knowledge, most of
them have their limitations such as small stokes shift [24-26] or
long respond time [27-31], which is not conducive to their practi-
cal use in bioimaging applications. Thus, it is particularly necessary
to develop efficient fluorescent probes for the detection of H2O2 in
biological systems with high sensitivity and large stokes shift.
In this work, a novel sensitive and selective ‘‘turn on” fluores-
cent probe (DCM-C) with a large stokes shift, was synthesized
(Scheme 1). 8-hydroxyquinoline-2-carbaldehyde conjugated ben-
zopyranonitrile group has been used as the fluorophore owing to
its excellent photophysical properties, and the borate ester moiety
has been introduced as the reactive group for high sensing H2O2.
Expectably, the probe expressed weak fluorescence at 617 nm
due to the intramolecular charge transfer (ICT) effect. After the
probe DCM-C reacting with H2O2, the boronate group was split, fol-
lowed by the generation of p-quinone-methide, and the fluo-
rophore DCM-OH was formed. The probe showed significant
fluorescent turn-on signals with a remarkable large stokes shift
(187 nm) for the detection of H2O2. Moreover, the probe was suc-
cessfully applied to HeLa cells and zebrafish to detect H2O2 by flu-
orescence imaging. All the results indicate that the probe can be
used effectively as a sensor to detect H2O2 both in vitro and in vivo.
2.1. Reagents and apparatus
All reagents and commercial chemicals were purchased from
commercial suppliers and were used without further purification
unless otherwise. The living HUVEC and HeLa cells were purchased
from the Chinese Academy of Sciences Cell Bank, and the zebrafish
were purchased from the Experimental Research Zebrafish Supply
Center (Shanghai, China). NMR spectra were measured with a Bru-
ker AVIII-500 MHz spectrometer. High resolution mass spectra
(HRMS) were recorded on Thermo (orbitrap Elite). Absorption
and fluorescence spectra were carried out with a Thermo (BioMate
3S) UV/Vis spectrophotometer and a F97pro fluorospectropho-
tometer, respectively. Fluorescent images of cells were obtained
with a Olympus confocal laser scanning microscope (FV1200).
2.2. Synthesis of DCM-C
The synthetic route of DCM-C was displayed in Scheme 2. 3 and
4 were prepared according to the literature [24,32,33]. DCM-C was
synthesized using classical Aldol condensation between 3 and 4 in
acetonitrle with piperidine as the catalyst [34]. The synthesis
details and structural identifications are shown in the Supplemen-
tal Information.
Scheme 1. Proposed reaction mechanism of H2O2 and borate ester-functionalized probes.
2

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
Scheme 2. The synthetic route for probe DCM-C.
DCM-C red solid. 1H NMR (500 MHz, DMSO d6)d7.71 (d,
J = 10.0 Hz, 1H), 7.38 (d, J = 10.0 Hz, 1H), 8.07 (d, J = 10.0 Hz, 2H),
7.92–7.90 (m, 2H), 7.86 (s, 2H), 7.69 (d, J = 10.0 Hz, 2H), 7.53 (d,
J = 10.0 Hz, 2H), 7.48 (t, J = 5.0 Hz, 2H), 7.23–7.2 (m, 1H), 7.15 (s,
1H), 5.40 (s, 2H), 1.26 (s, 12H); 13C NMR (125 MHz, DMSO d6)
d159.0, 157.5, 153.4, 152.6, 139.8, 136.2, 135.9, 130.9, 130.7,
130.1, 130.0, 127.5, 126.6, 125.1, 124.4, 119.9, 119.6, 118.7,
117.6, 116.4, 109.6, 106.9, 60.2, 35.6, 31.8, 29.6, 29.1, 27.0, 22.6,
14.4. HRMS (ESI) m/z calcd for C36H31O4N3B (M + H): 580.24021,
Found: 580.24009, error: 0.218 ppm.
the control group was treated with the probe DCM-C (5
30 min, and the experimental group was treated with H2O2
(100 M) for 30 min, and then treated with DCM-C (5 M) for
another 30 min. For imaging, zebrafish were anaesthetized with
MS-222 and imaged on a fluorescence microscope (TI2-U Nikon).
