Near-infrared fluorescent probe with rapid response and large Stokes shift for imaging peroxynitrite in living cells, zebrafish and mice

Article abstract of DOI:10.1016/j.dyepig.2019.107820

Detection and imaging of peroxynitrite (ONOO^?) in living systems has attracted considerable attention in recent years due to its biological significance. In this paper, a new highly selective and sensitive near-infrared (NIR) fluorescent turn-on probe for ONOO^? was developed. This probe uses a dicyanoisophorone-based fluorescent dye as the fluorophore and α-ketoamide as the reaction site. In the presence of ONOO^?, the probe exhibits notable fluorescence enhancement at 660 nm with rapid response (<2 s), large Stokes shift (200 nm), and a low detection limit (81 nM). In addition, MTT assays indicated that this probe is of low cytotoxicity in cells, and bioimaging experiments demonstrated that the probe could be applied for imaging of ONOO^? in living cells, zebrafish and mice with good imaging performance.

Full text of DOI:10.1016/j.dyepig.2019.107820

DyesandPigments172(2020)107820
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Near-infrared fluorescent probe with rapid response and large Stokes shift
for imaging peroxynitrite in living cells, zebrafish and mice
Xinyu Yin, Weiyong Feng, Shengyi Gong, Guoqiang Feng⁎
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, Chemical Biology Center, College of Chemistry, Central China Normal University, 152 Luoyu
Road, Wuhan, 430079, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Peroxynitrite
NIR fluorescent probe
Large Stokes shift
Bioimaging
Detection and imaging of peroxynitrite (ONOO−) in living systems has attracted considerable attention in recent
years due to its biological significance. In this paper, a new highly selective and sensitive near-infrared (NIR)
fluorescent turn-on probe for ONOO− was developed. This probe uses a dicyanoisophorone-based fluorescent
dye as the fluorophore and α-ketoamide as the reaction site. In the presence of ONOO−, the probe exhibits
notable fluorescence enhancement at 660 nm with rapid response (< 2 s), large Stokes shift (200 nm), and a low
detection limit (81 nM). In addition, MTT assays indicated that this probe is of low cytotoxicity in cells, and
bioimaging experiments demonstrated that the probe could be applied for imaging of ONOO− in living cells,
zebrafish and mice with good imaging performance.
Living cells and animals
1. Introduction
valuable in practical applications both in vitro and in vivo, because
such probes provide minimal photodamage, deep tissue penetration
Peroxynitrite (ONOO−) is one of the most important endogenous
reactive oxygen species (ROS) in living systems, playing vital roles in
various physiological and pathological processes such as in signal
transduction and antimicrobial activities [1–3]. However, due to its
strong oxidizing and nitrating capabilities, the elevated level of ONOO−
can cause damage to many biomolecules including lipids, DNA, pro-
teins, and enzymes, which may further affect cellular processes and
even cause cell necrosis and apoptosis, eventually lead to the appear-
ance of various diseases, such as cardiovascular disease and injury,
autoimmune and inflammatory diseases, Alzheimer's disease, and even
cancer [4–6]. Even these are known facts, many aspects of ONOO− in
living systems are still unclear due to its elusive nature in vivo with low
concentration, high reactivity, and short lifetime [7]. Therefore, the
development of rapid, selective and sensitive detection methods for
ONOO− in living systems is of great significance to further understand
ability, and minimal interference form excitation/scattering light and
autofluorescence of biomaterials [10–14]. So far, a variety of fluor-
escent probes have been reported for ONOO− detection [15] via dif-
ferent reaction approaches, including boronate oxidation [16–24],
oxidative N-dearylation [25–30], oxidation of hydrazides [31–36],
oxidation of chalcogenides [37,38], cleavage of the C]C double bonds
[39–41], cleavage of α-ketoamide [42–45], cleavage of trifluoromethyl
ketone [46], and others [47–52]. However, most of these probes
showed fluorescence responses to ONOO− in the visible region, which
are suitable for in vitro detections but not suitable for applications in
vivo. Although several NIR fluorescent probes have been reported to
detect ONOO− [37,38,53–58], most of them suffered from small Stokes
Shift [37,38,55,57,58] or turn-off NIR fluorescence [55,56], which also
limited their practical use in bioimaging applications. In addition, many
reported probes take a few minutes or even dozens of minutes to re-
spond to ONOO−, which might be inefficient to real-time track ONOO−
in living systems due to its transient nature. To overcome these short-
comings, it is particularly necessary to develop rapid responsive NIR
the biological ONOO−
.
