A sensitive and selective fluorescence probe for the detection of superoxide radical anion in vivo based on a protection-deprotection process

Article abstract of DOI:10.1016/j.dyepig.2021.109614

Superoxide radical anion (O₂^·?) plays vital roles in numerous physiological and pathological processes. Thus, it is in great demand to develop an efficient fluorescence probe for the measurement of O₂^·?. In the investigation, a new fluorescence probe NAP-SCM was developed for detection of O₂^·? on the basis of a protection-deprotection process. This probe, which utilizes coumarin as the fluorescent platform and 1,8-naphthalimidesulfonyl as the masking moiety, could monitor O₂^·? with good specificity, rapid response and low detection limit. More importantly, probe NAP-SCM displayed negligible cytotoxicity, good bio-compatibility and excellent capability of tracking the endogenous superoxide radical anion in live cells and live animals. The favorable observations illustrated that this probe has the promising potential to provide a valuable support for exploring the role(s) played by O₂^·? in the biologically functional processes.

Full text of DOI:10.1016/j.dyepig.2021.109614

Dyes and Pigments 194 (2021) 109614
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A sensitive and selective fluorescence probe for the detection of superoxide
radical anion in vivo based on a protection-deprotection process
,
,**
Liyan Chena *, Xin Lua, Fengping Xiaoa, Di Wub
a CCNU-uOttawa Joint Research Centre, Key Laboratory of Pesticides & Chemical Biology Ministry of Education, College of Chemistry, Central China Normal University,
Wuhan, 430079, China
b School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan, 430070, China
A R T I C L E I N F O
A B S T R A C T
Superoxide radical anion (O⋅2ꢀ ) plays vital roles in numerous physiological and pathological processes. Thus, it is
in great demand to develop an efficient fluorescence probe for the measurement of O⋅2ꢀ . In the investigation, a
new fluorescence probe NAP-SCM was developed for detection of O⋅2ꢀ on the basis of a protection-deprotection
process. This probe, which utilizes coumarin as the fluorescent platform and 1,8-naphthalimidesulfonyl as the
masking moiety, could monitor O⋅2ꢀ with good specificity, rapid response and low detection limit. More
importantly, probe NAP-SCM displayed negligible cytotoxicity, good bio-compatibility and excellent capability
of tracking the endogenous superoxide radical anion in live cells and live animals. The favorable observations
illustrated that this probe has the promising potential to provide a valuable support for exploring the role(s)
played by O⋅2ꢀ in the biologically functional processes.
Keywords:
Superoxide radical anion
Fluorescent detection
Protection-deprotection strategy
Coumarin
Bioimaging
1. Introduction
formance liquid chromatography (HPLC), electrochemical sensors,
electron paramagnetic resonance (EPR) and several optical methods
Reactive oxygen species (ROS) including superoxide radical anion
(O⋅2ꢀ ), singlet oxygen (1O2), hypochlorous acid/hypochlorite
(HOCl/ꢀ OCl), hydrogen peroxide (H2O2) and hydroxyl radical (•OH)
play important roles in cell signaling and homeostasis, such as pathogen
response, aging and anti-inflammation regulation [1–3]. Among them,
O⋅2ꢀ has received special attention because it is acted as the major pre-
cursor of many ROS and can be functioned as the marker for early ROS
generation [4,5]. In addition, as a well-known mediator in oxidative
chain reaction, O⋅2ꢀ involves in numerous pathologies such as intercel-
lular signaling and non-specific immunity against bacteria, viruses and
parasites [6–8]. However, a large amount of evidences indicate that the
excessive generation of O⋅2ꢀ will lead to many photoreceptor cell damage
and oxidative-stress-induced diseases including inflammation, autoim-
mune disorders, neurodegeneration and ischemia-reperfusion injury in
surgery [9–12]. Therefore, it is critically meaningful to develop an
efficient analytical method with high sensitivity and selectivity to
monitor O⋅2ꢀ in vitro and in vivo.
