A novel fluorescent probe for hydrogen peroxide and its application in bio-imaging

Article abstract of DOI:10.3390/molecules26113352

Hydrogen peroxide (H₂O₂) plays an important role in the human body and monitoring its level is meaningful due to the relationship between its level and diseases. A fluorescent sensor (CMB) based on coumarin was designed and its ability for detecting hydrogen peroxide by fluorescence signals was also studied. The CMB showed an approximate 25-fold fluorescence enhancement after adding H₂O₂ due to the interaction between the CMB and H₂O₂ and had the potential for detecting physiological H₂O₂. It also showed good biocompatibility and permeability, allowing it to penetrate cell membranes and zebrafish tissues, thus it can perform fluorescence imaging of H₂O₂ in living cells and zebrafish. This probe is a promising tool for monitoring the level of H₂O₂in related physiological and pathological research.

Full text of DOI:10.3390/molecules26113352

molecules
Article
A Novel Fluorescent Probe for Hydrogen Peroxide and Its
Application in Bio-Imaging
Yingying Zuo, Yang Jiao * , Chunming Ma and Chunying Duan
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China;
z943877035@mail.dlut.edu.cn (Y.Z.); sxcm98@gmail.com (C.M.); cyduan@dlut.edu.cn (C.D.)
* Correspondence: jiaoyang@dlut.edu.cn
Abstract: Hydrogen peroxide (H2O2) plays an important role in the human body and monitoring its
level is meaningful due to the relationship between its level and diseases. A fluorescent sensor (CMB
based on coumarin was designed and its ability for detecting hydrogen peroxide by fluorescence
signals was also studied. The CMB showed an approximate 25-fold fluorescence enhancement after
adding H2O2 due to the interaction between the CMB and H2O2 and had the potential for detecting
physiological H2O2. It also showed good biocompatibility and permeability, allowing it to penetrate
cell membranes and zebrafish tissues, thus it can perform fluorescence imaging of H2O2 in living cells
and zebrafish. This probe is a promising tool for monitoring the level of H2O2 in related physiological
and pathological research.
)
Keywords: hydrogen peroxide; reactive oxygen species; fluorescence; imaging
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ꢀꢁꢂꢃꢄꢅꢆ
1. Introduction
Citation: Zuo, Y.; Jiao, Y.; Ma, C.;
Duan, C. A Novel Fluorescent Probe
for Hydrogen Peroxide and Its
Application in Bio-Imaging. Molecules
2021, 26, 3352. https://doi.org/
10.3390/molecules26113352
Reactive oxygen species (ROS) play important roles in a wide variety of biological
functions [
physiological and pathological processes [
cells is much higher than that in normal cells. Hydrogen peroxide (H2O2), the least reactive
and mildest oxidant among reactive oxygen species [ ], can regulate many physiological
1,
2], and its abnormal production or accumulation is also closely related to many
3,4], especially, the concentration of ROS in cancer
5
processes in organisms and even affect the growth and development of cell [6–8]. It is
produced in low levels during the metabolism of normal living cells and excessive H2O2
production or accumulation in vivo is also considered as the key contributor to many
Academic Editor: Mei Pan
diseases [
of antioxidants is severely reduced, which may be found in numerous diseases, such as
cardiovascular disease and cancer [12 14]. Thus, it is significant to find a method that
9–11]. H2O2 is overproduced when there is an exogenous stimulus or the content
Received: 1 May 2021
Accepted: 28 May 2021
Published: 2 June 2021
–
can detect and quantify the production of H2O2 to facilitate the diagnosis and treatment
of diseases.
In recent years, various analytical methods that can detect hydrogen peroxide have
been reported, such as fluorescence spectroscopy, electroanalysis, chemiluminescence,
etc. [15]. The fluorescence detection method is paid much attention among many other
analytical methods due to its characteristics of good selectivity, high sensitivity, quick
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
response rate, as well as real-time detection [16
tracking certain biomolecules in cells or organisms [19] because of its non-invasiveness
and ease of operation [20 22], and thus has great potential in detecting small molecules’
markers to help in the diagnosis of some diseases. Some fluorescent small-molecule probes
for detecting ROS have been reported [23 27]. These studies proved the feasibility of using
fluorescent small-molecule probes to detect H2O2 and can promote the development and
progress of H2O2 probes, which may facilitate the realization of its applications in medicine.
Herein, a smart small-molecule fluorescent probe, CMB, was designed and syn-
thesized for H2O2 imaging in solution, cells, and zebrafish by employing coumarin as
–18]. It tends to be used for detecting and
–
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
–
fluorophore and the boronate as the recognition group [28–30] (Scheme 1). A short alkyl
Molecules 2021, 26, 3352. https://doi.org/10.3390/molecules26113352
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3352
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chain is used to connect the morpholine group with coumarin to improve the biological
applicability of the probe. The fluorescence signal of CMB would be enhanced in the
presence of H2O2 due to the reaction between probe CMB and H2O2, and the good lin-
ear relationship between the fluorescence intensity and the concentration of H2O2 also
facilitates its detection. Probe CMB can not only shows enhanced fluorescence signal to
H2O2 with good selectivity, but also exhibits desirable imaging effects on H2O2 in cells and
zebrafish, which indicates that CMB has the potential to monitor the content and level of
hydrogen peroxide in biological systems.
Scheme 1. The mechanism of CMB to detect H2O2.
2. Results and Discussion
2.1. Design Strategy of Probe CMB
The probe CMB was designed and synthesized in three steps from the coumarin moi-
ety (Scheme 2) that possesses excellent photo-physical properties [31,32]. The morpholine
group can increase the solubility of the probe in water, thereby increasing biocompatibility
and making it more beneficial for imaging in biological environments. The aryl boronate
can be bound to the hydroxyl group of CM by the ether linkage strategy to form CMB
and react with H2O2 as well as providing the specificity for H2O2 over other interference
species [33,34]. The synthetic route of probe CMB was provided and shown in Scheme 2.
The structure of CMB was accurately validated using the 1H NMR and HRMS analyses
presented in the Supporting Information (Supplementary Figures S1–S4).
Scheme 2. The synthesis route of probe CMB.
2.2. Optical Response of CMB to H2O2
The UV-vis absorption and fluorescence emission spectra were both studied in ace-
tonitrile/phosphate buffer (1:9 v/v, 10 mM, pH 7.4) at room temperature to test the optical
properties of CMB in the presence or absence of H2O2. The UV-vis spectrum of the probe
CMB (6 µM) showed a absorption band at 350 nm, then a new band appeared at 405 nm
after being treated with H2O2 (Figure 1a), which may be attributed to the chemical structure
change from CMB to CM. As shown in Figure 1b, CMB had only a very weak fluorescence
when excited at 400 nm, while a significant increase in fluorescence was observed at 450 nm
after adding H2O2. With the increase of hydrogen peroxide concentration, the fluorescence
intensity of CMB increased by about 25-fold (Figure 1c). There is a good linear relationship