Among various detection methods, small molecular probe-based
fluorescent detection has many advantages such as high sensitivity,
non-invasiveness, great bio-compatibility, and real-time monitoring
ability with high spatio-temporal resolution, and thereby been re-
cognized as one of the most attractive methods for the detections in
living systems [8,9]. Particularly, near-infrared (NIR, 650–900 nm)
fluorescent probes with a large Stokes shift have been proved highly
fluorescent probes with large Stokes shift for detection of ONOO−
.
Recently, dicyanoisophorone (DCI)-based fluorescent dyes have at-
tracted great attention due to their excellent optical properties in-
cluding NIR fluorescence, large Stokes shift, good biocompatibility and
facile acquirability [59]. However, the potential of DCI-based
⁎ Corresponding author.
E-mail address: gf256@mail.ccnu.edu.cn (G. Feng).
https://doi.org/10.1016/j.dyepig.2019.107820
Received 10 July 2019; Received in revised form 22 July 2019; Accepted 16 August 2019
Availableonline17August2019
0143-7208/©2019ElsevierLtd.Allrightsreserved.

X. Yin, et al.
DyesandPigments172(2020)107820
Scheme 1. A new NIR fluorescent probe DCI-NPG-
ONOO for detection of ONOO−
.
fluorescent dyes for detecting ONOO− has not been reported. In this
work, by taking advantage of the reactivity of α-ketoamides towards
ONOO− [42–45], we attached an α-ketoamide moiety to an easily
obtained DCI-based fluorophore (DCI-NIR-NH2 in Scheme 1) and de-
veloped a novel NIR fluorescent probe (DCI-NPG-ONOO in Scheme 1)
for ONOO−. This probe was found to show rapid response (response
time < 2 s), high selectivity and sensitivity, and significant NIR
2.3. Optical measurements
Stock solution of probe DCI-NPG-ONOO (1 mM) was prepared in
HPLC grade DMSO. ONOO− was prepared from isoamyl nitrite and
H2O2 according to the literature method [61], and its concentration was
estimated by the absorption at 302 nm in 0.1 M aqueous NaOH solution
with extinction coefficient of 1670 cm−1M−1. Other reactive oxygen/
nitrogen species (ROS/RNS) were prepared and used freshly according
to our reported methods [62,63]. Stock solutions of the other analytes
including tyrosine (Tyr), alanine (Ala), aspartic acid (Asp), threonine
(Thr), methionine (Met), isoleucine (Ile), phenylalanine (Phe), cysteine
(Cys), homocysteine (Hcy), and glutathione (GSH), NaHCO3, NaHSO3,
NaHS, NaCl, NaF, NaI, KCl, FeCl3, and CuCl2 were prepared in ultrapure
water and used freshly. As a general measurement, a 3.0 mL solution of
probe DCI-NPG-ONOO (10 μM) was first prepared in PBS buffer
(10 mM, pH 7.4, with 30% EtOH, v/v) in a quartz cuvette. When the
temperature of solution reached to 37 °C, a certain amount of ONOO−
(or other analyte) was added with a micropipette (the total volume
change of the test solution: < 2%), and then the UV–vis and fluores-
cence spectra were recorded.
fluorescent turn-on signals with
a remarkable large Stokes shift
(200 nm) for ONOO−. Moreover, this probe shows good performance
for imaging ONOO− in living cells and living animals. All the results
indicate that this new probe can be used as an effective tool for de-
tection and imaging of ONOO− both in vitro and in vivo.
