Aromatic primary monoamine-based fast-response and highly specific fluorescent probes for imaging the biological signaling molecule nitric oxide in living cells and organisms

Article abstract of DOI:10.1039/c6tb03382b

Nitric oxide (NO) is an important cellular signaling molecule involved in many physiological and pathological processes. To probe its spatiotemporal information in biosystems, a large number of NO fluorescent probes have been exploited in the past ten yea

Full text of DOI:10.1039/c6tb03382b

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Jo uP rl ne aa sl eo fd oM na ot et r ai ad l jsu sCt hme am r gi si nt rs y B
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DOI: 10.1039/C6TB03382B
Journal Name
ARTICLE
Aromatic Primary Monoamine-Based Fast-Response and Highly
Specific Fluorescent Probes for Imaging Biological Signaling
Molecule Nitric Oxide in Living cells and Organisms
Received 00th January 20xx,
Accepted 00th January 20xx
a,†
a,†
a
b
a
a,
DOI: 10.1039/x0xx00000x
Yingying Huo, Junfeng Miao, Yaping Li, Yawei Shi, Heping Shi and Wei Guo *
www.rsc.org/
Nitric oxide (NO) is an important cellular signaling molecule involved in many physiological and pathological processes. To
probe its spatiotemporal information in biosystems, a large number of NO fluorescent probes have been exploited in the
past ten years. Among them, the o-phenylenediamine-based ones are the earliest developed and most versatile NO
fluorescent probes to date. Even so, there are still limitations that need to be solved, such as the relatively long response
time, possible interference by dehydroascorbic acid (DHA)/methylglyoxal (MGO), and pH-sensitivity of their fluorescence
signals. In this work, we present two aromatic primary monoamine-based NO fluorescent probes MA and NIR-MA by
exploring the reductive deamination reaction of the electron-rich p-methoxyaniline group with NO in aerobic condition.
The superiority of the two probes could be illustrated by their quick, stable, sensitive, and specific fluorescence offon
responses for NO over a series of biologically relevant interfering species, including reactive oxygen species, DHA/MGO,
biothiols, and metal ions. Coupled with the good cell permeability and low cytotoxicity, the two probes have successfully
been applied to image the endogenous NO in RAW264.7 macrophages stimulated by LPS/IFN-. Moreover, the
fluorescence response of NIR-MA for NO occurs in the physiologically favorable NIR region, enabling its fruther use to
image endogenous NO in an inflamed mouse model.
severely damage a wide variety of molecular targets, including
lipid, DNA, protein and enzyme, to elicit mitochondria
dysfunction and cell death. Therefore, real-time imaging of
NO generation in living cells or bodies is of particular
significance for understanding its diverse pathophysiological
roles.
Introduction
7
,8
Nitric oxide (NO), a ubiquitous messenger molecule in
biological systems, is endogenously produced from L-arginine
mainly by three NO synthases, i.e. endothelial NO synthase
(eNOS), neuronal NO synthase (nNOS), and inducible NO
1-3
synthase (iNOS). NO produced via eNOS and nNOS pathways
is in physiologic level (nM range), and plays various
physiological roles in human body, such as inhibition of
platelet aggregation and adhesion, blood vessel relaxation and
angiogenesis, and neurotransmission. However, upon induced
by tumor necrosis factor TNF-, lipopolysaccharide (LPS), and
high glucose levels, the high level of intracellular NO could be
produced via iNOS pathway, which completely transforms the
Among various cellular biology tools, fluorescence
techniques have become the gold standard for the detection
and imaging of various biological species in terms of their
convenience, sensitivity, visualization, and noninvasiveness.
Therefore, in the past ten years, a large number of NO
fluorescent probes have been exploited, which typically
9
-17
include o-phenylenediamine
and metal
ligand complexe
18-22
based ones.
Among them, the o-phenylenediamine-based
biological actions of NO either by its autoxidation with O to
2
ones are the earliest developed and most versatile to date,
some of which are commercially available. However, despite
great progress they have made, there are still limitations that
need to be addressed, such as the possible interference by

produce nitrosating agent N O or by its fast reaction with O
2
2
3

to produce extremely powerful oxidant peroxynitrite (ONOO ).
N O can directly damage DNA by deamination of DNA bases
2
3
2
3-26
following the nitrosation of their primary amine
dehydroascorbic acid (DHA) and methylglyoxal (MGO),
4
,5