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between the fluorescence intensity and the concentration of H2O2 in the range of 0 to 50
with a correlation coefficient of 0.9979 (Figure 1d) and the detection limit was 0.13
µM
µM,
thus being able to be used for direct imaging of physiological H2O2 [23]. This fluorescence
change may be attributed to the structural change from CMB to CM caused by the reaction
with H2O2. The good “off–on” response of CMB toward H2O2 showed its potential for
detection of H2O2 and may realize the detection of H2O2 level.
Figure 1.
addition of H2O2. (
(
a
) UV-vis absorption and (
b
) fluorescence emission spectra of CMB before and after
M) to various concentrations of H2O2.
c) Fluorescence responses of CMB (2
µ
(d) The linear correlation between the fluorescence intensity at 450 nm and H2O2 concentration.
The response time of probe CMB toward H2O2 was also investigated by the time-
dependent fluorescence intensity. After adding H2O2 to the solution of probe CMB, the
fluorescence intensity at 450 nm gradually increased with time and stabilized finally. The
selectivity study of probe CMB was carried out in the presence of various biologically
relevant possible competing species, mainly including ions (K−+, Mg2+, Fe3+, Cl−, etc.),
amino acids (Cys, Pro), reactive oxygen species ( OH, TBHP, ClO , etc.) [35,36]. As shown
•
in Figure 2b, ions, amino acids, and most of the reactive oxygen species showed negligible
−
changes in fluorescence and 1O2 and NO2 caused a slight response. The fluorescence
enhancement of CMB to H2O2 is more obvious relative to the interferences. In order to
further verify the specific reaction between the probe and H2O2, the catalase (hydrogen
peroxide quencher) and glucose oxidase (hydrogen peroxide producer) were used to
perform the fluorescence experiment. When H2O2 is treated with catalase, CMB no longer
produced a significant increase in fluorescence toward it, but when glucose reacted with
glucose oxidase was added, the obvious fluorescence enhancement was observed due to
the H2O2 produced by the reaction, which proved the specificity of CMB to H2O2. The
obvious fluorescence response and extended linear range as well as good biocompatibility
and permeability make CMB an excellent candidate and can serve as the indicators for
H2O2 detection compared with other candidates (Supplementary Table S1). All the results
illustrated that CMB can react with hydrogen peroxide selectively to achieve fluorescence
enhancement and we hope that it is suitable in a bio-environment.