2. Materials and methods
2.1. Materials and instrumentation
All chemicals and solvents were purchased from commercial sup-
pliers and used as granted. Water was purified by a Millipore-Q ultra-
purification system before use. NMR spectra were recorded on a Varian
MercuryPlus 400 spectrometer. Low-resolution mass spectra were re-
corded on a Thermo Scientific DSQ II GC/MS spectrometer or a Waters
E2695 LC-MS system. High-resolution mass spectrometry (HR-MS) was
obtained on an Agilent 6224 TOF LC/MS instrument. UV–Vis and
fluorescence spectra were recorded on a Cary 100 UV–Vis spectro-
photometer and an Agilent Cary Eclipse fluorescence spectro-
photometer, respectively. Standard quartz cuvettes with a 10 mm
lightpath were used for all optical measurements. Cell imaging and
zebrafish imaging were performed in a Leica laser scanning confocal
microscope. Mice imaging were taken in a Bruker In Vivo-Xtreme
imaging system.
2.4. Imaging of ONOO− in living cells
Before cell imaging experiments, HeLa cells and RAW264.7 cells
were cultured in Dulbecco's Modified Eagle's Medium (DMEM) sup-
plemented with 10% FBS (Fetal Bovine Serum), 100 μg/mL penicillin
and 100 μg/mL streptomycin in a 5% CO2, water-saturated incubator at
37 °C [53]. The cytotoxicity of DCI-NPG-ONOO (0–50 μM) was eval-
uated by the standard MTT assays. In cell imaging experiments, part of
cells were incubated with probe DCI-NPG-ONOO (10 μM, with 1%
DMSO, v/v) at 37 °C for 30 min and then imaged as a control after
washing with PBS for three times. For imaging exogenous ONOO−
,
HeLa cells were incubated with DCI-NPG-ONOO (10 μM) for 30 min at
37 °C, and then incubated with ONOO− (10, 30 and 50 μM, respec-
tively) for 30 min at 37 °C. Cells were also incubated with probe DCI-
NPG-ONOO (10 μM) for 30 min, and then with ONOO− (50 μM) for
another 1, 2, 5, 10, 15, and 30 min, respectively. Imaging was then
taken after washing the cells with PBS buffer. For imaging endogenous
2.2. Synthesis of probe DCI-NPG-ONOO
Compound DCI-NIR-NH2 was first prepared according to the re-
ported method [60], and then it was used to synthesize probe DCI-NPG-
ONOO as follows.
A mixture of 4-nitrophenylglyoxylic acid (78 mg, 0.4 mmol), 2-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
ONOO−
, RAW264.7 cells were treated with interferon-γ (IFN-γ,
100 ng/mL) and lipopolysaccharide (LPS, 1 μg/mL) for 8 h. Meanwhile,
a control experiment was also performed by treating RAW264.7 cells
with IFN-γ (100 ng/mL), LPS (1 μg/mL), and minocycline (a scavenger
for ONOO−, 200 μM) for 8 h. After that, the two group of cells were
further incubated with DCI-NPG-ONOO (10 μM) for 30 min at 37 °C,
respectively. After washing the cells with PBS buffer, the cells were
imaged. For all of the fluorescence imaging, λex = 488 nm and emis-
sions were collected at 600–700 nm.