[13–17]. Though effective, there are still limitations existed. For
example, electrochemical sensors and HPLC could recognize O⋅2ꢀ with
high specificity, but they are complicated to fabricated. EPR and MS
suffer the expensive instrument and only suitable for the detection of
extracellular O⋅2ꢀ . In contrast, fluorescent imaging technology becomes a
vital method for the measurement of O⋅2ꢀ because of its high sensitivity,
good selectivity and powerful potential for biological research [18–22].
During the past several years, a variety of elegant fluorescent probes
have been implemented to detect O⋅2ꢀ via redox mechanism using
hydroethidine (HE) and its mitochondria-targeting analogue (Mito-HE
or MitoSOX) [23–26]. However, HE and its analogue are easy to be
interfered by other intracellular species like Fe2+, ascorbic acid and
glutathione, resulting in the limitation of applications in bioimaging. In
order to improve the degree of specificity for O⋅2ꢀ , a series of fluorescence
probes on the basis of protection-deprotection mechanism have been
developed utilizing 2,4-dinitrobenzenesulfonyl [27–30], 4,5-dimethox-
y-2-nitro-phenylsulfonyl [31], trifluoromethanesulfonyl [32–34] or
diphenyl phosphinyl [35–40] as the representative reactive moieties.
Though available, there is still plenty of room to improve such as the
To date, various analytical technologies have been applied to
recognize the level of O⋅2ꢀ , such as mass spectrometry (MS), high per-
* Corresponding author. .
** Corresponding author. .
E-mail addresses: clyan@mail.ccnu.edu.cn (L. Chen), chemwd@whut.edu.cn (D. Wu).
https://doi.org/10.1016/j.dyepig.2021.109614
Received 16 May 2021; Received in revised form 21 June 2021; Accepted 28 June 2021
Available online 29 June 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

L. Chen et al.
Dyes and Pigments 194 (2021) 109614
problems of high detection limit, complicated synthetic pathway, poor
narrow linear, water-solubility and long reaction time. In this sense,
more efforts should be made in constructing efficient fluorescent probes
with novel masking moiety for the detection and imaging of O⋅2ꢀ in the
living systems.
Glutathione (GSH), cysteine (Cys), homocysteine (Hcy), hydroquinone
(HQ) and ascrobic acid (AA) were prepared by adding relative chemicals
to deionized water.
2.3. Cell-culture
In the investigation described below, we would like to present a
fluorescence probe for the detection of O⋅2ꢀ on the basis of a protection-
deprotection process. The probe is constructed by the incorporation of 7-
hydroxycoumarin platform and 1,8-naphthalimidesulfonyl unit. The
novel masking moiety 1,8-naphthalimidesulfonyl unit is served for O2⋅ꢀ
detection via protection-deprotection mechanism. We proposed that O2⋅ꢀ
could induce the cleavage of S–O band and release the strong fluores-
cence emitted by 7-hydroxycoumarin (HYCA) (Scheme 1).
The macrophage cell line murine (RAW 264.7) were plated on the
surface of a glass slide in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with penicillin (100 units/mL), 10% heat-inactivated
Fetal Bovine Serum (FBS) and streptomycin (100
μg/mL). The cells
were maintained in a humidified atmosphere of 5% CO2/air at 37 ◦C.
