A dual-emission and large Stokes shift fluorescence probe for real-time discrimination of ROS/RNS and imaging in live cells

Article abstract of DOI:10.1039/c3cc38471c

A novel dual-emission fluorescence probe has been developed for specific and sensitive detection of hypochlorite (ClO^-). Upon addition of ClO^-, significant changes in fluorescence emission intensity at two discrete wavelengths were observed. Meanwhile OONO^- led to only a single-channel fluorescence enhancement. This feature makes it a clear advantage in distinguishing ClO^-, RNS from other ROS.

Full text of DOI:10.1039/c3cc38471c

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Dual-Emission and Large Stokes Shift Fluorescence Probe for Real-time
Discrimination of ROS/RNS and Its Imaging in Living Cells
Ting Guo, Lei Cui, Jiaoning Shen, Rui Wang, Weiping Zhu, Yufang Xu* and Xuhong Qian*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
been developed for ClOꢀ. However, the poor water solubility of
A novel dual-emission fluorescence probe has been developed
50 the probe may limit their use in some biological applications.13 In
this work, we described a novel dual channel fluorescence probe
HA, which exhibits a good aqueous solubility, quantitative
detection of ClOꢀ from other ROS/RNS and the potentials for
living cell imaging.
for specific and sensitive detection of hypochlorite (ClO-).
Upon addition of ClO-, significant changes of fluorescence
emission intensity at two discrete wavelengths were observed.
10 Meanwhile OONO- only led to a single-channel fluorescence
enhancement. This feature makes it a clear advantage in
distinguishing ClO-, RNS between other ROS.
55 HA is constructed by tethering 4ꢀaminophenoxyl to 1,8ꢀ
naphthalimide via carbamate linkage (Scheme 1). This is because
4ꢀaminophenyl ether has been proved to be a viable substrate for
Reactive oxygen species (ROS) are produced mainly through
electron transfer reaction from oxygen, which play crucial roles
15 in many physiological processes and are also implicated in many
human diseases, including cancer, neurodegenerative disorders,
and inflammatory processes.1 Reactive nitrogen species (RNS)
are another group of reactive chemical species, which also play
key roles in cell signalling during many physiological processes.2
20 The more powerful oxidant peroxynitrite (ONOOꢀ), formed by
nitric oxide (NO) and superoxide (O2•ꢀ) in cells, is implicated
with various human diseases, such as inflammatory,
cardiovascular disease, neurodegeneration and cancer.3
oxidant detection and 1,8ꢀnaphthalimide is
a bright and
photostable fluorophore with a large Stokes shift.14, 15 The probe
60 HA was easily prepared from 3ꢀaminoꢀ1,8ꢀnaphthalimide HA-3
in oneꢀpot. HA-3 was first stirred with 4ꢀnitrophenyl
chloroformate in anhydrous acetonitrile for 12 hours prior to
Pd/C catalyzed hydrogenation to yield HA. The structure was
unambiguously confirmed by NMR and HRMS (Scheme S1,
65 ESI†). The probe HA exhibits good aqueous solubility. This is a
direct benefit of the incorporation of an ethylene glycol moiety.
Probe HA displays a maximum absorption at 340 nm and is
essentially nonꢀfluorescent (Fig.S1, ESI†). This is expected since
4ꢀaminophenol moiety is electronꢀrich and can quench the
70 fluorescence via the photoinduced electron transfer (PeT)
mechanism. The fluorescence response of HA to different pH
was investigated (Fig.S2, ESI†). HCl was slowly introduced into
a solution of HA at pH 11.0 and the fluorescence of the solution
was monitored with excitation at 340 nm. The fluorescence of the
Hypochlorite anion (ClOꢀ) is an important ROS converted from
25 H2O2 and chloride catalyzed by the enzyme myeloperoxidase
(MPO) in phagosomes.4 The highly active hypochlorite can
mediate the chemical modification of various biomolecules such
as proteins, lipids, DNA and RNA.5 Hypochlorite plays critical
roles in hosting innate immunity during microbial invasion.6
30 However, disorder of MPO or other reasons could cause
uncontrollable production of hypochlorite, which in turn leads to
a
variety of diseases including cardiovascular diseases,
neurondegeneration and osteoarthritis.7 Therefore, it is of great
importance to develop an effective method to detect ClOꢀ for
35 understanding the role of hypochlorite in immunization and
diseases.