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Figure 2.
various competing species (100
(Cys), proline (Pro), hypochlorite (ClO ), tert-butyl hydroperoxide (TBHP), hydroxyl radical (
peroxynitrite (ONOO−), singlet oxygen (1O2), nitrite (NO2−), H2O2 + catalase,
(
a
) The time response of the probe CMB. (
b
) Fluorescence response of CMB−(2
µM) to
−
−
µ
M), including K+, Mg2+, Fe2+, Fe3+, Cl , HCO3 , NO3 , cysteine
−
•
OH),
-D-
β-D-glucose,
β
glucose + glucose oxidase (GOD), and H2O2.
2.3. Response Mechanism of CMB to H2O2
According to the fluorescence response of CMB to H2O2, the possible response mech-
anism of H2O2 detection can be preliminarily assumed (Scheme 1). When the fluorophore
was connected with the aryl boronate, CMB showed almost no fluorescence due to the
intramolecular interaction. The added H2O2 will react with the aryl boronate group, caus-
ing the hydroxyl of the fluorophore to be released, thereby revealing a strong fluorescent
signal. In order to confirm the mechanism, high-resolution mass spectrometry (HRMS) was
performed. CMB showed an intense peak at m/z = 535.2606 (Supplementary Figure S4).
After adding H2O2, the peak of CMB had almost disappeared, while a new peak at m/z =
319.1291 appeared accompanied by the peak of the intermediate product at m/z = 425.1710,
proving the transformation of CMB into CM by the reaction of aryl boronate group and
H2O2. The liquid chromatography experiments were also done. As shown in Figure 3b,
the CMB had a signal peak at 20.528 min. After adding H2O2, a new peak appeared at
9.116 min, which had the same as the retention time of CM. Thus, the experiments both
supported the fact that the structural transformation caused by the reaction of CMB with
H2O2 triggers the enhanced fluorescence signal, proving our inference about the response
mechanism of CMB to H2O2 proposed in Scheme 1.
2.4. Cytotoxicity and Fluorescence Imaging in Living Cells
Taking into account the particularity of the biological environment, the viability of
cells against the probe was determined using the CCK8 assay technique [37] to evaluate the
cytotoxicity of the CMB. The relative growth rate of cells was detected upon exposure to
CMB with concentrations of 0 to 20
µM for 24 h. As is shown in Figure 4, the cell survival
rate was greater than 90% at probe concentrations up to 20
µM, demonstrating that CMB
has an acceptable cytotoxicity to cells within this concentration range and can be used in
biological systems. Thus, it can perform the subsequent cell imaging experiment.

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Figure 3.
(
a) HRMS of CMB after adding H2O2. (
b) Liquid chromatography of CMB, CMB treated
with H2O2, and CM.
Then, we carried out the imaging experiment to test the practical feasibility of CMB
for exogenous and endogenous H2O2 detection. When MCF-7 cells were stained with
CMB alone, there was almost no fluorescence, but was observed that the fluorescence
signal increased significantly and dose-dependently after treating cells with different
concentrations of H2O2 (0 to 50 µM) (Figure 5a), which also proved that the CMB could
effectively penetrate the cell membrane and disperse in the cytoplasm. In addition, the cells
were incubated with H2O2 and then treated with CMB for imaging at different time points.
It was found that the fluorescence intensity gradually increased over time (Figure 5b)
,
which further indicates that CMB can be used to detect exogenous H2O2.

