Strong red fluorescent probes suitable for detecting hydrogen peroxide generated by mice peritoneal macrophages

Article abstract of DOI:10.1039/b512440a

This paper reports the synthesis, fluorescence properties, and biological applications of naphthofluorescein disulfonate (NFDS-1), as a red fluorescence imaging probe to detect intracellular H₂O₂. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b512440a

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Strong red fluorescent probes suitable for detecting hydrogen peroxide
generated by mice peritoneal macrophages{
a
a
a
b
a
a
Kehua Xu, Bo Tang,* Hui Huang, Guiwen Yang, Zhenzhen Chen, Ping Li and Liguo An
b
Received (in Cambridge, UK) 2nd September 2005, Accepted 25th October 2005
First published as an Advance Article on the web 8th November 2005
DOI: 10.1039/b512440a
1
5
This paper reports the synthesis, fluorescence properties, and
biological applications of naphthofluorescein disulfonate
of monopentafluorobenzenesulfonyl fluorescein to fluorescein.
The method relies on simple deprotection, not on oxidation, which
allows the highly specific and peroxidase-independent detection of
(NFDS-1), as a red fluorescence imaging probe to detect
2 2
H O under the complicated oxidative circumstances. However,
2 2
intracellular H O .
there are two disadvantages in the fluorescein-based probe: the
weak fluorescence of the probe itself and the high background
fluorescence interference in biological systems reduced measurable
sensitivity (the detection limit is 92.3 nmol or higher). In vivo,
hydrogen peroxide concentrations are usually considered to be in
the lower nanomolar range. Therefore, it is necessary to develop
probes that have higher selectivity and enough sensitivity for H O
The far-visible and the near-IR spectral regions (600–1000 nm),
where only a few classes of molecules exhibit significant
absorption, and they do not contribute to the fluorescence signal,
are areas of low background fluorescence interference in biological
1,2
systems. The features of the particular spectral region make it
ideal for using a fluorogenic probe to detect reactive oxygen species
2
2.
(
ROS) in living cells. However, as far as we know, only one probe
2
We designed and synthesized naphthofluorescein disulfonates
(NFDS-1, NFDS-2, Scheme 1) as fluorescence imaging probes for
intracellular H O , which were characterized with elemental
in this region has been reported so far, for detecting nitric oxide.
In the current work, we aim to develop molecular probes that
could be used to detect reactive oxygen species in this spectral
region. Reactive oxygen species such as superoxide radical anion,
hydrogen peroxide, hydroxyl radical, nitric oxide and peroxynitrite
2
2
1
analysis, IR, and H NMR. NFDS are closed, colorless, and
non-fluorescent lactones. Upon treatment with H O , hydrolytic
2
2
deprotection of NFDS would subsequently generate open, colored,
and fluorescent products.
3
play a vital role in physiology. A rapid rise in intracellular oxidant
levels under oxidative stress could cause damage to biological
The NFDS-2 was chosen for the following reasons: First, owing
to the effect of perfluorooctanesulfonic acid, a surfactant generated
from the hydrolysis of NFDS-2 in the reactive circumstances, the
excitation and emission spectra of the product, naphthofluorescein,
generated from reaction of NFDS-2 with H O would have a red
4–6
molecules and result in various diseases. Hydrogen peroxide is
the precursor to other ROS, and its homeostasis can have diverse
7
physiological and pathological consequences. However, how this
damage occurs is insufficiently understood even in the simplest
8
eukaryotic organisms.
2
2
shift compared with NFDS-1, theoretically. Second, the perfluoro-
octane chain would enhance the reactivity of the NFDS-2 toward
H O owing to the lipophilic characteristics of fluorine atoms and
In order to explore the role of hydrogen peroxide in toxicology
and human diseases, it is necessary to establish an accurate way of
detecting the ROS, especially in living cells. Many probes such as
dihydro-analogues of fluorescent dyes [e.g. dichlorofluorescin
2
2
hydrogen peroxide.
