Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells

Article abstract of DOI:10.1016/j.cclet.2013.11.024

A new two-photon fluorescent probe, ADNO, for nitric oxide (NO) based on intramolecular photoinduced electron transfer (PET) mechanism displays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity

Full text of DOI:10.1016/j.cclet.2013.11.024

Chinese Chemical Letters 25 (2014) 19–23
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Highly selective two-photon fluorescent probe for imaging of nitric
oxide in living cells
a,b,
Qing Liu a,c, Lin Xue a, Dong-Jian Zhu a,c, Guo-Ping Li a,c, , Hua Jiang
*
*
a Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100190, China
b College of Chemistry, Beijing Normal University, Beijing 100875, China
c University of Chinese Academy of Sciences, Beijing 100049, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A new two-photon fluorescent probe, ADNO, for nitric oxide (NO) based on intramolecular photoinduced
electron transfer (PET) mechanism displays a rapid response to NO with a remarkable fluorescent
enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS (reactive
oxygen species or nitrogen species) and common metal ions was observed. Moreover, ADNO has been
successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy
(OPM) and two-photon microscopy (TPM).
Received 12 October 2013
Received in revised form 30 October 2013
Accepted 8 November 2013
Available online 20 November 2013
Keywords:
ß 2013 Guo-Ping Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Fluorescent probe
Nitric oxide
Fluorescence imaging
One-photon microscopy
Two-photon microscopy
1. Introduction
central tool for visualizing various ROS/RNS (reactive oxygen
species or nitrogen species) [3]. Compared with standard laser
Nitric oxide (NO), an important and ubiquitous endogenous
second messenger or cytostatic/cytotoxic agent, has attracted
more and more attention in recent years [1]. It is believed that NO
is, at low concentrations, essential for many normal physiological
processes and plays an important role in several physiological
functions, such as regulation of vascular tone, neurotransmission,
anti-viral defense, immune response and memory. Whereas
excessive generation of NO in physiological processes can cause
damage to biomolecules, such as DNA, proteins and lipids, and
consequently lead to cellular necrosis or apoptosis. Therefore,
sensitive and specific detection of NO in living systems is indeed
indispensable for understanding the biological roles of NO. The
development of techniques to analyze NO in organisms has
dramatically increased over the last decades [2], especially,
fluorescence microscopy, which features non-destruction, high
sensitivity and spatiotemporal resolution, has been proved to be a
confocal approaches which employ one photon of higher energy
for the excitation, two-photon microscopy of lower energy as the
excitation source has significant advantages by providing deeper
sectioning, less phototoxicity, and lower background fluorescence
[4]. Although a number of NO-specific fluorescent probes have
been reported to date [5,3b,c,e], two-photon fluorescent probes for
imaging of NO suitable for biological applications are still rare.
In this report, we designed and synthesized a new two-photon
fluorescent probe, ADNO (2-(a-(3,4-diaminophenoxy)acetyl)-6-
(dimethylamino)naphthalene), for monitoring NO based on
photoinduced electron transfer (PET) mechanism. As shown in
Scheme 1, the probe is composed of two moieties: o-phenylene-
diamine as the NO-sensitive fluorescence modulator and 2-acetyl-
6-(dimethylamino)naphthalene (Acedan) as the two-photon
fluorophore. We anticipated that detection of NO can be well
achieved by altering the electron-donating capacity of the o-
phenylenediamine moiety. The electron-rich o-phenylenediamine
group should quench the fluorescence of Acedan due to the
existence of electron transfer from electron-rich diamine moiety to
the excited fluorophore. However, the presence of NO under
aerobic conditions leads to the transformation of o-phenylene-
diamine to benzotriazole, which consequently suppresses the PET
process and revives the strong fluorescence response of the probe.
*
Corresponding authors at: Beijing National Laboratory for Molecular Sciences,
CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China.
E-mail addresses: liguoping08@iccas.ac.cn (G.-P. Li), hjiang@iccas.ac.cn
(H. Jiang).
1001-8417/$ – see front matter ß 2013 Guo-Ping Li. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.11.024