lM) for
l
l
3. Results and discussion
3.1. Optical properties of DCM-C to H2O2
2.3. General method for fluorescence detection
Solvents have a large effect on the fluorescence response of the
probe, and the selectivity varies depending on the solvent. In order
to explore the most suitable solvent for the probe DCM-C to detec-
tion of H2O2. The solvent selection experiment was carried out by
the fluorescence spectrum. As shown in Fig. S11, we found that the
reaction of DCM-C and H2O2 has the best effect in DMSO. There-
fore, we chose DMSO that exhibited the strongest fluorescence
after DCM-C reacting with H2O2 as the solvent. At the same time,
we tried to add PBS buffer into DMSO to explore the possibility
of use in physiological environment. As described in Fig. S12, upon
increasing the volume of DMSO percentages from 5% to 20%, the
fluorescence intensity of DCM-C increased. Then, for practical pur-
pose, the concentration of the probe which is safer and applies
more friendly. We tested the fluorescence response of DCM-C (5–
The fluorescence emission spectra were recorded in a range
from 550 to 750 nm at excitation wavelength of 430 nm. Dimethyl
sulfoxide (DMSO) was used to dissolve the DCM-C to prepare
2 mM stock solutions. Dilute the H2O2 (30%) with PBS buffer
(10 mM, pH 7.4) to obtain the H2O2 stock solution, and further
dilute it to 40–800
stock solution (20
concentrations of H2O2 stock solution (1 mL) and PBS buffer
lM. For spectral measurements, the DCM-C
lL) was diluted with DMSO (380 L) , different
l
(10 mM, pH 7.4) to a final concentration of 20 lM (V = 2 mL).
2.4. Fluorescence imaging in living cells
The HeLa cells (a cervical cancer cell line) and HUVEC cells (a
normal human umbilical vein cells line) were cultured in DMEM
culture medium supplemented with 1% (v/v) penicillin, and 10%
(v/v) fetal bovine serum at 37 ℃ in the atmosphere of 5% CO2.
The cells were plated on cultured dish and allowed to adhere for
24 h before the experiments. For fluorescence imaging of endoge-
30
fore, we chose PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v) as
the testing solvent and 20 M DCM-C as the testing concentration
lM) towards H2O2 and the results are shown in Fig S13. There-
l
for optical properties research.
In PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v), the
response of probe DCM-C for H2O2 was investigated. As shown in
Fig. 1A and 1B, the DCM-C itself exhibited obvious absorption at
430 nm and essential non-fluorescent because the inhibition of
the intramolecular charge transfer (ICT) effect is blocked by the
borate ester group (Scheme 1). After the addition of H2O2, the
absorption peak at 430 nm obviously weakened as the color of
the solution changed from yellow to colorless (the inset of
Fig. 1A), which is easy to detect H2O2 by the ‘‘naked-eye”. At the
same time, the fluorescence emission of the DCM-C at 617 nm
nous H2O2, HUVEC cells were treated with probe DCM-C (5
for 30 min as the control group. In the experiment group, one
group of HeLa cells were only treated with probe DCM-C (5 M)
for 30 min, the other group of HeLa cells were pretreated with
NAC for 30 min, followed incubated with DCM-C (5 M) for
another 30 min. Procedures for imaging exogenous H2O2: first,
cells were incubated with H2O2 (100 M) for 30 min; second, cells
were treated with DCM-C (5 M) for another 15 min, 30 min and
45 min respectively; finally, all cells were added 1 mL of DAPI
dye (1 g/mL) to stain the nucleus for 15 min, and then washed
lM)
l
l
l
l
drastically enhanced. The results indicated that DCM-C is
a
l
‘‘turn-on” fluorescent probe for H2O2 with a large stokes shift
(187 nm) (Fig. S14) which is in favour of reducing self-quenching
[13,15]. It may be explained that H2O2 can trigger oxidation of
the p-phenyl boronic acid ester moiety and a strong ICT process
appeared from benzopyranonitrile to 8-hydroxyquinoline
(Scheme 1). To test the sensitivity of DCM-C, the fluorescence spec-
tral of DCM-C to various concentrations of H2O2 were studied. As is
seen, when excited at 430 nm, the fluorescence intensity enhanced
about 42-fold within 18 min as the concentration of H2O2 gradu-
with PBS buffer for cell imaging experiments. The fluorescence
images were acquired by Olympus confocal laser scanning
microscope.