Fluorescence probes with high sensitivity and spatial and
temporal resolutions have become
a powerful tool for
noninvasive and realꢀtime monitoring of bioꢀrelevant species in
40 living systems.8 Recently, several small fluorescence probes have
been reported for ClOꢀ. These probes showed excellent selectivity
for ClOꢀ over other ROS/RNS, whereas most of them displayed
fluorescent intensity fluctuation at a single channel.9,10,11 Dual
emission fluorescence probes enable a selfꢀcalibration for other
45 actors, including unstable excitation source, photobleaching of
fluorophores or uneven probe loading. Thus, this kind of
fluorescence probes has potential application in quantitative
measurement.12 Several ratiometric fluorescence probes have
75
Scheme 1 Proposed detection mechanism of HA
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60
54
44
36
28
20
16
12
8
µM
-
ClO
250
200
150
100
50
300
250
200
150
100
50
a)
µM
a)
-
ONOO
µM
H
.
O
2
µM
2
µM
µM
µM
µM
µM
µM
µM
OH
-
O
2
ROO?
blank
4
2
0
0
400
0
400
450
500
550
600
650
450
500
550
600
650
Wavelength / nm
Wavelength / nm
b)
180
160
140
120
100
80
466 nm
570 nm
Y = 5.8343+4.9981X
R2 = 0.99162
60
Y = 3.2767+2.4659X
2
40
R
= 0.99957
20
0
0
5
10 15 20 25 30 35 40
[OCl-] /
µ
M
Fig. 2 (a) Fluorescence changes of probe HA (20 ꢁM) in response of
45 various ROS. ClOꢀ (60 ꢁM), ONOOꢀ (60 ꢁM), H2O2 (1 mM), •OH (300
ꢁM Ferrous perchlorate and 1 mM H2O2), O2•ꢂ (KO2, 200 ꢁM), ROO•
(200 ꢁM tertꢀButyl hydroperoxide). The fluorescent assays of probe HA
for various ROS were carried out in phosphate buffer (0.1 M, pH 7.4,
with 1% DMF) at room temperature. (b) Emission intensity ratio (I570/I0)
50 of probe HA with various ROS, measured at 570 nm with excitation at
340 nm.
Fig. 1 (a) The emission spectra of probe HA (20 ꢁM) upon addition of
hypochlorite anion (0 to 3 equiv) in 0.1 M phosphate buffer (pH 7.4, with
1% DMF). (b) A liner calibration graph of the response to ClOꢀ
concentration. Excitation wavelength was 340 nm.
5
solution remained stable until the pH reaches 6.5, from which
point the emission intensity at 460 nm started to rise steadily.
10 When the solution went near or below the pKa of amine, more
and more NH2 got protonated. This prohibits the PET process and
restores the fluorescence. The pKa of HA was determined to be
5.38, which is 2.0 pH unit lower than the physiological pH. This
verifies that the probe can be used in biological system.
at 340 nm. Compared with the fluorophore HA-3, the acylation of
3ꢀamino group makes it a weaker electron donating group, which
explains the hypochromatic shift of emission maximum to 460
55 nm. Second, variously acylated 3ꢀaminonaphthalimides display
emission maximum at 460 nm in the literatures.15 Compound CA
from acetylation of the fluorophore HA-3 was synthesized and
also displayed an emission band at 460 nm when excited at 340
nm (Scheme S2, Fig.S1 ESI†). Two such acylated derivatives
60 from HA oxidation would be HA-1 and HA-2.16 We propose that
the species is likely HA-1 since carbamic acids like HA-2 are
known to be unstable.17
The selectivity of probe HA was also studied. The probe HA (20
ꢁM) was treated with various ROS/RNS in sodium phosphate
65 buffer (0.1 M, pH 7.4, 1% DMF) at room temperature. As shown
in Fig. 2, when HA reacted with ClOꢀ, the two characteristic
fluorescence peaks at 460 nm and 570 nm were observed. In
contrast, other ROS species, including superoxide (O2•ꢀ, 10
equiv), hydrogen peroxide (H2O2, 50 equiv), lipid peroxides
70 (ROO•, 10 equiv) and hydroxyl radical (•OH, 15 equiv), induced
nearly no fluorescence responses even after 30 min at 37 oC.
HPLC studies verified that the probe was not oxidized (Fig. S6,
ESI†). Whereas upon addition of peroxynitrite (ONOOꢀ, 3 equiv),
a large and immediate increase of fluorescence intensity at 460
75 nm (ca. 68ꢀfold) was observed (Fig. S7, S8, ESI † ). Singleꢀ
channel response only at 460 nm for OONOꢀ makes it nonꢀ
interfering to ClOꢀ, which induces a dualꢀchannel signal at 460
nm and 570 nm, respectively. The mechanism of the probe
oxidation with OONOꢀ is currently under investigation.