hexafluoropho-
sphate (HATU, 152 mg, 0.4 mmol), and triethylamine (40 μL) in 3 mL of
anhydrous dichloromethane was stirred for 30 min, and then DCI-NIR-
NH2 (58 mg, 0.2 mmol) was added under a nitrogen atmosphere. The
mixture was stirred at room temperature for 12 h. Then the solvent was
removed under vacuum, and the residue was purified by column
chromatography on silica gel with petroleum ether/dichloromethane
(1:10, v/v) to afford DCI-NPG-ONOO as a brick red solid (30 mg, yield
33%). Mp: 233–235 °C. 1H NMR (400 MHz, CDCl3) δ 9.14 (s, 1H), 8.67
(d, J = 8.7 Hz, 2H), 8.41 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 8.4 Hz, 2H),
7.63 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 16.1 Hz, 1H), 7.03 (d,
J = 16.2 Hz, 1H), 6.91 (s, 1H), 2.67 (s, 2H), 2.53 (s, 2H), 1.15 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 186.39, 169.26, 159.64, 154.01, 150.72,
138.33, 137.74, 136.30, 132.45, 132.04, 128.62, 128.35, 123.48,
123.31, 120.72, 113.48, 112.71, 78.15, 77.47, 42.90, 39.05, 31.94,
2.5. Imaging of ONOO− in zebrafish
The Larval zebrafish (4 days post fertilization) were purchased from
Zernike Company (Wuhan, China). Zebrafish were randomly divided
into five groups. The first group was the zebrafish blank. The second
group was incubated with DCI-NPG-ONOO (10 μM) for 15 min as the
probe blank. The third group was prestimulated with LPS (1 μg/mL)
and IFN-γ (50 ng/mL) for 30 min, and then incubated with DCI-NPG-
27.92. MS(EI): m/z found 466.30 (M+). HR-MS (ESI) calcd. for
+
C
27H22N4NaO4 (M + Na+) 489.1533, found 498.1547.
2

X. Yin, et al.
DyesandPigments172(2020)107820
ONOO (10 μM) for 15 min. The fourth group was prestimulated with
LPS (3 μg/mL) and IFN-γ (150 ng/mL) for 30 min, and then incubated
with DCI-NPG-ONOO (10 μM) for 15 min. The fifth group was presti-
mulated with LPS (3 μg/mL), IFN-γ (150 ng/mL) and uric acid (200 μM)
for 30 min, and then incubated with DCI-NPG-ONOO (10 μM) for
15 min. After that, all experimental samples were imaged after washing
three times with PBS buffer to remove the remaining medium. For
fluorescence imaging, λex = 488 nm and emissions were collected at
600–700 nm.
range (Fig. 1d). Obviously, all these results indicate that probe DCI-
NPG-ONOO can be used as an effective NIR fluorescent turn-on probe
for rapid detection of ONOO− in aqueous solution.
Under the above test conditions, DCI-NIR-NH2 also shows a strong
fluorescence (Ф = 0.156, using Rhodamine B as standard) around
660 nm (Fig. S1). Based on this and according to the previous reports of
ONOO− probes using α-ketoamide as the reaction site [42–45], the
sensing mechanism of probe DCI-NPG-ONOO for ONOO− should be a
process as shown in Scheme 1, and the formation of DCI-NIR-NH2
should be responsible for the fluorescence signal changes. To confirm
this, the reaction mixture of probe DCI-NPG-ONOO and ONOO− in PBS
buffer with 30% EtOH was analyzed by mass spectroscopy. As shown in
Fig. S2, the reaction produced two main peaks with mass of 288.15 and
166.11, which can be assigned to DCI-NIR-NH2 and 4-nitrobenzoate,
respectively, suggesting DCI-NIR-NH2 and 4-nitrobenzoate were
formed. An analysis of the reaction mixture with the most conventional
thin layer chromatography (TLC) also proved the formation of DCI-
NIR-NH2 (Fig. S3). Notably, DCI-NIR-NH2 possesses a remarkable large
Stokes shift of 200 nm (Fig. S1), indicating that the detection of
ONOO− with probe DCI-NPG-ONOO can be well protected from the
interference of excitation/scattering light. Thus, we established a new
probe for ONOO− with rapid response, turn-on NIR fluorescence
changes, and large Stokes shift at the same time. It should be noted that
such kind of probe for ONOO− is rarely reported in the literature (Table
S1).