2.5. Fluorescence imaging in zebrafish
The zebrafish embryos were cultured in the medium at 29 ℃ for
3 days. Before imaging experiments, the zebrafish were placed in
petri dishes. The blank group was only treated with PBS buffer,
ally increases from 0 to 400 lM (Fig. 1C and 1D, Fig. S15), and
3

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
Fig. 1. Optical response of DCM-C to H2O2. (A) Absorption spectra of DCM-C (20
l
M) before and after incubating with H2O2 (200
M) with the concentration of H2O2 (0–400
lM). (E) The fitting straight line between the fluorescence intensity at 617 nm and the concentrations of H2O2. (F) The
l
M). (B) Fluorescence spectra of DCM-C
(20
lM) before and after incubating with H2O2 (200
l
M). (C) Fluorescence spectra of DCM-C (20
l
lM). (D) Time-dependent
fluorescence changes of DCM-C (20
lM) to H2O2 (200
linear relationship between ln[(Fmax - Ft)/Fmax] and time (Ft: the fluorescence intensity at time t, Fmax: the steady-state fluorescence intensity). Conditions: PBS buffer (10 mM,
pH 7.4, with 20% DMSO, v/v); kex/em = 430/617 nm. The error bars indicate the standard deviation (n = 3).
the fluorescence is linear relationship (Fig. 1E). The detection limit
was estimated to 35.5 nM by the equation of 3 /k (Fig. 1E). As a
straight line, the pseudo-first-order constant was calculated to be
1.17 ꢁ 10ꢂ3 sꢂ1. To further assess the feasibility of the sensing sys-
tem, the fluorescence spectral changes of DCM-C to H2O2 at differ-
ent pH value were examined. As shown in Fig. S16, probe DCM-C
itself was not pH sensitive, suggesting its good stability in the wide
range of pH. However, when added H2O2, the fluorescence was evi-
dently increased in the pH range of 7.0–10.0. In acidic conditions
(pH < 6), the weak response of DCM-C towards H2O2 may be due
to the inhibition of nucleophilic reaction by strong acid. Mean-
r
comparison, the performance of reported H2O2 fluorescent probes
was showed in the Table S1, indicating the probe DCM-C exhibited
high sensitivity to H2O2 with a low detection limit and a large
stokes shift. Moreover, the fluorescence intensity changes with
time was tested. It showed that the reaction of DCM-C with H2O2
was a pseudo-first order due to the linear coefficient of 0.9905
between ln[(Fmax - Ft)/Fmax] and time (Fig. 1F). Based on the fitting
4

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
while, DCM-C also showed a weak response to H2O2 in an alkaline
conditions (pH > 11), which may be caused by the decomposition
of H2O2. Thus, the detection abilities of DCM-C to H2O2 can be
achieved in the pH range from 6 to 11, which indicates that
DCM-C can be applied in the physiological pH for the detection
of H2O2.