20
Q. Liu et al. / Chinese Chemical Letters 25 (2014) 19–23
PET
NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Gibco) supplemented with 10% fetal calf serum
(FCS, Gibco), 50 g/mL penicillin/streptomycin (Hyclone) at 37 8C
PET
m
O
O
H
N
in a humidified incubator containing 5% CO2 gas. The cells were
plated in a 35 mm glass-bottomed dish and cultured for 2 days
before dye loading. Then the cells were washed with phosphate-
buffered saline (PBS) and bathed in corresponding serum-free
O
O
NH2
NH2
NO
O2
N
N
N
N
ADNO
weak fluorecence
strong fluorecence
DMEM medium with 4
mmol/L ADNO for 20 min at 25 8C, washed
Scheme 1. Design strategy of probe ADNO.
with PBS three times to remove the excess probe and bathed in PBS
(2 mL) before imaging.
The synthetic procedures are outlined in Scheme 2. The
compounds 1 and 2 were synthesized according to previously
published methods [8]. Compound 3 can be easily obtained in a
good yield by coupling of the compound 2 with 4-amino-3-
nitrophenol under basic conditions. Then reduction of compound 3
with sodium dithionite gave the final product ADNO.
2. Experimental
All chemicals were purchased from Alfa Aesar or Sigma–Aldrich
and used as received. All solvents were purified and dried by
standard methods prior to use. Pure water (18.2
V) was used to
prepare all aqueous solutions. Various reactive oxygen species
were prepared according to the reported literature [6]. The 1H NMR
spectra were recorded on a Bruker AVANCE-400 spectrometer, and
13C NMR spectra were recorded on a Bruker AVANCE-600 MHz
spectrometer. All chemical shifts are reported in the standard
notation of parts per million using residual solvent protons as
internal standard. Mass spectra (ESI) were obtained on Bruker
Apex IV FTMS. UV–vis spectra were recorded on SHIMADZU UV-
2550 UV–vis spectrometer. Fluorescence spectra were recorded
using a HITACHI F-4600 spectrometer. OPM imaging experiments
were performed with an Olympus FV-1000 laser scanning
microscopy system, based on an IX81 (Olympus, Japan) inverted
microscope. TPM imaging experiments were performed with an
Olympus FV1000MPE two-photon excitation fluorescence micros-
copy system. The quantum yields for fluorescence were calculated
by comparison of the integrated area of the corrected emission
spectrum of the samples with that of a solution of quinine sulfate in
3. Results and discussion
To evaluate whether our probe can monitor NO, we first
examined the spectral properties of ADNO in PBS buffer (20%
DMSO was added as a co-solvent). As predicted, ADNO only
displayed weak fluorescence emission (
at 386 nm,
e
= 1.31 Â 104 L molÀ1 cm
F
0 = 0.01) owing to the PET process between o-
phenylenediamine group and fluorophore Acedan (Fig. 1a). Addi-
tion of NO (2 mmol/L saturated solution in oxygen-free water) to
ADNO in PBS buffer induced a rapid and remarkable enhancement
on fluorescence intensity (
e
= 1.79 Â 104 L molÀ1 cmÀ1 at 356 nm,
F
= 0.08, F/F0 = 8). Meanwhile, the absorption spectrum of ADNO
centered at 390 nm also blue-shifted to 358 nm (Fig. S1 in
Supporting information). It can be well explained that the
formation of benzotriazole (Fig. S2 in Supporting information)
mediated by NO under aerobic conditions has suppressed the PET
0.1 mol/L H2SO4
(
F
= 0.54) [7].
O
O
Br
Br2
4-amino-3-nitrophenol
HBr 45% in AcOH
N
N
K2CO3, acetone
2
1
O
O
O
NO2
O
NH2
NH2
Na2S2O4
THF/MeOH/H2O
N
NH2
N
ADNO
3
Scheme 2. Synthetic procedure for probe ADNO.
(b)
(a)
2400
2400
2000
1600
1200
800
ADNO
ADNO + NO
2000
1600
1200
800
400
0
+ SNOC
+ NO
400
0
450
500
550
600
650
700
450
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Fig. 1. (a) Fluorescence emission spectra of 4
mmol/L ADNO upon addition of 40
mmol/L NO. (b) Fluorescence emission spectra of 4
mmol/L ADNO upon addition of 100 mmol/L
SNOC changing with time (every 5 min). Spectra were acquired in PBS buffer (20% DMSO, pH 7.40),
l
ex = 405 nm, em = 546 nm.
l