9-12
functionalities. ONOO , despite its positive functions in
unfavorable pH-sensitivity of their fluorescence signals, and
relatively slow response rate. As such, in recent years, some
new strategies have been developed, such as the diazo ring
6
immune response and redox regulation of cell signal, could
27
28-
formation, monoprotection of o-phenylenediamine group,
35
36,37
38
reductive deamination,
formation of Se-NO bond, and
3
9
aromatization of Hantzsch ester. Due to avoiding the use of
o-phenylenediamine reaction group, these probes commonly
have improved selectivity and pH stability. However, the issue
on response rate has not been well-settled, and, in fact, most
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ARTICLE
Journal Name
of them have a relatively long response time (commonly more FV1000 confocal microscope, and emission wasVicewolAlerticctleeOdnlianet
than 5 min), which is disadvantageous for the real-time 645−780 nm (ex: 633 nm). The living aDnOimI: 1a0l.1i0m3a9/gCin6TgB0a3s3s8a2yBs
tracking of the transient small molecule in biosystems (half-life were performed in a Bruker In-Vivo FX Pro small animal optical
4
0
of NO: t1/2 = 0.15 s). In this work, we present two aromatic imaging system with an excitation filter 720 nm and an
primary monoamine-based NO fluorescent probes MA and emission filter 790 nm. The synthetic details for all compounds
NIR-MA by exploring the reductive deamination reaction of in this work were shown in ESI†
the electron-rich p-methoxyaniline group with NO in aerobic
Quantum yield determination
condition in combinaiton with the photoinduced electron
transfer (PeT) fluorescence switching mechanism (Scheme 1). Fluorescence quantum yields of 1a
e as well as the
The fluorescence response of two probes toward NO is fairly deamination product dA-1c dA-MA) were determined in PBS
(
fast, and could be completed within seconds. The commonly (50 mM, pH 7.4, containing 20% DMSO) with fluorescein (0.1
coexisting biological species, such as reactive oxygen species M NaOH, Φ = 0.95) as reference. The quantum yield was
F
(
ROS), DHA/MGO, biothiols, and metal ions, do not interfere calculated using Eq.1:
2
2
with their detection for NO. Moreover, the fluorescence of Φ = [(A FAu )/ (A FAs0 )]Φs. (Eq.1)
u
s
u
their deamination products is pH-stable. Coupled with the Where A and A are the absorbance of the reference and
s
u
good cell permeability and low cytotoxicity, the two probes sample solution at the reference excitation wavelength, FAs
have been successfully applied to image the endogenous NO in and FAu are the corresponding integrated fluorescence
the activated RAW264.7 macrophages. Notably, the intensity, and
 and 0 are the solvent refractive indexes of
fluorescence response of NIR-MA for NO occurs in the sample and reference, respectively. Absorbance of sample and
physiologically favorable NIR region, enabling its further use to reference at their respective excitation wavelengths was
image endogenous NO in an inflamed mouse model.
controlled to be lower than 0.05.
Preparation of the test solution
Deionized water and spectroscopic grade DMSO and CH CN
3
were used for spectroscopic studies. Probe MA or NIR-MA was
dissolved in DMSO or CH CN to obtain its stock solution. The
3
NO stock solution in deionized water was fleshly prepared by
bubbling NO gas into a NaOH solution to eliminate NO2
generated from the reaction of NO and O , and then into
2
deoxygenated deionized water for 30 min under N , and its
2
concentration (1.8 mM) was determined by Griess method.
For intracellular imaging assays, NOC-9 (a commercially

available NO donor) was used. ONOO was synthesized as
41
reported, and its concentration was estimated by using an
-
1
-1
Scheme 1 Chemical structures of fluorescent probes MA and NIR-MA as well as the
proposed fluorescence sensing mechanism of the two probes for NO.
extinction coefficient of 1670 M cm at 302 nm [CONOOˉ
=
Abs302 nm/1.67 (mM)]. For intracellular imaging assays, SIN-1 (a

commercially available ONOO donor) was used. Superoxide


Experimental Section
General information
solution (O
) was prepared by adding KO (1 mg) to dry
2 2
dimethyl sulfoxide (1 mL) and stirring vigorously for 10 min.