15 To investigate the reactivity and spectral properties of probe HA
with ClOꢀ, spectroscopic titration was carried out at pH 7.4 (0.1
M sodium phosphate buffer, with 1% DMF) at room temperature.
Upon oxidation by ClOꢀ, two emission bands at 460 nm and 570
nm appeared from the dark background in a dose dependant
20 manner with excitation at 340 nm (Fig.1a) and levelled off in a
few seconds (Fig. S3, ESI†). The absorption spectra of the
solution displayed a characteristic redꢀshift to 410 nm (Fig. S4,
ESI†). This suggested that the probe HA has potentials for realꢀ
time detection of ClOꢀ. The emission intensity was enhanced by
25 71ꢀfold at around 460 nm and 63ꢀfold at around 570 nm with 3
equiv of ClOꢀ. A linear response to the ClOꢀ concentration was
found from 0 ꢁM to 32 ꢁM (Fig.1b) and the detection limit was
estimated to be 0.7 ꢁM.
We studied the oxidation mechanism of the probe HA.
30 Compound HA-3 (3ꢀaminoꢀ1,8ꢀnaphthalimide) was confirmed to
be generated from the solution upon oxidation and responsible for
the emission at 570 nm, by comparison of spectral properties and
HPLC retention time (Fig.S5, ESI†) with independently
synthesized HA-3. However, we could not unambiguously
35 determine the identity of the species with an emission at 460 nm.
Attempts of isolation by HPLC or direct detection by LCꢀMS
were not successful. We suppose it is an analog of HA-3 with the
amino group acylated based on the following facts. First, amino
group of 4ꢀaminophenoxyl moiety in probe HA was protonated in
40 acidic media (pH < 6.5) in which the PET process was inhibited,
and HA showed an emission maximum at 460 nm when excited
80 The bioimage application of the probe HA was investigated.
HeLa cells were first incubated with probe HA (50 ꢁM) for 20
min at 37 oC and washed with phosphate buffer (pH 7.4). Then,
the cells were treated with (or without, as the control) ClOꢀ (250
2
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Fig. 3 Fluorescence and brightꢀfield images of HeLa cells. (a) Brightꢀfield
image of HeLa cells incubated with HA (50 ꢁM) for 20 min. (b) (c)
Fluorescence image of HeLa cells incubated with HA (50 ꢁM) for 20
min. (d) Brightꢀfield image of HeLa cells preꢀtreated with HA (50 ꢁM)
for 20 min and then incubated with ClOꢀ (5 eq) for another 30min. (e) (f)
Fluorescence image of preꢀtreated cells with HA (50 ꢁM) for 20 min and
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10
o
ꢁM) for another 30 min at 37 C and washed three times with
phosphate buffer (pH 7.4). As shown in Fig. 3b and 3c, HeLa
cells without ClOꢀ showed nearly no fluorescence, whereas the
cells treated with ClOꢀ displayed strong fluorescence in both blue
15 and green channels (Fig. 3e and 3f). Fluorescence enhancements
at both channels are doseꢀdependent with the added ClOꢀ (Fig.
S9). 3ꢀmorpholinosydnonimine (SINꢀ1, an OONOꢀ donor)
induced moderate signal only at the blue channel (Fig. S10, ESI†)，
while other ROS did not yield any noticeable signals in both
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ClOꢀ in living cells.
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In summary, we have designed and synthesized
a novel
fluorescence probe HA. Good water solubility and inertness of its
25 fluorescence to pH from 6.5 to 11.0 made the probe suitable for
applications under physiological conditions. With the addition of
ClOꢀ, probe HA showed a dualꢀchannel emission at 460 nm and
570 nm. OONOꢀ led to a singleꢀchannel enhancement at 460 nm
only and other ROS/RNS induced no spectral changes. These
30 mean that the probe HA could distinguish ClOꢀ from ROS/RNS
by this dualꢀchannel signal. Fluorescence imaging of HeLa cells
also showed that the probe HA can be used as hypochlorite probe
in living cell imaging.
The authors are grateful for the financial support from the State
35 Key Program of National Natural Science of China (21236002),
the National Basic Research Program of China (2010CB126100),
the National High Technology Research and Development
Program of China (2011AA10A207), the Fundamental Research
Funds for the Central Universities.
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State key Laboratory of Bioreactor Engineering, Shanghai Key
Laboratory of Chemical Biology, School of Pharmacy, East China
University of Science and Technology, 130 Meilong Road, Shanghai,
200237, China. Fax: (+86) 21-64252603; E-mail: xhqian@ecsut.edu.cn.:
45 yfxu@ecust.edu.cn;
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
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