Q. Liu et al. / Chinese Chemical Letters 25 (2014) 19–23
21
ADNO + NO (F )
2500
2000
1500
1000
500
0
2400
10
8
ADNO + NO (F)
ADNO
F / F
ADNO+ROS/RNS
2000
1600
1200
800
400
0
0
6
4
2
0
0
6
7
8
9
10
11
NO SNOC 1O2 O2 ·OH ClO H2O2 NO2 NO3
-
-
-
-
pH
Fig. 3. Fluorescence responses of ADNO (4
mmol/L) to NO (40 mmol/L) in PBS buffer
Fig. 2. Fluorescence responses of ADNO (4
(100 mol/L) and other ROS/RNS (200 mol/L). All the data were acquired 5 min
after addition of each ROS/RNS except for SNOC (1 h) in PBS buffer (20% DMSO, pH
7.40). ex = 405 nm, em = 546 nm.
mmol/L) to NO (40 mmol/L), SNOC
(20% DMSO, pH 7.40) at various pH values. F/F0 means the ratio ( ) between
fluorescence intensity of ADNO in the absence (&, F0) and presence of NO (*, F),
m
m
lex = 405 nm, lem = 546 nm.
l
l
Fig. 4. OPM images of NO in ADNO-labeled NIH 3T3 cells. (a) Bright-field image of cells stained with 4
of ADNO-labeled cells treated with 5 mmol/L SNOC for 15 min and (d) 30 min at 25 8C. Emission intensities were collected in an optical window 460–560 nm,
Scale bar: 30 m, intensity bar: 100–3000.
m
mol/L ADNO at 25 8C for 20 min. (b) OPM images of (a). (c) OPM images
lex = 405 nm,
m
process. Considering that NO has been discovered to circulate as an
S-nitroso adduct of serum albumin, [9] we then applied the probe
to monitor the process of S-nitrosocysteine (SNOC, a NO donor)
spontaneously releasing NO in aqueous solution (Fig. 1b). After
including singlet oxygen (1O2), superoxide (O2À), hydroxyl radical
(ꢀOH), hypochlorite (ClOÀ), hydrogen peroxide (H2O2), nitrite
(NO2À) and nitrate (NO3À), gave out no obvious changes in
fluorescence emission intensity. Common metal ions in organisms,
such as Na+, K+, Ca2+, Mg2+, Zn2+, Fe2+, Cu2+, Co2+, Ni2+, had no effect
on the fluorescence emission of ADNO as well (Fig. S3 in Supporting
information). Therefore, we conclude that the probe ADNO has
excellent chemoselectivity for NO over other competing ROS/RNS
and common metal ions in PBS buffer.
100
mmol/L SNOC was added, the fluorescence intensity increased
gradually and reached the same level as NO solution after 1 h.
These results clearly indicate that ADNO can rapidly respond to NO
and monitor the generation and accumulation of NO in aqueous
solution.
Next, we investigated the chemoselectivity of ADNO for NO by
fluorimetric titration of various ROS/RNS and common metal ions
in the organism. Only the NO solution and NO donor can induce
remarkable fluorescence response (Fig. 2). Other ROS/RNS,
In addition, we also assessed the NO-sensing ability of ADNO at
different pH values. As shown in Fig. 3, probe ADNO is stable and
shows weak fluorescence within a wide pH range from 6.5 to 10.5.
The change of pH did not obviously affect the fluorescence
Fig. 5. TPM images of NO in ADNO-labeled NIH 3T3 cells. (a) Bright-field image of cells stained with 4
of ADNO-labeled cells treated with 5 mmol/L SNOC for 15 min and (d) 30 min at 25 8C. Emission intensities were collected in an optical window 520–560 nm,
Scale bar: 30 m, intensity bar: 100–2000.
m
mol/L ADNO at 25 8C for 20 min. (b) TPM images of (a). (c) TPM images
lex = 820 nm,
m

22
Q. Liu et al. / Chinese Chemical Letters 25 (2014) 19–23
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pH > 7.5, the change of fluorescence intensity at pH 6.5–7.5 is still
adequate enough for the signal output in sensing NO. These
findings suggested that probe ADNO is suitable for the detection of
NO within biological pH range.
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incubated with 4
mmol/L of ADNO for 20 min at 25 8C and then
washed three times with PBS buffer to remove the remaining dyes.
The ADNO-labeled cells exhibit only faint fluorescence in the
optical window 460–560 nm by one-photon microscopy (OPM)
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and TPM.
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In summary, on the basis of PET mechanism, we present a new
two-photon fluorescent probe for detection of NO. The probe
ADNO can rapidly respond to NO with significant turn-on
fluorescence, high selectivity, and less pH-dependency. More-
over, we have successfully applied ADNO to monitor the
generation and accumulation of NO in living cells by both
OPM and TPM. We also expect that this new two-photon
fluorescent probe will be a practical tool for NO-related biological
research.
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We thank the National Natural Science Foundation of China
(Nos. 21102148 and 21125205), National Basic Research Program
of China (No. 2011CB935800), and the State Key Laboratory of Fine
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
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