2+
Hydroxyl radical ( OH) was generated in situ by addition of Fe
based on Fenton reaction, and its
All reagents and solvents were purchased from commercial to 10 equiv of H O
2 2
2+
sources and were of the highest grade without further concentration is equal to the Fe concentration. Singlet
1

purification. Solvents were dried according to standard oxygen ( O
) was generated from ClO and H O . The source of
2
2 2
procedures. All reactions were magnetically stirred and NaClO was commercial bleach, and the concentration of the

monitored by thin-layer chromatography (TLC). Flash ClO stock solution was determined by using an extinction
-
1
-1
chromatography (FC) was performed using silica gel 60 (200– coefficient of 350 M cm at 292 nm. H
O
solution was
solution in deionized
000 spectrophotometer. Fluorescence spectra were taken on water, and its concentration was determined by using an
2
2
3
4
00 mesh). Absorption spectra were taken on Varian Cary prepared by dilution of commercial H O
2 2
1
13
-1
-1
Hitachi F-7000 fluorescence spectrometer. The H NMR and
NMR spectra were taken on a Bruker spectrometer, and solutions of NaNO
recorded at 600 and 150 MHz, respectively. High resolution source. The aqueous solutions of K , Ca , Na , Mg , Al , Zn ,
C
extinction coefficient of 43.6 M cm at 240 nm. The aqueous
−
was freshly prepared and used as NO
2
2
+
2+
+
2+
3+
2+
2+
3+
2+
mass spectra were obtained on a Varian QFT-ESI mass Fe , Fe , and Cu were prepared from their chloride salts.
spectrometer. The imaging assays for MA in cells were The aqueous solutions of Cys/Hcy/GSH and the DMSO
performed in an EVOS FL Auto imaging system, and the solutions of DHA/MGO were freshly prepared. For spectra
excitation (470/22 nm) and emission (510/42) bandpasses of studies, various analytes, except OH and O
the standard GFP filter set were used. The imaging assays for the solution of MA or NIR-MA (4.0 μM) in PBS (50 mM, pH 7.4,
NIR-MA in cells were performed in an Olympus FluoView™ containing 20% DMSO or 20% CH CN), respectively, and then

1
, were added to
2
3
2
| J. Name., 2012, 00, 1-3
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fluorescence spectra were recorded. As for OH and O , MA Cyotoxicity assays
ARTICLE

1
2
View Article Online
DOI: 10.1039/C6TB03382B
2+
(
or NIR-MA) and H O were first premixed, and then Fe or
2 2
HeLa Cells were seeded in 96-well microplates in DMEM
medium supplemented with 10 % FBS (Fetal Bovine Serum)
and 1% antibiotics at 37 ºC in humidified environment of 5%

ClO was added to the mixture.
Cell culture
CO . After 24 h of cell attachment, the plates were washed
2
The HeLa cells and RAW264.7 cells were kindly provided by
Key Laboratory of Chemical Biology and Molecular Engineering
of Ministry of Education (China). Cells were grown in
Dulbecco's modification of Eagle's medium (DMEM)
supplemented with 10 % FBS (Fetal Bovine Serum) and 1%
antibiotics at 37 ºC in humidified environment of 5% CO2.
Grow cells in the exponential phase of growth on glass-bottom
cell culture dishes (Φ 15 mm) for 1 day.
with PBS, followed by addition of increasing concentrations of
MA (120 M) or NIR-MA (120 M) in DMEM. The cells were
then incubated at 37 ºC in an atmosphere of 5% CO and 95%
2
air for 24 h, followed by standard MTT assays (n= 6). Untreated
assays (n = 6) were also conducted under the same conditions.
Results and Discussion
Design rationale and probe screening
Imaging exogenous NO in HeLa cells by MA
4
Inspired by the genotoxic effects of NO and taking into
Before the experiments, HeLa cells were washed with PBS 3
times. Then, the cells were pretreated with MA (2.0
min in DMEM medium at 37 °C . After washed three times with
PBS buffer, the MA-loaded cells were incubated with 10 equiv
of NOC-9 for 20 min in PBS at 37 °C . After washed with PBS 3
times, the fluorescence images were performed. For
account the reductive deamination reaction of aromatic