2.4. Bioimaging in live cells and zebrafish
For confocal microscopy experiments of control experiment, RAW
264.7 cells or 72 h post-fertilization zebrafish were treated with probe
2. Experimental section
NAP-SCM (10 μM) for 30 min, washed with DPBS. The experiments for
2.1. Reagents and instruments
monitoring endogenous superoxide radical anion, RAW 264.7 cells or
zebrafish were treated with PMA (1 g/mL) for 12 h or LPS (5 g/mL) for
8 h, washed with DPBS buffer and then treated with probe NAP-SCM
(10 M) for 30 min. For a blocking experiment, cells or zebrafish were
pre-cultured with Tiron (100 M) for 1 h, then loaded with PMA or LPS,
washed with DPBS buffer and then treated with probe NAP- SCM (10
μ
μ
All chemicals were obtained from commercial sources and used as
received. 1H NMR and 13C NMR spectra were recorded using a Bruker
600 MHz instrument. Chemical shifts are expressed in ppm using tet-
ramethylsilane as an internal reference, and coupling constants (J) are
reported in Hz. High-resolution mass spectra were obtained via a Milli-Q
water system (Agilent, USA). Fluorescence emission spectra were
recorded using a fluorescence spectrophotometer (Agilent Cary Eclipse,
USA). UV absorption spectra were conducted from an UV–Vis spectro-
photometer (Agilent Cary 60, USA). Fluorescent images were acquired
on laser confocal microscope (Leica TCS SP8X), and 50% of laser power
was applied. All spectroscopic experiments were performed in a 1 × 1
cm quartz cell. Deionized water was used to prepare all aqueous
solutions.
μ
μ
μ
M) for 30 min. Images were collected at 465–495 nm with an excitation
wavelength of 405 nm using a confocal imaging system.
2.5. Synthesis of intermediate NAP-SOK
The commercially available 4-potassiosulfo-1,8-naphthalenedicar-
boxylic anhydride (632 mg, 2.0 mmol) was added to 30 mL anhydrous
ethanol under N2 atmosphere and then n-butylamine (176 mg, 2.4
mmol) was added. The mixture was heated to reflux and vigorously
stirred overnight. The reaction was cooled to room temperature and the
ethanol was evaporated. The residue was washed with cold ethanol for
several times and used for the next step without further purification.
2.2. Preparation of reactive analytes
Superoxide radical anion solution (O⋅2ꢀ ) was prepared by adding KO2
to dry dimethylsulfoxide. Hypochlorous acid (HOCl) was prepared by
dilution of a 10% NaOCl solution in deionized water. Hydrogen peroxide
(H2O2) was generated by dilution of a 28% solution in deionized water.
Nitric oxide (NO∙) was generated from SNP (sodium nitroferricyanide
(III) dihydrate). ONOOꢀ was prepared using the reported procedure and
the concentration is confirmed using extinction co-efficient of 1670
cmꢀ 1Mꢀ 1 at 302 nm in 0.1 M sodium hydroxide aqueous solutions [41].
Hydroxyl radical (∙OH) was generated by mixing the solution of
2.6. Synthesis of probe NAP-SCM
The intermediate NAP-SOK (371 mg, 1.0 mmol) was dissolved in 20
mL SOCl2 under nitrogen atmosphere. Then three drops of DMF were
added to the mixture as the catalyst. The reaction was heated to reflux
for 12 h. Then the solvent was evaporated under reduced pressure to
obtain the yellow solid for the next step. The commercially available 7-
hydroxycoumarin (162 mg, 1.0 mmol) was dissolved in 20 mL anhy-
drous THF under nitrogen atmosphere followed by the addition of 2.5
mL pyridine. The mixture was cooled to 0 ◦C and stirred for 10 min. The
obtained yellow solid was dissolved in 15 mL anhydrous THF and added
to the mixture dropwise at 0 ◦C. The reaction was then heated to reflux
for 12 h. The reaction was cooled to room temperature and the solvent
was removed under reduced pressure. The residue was purified by silica
column to yield probe NAP-SCM as white solid. 1H NMR (600 MHz,
Chloroform-d) δ 9.12 (d, J = 8.6 Hz, 1H), 8.79 (d, J = 7.3 Hz, 1H), 8.58
(d, J = 7.7 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.07 (t, J = 8.0 Hz, 1H),
7.62 (d, J = 9.6 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 6.98 (dd, J = 8.6, 2.3
Hz, 1H), 6.71 (d, J = 2.3 Hz, 1H), 6.38 (d, J = 9.5 Hz, 1H), 4.19 (t, J =
7.7 Hz, 2H), 1.73 (h, J = 7.6 Hz, 2H), 1.46 (h, J = 7.4 Hz, 2H), 0.98 (t, J
= 7.4 Hz, 3H); 13C NMR (150 MHz, Chloroform-d) δ 162.99, 162.48,
159.38, 154.31, 151.00, 142.27, 135.30, 132.37, 131.14, 130.60,
130.50, 129.84, 129.11, 128.93, 128.49, 127.07, 123.57, 118.50,
118.02, 117.09, 110.45, 40.70, 30.07, 20.33, 13.79. HRMS (ESI) calcd.