To measure the specificity of DCM-C, the research of DCM-C to
the selective recognition of various biologically relevant species
(Na+, Mg2+, Ca2+, Zn2+, NO2–, CO32–, CH3COO–, ClO-, Cys, Hcy, GSH,
ꢃOH , Oꢃ2-, ONOO– and H2O2) by fluorescent spectra was carried
out. As expected, only H2O2 significantly increased the fluores-
cence intensity at 617 nm, while other substances did not induce
Based on the above results and the previous reports of H2O2 flu-
orescence probes using borate ester as the reaction site
[12,14,15,17,24,35,36], the proposed reactive mechanism of probe
DCM-C towards H2O2 was shown in Scheme 1. The H2O2-mediated
deprotection of hydroxyl group produces the fluorophore DCM-
OH, resulting in a turn-on fluorescence signal. To verify this pro-
posed process, ESI-MS spectral studies were performed. As is seen,
the peak at m/z = 362.09374 corresponding to [DCM-OH - H]- (m/z
calcd: 362.1008), was clearly observed (Fig. S10), and the peak of
580.24009 [DCM-C + H]+ (Fig S9) disappeared, indicating the fluo-
rophore DCM-OH was formed. Furthermore, the DFT (density func-
tional theory) calculations were used to analyze the sensing
mechanism of DCM-C with H2O2. The molecular frontier orbitals
of probe DCM-C and DCM-OH were calculated (Fig. 2). The results
confirmed that the p-phenyl boronic acid ester moiety inhibited
the electron transport of quinoline ring in DCM-C, and the extent
of intramolecular charge transfer (ICT) in DCM-OH were sufficient
to enhance its fluorescence. Moreover, the energy gap (HOMO and
LUMO) of DCM-C was calculated to be 2.9498 eV and that of DCM-
OH was 2.8981 eV. Hence, it can be concluded that DCM-OH is con-
sidered favorable for ICT transition in comparison with DCM-C.
obvious change even upon treatment with 100 lM (Fig. 3A). More-
over, when these interfering species were added to the solution of
DCM-C and H2O2, the fluorescence intensity was almost not
affected (Fig. 3B). All these results indicated that DCM-C has high
selectivity and good anti-interference ability, and is utilized for
the selective detection of H2O2 in complicated biological systems.
H2O2, as a byproducts of enzymatic reactions, was the main pro-
duct of glucose oxidation catalyzed by GOX: glucose + O2 + H2O ?
GOX ? gluconic acid + H2O2. To further investigate the potential
implications of DCM-C in biological system, H2O2 induced by
enzyme catalyzed oxidation between glucose and glucose oxidase
was detected (Fig. 4A, Fig S17). Not surprisingly, the fluorescence
intensity at 617 nm markedly increased with the concentration
of glucose, in which a linearly fluorescence response range for glu-
cose in 50–250 lM was found. These data proved that DCM-C can
be a potential fluorescent probe for endogenous H2O2. Further-
more, in order to explore the universality of probe in biological sys-
tem, DCM-C was applied to the detection of H2O2 in the viscosity
system (Fig. 4B and Fig. S18). The results demonstrated that
DCM-C showed a good optical response to H2O2 in viscous environ-
ment, and is utilized for the detection of H2O2 in biological system.
Fig. 2. Calculated the energy gap between HOMO and LUMO of DCM-C and DCM-OH.
5

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
Fig. 3. (A) Fluorescence intensity of 20 lM probe DCM-C to various biologically relevant species: H2O2 200 lM, other species 100 lM. (B) Fluorescence intensity of 20 lM
DCM-C with 200
lM H2O2 and 100 lM of potential interferences. Conditions: PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v); kex/em = 430/617 nm. The error bars indicate
the standard deviation (n = 3).
Fig. 4. (A) Fluorescence spectra of DCM-C (20
lM) in the presence of GOX and different concentrations of glucose. (B) Fluorescence spectra of DCM-C (20 lM) with H2O2
(200 M) in the presence of glycerol. Conditions: PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v); kex/em = 430/617 nm.
l
3.2. Imaging of H2O2 with DCM-C in living cells
addition, after the preincubation with H2O2 (100
the HeLa cells were incubated with probe DCM-C (5
l
M) for 30 min,
M) for differ-
l
Encouraged by the excellent fluorescence response, we further
explored the practical application in living cells. A cytotoxic assay
of the DCM-C probe was tested in HeLa cells by the methyl thia-
zolyl tetrazolium (MTT) assay before cellular application
(Fig. S19). The results revealed that > 80% of cells were alive upon
ent time. As shown in Fig. 5.B, a significant time-dependent was
observed. The results clearly indicate that DCM-C is capable of
imaging exogenous and endogenous H2O2 in living cells.