M) for 20
42,43
primary monoamines with NO,
Wang and coworkers, in
2
012, reported the first aromatic primary monoamine-based
36
NO fluorescent probe FA-OMe
.
Compared with the o-
phenylenediamine-based ones, the probe possesses only an
amino group and can not react with DHA to form quinoxaline
product, thus avoiding the interference of the species.
However, the response time of the probe for NO is relatively
intracellular selectivity evaluation, the MA (2.0
M)-loaded


cells were treated with 20 M of SIN-1, H O , ClO , and OH

2
2
2+
(
Fe +H O ), respectively, for 20 min in PBS, and after washed
2 2
long ( 20 min), which is disadvantageous for the real-time in
with PBS 3 times, the fluorescence images were performed.
vivo detection of NO. The concern prompted us to exploit the
new aromatic primary monoamine-based NO fluorescent
probes with improved response rate.
It is known that in o-phenylenediamine-based NO
fluorescent probes, the electron-rich o-phenylenediamine
Imaging endogenous NO in Raw 264.7 macrophage cells by
MA or NIR-MA
Before the experiments, the cells were washed with PBS 3
times. The cells were then pretreated with 20
50 units/mL INF- in DMEM medium at 37 C. After an
indicated time, the cells were washed 3 times with PBS, and
then incubated with MA (2.0 M) or NIR-MA (2.0 M) for 20
g/mL LPS and
groups are more efficient than the electron-deficient ones in
term of their higher reactivity towards NO.
o
1

12,13
We speculated
that the same principle should also be applicable for aromatic
primary monoamine-based ones. Thus, the slow response rate
of FA-OMe for NO is probably due to its electron-deficient
methyl m-aminobenzoate reaction group. With the
consideration in mind, as shown in Scheme 2, we designed and
synthesized a new series of potential NO fluorescent probes


min in DMEM medium. After washed with PBS 3 times, the
fluorescence images were performed. For inhibition assay, the
cells were pretreated with 20
and 0.5 mM AG (or 10 M L-NAME) in DMEM medium at 37 C
for 12 h, and then incubated with MA (2.0 M) or NIR-MA (2.0
M) for 20 min. After washed with PBS 3 times, the
fluorescence images were performed.
g/mL LPS, 150 units/mL INF-,
o


1
a

e
that contain more electron-rich aniline, o-methoxyaniline,
p-methoxyaniline, o-hydroxyaniline, and p-hydroxyaniline as
potential NO reaction groups, respectively, and
boron dipyrromethene (BODIPY) dye as fluorescence reporter.


Imaging exogenous and endogenous NO in inflamed mouse
models by NIR-MA
These probes were expected to inherit the advantage of the
high selectivity of FA-OMe, but to be more reactive for NO due
to the improved nucleophilicity of these electron-rich aromatic
primary monoamine groups. The detailed synthetic procedures
The mice were used according to the guidelines approved by
the Institutional Animal Care and Use Committee of Shanxi
University. A Bruker In-Vivo FX Pro small animal optical
imaging system with an excitation filter 720 nm and an
emission filter 790 nm was used for the living animal imaging
assays. For evaluating the selectivity of NIR-MA, the mice were
and characterization data of 1ae were shown in ESI†.
first i.p. injected with NIR-MA (10

M, 100
L) for 30 min, and

then i.p. injected with 100 L (100

M) H O , ClO , and NOC-9
2
2
for 30 min, respectively. Then, the mice were anesthetized and
applied to imaging system. For imaging endogenous NO, the
mouse was first i.p. injected with LPS (1 mg/ml, 100
L) for 24
h, and then i.p. injected with NIR-MA (10 M, 100 L) for 30 Scheme 2 Design of a series of aromatic primary monoamine-based potential NO

min. Then, the mouse was anesthetized and applied to the fluorescent probes 1ae for screening purpose.
imaging system.
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Fluorescence sensing performances of 1a
Journal Name
37

e
toward NO fluorescence off

on response for NO. In fact, the pyrylium
View Article Online
DOI: 10.1039/C6TB03382B
were subsequently evaluated in PBS (100 mM, pH 7.4, salt is not so sensitive for NO, because a high concentration of
o
containing 20% DMSO) at 25 C. As expected, all these NO solution (up to 1000

M) was needed to reach spectra
10 saturation, and when the probe was treated with 70 M NO,
9.610 , 6.310 , and 9.510 for 1ae, respectively) likely only a small fluorescence enhancement was observed. Thus, o-
-
3
-
compounds were almost nonfluorescent (
,