Molecules 2021, 26, 3352
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Figure 4. Relative growth rate (%) of cells that were cultured in the presence of a 0 to 20
CMB for 24 h and estimated by the CCK8 assay.
µM probe
Figure 5. Confocal images after the addition of exogenous H2O2 in MCF-7 cells. (
stained with different concentrations of H2O2 and then incubated with CMB (2 µM). (b) Cells were
pretreated with H2O2 (50 M), and then incubated with CMB (2 M) at different time points (0 to
a) Cells were
µ
µ
70 min). The excitation wavelength was 405 nm and the emission was collected at 420 to 520 nm.
Scale bar = 20 µm.
In addition, endogenous hydrogen peroxide is generated by the stimulation of
rotenone [38,39], so the imaging ability of CMB to the endogenous H2O2 level of MCF-7
cells could be studied by adding rotenone. When cells were treated with rotenone with the
addition of CMB, an obvious fluorescence could be observed (Figure 6). After incubating
the rotenone-pretreated cells with N-acetylcysteine (NAC) [40], a common H2O2 inhibitor,
the intracellular fluorescence was basically negligible, indicating the H2O2 produced by the
stimulation of rotenone was eliminated. These results proved the ability of CMB to detect
endogenous H2O2. The cell imaging experiment demonstrated that the CMB has favorable
membrane-permeability, showing its potential for fluorescence imaging of exogenous and
endogenous H2O2 levels in cells.

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Figure 6. Confocal images for exogenous and endogenous H2O2 in MCF-7 cells. (
stained with CMB (2 M). (
(2 M). ( ) Cells were incubated with rotenone and then stained with CMB (2
were incubated with rotenone, treated with NAC, and then stained with CMB (2 M). The excitation
a
–
c) Cells were
µ
d
–f
) Cells were incubated with H2O2 and then stained with CMB
M). ( ) Cells
µ
g–
i
µ
j–l
µ
wavelength was 405 nm and the emission was collected at 420 to 520 nm. Scale bar = 20 µm.
2.5. Zebrafish Imaging
To further investigate the availability of the probe CMB in biological systems, a ze-
brafish imaging experiment was carried out to detect its fluorescence imaging performance.
As shown in Figure 7, zebrafish incubated with H2O2 and then treated with CMB showed
obvious fluorescence, while the control group treated only with CMB exhibited negligible
fluorescence. Besides, the fluorescence intensity of zebrafish treated with rotenone also
increased significantly, and after further treatment with NAC, its weak fluorescence was
similar to that of the control group. In addition, the z-scan mode of the confocal microscope
was used to record the fluorescence intensity at different depths in the body of the zebrafish
to explore the penetration capability of the probe in tissue. As shown in Supplementary
Figure S5, bright fluorescence was observed even at a depth of up to 180 µm, showing
the probe CMB had tissue penetrating and staining capabilities. Thus, CMB has excellent
penetration and exhibits good imaging feasibility for endogenous and exogenous H2O2
in zebrafish.

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Figure 7. Confocal images for exogenous and endogenous H2O2 in zebrafish. (
stained with CMB (2 M). (
(2 M). ( ) Zebrafish were incubated with rotenone and then stained with CMB (2
Zebrafish were incubated with rotenone, treated with NAC, and then stained with CMB (2
a
–
c
) Zebrafish were
µ
d–
f
) Zebrafish were incubated with H2O2 and then stained with CMB
M). (j–l)
µ
g–
i
µ
µM).
The excitation wavelength was 405 nm and the emission was collected at 420 to 520 nm. Scale bar =
300 µm.
3. Conclusions
In summary, a novel fluorescence probe CMB based on coumarin was designed
and developed successfully, that could interact with H2O2 and realize its detection by
fluorescence. It can react with H2O2 selectively, thus producing an enhanced fluorescence
signal. The probe CMB has low toxicity and good biocompatibility, and it can penetrate
the cell membranes and zebrafish tissues to image endogenous and exogenous H2O2. The
good penetration and staining ability further prove the feasibility of CMB to accurately
monitor H2O2 in biological systems. It is expected that CMB will become a promising
tool to detect the H2O2 and exhibits important potential applications in the diagnosis of
H2O2-related diseases.
4. Materials and Methods
4.1. Materials and Instruments
All chemicals used were of reagent grade or purchased as high-grade commercial
products and used without further purification. Solvents used were purified via standard
1
methods. H NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer.
Mass spectrometric data were performed on an LTQ Orbitrap XL spectrometer. The high-
performance liquid chromatography (HPLC) experiment was recorded on an Agilent
1260 LC system (USA). Fluorescent spectra were achieved with an F7000 fluorescence
spectrophotometer. The UV–vis spectra were recorded on a TU 1900 UV–vis spectrometer.
The Dulbecco’s modified Eagle’s medium (DMEM) that was added with 10% fetal bovine
serum (FBS), was used to culture all the cell lines. The OD values of the CCK8 assay
were measured by BIO-RAD xMark microplate spectrophotometer. Confocal fluorescence
imaging was performed on an OLYMPUS FV1000 confocal microscopy.
4.2. Synthesis of Compound 1
Compound
1 was synthesized according to a previously published article [41]. 2,4-
dihydroxybenzaldehyde (1.38 g, 10.0 mmol) and diethyl malonate (1.92 g, 12.0 mmol) were