2.6. Imaging of ONOO− in living mice
Kunming mice (weight 20
2 g) were purchased from the Center
for Disease Control and Prevention of Wuhan, China. The mice were
anesthetized by intraperitoneal (i.p.) injection of 1% pelltobarbitalum
natricum solution (0.01 mL/g) before imaging experiments. As controls,
one group of mouse was only given an i. p. injection of saline (200 μL),
and another group was given an i. p. injection of DCI-NPG-ONOO
(1 mM, 200 μL) in the peritoneal cavity, and after 5 min, the mice were
imaged. For imaging of ONOO−, the third group of mouse was given an
i. p. injection of DCI-NPG-ONOO (1 mM, 200 μL) and then ONOO−
(20 mM, 200 μL). The mouse was then imaged after 5 min. An excitation
filter of 460 nm and an emission filter of 700 nm were used for fluor-
escence imaging. All the animal experiments were approved by the
Animal Ethical Experimentation Committee of Wuhan University and
were carried out strictly in accordance with the requirements of the
National Act on the use of experimental animals (China).
3.3. The selectivity and sensitivity of DCI-NPG-ONOO
3. Results and discussion
With the above promising results, we next investigated the se-
lectivity of probe DCI-NPG-ONOO toward ONOO−. Thus, the fluores-
cence responses of the probe solution toward various biologically re-
levant analytes were examined under the same conditions. As shown in
Fig. 2, upon addition of various analytes including other ROS/RNS
3.1. Probe synthesis
Probe DCI-NPG-ONOO was prepared from the reaction of DCI-NIR-
NH2 and 4-nitrophenylglyoxylic acid in the presence of condensation
agent HATU (Scheme 2). DCI-NIR-NH2 is a dicyanoisophorone-based
fluorescent dye, which can be readily prepared according to the lit-
erature method. Probe DCI-NPG-ONOO was purified by silica gel
column chromatography and characterized by 1H NMR, 13C NMR, and
mass analyses (see Supplementary Data).
species (such as H2O2, ClO−, NO2−, BuOO•, HNO, NO, O2•−, OH•),
t
amino acids and biothiols (such as Tyr, Ala, Asp, Thr, Met, Ile, Phe, Cys,
Hcy, GSH), and anions and metal ions (such as HCO3−, HSO3−, HS−
,
Cl−, F−, I−, Na+, K+, Fe3+, Cu2+), the probe solution showed no
evident fluorescence enhancement, which is significantly different from
the fluorescent turn-on changes caused by ONOO−, indicating DCI-
NPG-ONOO is highly selective for ONOO−. Moreover, the results of
competition tests showed that the presence of these potential inter-
ference species did not interfere probe DCI-NPG-ONOO to detect
ONOO− (Fig. S4). All these results indicate that DCI-NPG-ONOO has
high selectivity and good anti-interference ability and therefore is
guaranteed for the detection of ONOO− in complicated systems.
The sensitivity of probe DCI-NPG-ONOO for ONOO− was also in-
vestigated. As shown in Fig. 3a, with increasing concentrations of
ONOO− added, the fluorescence of the DCI-NPG-ONOO (10 μM) so-
lution showed a progressive enhancement until a saturation occurred at
the addition of about 200 μM of ONOO−. A good linearity between the
fluorescent intensity at 660 nm and the concentration of ONOO− in the
range of 0–10 μM was observed (Fig. 3b). The limit of detection (LOD)
of DCI-NPG-ONOO for ONOO− was calculated to be about 81 nM
based on the method of LOD = 3δ/k. This indicates that probe DCI-
3.2. The response of DCI-NPG-ONOO for ONOO−
The response of probe DCI-NPG-ONOO for ONOO− was in-
vestigated in PBS buffer (10 mM, pH 7.4, with 30% EtOH, v/v) at 37 °C.
As shown in Fig. 1, the DCI-NPG-ONOO solution displays a maximum
absorption peak at 418 nm and is nearly non-fluorescent (Ф < 0.01,
using Rhodamine B as standard) due to the fluorescent quenching effect
of the α-ketoamide group [42–45]. However, upon addition of ONOO−
,
the absorption of the probe solution at 418 nm decreased, accompanied
by a significant fluorescence turn-on changes in the far-red to NIR re-
gion (centred at 660 nm, about 30-fold enhancement). Notably, when
ONOO− was tested in a concentration range of 10–200 μM, the fluor-
escent enhancement finished almost immediately (< 2 s) (Fig. 1c),
which is significant and highly favorable for detection of the transient
ONOO−. Moreover, probe DCI-NPG-ONOO itself is not pH sensitive,
but shows obvious fluorescence enhancement at 660 nm when the pH
is > 6, indicating that it can be used to detect ONOO− over a wide pH
NPG-ONOO has high sensitivity for ONOO−
.