for C25H18NSO+7 [M + H+] 478.0960, found 478.0962.
ammonium iron (II) sulfate (100 μM) and H2O2 in deionized water.
Singlet oxygen (1O2) was generated by mixing H2O2 stock solution with
10 equivalents of HOCl. NOꢀ2 and hydrogen sulfide (H2S) were generated
by adding NaNO2 and Na2S to deionized water, respectively.
Scheme 1. Proposed reaction mechanism of probe NAP-SCM and O2⋅ꢀ
.
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L. Chen et al.
Dyes and Pigments 194 (2021) 109614
3. Results and discussion
3.1. Probe design and synthesis
In order to evaluate the suggestion proposed in Scheme 1, probe
NAP-SCM was synthesized according to the previous literature [42]. The
commercially available 4-potassiosulfo-1,8-naphthalenedicarboxylic
anhydride was converted into N-butyl-1,8-naphthalimide NAP-SOK.
The condensation of NAP-SCM with 7-hydroxylcoumarin in the pres-
ence of SOCl2 could produce probe NAP-SCM finally (Scheme 2). The
structure of NAP-SCM was characterized by 1H NMR, 13C NMR and
ESI-HRMS (Figs. S1–S3).
3.2. The spectroscopic properties and TD-DFT calculations
The capability of probe NAP-SCM to detect O⋅2ꢀ was first investigated
under simulated physiological conditions (1.0 mM PBS buffer, pH =
7.4). As the results shown in Fig. 1a, the probe NAP-SCM is non-
fluorescent in the absence of O⋅2ꢀ by the incorporation of 1,8-naphthali-
midesulfonyl unit. With the increasing concentrations of O⋅2ꢀ added in a
solution of NAP-SCM (10 μM in PBS buffer), the emission band peaked
at 454 nm is dramatically enhanced. The intensity arrived the maximum
when the concentration of O⋅2ꢀ is above 150
μM, which corresponds to a
fluorescence enhancement of about 55 folds (Fig. 1b). The fluorescence
intensity at 454 nm displayed an excellent linear relationship (R2
=
0.9915) towards the concentrations of O⋅2ꢀ ranging from 0 to 25
μM. The
result displayed that probe NAP-SCM is well suited for quantitatively
monitoring O⋅2ꢀ . The detection limit was calculated to be as low as 78 nM
according to the 3δ/k criterion, indicating the high sensitivity of probe
NAP-SCM for O⋅2ꢀ detection (Fig. 1b insert). Furthermore, the slight
increasing of UV absorption band centered at 340 nm takes place in the
correspondence with the occurrence of the response for O⋅2ꢀ (Fig. S4).
The time-dependent density functional theory (TD-DFT) calculations
were then conducted in order to shed light on the fluorescent change. As
shown in Fig. S5, the highest occupied molecular orbital (HOMO) was
mainly located on the coumarin moiety while the lowest unoccupied
molecular orbital (LUMO) mainly dispersed on the naph-
thalimidesulfonyl unit in the probe NAP-SCM. The frontier orbitals of
the fluorophore (HYCA) and the masking moiety (NAP-SOH) were also
illustrated in Fig. S6. The empty orbital (LUMO) of NAP-SOH located
between the HOMO and LUMO levels of HYCA, which facilitated the
photoinduced electron transfer (PET) process and consequently
switched off the fluorescence of fluorophore in the probe [43].