3.3. Imaging of H2O2 in living zebrafish
treatment with 40 lM DCM-C for 12 h, which indicated the low
cytotoxicity of DCM-C. Then, the usage of DCM-C for imaging in liv-
ing cells was studied. As shown in Fig. 5.A, when normal HUVEC
cells were incubated with DCM-C (5 lM) for 1 h, weak red fluores-
Inspired by the excellent performances of DCM-C in vitro, we
also studied the potential imaging for H2O2 in a zebrafish larvae
model (Fig. 6). As the blank controls, zebrafish themselves were
no red fluorescence. When the zebrafish were incubated with only
cence can be observed, indicating the probe DCM-C can be sensi-
tive to low-level cellular H2O2 (Fig. 5.A). However, under the
same test conditions, a stronger red fluorescence enhancement
was observed in HeLa cells (Fig. 5.A(b)), indicating the higher
endogenous H2O2 lever in HeLa cells than that in HUVEC cells. It
is evident that DCM-C exhibited favorable membrane permeability
and could sense the endogenous H2O2 in living cells. To further
confirm the above assumption, control experiments were per-
formed, in which N-acetyl-L-cysteine (NAC), a widely H2O2 effec-
tive scavenger, was added before incubation with probe DCM-C
to decrease cellular endogenous H2O2. As shown in Fig. 5.A, the
cells exhibited non-fluorescence, which may be attribute to the
inhibition effect of NAC on the cellular H2O2 production. Further-
more, we used DAPI (40,6-diamidino-2-phenylindole) to dye the
nucleus, found that the increased red fluorescence mainly existed
in cytoplasm (Fig. 5.B), indicating DCM-C did not enter the cell
nucleus and was mainly concentrated in the the cytoplasm. In
probe DCM-C (5
observed. However, when pretreated with 100
30 min, and then incubated with DCM-C (5 M) for another
lM) for 30 min, a weak red fluorescence was
lM
H2O2 for
l
30 min, a remarkable red fluorescence increase was obtained.
Herein, DCM-C has the potential for detection of H2O2 in vivo.
4. Conclusion
In conclusion, a novel turn-on fluorescent probe DCM-C for
H2O2 detection has been developed. The probe DCM-C exhibited
high selectivity for H2O2 over other competitive substances and
various coexisting species in PBS solutions with a large stokes shift
(187 nm) and a low detection limit of 35.5 nM. Moreover, fluores-
cence imaging experiments of DCM-C for H2O2 in living cells and
zebrafish demonstrated the potential of intracellular exogenous
and endogenous hydrogen peroxide imaging.
6

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
Fig. 5. (A) Fluorescence images of HUVEC cells and HeLa cells. Cells were treated with the DCM-C probe (5
then treated with DCM-C probe (5 M). (B) Fluorescence images of DCM-C to H2O2 in HeLa cells. Cells were incubated with H2O2 (100
with 5 M DCM-C for 15 min, 30 min, 45 min, respectively. kex = 430 nm,kem = 600–660 nm; Scale bar: 30 m.
l
M); cells were pretreated with the NAC (50
lM) for 30 min, and
l
lM) for 30 min, and then coincubated
l
l
Fig. 6. Fluorescence imaging of zebrafish (3-day old). (a) zebrafish itself; (b) only incubated with DCM-C (5
with DCM-C (5 M) for another 30 min. kex = 430 nm,kem = 600–660 nm; Scale bar: 30 m.
lM); (c) treated with 100 lM H2O2 for 30 min, and then incubated
l
l
CRediT authorship contribution statement
References
[1] J.R. Stone, S. Yang, Hydrogen peroxide: a signaling messenger, Antioxid. Redox.
Sign. 8 (2006) 243–270, https://doi.org/10.1089/ars.2006.8.243.
Yun Hua: Conceptualization, Methodology, Formal analysis,
Data curation, Resources, Writing – original draft, Writing – review
& editing. Yajing Shang: Investigation, Formal analysis, Data cura-
tion, Writing – review & editing, Supervision, Project administra-
tion, Funding acquisition. Mengjiao Gao: Formal analysis,
Validation, Data curation. Jin Li: Formal analysis, Validation. Yanfei
Kang: Supervision, Project administration, Funding acquisition. : .