=2.6

10 , 6.4


4
-4
-4
-4
due to the PeT quenching process from the electron-rich or p-hydroxyaniline group, although electron-rich, seems
aniline groups to the excited Bodipy fluorophore. Upon treated unfavourable to the reductive deamination reaction. Further,
with NO, 1a and 1b only showed a moderate increase in we tested the products of 1d and 1e treated with excessive NO
fluorescence intensity (Figs. 1A,B); by contrast, 1c gave a large solution by TLC analysis, and compared the results with that of
fluorescence enhancement of 155-fold (Fig. 1C), suggesting 1c. As shown in Fig. S1 (ESI†), the reaction of 1d with NO
that the electron-rich and less steric p-methoxyaniline group is resulted in a small amount of the unknown poorly fluorescent
more efficient than aniline or 2-methoxyaniline group. Notably, products, and most of it remained unreacted; by contrast, 1e
the fluorescence responses of 1ac for NO all could be was completely consumed by NO to give rise to the
completed within 1 min (Fig. 1F), revealing that these electron- nonfluorescent and highly polar products that are difficult to
rich aniline groups are more reactive than the electron- be separated; to our delight, the reaction of 1c with NO
3
6
deficient methyl m-aminobenzoate group in FA-OMe
.
generated a highly fluorescent product with negligible by-
Moreover, among the three compounds, 1c displayed the product. Obviously, these results are consistent with those
fastest fluorescence response for NO (within seconds), thus shown in Fig. 1. Even so, the detailed reason why o-
holding the large potential for real-time imaging of NO in hydroxyaniline group shows the poor reactivity for NO is still
biosystems.
unclear currently, and studies are underway in our lab. Taken
together, the above studies establish that 1c is the most
promising candidate to serve as a fast-response fluorescent
probe for NO.
To confirm that the fluorescence off-on response of 1c for
NO is indeed due to the NO-triggered deamination reaction,
we performed HPLC-MS assay on the reaction of 1c with NO,
and observed a main new peak, which could tentatively be
assigned to the deamination product dA-1c based on MS data
(m/z: calcd 355.1788; found 355.1791) (Fig. S2, ESI†). Further,
by bubbling NO gas to THF solution of 1c, a highly fluorescent
product was isolated with a high yield of 95%, which was
1
unambiguously identified to be dA-1c
(

: 0.76) based on H
13
NMR, C NMR, and HRMS data (ESI†). Moreover, the
absorption and fluorescence spectra of dA-1c matched well
with those of 1c treated with NO (Fig. S3, ESI†). Based on the
results, the possible reaction mechanism of 1c with NO in
aerobic condition was proposed as follows (Scheme 3): first,
+
the p-methoxyaniline group of 1c reacts with NO (formed
from NO and O ) to produce a diazonium salt intermediate;
2
the intermediate then accepts an electron from NO to give the
phenyl radical; finally, the phenyl radical abstracts a hydrogen
atom from solvent to give dA-1c
.
Fig. 1 (AE) Fluorescence spectra of 1ae (4.0 M) in the absence and presence of 10
equiv of NO. (F) Time-dependent fluorescence intensity changes of 1ae upon treated
by 10 equiv of NO. Conditions: PBS buffer (50 mM, pH 7.4, containing 20% DMSO); ex
=
o
475 nm; em = 519 nm; Slits: 5/5 nm; voltage: 650 V; T = 25 C.
However, with the same treatment, only a small
fluorescence response was observed for 1d, and almost no
fluorescence response for 1e (Figs. 1D,E). The result is
seemingly contradictory to a recent report, where an o-
hydroxyaniline-containing pyrylium salt showed the
Scheme 3 Proposed reaction mechanism of 1c with NO in aerobic condition.
4
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ARTICLE
To understand the fluorescence switching mechanism, we
performed the density functional theory (DFT) calculations on
both 1c and its deamination product dA-1c at the B3LYP/6-
View Article Online
DOI: 10.1039/C6TB03382B
3
1+G(d,p) level of Gaussian 09. As shown in Fig. S4 (ESI†), no
fluorescence of 1c could be rationalized by the higher HOMO
level of p-methoxyaniline group (PeT donor) than that of
BODIPY fluorophore (PeT acceptor), which promotes the
electron transfer from the former to the latter upon excitation;
the high fluorescence of dA-1c could be clarified by the lower
Fig. 2 (A) Fluorescence intensities of MA (4 M) treated various ROS (40 M),
HOMO level of the mothoxyphenyl group than that of BODIPY DHA/MGO (1 mM), Cys/Hcy (100 M), GSH (1 mM), and metal ions (100 M, 10 min)
fluorophore, which blocks the PeT quenching process and (black bar), respectively, and then treated with NO (40 M, 10 min) (red bar). (B)
Fluorescence spectra of MA (4 μM) treated with the varied concentrations of NO (0 –
restores the fluorescence of dA-1c
.
100 M). (C) Plots of the fluorescence intensities of MA at 519 nm vs NO
concentrations (0 – 16 M). Conditions: PBS buffer (50 mM, pH 7.4, containing 20%
Selectivity and sensitivity of 1c, named “MA” afterwards,
towards NO
o
DMSO); ex = 475 nm; em = 519 nm; Slits: 5/5 nm; voltage: 650 V; T = 25 C.
Subsequently, we evaluated the selectivity of MA for NO by
testing its fluorescence responses toward various biologically
relevant species, including reactive oxygen species (ROS),
DHA/MGO, biothiols, and metal ions. The obtained results
were summarized in Fig. 2A (details in Fig. S5, ESI†). From
these results, it could be concluded that the probe is (i) robust
To evaluate the sensitivity, the fluorescence titration assay
of MA for NO was performed. As shown in Fig. 2B, a dose-
dependent fluorescence intensity increase of MA for NO was
observed, which reached saturation when 20 equiv of NO was
used. In this case, an excellent linear correlation (R = 0.999)
between the fluorescence intensities of MA and NO
2