Fig. 1. a) Fluorescence spectra of probe NAP-SCM (10
μ
M) upon addition of
M) in PBS buffer (1.0 mM, pH 7.4); b)
M) at 454 nm to different
different amounts of O⋅2ꢀ (0–200
μ
Fluorescence response of probe NAP-SCM (10
μ
concentrations of O⋅2ꢀ (0–200
μM); b) insert: The linear relationship between
fluorescence intensity at 454 nm and O⋅2ꢀ . Spectra were recorded after incu-
bation with different concentrations of O⋅2ꢀ for 20 min λex = 390 nm, λem = 454
nm. Slits: 3/5 nm.
3.3. Selectivity of NAP-SCM towards O⋅2ꢀ and pH tolerance
homocysteine (Hcy), glutathione (GSH)) and other relative species such
as ascorbic acid (AA), ferrous ion (Fe2+) and 1,4-hydroquinone (HQ)
were explored (Fig. 2). In contrast to the significant fluorescent
enhancement (~55 folds) caused by O⋅2ꢀ , probe NAP-SCM suffered
hardly change in the presence of other analytes, which indicate NAP-
The good specificity towards the desired target is another important
component for an ideal probe. Therefore, the fluorescent response of
probe NAP-SCM to a variety of bioanalytes, including ROS (1O2, HOCl,
H2O2), RNS (NOꢀ2 , NO•, HNO, ONOO•-), RSS (H2S, cysteine (Cys),
Scheme 2. Route for synthesis of probe NAP-SCM.
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L. Chen et al.
Dyes and Pigments 194 (2021) 109614
Fig. 2. Relative fluorescence intensities of probe NAP-SCM in the absence and presence of O⋅2ꢀ and other reactive analytes (200
μ
M) in PBS buffer (1.0 mM, pH 7.4).
SCM is highly specific for O⋅2ꢀ .
enhancement could be witnessed. This broad pH tolerance revealed
NAP-SCM possesses the potential applications in biochemical system.
In addition, the pH-dependent fluorescent response of NAP-SCM for
O⋅2ꢀ is examined under different pH buffers in order to study the further
biological applicability. As shown in Fig. 3, NAP-SCM displayed supe-
rior stability over the pH 3–9. With the addition of O⋅2ꢀ to the NAP-SCM
containing solutions at pH value ranging from 4 to 11, the significant
3.4. The kinetic study and sensing mechanism of NAP-SCM for detection
of O⋅2ꢀ
Considering the rapid metabolism nature of O⋅2ꢀ in the living systems,
Fig. 3. Fluorescence intensity of probe NAP-SCM (10
μ
M) at 454 nm under different pH values in the absence and the presence of 15 equiv. of O⋅2ꢀ in PBS buffer (1.0
mM, pH = 7.4). Spectra were recorded after incubation of O⋅2ꢀ for 20 min.
4

L. Chen et al.
Dyes and Pigments 194 (2021) 109614
the rapid reaction rate is essential for the biological applications. Thus,
the rate constant for the reaction of probe NAP-SCM and O⋅2ꢀ was
investigated using the time-dependent fluorescence change. As the re-
sults shown in Fig. 4, the addition of 10 equiv. of O⋅2ꢀ leads to a rapid and
dramatic increasing of fluorescence intensity at 454 nm and the
maximum intensity was achieved after about 5 min. The results
demonstrated that response of probe NAP-SCM with O⋅2ꢀ is rapid and the
product of probe NAP-SCM with O⋅2ꢀ possesses the excellent photo-
systems were further examined. RAW 264.7 macrophages were utilized
as a bioassay model in result from its ability to generate ROS and RNS in
immunological and inflammatory processes. Initially, the survival rate
of RAW 264.7 macrophages under different amounts of NAP-SCM was
employed to estimate the cytotoxicity. As the results shown in Fig. S9,
over 85% RAW 264.7 cells could well survived in the concentrations of
NAP-SCM from 0 to 20 μM, indicating probe NAP-SCM possessed good
bio-compatibility and negligible cytotoxicity. Subsequently, the capa-
bility of probe NAP-SCM to monitor O⋅2ꢀ inside cells was employed.