[2] T.S. Gechev, J. Hille, Hydrogen peroxide as
a signal controlling plant
programmed cell death, J. Cell Biol. 168 (2005) 17–20, https://doi.org/
10.1083/jcb.200409170.
[3] S.G. Rhee, H2O2, a necessary evil for cell signaling, Science 312 (2006) 1882–
1883, https://doi.org/10.1126/science.1130481.
[4] J. Noh, B. Kwon, E. Han, M. Park, W. Yang, W. Cho, W. Yoo, G. Khang, D. Lee,
Amplification of oxidative stress by a dual stimuli-responsive hybrid drug
enhances cancer cell death, Nat. Commun. 6 (2015) 6907–6915, https://doi.
org/10.1038/ncomms7907.
[5] H. CAI, Hydrogen peroxide regulation of endothelial function: origins,
mechanisms, and consequences, Cardiovasc. Res. 68 (2005) 26–36, https://
doi.org/10.1016/j.cardiores.2005.06.021.
[6] S.B. Bankar, M.V. Bule, R.S. Singhal, L. Ananthanarayan, Glucose oxidase-An
overview, Biotechnol. Adv. 27 (2009) 489–501, https://doi.org/10.1016/j.
biotechadv.2009.04.003.
[7] S.Y. Ma, K.N. Wang, M.M. Xing, F. Feng, Q.L. Pan, D.X. Cao, A coumarin-boronic
ester derivative as fluorescent chemosensor for detecting H2O2 in living cells,
Inorg. Chem. Commun. 124 (2021) 108414–108418, https://doi.org/10.1016/j.
inoche.2020.108414.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Acknowledgements
[8] X. Liang, X. Xu, D. Qiao, Z. Yin, L. Shang, Dual mechanism of an intramolecular
charge transfer (ICT)-FRET-based fluorescent probe for the selective detection
of hydrogen peroxide, Chem. Asian J. 12 (2017) 3187–3194, https://doi.org/
10.1002/asia.201701382.
[9] H.I. Mahsa, K. Alireza, H. Javad, O. Yasin, A chemiluminescent method for the
detection of H2O2 and glucose based on intrinsic peroxidase-like activity of
WS2 quantum dots, Molecules 24 (2019) 689–703.
[10] A. Khataee, M. Haddad Irani-nezhad, J. Hassanzadeh, S. Woo Joo, Superior
peroxidase mimetic activity of tungsten disulfide nanosheets/silver
nanoclusters composite: Colorimetric, fluorometric and electrochemical
studies, J. Colloid Interf. Sci. 515 (2018) 39–49.
This work was supported by Tianyou innovation team of Lanz-
hou Jiaotong University (TY202004), the key projects of Hebei edu-
cational committee (ZD2020148), the basic scientific research
funds of Hebei province (JYT2020006, JYT 2020010), Hebei pro-
vince three three talent project (A201901062),Youth foundation
of Hebei educational committee (QN2020402).
Appendix A. Supplementary data
[11] K. Brieger, S. Schiavone, F.J. Miller Jr, K.H. Krause, Reactive oxygen species:
from health to disease, Swiss. Med. Wkly. 142 (2012) 13659–13672, https://
doi.org/10.4414/smw.2012.13659.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.saa.2021.120320.
7

Y. Hua, Y. Shang, M. Gao et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 265 (2022) 120320
[12] D.J. Zheng, Y.S. Yang, H.L. Zhu, Recent progress in the development of small-
molecule fluorescent probes for the detection of hydrogen peroxide, Trac-
Trend Anal. Chem. 118 (2019) 625–651, https://doi.org/10.1016/
j.trac.2019.06.031.