because it could resist oxidation by other ROS including ClO ,
concentrations from 0 to 16
M was achieved (Fig. 2C), and



1


H O , O , OH, O , NO , ONOO , (ii) more selective than the
2
2
2
2
2
the detection limit was estimated to be 19 nM based on S/N =
typical o-phenylenediamine-based NO fluorescent probes
because it could avoid the interference of DHA/MGO, and (iii)
highly reactive for NO because it could outcompete biothiols,
3
1
. Moreover, MA was poorly fluorescent in the pH range of 6–
2, and displayed the dramatic fluorescence off-on responses
for NO in the pH range of 6–9 (Fig. S7A, ESI†), indicating its
suitability for imaging applications in physiological condition.
Notably, unlike the triazole products of the o-
phenylenediamine-based NO probes, the deamination product
44
such as GSH, to react with NO. Moreover, the biologically
+
2+
+
2+
3+
2+
relevant metal ions, including K , Ca , Na , Mg , Al
，Zn ,
2+
3+
2+
Fe , Fe , and Cu , neither elicited the fluorescence
enhancement of MA, nor interfered with its detection for NO.
In addition, we also tested the fluorescence response of MA
towards OH and ClO in PBS with CH CN as cosolvent instead
of DMSO, because DMSO is known to be an effective OH Imaging exogenous and endogenous NO in living cells using
dA-MA
(dA-1c) showed stable fluorescence in a wide pH range
of 2 12 (Fig. S7B, ESI†).



3

4
5

46,47
scavenger and also highly reactive to ClO .
As shown in MA


Fig. S6 (ESI†), both OH and ClO was still incapable of eliciting
any measurable fluorescence change of MA in the condition,
and even in the presence of the two species, NO still worked
well; moreover, when other ROS were tested in the system,
similar results were also observed. Thus, the selectivity of MA
for NO is considerably high.
First, we tested the cytotoxicity of MA by MTT assays. As
shown in Fig. S8A (ESI†), when the cells were treated with MA
(120 M) for 24 h, the high cell viability ( 95%) was
observed for all cases, indicating that MA has fairly low
cytotoxicity. Subsequently, we evaluated the specificity of MA
for NO in cell environment. As shown in Fig. 3, when HeLa cells
were treated with MA, almost no fluorescence was found;
when MA-loaded HeLa cells were treated with NOC-9 (a
48
commercially available NO donor), a strong fluorescence was
clearly observed in green channel, indicating that MA is cell
membrane-permeable and could image exogenous NO in cell
environment; when MA-loaded HeLa cells were treated with
several representative ROS, such as SIN-1 (a commercially
available ONOO donor), H O , and ClO , respectively,
49