According to the previous report, the cellular endogenous O⋅2ꢀ could be
generated by phorbol myristate acetate (PMA) or lipopolysaccharide
(LPS) [46]. As can be seen by viewing Fig. 5a, when RAW 264.7 mac-
stability under visible light. The pseudo-first-order rate constant (kobs
)
for the reaction of probe NAP-SCM and O⋅2ꢀ is estimated to be 1.0 ×
10ꢀ 2 sꢀ 1 on the basis of the kinetic data (Fig. 4 insert).
According to well reported protection-deprotection processes
[32–34], the sensing mechanism proposed in Scheme 1 is verified by
monitoring the PBS buffer solution which contains the mixture of probe
rophages were loaded with probe NAP-SCM (10 μ
M) at 37 ◦C for 30 min,
only faint intracellular background fluorescence could be observed
under confocal microscopy. In sharp contrast, when RAW 264.7 mac-
NAP-SCM (10
μМ) and superoxide radical anion (100 μМ) under
rophages were pretreated with PMA (1 μg/mL) for 12 h, and then were
high-resolution mass spectrometry (HRMS). As the mass spectrum
shown in Fig. S7, the new peaks arised at 161.0250 (m/z) and 332.0613
(m/z) are well matched with the formation of the main product HYCA
(calcd m/z 161.0244 for C9H5O3) and NAP-SOH (calcd m/z 332.0598
for C16H14NSO5), respectively. The peak located at 478.0974 is consis-
tent with the remained probe NAP-SCM. In order to further confirm the
reaction mechanism, the 1H NMR titration experiment was conducted.
As the result shown in Fig. S8, the addition of 5.0 equiv of KO2 (dissolved
in DMSO d6) led to the weakening of signals (Ha1 at 6.46 ppm, Hb1 at
6.88 ppm, Hc1 at 7.13 ppm and Hd1 at 7.63 ppm) relevant to the aro-
matic protons of coumarin moiety in probe NAP-SCM. Meanwhile, the
new upfield peaks at about 5.30 (Ha2), 5.94 (Hb2), 5.68 (Hc2) and 7.41
ppm (Hd2) were witnessed, which attributed to the self-cleavage trig-
gered by O⋅2ꢀ . Moreover, the fluorescent spectrum of the mixture
following the reaction of NAP-SCM with O⋅2ꢀ is well matched the fluo-
rescent spectrum of 7-hydroxycoumarin (HYCA) which has been re-
ported previously [44,45].
incubated with probe NAP-SCM for 30 min, a notable enhancement in
fluorescence images was observed (Fig. 5b). Similarly, the distinct blue
fluorescence was observed within cells after treated with LPS (5 μg/mL)
(Fig. 5c). To further verify the specificity of probe NAP-SCM to measure
the endogenous O⋅2ꢀ , the PMA-stimulated or LPS-stimulated RAW264.7
macrophages were pre-treated with Tiron (1,2-dihydroxy-3,5-benzene-
disulfonic acid disodium salt), a common used cell-permeable O2⋅ꢀ
scavenger, for 1 h and then incubated with probe NAP-SCM for 30 min
[47]. As the results in Fig. 5d and e, the bright cellular fluorescence is
significantly attenuated under confocal microscopy. The combined re-
sults demonstrate that NAP-SCM can be utilized for specific and sensi-
tive imaging of endogenous O⋅2ꢀ in live cells.