[13] H. Ren, F. Huo, X. Wu, X. Liu, C. Yin, An ESIPT-induced NIR fluorescent
probe to visualize mitochondrial sulfur dioxide during oxidative stress
in vivo, Chem. Commun. 57 (2021) 655–658, https://doi.org/10.1039/
D0CC07398A.
systems, Analyst. 144 (2019) 3643–3648, https://doi.org/10.1039/
C9AN00385A.
[25] X. Huang, Z.P. Li, Z.X. Liu, C.C. Zeng, L.M. Hu, A near-infrared fluorescent probe
for endogenous hydrogen peroxide real-time imaging in living cells and
zebrafish, Dyes Pigments. 165 (2019) 518–523, https://doi.org/10.1016/j.
dyepig.2019.02.042.
[26] R.Q. Zhou, L.X. Niu, Y.F. Hu, Q.R. Qi, W.C. Huang, L. Yang, A novel dual-function
fluorescent probe for the rapid detection of bisulfite and hydrogen peroxide in
aqueous solution and living cells, Spectrochim. Acta. A. 248 (2021) 119226–
119234, https://doi.org/10.1016/j.saa.2020.119226.
[14] L.L. Xia, Y. Tong, L.S. Li, M.Y. Cui, Y.Q. Gu, P. Wang, A selective fluorescent turn-
on probe for imaging peroxynitrite in living cells and drug-damaged liver
tissues,
j.talanta.2019.06.032.
[15] W.X. Wang, W.L. Jiang, Y. Liu, Y.F. Li, J. Zhang, C.Y. Li, Near-infrared
fluorescence probe with large stokes shift for visualizing hydrogen
peroxide in ulcerative colitis mice, Sens. Actuators Chem. 320 (2020)
Talanta
204
(2019)
431–437,
https://doi.org/10.1016/
[27] H. Han, X. He, M.X. Wu, Y.B. Huang, L.H. Zhao, L.L. Xu, P.Y. Ma, Y. Sun, D.Q.
Song, X.H. Wang, A novel colorimetric and near-infrared fluorescence probe for
detecting and imaging exogenous and endogenous hydrogen peroxide in living
cells, Talanta 217 (2020) 121000–121005, https://doi.org/10.1016/
j.talanta.2020.121000.
a
B
128296–128304, https://doi.org/10.1016/j.snb.2020.128296.
[16] W.W. Wang, J.H. Xiong, X.J. Song, Z. Wang, F. Zhang, Z.Q. Mao, An Activatable
Two-Photon Near-Infrared Fluorescent Probe Tai-lored toward Peroxynitrite In
Vivo Imaging in Tumor, Anal. Chem. 92 (2020) 13305–13312, https://doi.org/
10.1021/acs.analchem.0c02587.
[17] H.C. Zhang, D.H. Tian, Y.L. Zheng, F. Dai, B. Zhou, Designing an ESIPT-based
fluorescent probe for imaging of hydrogen peroxide during the ferroptosis
process, Spectrochim. Acta. A 248 (2021) 119264–119272, https://doi.org/
10.1016/j.saa.2020.119264.
[18] S. Ye, J.J. Hu, D. Yang, Tandem Payne/Dakin reaction: a new strategy for
hydrogen peroxide detection and molecular imaging, Angew. Chem. Int. Ed. 57
(2018) 10173–10177, https://doi.org/10.1002/anie.201805162.
[19] S. Ye, N. Hananya, O. Green, H. Chen, A.Q. Zhao, J.G. Shen, D. Shabat, D. Yang, A
Highly Selective and Sensitive Chemiluminescent Probe for Rea-Time
Monitoring of Hydrogen Peroxide in Cells and Animals, Angew. Chem. Int.
Ed. 59 (2020) 14326–14330, https://doi.org/10.1002/anie.202005429.
[20] B. Siegel, J. Lanphear, Kinetics and mechanisms for the acid-catalyzed
oxidative decarboxylation of benzoylformic acid, J. Org. Chem. 44 (1979)
942–946, https://doi.org/10.1021/jo01320a010.
[28] Z. Li, Imaging of hydrogen peroxide (H2O2) during the ferroptosis process in
living cancer cells with a practical fluorescence probe, Talanta 212 (2020)
120804–120810, https://doi.org/10.1016/j.talanta.2020.120804.