Scheme 2. Synthetic route for DCI-NPG-ONOO.
3

X. Yin, et al.
DyesandPigments172(2020)107820
Fig. 1. (a) The UV–Vis and (b) fluorescence spectral responses of probe DCI-NPG-ONOO (10 μM) upon addition of ONOO− (200 μM). (c) Fluorescent reaction
kinetics of probe DCI-NPG-ONOO (10 μM) at 660 nm upon addition of ONOO− (10, 20, 50, and 200 μM). (d) Fluorescence intensity changes of probe DCI-NPG-
ONOO (10 μM) at 660 nm in the absence and presence of ONOO− (200 μM) at different pH values. All experiments were performed in PBS buffer (10 mM) with 30%
EtOH at 37 °C. For fluorescence measurement, λex = 460 nm, slit width: dex/dem = 10/10 nm.
3.4. Imaging of ONOO− with DCI-NPG-ONOO in living cells
When cells were incubated with probe DCI-NPG-ONOO (10 μM) for
30 min, and then with ONOO− (50 μM) for different time, a time-de-
pendent fluorescence increase was observed within 30 min (Fig. S6). All
these results clearly indicate that DCI-NPG-ONOO has the ability to
image exogenous ONOO− in living cells.
In order to explore the potential of DCI-NPG-ONOO for imaging
endogenously produced ONOO− in living cells, RAW264.7 cells were
stimulated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) ac-
cording to literature methods [55,56]. As shown in Fig. 5, when the
living RAW264.7 cells were only incubated with DCI-NPG-ONOO, al-
most no fluorescence can be observed (image A1). However, when cells
were pretreated with LPS and IFN-γ, and then incubated with DCI-NPG-
ONOO, an obviously enhanced red fluorescence can be observed
(images B1). In addition, when cells were pretreated IFN-γ and LPS in
the presence of minocycline (a known scavenger for ONOO−), and then
incubated with DCI-NPG-ONOO, the cells only showed very weak
Encouraged by the good sensing performance of DCI-NPG-ONOO in
solution, we next investigated its capability to image ONOO− in living
cells. The cytotoxicity of probe DCI-NPG-ONOO was first evaluated by
MTT assays, and the results showed both HeLa cells and
RAW264.7 cells exhibited high survival rate (> 90%) after incubation
with DCI-NPG-ONOO (0–50 μM) for 24 h. This indicates that 0–50 μM
of DCI-NPG-ONOO has very low cytotoxicity. Imaging ONOO− in
living cells with DCI-NPG-ONOO was then explored. As shown in
Fig. 4, when HeLa cells were incubated with DCI-NPG-ONOO (10 μM)
for 30 min, almost none fluorescence can be observed. However, when
the cells were incubated with probe DCI-NPG-ONOO and then exposed
to ONOO− (10, 30, and 50 μM, respectively), significant fluorescence
enhancement was observed inside the cells and the fluorescence dis-
played dose-dependent increases to the concentration of ONOO−
.
Fig. 2. (a) Fluorescent spectral changes of probe DCI-
NPG-ONOO (10 μM) upon addition of various ana-
lytes. (b) Fluorescence intensity changes of probe
DCI-NPG-ONOO (10 μM) at 660 nm toward various
analytes (200 μM of each unless otherwise stated)
including: (1) none, (2) H2O2, (3) ClO−, (4) NaNO2,
t
(5) BuOO•, (6) HNO, (7) NO, (8) O2•−, (9) OH•, (10)
Tyr, (11) Ala, (12) Asp, (13) Thr, (14) Met, (15) Ile,
(16) Phe, (17) Cys, (18) Hcy, (19) GSH (1 mM), (20)
NaHCO3, (21) NaHSO3, (22) NaHS, (23) NaCl, (24)
NaF, (25) NaI, (26) KCl, (27) FeCl3, (28) CuCl2, and
(29) ONOO−. All data were obtained in PBS buffer
(10 mM, pH 7.4, 30% EtOH, v/v) at 37 °C. Each data
was obtained 1 min after addition of the specific
analyte.