Molecules 2021, 26, 3352
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dissolved in anhydrous EtOH, and then an appropriate amount of piperidine was added.
The reaction mixture was refluxed for 12 h and then cooled. The precipitate was collected
and washed with cold EtOH to obtain the yellow compound
1 (1.87 g) with a yield of 80%.
1H NMR (400 MHz, DMSO-d6)
δ
11.07 (s, 1H), 8.65 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 6.82 (dd,
J = 8.6, 2.1 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H).
4.3. Synthesis of Compound CM
Compound 1 (0.41 g, 2.0 mmol) was dissolved in ethanol, then N-(2-methylamino)-
morpholine (0.31 g, 2.4 mmol) and triethylamine (2.4 mmol) were added. The mixture was
heated to reflux for 8 h and cooled after the reaction was complete. Then the precipitate
was filtered, and the filtrate was extracted with dichloromethane and water. The organic
layers were combined and dried over anhydrous sodium sulfate and column chromatogra-
1
phy (CH2Cl2:CH3OH = 100:1) to obtain compound CM (0.417 g, yield 65.4%). H NMR
(400 MHz, DMSO-d6)
δ 11.03 (s, 1H), 8.85 (t, J = 5.3 Hz, 1H), 8.79 (s, 1H), 7.82 (d, J = 8.6 Hz,
1H), 6.88 (dd, J = 8.6, 2.2 Hz, 1H), 6.84–6.73 (m, 1H), 3.74–3.54 (m, 4H), 3.43 (q, J = 6.2 Hz,
2H), 2.48 (d, J = 6.4 Hz, 2H), 2.42 (s, 4H).
4.4. Synthesis of Compound CMB
CM (0.32 g, 1.0 mmol) and K2CO3 (0.42 g, 3.0 mmol) were dissolved in 5 mL CH3CN
and then 2-(4-(bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.42◦g,
1.4 mmol) was also added to the mixture. The reaction mixture was heated at 80 C
for 24 h and cooled to room temperature. Then, the mixture was diluted with CH2Cl2 and
water and the aqueous layer was extracted with CH2Cl2. The combined organic phase was
washed with water and dried with anhydrous sodium sulfate, then purified by column
chromatography (CH2Cl2:CH3OH = 100:1) to obtain a white solid (0.25 g) with the yield of
1
46.7%. H NMR (400 MHz, DMSO-d6)
δ 8.85 (d, J = 3.8 Hz, 2H), 7.92 (d, J = 8.7 Hz, 1H),
7.72 (d, J = 7.8 Hz, 2H), 7.49 (d, J = 7.8 Hz, 2H), 7.18 (d, J = 2.1 Hz, 1H), 7.12 (dd, J = 8.7,
2.2 Hz, 1H), 5.32 (s, 2H), 3.59 (t, J = 4.6 Hz, 4H), 3.44 (q, J = 6.1 Hz, 2H), 2.47 (d, J = 6.4 Hz,
2H), 2.42 (s, 4H)„ 1.30 (s, 12H).
4.5. Preparation of Related Species and Configuration of Solutions
Probe CMB was dissolved with DMSO to prepare stock solutions. Adding 4
µL probe
stock solution into 2 mL acetonitrile/phosphate buffer (1:9 v/v, 10 mM, pH 7.4) with the
−
−
,
final concentration of probe as 2
µ
M for fluorescent spectra test. H2O2, ClO , TBHP, NO2
−
and NO3 obtained from commercial sources were diluted or dissolved in water. Other
reactive oxygen species are prepared according to the literature [42
,
43]. ONOO− was
prepared by mixing pre-cooled 0.6 M NaNO2, 0.6 M HCl, and 0.7 M H2O2 into 1.5 M NaOH
◦
at 0 C. Manganese dioxide was then added to the solution to eliminate the residual H2O2
and was removed by filtration. Its concentration was estimated by its extinction coefficient
of 1670 M−1 cm−1 at 302 nm. A singlet oxygen solution was prepared by the reaction of
H2O2 and sodium hypochlorite solution. Hydroxyl radical was generated by the Fenton
reaction and can be acquired by adding Fe2+ to H2O2.
4.6. Measurement of Detection Limit
The detection limit was obtained from the fluorescence titration curve and was calcu-
lated according to the following equations [44]:
Detection limit = 3σ/k
Here
σ represents the deviation of blank and k is the slope of the linear regression
equation.
4.7. Cell Culture and Cytotoxicity Assay
The Michigan Cancer Foundation-7 (MCF-7) cells were allowed to culture for 24 h
◦
at 37 C with medium supplemented with 10% fetal bovine serum (FBS) in a 5% CO2