2
2
almost no fluorescence was observed for all cases, confirming
that MA still possesses the high specificity for NO in cell
environment.
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J. Name., 2013, 00, 1-3 | 5
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Jo uP rl ne aa sl eo fd oM na ot et r ai ad l jsu sCt hme am r gi si nt rs y B
Page 6 of 9
ARTICLE
Journal Name
The fluorescence sensing performances of NIR-MA toward
View Article Online
DOI: 10.1039/C6TB03382B
NO were evaluated in PBS buffer (50 mM, pH 7.4, containing
o
2
0% CH CN) at 25 C. As shown in Fig. 5A, NIR-MA showed a
3
poor fluorescence at 794 nm in the solution; upon treated with
NO, a dramatic fluorescence off-on response was immediately
observed, indicating that the p-methoxyaniline reaction group
and PeT switching mechanism are still efficient in the probe.

When NIR-MA was treated with other ROS, such as ClO , H O ,
2
2



1


Fig. 3 Fluorescence images of MA-loaded HeLa cells treated with various ROS. (A) Cells
O , OH, O , NO , and ONOO , as well as DHA/MGO, almost
2 2 2
treated with MA (2.0 M, 20 min) only; (BF) Cells pre-treated with MA (2.0 M, 20 no fluorescence enhancement was observed (Fig. 5B),
min), and then treated with NOC-9 (20 M, 20 min), SIN-1 (20 M, 20 min), H
2
O
2
(20
confirming that the probe still has good selectivity for NO.
Further, the titration experiment revealed a dose-dependent
fluorescence intensity increase (Fig. 5C), which reached
saturation when 10 equiv of NO was used. In the case, a good
M, 20 min), ClO (20 M, 20 min), and OH (20 M Fe + 200 M H

2+
O , 20 min),
2 2

respectively. Scale bar: 45 m.
Given that RAW 264.7 macrophages can produce the high
concentration of NO upon stimulation with interferon- (IFN-
we further tested the ability
of MA to image endogenous NO in the cells. As shown in Fig.
A, when the cells were stimulated with LPS/IFN- for different
2
linear correlation (R = 0.996) between the fluorescence

)
intensities and NO concentrations from 0 to 25
achieved (Fig. 5D), and the detection limit was estimated to be
5 nM based on S/N = 3. In addition, NIR-MA showed the best
M was
5
0,51
and lipopolysaccharide (LPS),
9
4

fluorescence off-on response for NO at pH = 7.4 (Fig. S9, ESI†),
thus suitable for applications in physiological condition.
durations (2 h, 4 h, 8 h, and 12 h), respectively, and then
incubated with MA for 20 min, a time-dependent fluorescence
increase was observed with detectable fluorescence signal at
the time point of 2 h and maximal signal at 12 h. When
RAW264.7 cells were pretreated with IFN-
presence of aminoguanidine (AG), a NO synthase inhibitor,
/LPS in the
52
for 12 h, and then treated with MA for 20 min, almost no
cellular fluorescence was observed (Fig. 4B). Thus, the
intracellular green fluorescence indeed originates from the
reaction of MA with the endogenous NO in the cells.
Fig. 4 (A) Fluorescence images of RAW264.7 cells pretreated with LPS (20 g/mL)/INF-
Fig. 7 (A) The time course of fluorescence intensity of NIR-MA (4 M) at 794 nm upon
treated with NO (40 M). (B) Fluorescence intensities of NIR-MA (4 M) treated with
(
150 units/mL) for 2 h, 4 h, 8 h, and 12 h, respectively, and then treated with MA (2.0
μM, 20 min). (B) Fluorescence images of RAW264.7 cells pretreated with LPS (20
g/mL)/INF- (150 units/mL)/AG (0.5 mM) for 12 h, and then treated with MA (2.0 μM,
0 min). Scale bar: 45 m.
40 M NO, 100 M other ROS, and 1 mM DHA and MGO. (C) Fluorescence spectra of