10 nm.
λex = 460 nm, slit width: dex/dem = 10/
4

X. Yin, et al.
DyesandPigments172(2020)107820
Fig. 3. (a) Fluorescent spectral changes of DCI-NPG-
ONOO (10 μM) upon addition of different con-
centrations of ONOO− in PBS buffer (10 mM, pH 7.4,
30% EtOH, v/v) at 37 °C.
dex = dem = 10 nm. Each spectrum was obtained
1 min after ONOO− addition. Inset:
saturation
λex = 460 nm,
a
curve of fluorescence intensity at 660 nm against
concentrations of ONOO−. (b) Linear relationship of
the fluorescence intensity changes at 660 nm against
the concentration of ONOO− from
0 to 10 μM
λ
ex = 460 nm, dex = dem = 20 nm.
Fig. 4. Confocal fluorescent imaging of exo-
genous ONOO− in HeLa cells. Top: bright field
images.
Middle:
fluorescent
images
(λex = 488 nm,
λ
em = 600–700 nm). Bottom:
merged images. (A, A1 and A2): Cells were in-
cubated with probe DCI-NPG-ONOO (10 μM)
for 30 min as control; (B, B1 and B2): Cells were
incubated with probe DCI-NPG-ONOO (10 μM)
for 30 min, and then with ONOO− (10 μM) for
another 30 min; (C, C1 and C2): Cells were in-
cubated with probe DCI-NPG-ONOO (10 μM)
for 30 min, and then with ONOO− (30 μM) for
another 30 min (D, D1 and D2): Cells were in-
cubated with probe DCI-NPG-ONOO (10 μM)
for 30 min, and then with ONOO− (50 μM) for
another 30 min. (E). The histogram shows the
quantitative analysis of the average fluorescence
in A1-D1, respectively.
ONOO in living zebrafish. As shown in Fig. 6, as blank controls, the
zebrafish itself and the zebrafish incubated with probe DCI-NPG-ONOO
(10 μM) were imaged and both of them showed very weak NIR fluor-
escence (images A1 and B1). However, when zebrafish was stimulated
by, followed by incubation with DCI-NPG-ONOO, the zebrafish ex-
hibited obviously enhanced fluorescence, and the fluorescence was
enhanced as the concentration of IFN-γ/LPS increased (Fig. 6, images
C1 and D1). In another control group, when zebrafish was pretreated
with IFN-γ/LPS in the presence of uric acid (a known scavenger for
ONOO−), and then incubated with DCI-NPG-ONOO, the zebrafish only
showed very weak fluorescence (image E1). These results indicate that
DCI-NPG-ONOO can be used as an effective tool for imaging en-
dogenous ONOO− in live zebrafish.
Fig. 5. Confocal fluorescent imaging of endogenous ONOO− in RAW
264.7 cells with DCI-NPG-ONOO (10 μ M). Top: bright field images. Middle:
3.6. Imaging of ONOO− in living mice
fluorescent images (λex = 488 nm,
λem = 600–700 nm). Bottom: merged
With the advantage of NIR fluorescence turn-on responses for
ONOO−, the potential of DCI-NPG-ONOO for in vivo detection of
ONOO− was further explored with living mice as model. As shown in
Fig. 7, when the mouse was given an intraperitoneal (i.p.) injection of
saline or DCI-NPG-ONOO as controls, the mouse only showed very
weak NIR fluorescence. However, when the mouse was given an in-
jection of DCI-NPG-ONOO and followed with ONOO−, the mouse
started to show strong NIR fluorescence. Obviously, this result indicates
images. (A, A1 and A2): cells were incubated with probe DCI-NPG-ONOO
(10 μM) for 30 min as control; (B, B1 and B2): cells were incubated with LPS
(1 μg/mL) and IFN-γ (100 ng/mL) for 8 h, and then with probe DCI-NPG-ONOO
(10 μM) for 30 min; (C, C1 and C2): cells were incubated with LPS (1 μg/mL),
IFN-γ (100 ng/mL) and minocycline (200 μM) for 8 h, and then with DCI-NPG-
ONOO (10 μM) for 30 min. (D) the histogram shows the quantitative analysis of
the average fluorescence in A1-C1, respectively.