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humidified incubator. Cells were seeded in 96-well microplates for 24 h and then cultured
in medium with 0, 1, 2, 5, 10, and 20 µM of probe CMB for 24 h. Cells in culture medium
without probe were used as the control. The solution of CCK-8 reagent was added to
each well and the plates were incubated for another 1 h. The medium was then removed
carefully, and the absorbance of solutions was determined on a microplate reader at 450 nm.
4.8. Cell Imaging
The MCF-7 cells were treated with different concentrations of H2O2 (0◦
µM, 10 µM,
20 µM, 30 µM, 40 µM, 50 µM) and then incubated with CMB (2 µM) at 37 C to detect
exogenous H2O2. In addition, MCF-7 cells were stimulated with rotenone (2
to produce endogenous H2O2, and then treated with CMB (2 M). Then, another group
µ
M) at 37 ◦C
µ
of MCF-7 cells was first stimulated with rotenone (2
to eliminate the endogenous H2O2, and treated with CMB (2
µ
M), incubated with NAC (1 mM)
◦
µM) at 37 C. The cells
were washed three times with PBS after incubation, and then imaged on a confocal laser
microscope with a 60× objective lens.
4.9. Zebrafish Imaging
◦
Zebrafish embryos were maintained at 28.5 C. First, the zebrafish were only incubated
with CMB (2
rotenone (2
stimulated with rotenone (2
H2O2, and treated with CMB (2
µ
M) as the control group. Another group of zebrafish was stimulated with
M), and then treated with CMB (2 M). Then the last group of zebrafish was
M), incubated with NAC (1 mM) to eliminate the endogenous
M). The zebrafish were washed three times with PBS
µ
µ
µ
µ
after incubation and imaged on a confocal laser microscope with a 4× objective lens.
Supplementary Materials: The following are available online, Figure S1: The 1H NMR of com-
pound
1 in DMSO-d6 solution, Figure S2: The 1H NMR of compound CM in DMSO-d6 solution,
Figure S3: The 1H NMR of compound CMB in DMSO-d6 solution, Figure S4: The HRMS of com-
pound CMB, Table S1: Properties of the previously developed fluorescent H2O2 probes and the probe
CMB, Figure S5: The confocal z-scan images of zebrafish treated with probe and H2O2. Zebrafish
were incubated with H2O2 and then stained with CMB (2 µM). The excitation wavelength was 405
nm and the emission was collected at 420 to 520 nm.
Author Contributions: Conceptualization, Y.J.; methodology, Y.J. and C.D.; formal analysis, Y.Z. and
C.M.; writing—original draft, Y.Z. and Y.J.; writing—review and editing, Y.Z., Y.J., and C.D.; project
administration, Y.J.; supervision, C.D. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (21977015
and 21820102001), and the Fundamental Research Funds for the Central Universities (DUT20LK12).
Acknowledgments: We acknowledge Jiqiu Yin (from Dalian Medical University) for her assistance
in cytotoxicity experiments.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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