9
,10
diacetate (DCFDA), dihydrorhodamine 123,
phosphine-based
11,12
13
fluorophores,
mophores with ROS-cleavable protecting groups
developed in recent years. DCFDA is the most popular one, and
lanthanide coordination complexes, and chro-
14,15
] have been
9
2 2
has been used frequently to detect cell-derived H O , but it suffers
from a major drawback in that it is poorly selective toward H O
2
2
owing to its autoxidation and reaction with other ROS or
peroxidase.
2 2
Recently, an interesting method for H O detection was
developed based on the selective H O -mediated transformation
2
2
a
College of Chemistry, Chemical Engineering and Materials Science,
Shandong Normal University, Jinan, 250014, China.
E-mail: tangb@sdnu.edu.cn; Fax: (+86) 531-6180017;
Tel: (+86) 531-6180010
b
College of Life Science, Shandong Normal University, Jinan, 250014,
China. E-mail: yanggw@sdnu.edu.cn; Fax: (+86) 531-6180107;
Tel: (+86) 531-61880143
{
Electronic supplementary information (ESI) available: detailed synthetic
Scheme 1 The synthesis of fluorescent probes and their reaction with
procedures and confocal fluorescence imaging. See DOI: 10.1039/
b512440a
2 2
H O .
5
974 | Chem. Commun., 2005, 5974–5976
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NFDS-1 and NFDS-2 were evaluated under chemical circum-
stances. A solution of NFDS-1 (50 mM) or NFDS-2 (50 mM) in
DMSO was diluted 10 times with 2-[4-(hydroxyethyl)-1-piperazi-
nyl]ethanesulfonic acid (HEPES) buffer (0.1 M, pH 7.4).
Treatment of each of the solutions (5 mM) containing the probe
the following experiments, we have focused our attention on
NFDS-1.
The reactivity of NFDS-1 toward various ROS, reductant,
glutathione (GSH), and esterase was studied in detail. The
fluorescent response from the solution of NFDS-1 (10 mM, in
compounds with H
2
O
2
(1 mM) at 37 uC for 40 min gave
fluorescence responses, Fig. 1. With different concentrations of
HEPES buffer) with H O (2 mM) at 662 nm with excitation at
2 2
602 nm after incubation at 37 uC for 40 min was compared to
those of reactions with NaOCl, t-BuOOH, esterase, 1,4-hydro-
quinone, GSH, 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-
triazene (NOC-5), 3-morpholinosydnonimine (SIN-1), and
ascorbic acid (Vc) (final 10 mM each), and with superoxide radical
anion or hydroxyl radical. Superoxide radical anions were
generated by the enzymatic reaction of hypoxanthine (HPX,
H O we obtained the NFDS-1 linear calibration curve from
2
2
2
9
26
6
.0 6 10 to 4.0 6 10 M and the detection limit (81.5 pM)
15
which was lower than for reported probes (92.3 nM). We believe
that the lower detection limit of NFDS-1 for H O was based on
2
2
its low background interference and the non-fluorescence of the
probe itself. The experiments also showed that the fluorescence
property of the product generated from reaction of NFDS-2 with
2
1
1
ml, 10 mM) with xanthine oxidase (XO, 0.2 ml, 1 U ml ) or
2
+
2 2
KO (10 mM). The Fenton reaction between H O (5 mM) and
2 2
H O is not stable enough owing to the concentration change
2
?
Fe ion (10 mM) was used as the source of OH. All results are
summarized in Table 1. The experiments showed that NFDS-1
of the surfactant, perfluorooctanesulfonic acid, generated from
the hydrolysis of NFDS-2, as shown in Fig. 1(b). Therefore, in
2 2
provided a highly specific fluorescent response toward H O , while
giving a small response to ascorbic acid, glutathione, esterase, and
other ROS, especially to hydroxyl radical. We suggest that the
2 2
observed selectivity of NFDS-1 for H O over more oxidizing
ROS is based on simple deprotection, not on an oxidative
mechanism.