that DCI-NPG-ONOO has potential for in vivo detection of ONOO−
.
fluorescence (image C1). These results demonstrated that DCI-NPG-
ONOO could be used to image endogenously produced ONOO− in
living cells.
4. Conclusions
3.5. Imaging of ONOO− in living zebrafish
In summary, we have developed a new NIR fluorescent probe (DCI-
NPG-ONOO) for detection of ONOO−. This probe uses a dicyanoiso-
phorone-based NIR fluorescent dye DCI-NIR-NH2 as the fluorophore
We also investigated the imaging of ONOO− with probe DCI-NPG-
5

X. Yin, et al.
DyesandPigments172(2020)107820
Fig. 6. Imaging of endogenous ONOO− in zebrafish
with probe DCI-NPG-ONOO. (A–E) bright field
images. (A1-E1) fluorescent images (λex = 488 nm,
λ
em = 600–700 nm). (A2-E2) merged images. (A, A1
and A2) zebrafish blank. (B, B1, B2) zebrafish was
incubated with DCI-NPG-ONOO (10 μM) for 15 min
as probe blank. (C, C1, C2) Zebrafish was stimulated
with LPS (1 μg/mL) and IFN-γ (50 ng/mL) for
30 min, and then stained with DCI-NPG-ONOO
(10 μM) for 15 min (D, D1, D2) Zebrafish was sti-
mulated with LPS (3 μg/mL) and IFN-γ (150 ng/mL)
for 30 min, and then incubated with DCI-NPG-
ONOO (10 μM) for 15 min (E, E1, E2) Zebrafish were
prestimulated with LPS (3 μg/mL), IFN-γ (150 ng/
mL) and uric acid (200 μM) for 30 min, and then
incubated with DCI-NPG-ONOO (10 μM) for 15 min.
(F) The histogram shows the quantitative analysis of
the average fluorescence in A1-E1, respectively.
Fig. 7. Fluorescent imaging of ONOO− in live mice
with probe DCI-NPG-ONOO. (A) As a blank, mouse
was only given an i. p. injection of 200 μL of saline in
the peritoneal cavity; (B) Mouse was given an i. p.
injection of DCI-NPG-ONOO (1 mM, 200 μL) as the
probe blank; (C) Mouse was given an i. p. injection of
DCI-NPG-ONOO (1 mM, 200 μL) and followed by i.
p. injection with ONOO− (20 mM, 200 μL); (D)
Quantification of fluorescent intensity from the ab-
dominal area of mice A-C. An excitation filter of
460 nm and an emission filter of 700 nm were used
for fluorescence imaging.
and α-ketoamide as the recognition unit. More importantly, this probe
displays excellent detecting performance for ONOO−, including rapid
response (< 2 s), high selectivity and sensitivity, large NIR fluorescence
enhancement with a remarkable large Stokes shift. Moreover, this probe
shows low cytotoxicity and excellent imaging ability for both exo-
genous and endogenous ONOO− in living cells, zebrafish and mice.
Taken together, this work provided a valuable new NIR fluorescent
turn-on probe for rapid detecting ONOO− in living systems.
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Acknowledgment
[13] Feng W, Li M, Sun Y, Feng G. Near-infrared fluorescent turn-on probe with a re-
markable large Stokes shift for imaging selenocysteine in living cells and animals.
Anal Chem 2017;89:6106–12.
Financial support from the National Natural Science Foundation of
China (21672080 and 21472066) and the Fundamental Research Funds
for the Central Universities (CCNU19TS007) is gratefully acknowl-
edged.
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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.2019.107820.
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