3.6. Imaging endogenous O∙2ꢀ in live zebrafish
Moreover, we also investigated the capability of tracking endoge-
nous O⋅2ꢀ in live zebrafish. As the results shown in Fig. 6, when zebrafish
was incubated with probe NAP-SCM, a very weak fluorescence was
observed (Fig. 6b). The distinct fluorescence distribution was then
observed after zebrafish was pre-treated with PMA (Fig. 6c). In contrast,
the fluorescence was significantly diminished when Tiron was added
3.5. Imaging endogenous O∙2ꢀ in RAW 264.7 cells
Encouraged by good performance in the simulated physiological
conditions, the potential applications of probe NAP-SCM in biological
Fig. 4. Time course experiment of probe NAP-SCM (10
μ
M) with O⋅2ꢀ (100
μ
M) in PBS buffer (1.0 mM, pH = 7.4). Insert: Pseudo first-order kinetic plot of NAP-SCM
(10
μ
M) with the addition of 10 equiv. of O2∙ꢀ
.
5

L. Chen et al.
Dyes and Pigments 194 (2021) 109614
Fig. 5. Detection of superoxide radical anion produced by an immune reaction in the macrophage RAW 264.7 cells. RAW 264.7 cells were treated with (a) 0; (b) 1
g/mL PMA for 12 h; (c) 5 g/mL LPS for 8 h; (d) RAW 264.7 cells were pre-treated with Tiron (100 M) for 1 h, then 1 g/mL PMA for 12 h; (e) RAW 264.7 cells
were pre-treated with Tiron (100 M) for 1 h, then 5 g/mL LPS for 8 h. The cells were incubated with 10 M probe NAP-SCM for 30 min and washed with DPBS and
imaged by confocal microscopy; (d) bar graphs about mean fluorescent intensities in (a–c); λex 405 nm/λem 465–495 nm. Scale bar: 10 m.
μ
μ
μ
μ
μ
μ
μ
μ
after the treatment of PMA (Fig. 6d). These results indicate that NAP-
SCM can be utilized as a promising fluorescence imaging agent for the
detection of O•2- in live animals.
4. Conclusion
In summary, a novel non-redox strategy based fluorescence probe
NAP-SCM for the measurement of O⋅2ꢀ is designed and synthesized uti-
lizing coumarin as the fluorescent platform and 1,8-naphthalimidesul-
fonyl as the masking moiety. Probe NAP-SCM displayed excellent
detection performance for O⋅2ꢀ in vitro including the good specificity to
O⋅2ꢀ over other reactive species, the rapid response, the high light sta-
bility, the high sensitivity with the detection limit as low as 78 nM, the
good linearship in a wide range of O⋅2ꢀ concentrations and a wide pH
tolerance. Furthermore, the biologically potential of probe NAP-SCM is
demonstrated by its low cytotoxicity, good bio-compatibility and the
capability to monitor the endogenous O⋅2ꢀ in RAW 264.7 cells and live
zebrafish with high specificity. Collectively, the favorable observations
in this investigation demonstrate the high practical value of probe NAP-
SCM for elucidating the biological roles of O⋅2ꢀ in future studies.
CRediT authorship contribution statement
Liyan Chen: Formal analysis, Investigation, Writing – original draft,
Writing – review & editing. Xin Lu: Formal analysis, Investigation, NMR
experiment. Fengping Xiao: Spectroscopic experiments. Di Wu: Su-
pervision, Writing – review & editing, All authors discussed the results
and contributed to the final manuscript.
Fig. 6. Confocal imaging of endogenous O•2- in 72 h post-fertilization zebrafish
Declaration of competing interest
embryos. (a) Blank; (b) Zebrafish was incubated with probe NAP-SCM (10
μ
M)
g/mL) for 12 h after
M) for 30 min; (d) Zebrafish initially
g/mL) for 30 min and treated with Tiron (100 M) for
1 h was loaded with probe NAP-SCM (10 M) for 30 min λex 405 nm/λem
465–495 nm. Scale bar = 10 m.
for 30 min; (c) Zebrafish briefly challenged with PMA (1
preloaded with probe NAP-SCM (10
stimulated with PMA (1
μ
μ
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
μ
μ
μ
μ
6

L. Chen et al.
Dyes and Pigments 194 (2021) 109614
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109614.
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