2 2
We next assessed the reaction of NFDS-1 for H O generated in
living cells. Separated mice peritoneal macrophages (PM) were
seeded onto a glass slide. Then the cells were loaded with NFDS-1
(10 mM, DMSO–HEPES buffer, pH 7.4) by incubation at 37 uC
for 30 min and showed negligible intracellular background
fluorescence, Fig. 2(a). Probe-loaded macrophages were stimulated
2
1
with phorbol myristate acetate (PMA: 2 ng ml ) at 37 uC for
0 min, and a strong signal was observed, Fig. 2(b). In addition,
1
prompt fluorescent increases in probe-loaded macrophages treated
with exogenous H O (10 or 100 nM, respectively) were also
2
2
observed, Fig. 2(c) and (d). Brightfield transmission measurements
after NFDS-1 incubation and 100 nM H O addition confirm that
2
2
the cells are viable throughout the imaging experiments, Fig. 2(e).
These data establish that NFDS-1, a small lipophilic molecule, is
membrane-permeable, and can respond to nanomolar change in
2 2
H O concentration within living cells, while native cellular species
such as GSH and ascorbic acid, as shown in Fig. 2(a), do not
contribute to the fluorescence imaging.
In conclusion, we have presented in this article the synthesis,
fluorescence properties, and biological applications of NFDS-1, as
a fluorescence imaging probe to detect intracellular H
naphthofluorescein-based reagent features high selectivity for
over other intracellular ROS and some biological
2 2
O . This
2 2
H O
compounds, a wide dynamic response range and low detection
Table 1 Relative fluorescence intensity (RFI) observed upon reaction
of NFDS-1 with various ROS, and biological compounds
a
a
Compounds
RFI
Compounds
RFI
2
ONOO (SIN-1)
t-BuOOH
1,4-hydroquinone
GSH
Ascorbic acid
Esterase
Blank
10
155
20
42
5
8
35
8
8
11
10
H
2
O
2
?
2
O
2
(XO/HPX)
)
2
?
O
OH
2
(KO
2
Fig. 1 (a) Emission spectra of 5 mM NFDS-1 with 1 mM H
blank (B) in HEPES (pH = 7.4) at 37 uC for 40 min (lem = 662 nm). (b)
Emission spectra of 5 mM NFDS-2 with 1 mM H (A) at 37 uC
for 40 min, 45 min and 50 min, and blank (B) at 37 uC for 40 min
em = 692 nm).
2 2
O (A) and
?
2
OCl
?
34
7
2
O
2
NO (NOC-5)
a
All data obtained at 662 nm after incubation at 37 uC for 40 min.
(l
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Fig. 2 Confocal fluorescence and phase contrast images of live PM. (a) Fluorescence image of PM incubated with 10 mM NFDS-1 at 37 uC for 30 min.
2
1
(
b) Fluorescence image of probe-stained PM stimulated with PMA (2 ng ml ) at 37 uC for 10 min. (c) Fluorescence image of probe-stained PM treated
with H (10 nM) at 37 uC for 10 min. (d) Fluorescence image of probe-stained PM treated with H (100 nM) at 37 uC for 10 min. (e) Brightfield image
of live PM after NFDS-1 incubation and 100 nM H addition to confirm viability (Scale bar = 10 mM).
2
O
2
2 2
O
2 2
O
limit owing to its nonredox mechanism, and far-visible excitation
and near-IR fluorescence emission profiles to minimize cell and
tissue damage while avoiding native fluorescence from native
cellular species. Furthermore, we have demonstrated the value of
this probe by measuring living cell-derived H O and the
Notes and references
1
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3
4
5
2
2
nanomolar concentration of exogenous H
macrophages. We found that NFDS-1 is an excellent fluorescence
reagent in detecting intracellular H and can respond to
nanomolar change in H O concentrations within living cells.
2 2
O within living
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2
2
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biological systems to explore its potential applications. We believe
that such a naphthofluorescein-based fluorescent dye will have an
important application in detecting oxidative stress through direct
intracellular imaging.
8
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We thank Dr Chun-ming Liu from Plant Research
International, Wageningen, The Netherlands for critical comments
on the manuscript. This work was supported by research grants
from the Important Project of National Natural Science
Foundation of P. R. China (Nos. 20335030 and 90401019) and
the Important Project of Natural Science Foundation of Shandong
Province in China (No. Z2003B01).
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976 | Chem. Commun., 2005, 5974–5976
This journal is ß The Royal Society of Chemistry 2005