NIR-MA (4 μM) treated with the varied concentrations of NO (0 – 70 M). (D) Plots of
the fluorescence intensities of NIR-MA at 794 nm vs NO concentrations (0 – 25 M).
2
Conditions: PBS buffer (50 mM, pH 7.4, containing 20% CH
Aromatic primary monoamine based near-infrared (NIR) nm; Slits: 5/10 nm; voltage: 650 V; T = 25 C.
3
CN); ex = 750 nm; em = 794
o
fluorescent probe NIR-MA for NO
Subsequently, the ability of NIR-MA to image endogenous
NO in RAW 264.7 macrophages was tested. As shown in Fig. 6,
when the cells were incubated with NIR-MA, only a negligible
fluorescence was observed; when the cells were stimulated
Fluorescent dyes that have excitation and emission
wavelengths in NIR region (650−900 nm) are especially
favorable for tissue and living animal imaging due to the
improved tissue penetration depth, minimum photodamage,
and reduced background autofluorescence of biomolecules.
However, as far as we know, only a few far-red or NIR NO
with LPS/IFN-
obvious intracellular fluorescence was observed; when the
cells were pretreated with LPS/INF- in the presence of NO
 for 12 h and then treated with NIR-MA, an
16,53-56

fluorescent probes were reported to date.
Therefore, we
synthase inhibitors L-NAME or AG, and then treated with NIR-
MA, almost no intracellular fluorescence was found. Thus, NIR-
MA could be used to trace the endogenous NO in cell
environment. Moreover, similar to that of MA, the cytotoxicity
further developed a NIR NO fluorescent probe NIR-MA by
attaching the p-methoxyaniline reaction group to the widely
used NIR cyanine dye (Scheme 1). The synthetic procedures
and characterization data of NIR-MA were shown in ESI†.
6
| J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Page 7 of 9
Jo uP rl ne aa sl eo fd oM na ot et r ai ad l jsu sCt hme am r gi si nt rs y B
Journal Name
ARTICLE
of NIR-MA was considerably low, as indicated by MTT assays Conclusions
Fig. S8B, ESI†).
View Article Online
DOI: 10.1039/C6TB03382B
(
In summary, we have designed and synthesized two new NO
fluorescent probes, MA and NIR-MA, based on the reductive
deamination reaction of the electron-rich p-methoxyaniline
group with NO in aerobic condition. Compared with the widely
used o-phenylenediamine-based NO fluorescent probes, the
two probes possess two obvious advantages, i.e. fast response
rate and high selectivity. Their practicability has been
demonstrated by the real-time imaging of the endogenous NO
in RAW264.7 macrophages stimulated by LPS/IFN-
. Moreover,
NIR-MA could operate in the physiologically favorable NIR
Fig. 6 The imaging of endogenous NO in RAW 264.7 macrophages. (A) Blank; (B) NIR-
MA (2 M) only; (C) Stimulated by LPS (20 g/mL)/INF- (150 units/mL) for 12 h, and region, allowing its further application to image NO in an
then treated with NIR-MA (2.0 M, 20 min); (D, E) Stimulated by LPS (20 g/mL)/INF- inflamed mouse model. It is expected that the p-
(
150 units/mL) in the presence of NO synthase inhibitors L-NAME (10 M) or AG (0.5
methoxyaniline group reported here could be utilized to
develop various practical NO fluorescent probes in the future.
mM) for 12 h, and then treated with NIR-MA (2.0 M, 20 min). Scale bar: 20 m.
Given that NIR-MA could operate in the NIR region, we
further investigated its potential to image exogenous and Acknowledgements
endogenous NO in living nude mice. The images were obtained
using a Bruker In-Vivo FX Pro small animal optical imaging
system with an excitation filter 720 nm and an emission filter
We acknowledge the Natural Science Foundation of China (Nos.
1572121, 21302114, and 21502108) for support of this work.
2
7
90 nm. As shown in Fig. 7, when the mouse was
intraperitoneally (i.p.) injected with NIR-MA for 30 min, almost
Notes and references
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Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C6TB03382B
A table of contents entry
Aromatic Primary Monoamine-Based Fast-Response and Highly Specific
Fluorescent Probes for Imaging Biological Signaling Molecule Nitric Oxide in
Living cells and Organisms
Yingying Huo, Junfeng Miao, Yaping Li, Yawei Shi, Heping Shi and Wei Guo*
Two fast-response and highly specific NO fluorescent probes were developed based on the reductive
deamination reaction of p-methoxyaniline with NO in areobic condition.