[29] J.C. Xiong, L.L. Xia, L.S. Li, M.Y. Cui, Y.Q. Gu, P. Wang, An acetate-based NIR
fluorescent probe for selectively imaging of hydrogen peroxide in living cells
and in vivo, Sens. Actuators. B Chem. 288 (2019) 127–132, https://doi.org/
10.1016/j.snb.2019.02.100.
[30] Z. Han, X. Liang, X. Ren, L. Shang, Z. Yin, A 3,7-dihydroxyphenoxazine-based
fluorescent probe for selective detection of intracellular hydrogen peroxide,
Chem.Asian J. 11 (2016) 818–822, https://doi.org/10.1002/asia.201501304.
[31] Q. Chen, K.e. Cheng, W. Wang, L. Yang, Y. Xie, L. Feng, J. Zhang, H. Zhang, H. Sun,
A pyrene-based ratiometric fluorescent probe with a large Stokes shift for
selective detection of hydrogen peroxide in living cells, J. Pharm. Anal. 10
(2020) 490–497, https://doi.org/10.1016/j.jpha.2020.07.003.
[32] R.R. Nawimanage, B. Prasai, S.U. Hettiarachchi, R.L. McCarley, Cascade
Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the
Selective Detection of Cysteine, Anal. Chem. 89 (2017) 6886–6892, https://
doi.org/10.1021/acs.analchem.7b01384.
[33] Y.L. Jin, R. Liu, Z.X. Zhan, Y. Lv, Fast response near-infrared fluorescent probe
for hydrogen sulfide in natural waters, Talanta 202 (2019) 159–164, https://
doi.org/10.1016/j.talanta.2019.04.067.
[21] B.S. Li, J.B. Chen, Y. Xiong, X. Yang, C.D. Zhao, J. Sun, Development of turn-on
fluorescent probes for the detection of H2O2 vapor with high selectivity and
sensitivity, Sens. Actuators B Chem. 268 (2018) 475–484, https://doi.org/
10.1016/j.snb.2018.04.140.
[34] F.Y. Xu, W. Tang, S.S. Kang, J.S. Song, X.R. Duan, A highly sensitive and photo-
stable fluorescent probe for endogenous intracellular H2O2 imaging in live
cancer cells, Dyes Pigments. 153 (2018) 61–66, https://doi.org/10.1016/j.
dyepig.2018.02.015.
[22] M. Abo, R. Minakami, K. Miyano, M. Kamiya, T. Nagano, Y. Urano, H. Sumimoto,
Visualization of phagosomal hydrogen peroxide production by
a novel
fluorescent probe that is localized via SNAP-tag labeling, Anal. Chem. 86
(2014) 5983–5990, https://doi.org/10.1021/ac501041w.
[23] A.L. Perkel, S.G. Voronina, G.G. Borkina, The role of the Baeyer-Villiger reaction
in the liquid-phase oxidation of organic compounds, Russ. Chem. B 67 (2018)
779–786, https://doi.org/10.1007/s11172-018-2137-0.
[35] C.Y. Liu, C.X. Shao, H.F. Wu, B.P. Guo, B.C. Zhu, X.L. Zhang, A fast-response,
highly sensitive and selective fluorescent probe for the ratiometric imaging of
hydrogen peroxide with a 100 nm red-shifted emission, RSC Adv. 4 (2014)
16055–16061, https://doi.org/10.1039/c4ra01039f.
[36] L. Yuan, W. Lin, Y. Xie, B. Chen, S. Zhu, Single Fluorescent Probe Responds to
H2O2, NO, and H2O2/NO with Three Different Sets of Fluorescence Signals, J.
Am. Chem. Soc. 134 (2) (2012) 1305–1315, https://doi.org/10.1021/ja2100577.
[24] J. Zhang, L. Shi, Z. Li, D. Li, X. Tian, C. Zhang, Near-infrared fluorescence probe
for hydrogen peroxide detection: design, synthesis, and application in living
8