A ratiometric fluorescent probe for the detection of hydroxyl radicals in living cells

Article abstract of DOI:10.1039/c4cc00975d

A naphthalimide-naphthyridine derivative has been synthesized for the detection of hydroxyl radicals. It can distinguish hydroxyl radicals from other reactive oxygen species with high selectivity and short response time. Moreover, it has no cellular toxicity, and can be effectively used for intracellular detection of hydroxyl radicals. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c4cc00975d

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A ratiometric fluorescent probe for the detection
of hydroxyl radicals in living cells†
Cite this: Chem. Commun., 2014,
0, 4843
5
Luyan Meng, Yongquan Wu and Tao Yi*
Received 6th February 2014,
Accepted 21st March 2014
DOI: 10.1039/c4cc00975d
www.rsc.org/chemcomm
A naphthalimide–naphthyridine derivative has been synthesized for the By contrast, ratiometric fluorescent probes are not affected by
detection of hydroxyl radicals. It can distinguish hydroxyl radicals from factors, such as probe concentration and environmental condi-
other reactive oxygen species with high selectivity and short response tions, which makes them ideal fluorescent probes. To date, only
time. Moreover, it has no cellular toxicity, and can be effectively used a few examples of hydroxyl radical ratiometric probes have been
1
4–17
for intracellular detection of hydroxyl radicals.
reported, including an inorganic–organic hybrid nanoprobe
and a pure organic probe.
1
8,19
However, these probes are rarely
Reactive oxygen species (ROS) are associated with a wide range applied in the detection of hydroxyl radicals in living cells.
of physiological and pathological processes, such as signal Hence, designing a ratiometric probe for intracellular detection
transduction, inflammation, ischemic or traumatic brain injury, of hydroxyl radicals would be very useful.
1,2
carcinogenesis, and neurodegenerative diseases. The primary
1,8-Naphthalimide derivatives possess excellent spectroscopic
ROS include hydrogen peroxide (H O ), hypochlorous acid (HOCl) properties, such as good photostability, high fluorescence quantum
2
2
À
1
20–23
and hypochlorite (ClO ), singlet oxygen ( O The fluorescence
À
(O ), peroxynitrite (ONOO ), and hydroxyl radical ( OH). Among spectra of 1,8-naphthalimide derivatives depend mainly on the
2
2
), superoxide anion yield, and tuneable fluorescence emission.
À
ꢀ
1
these, the hydroxyl radical possesses high reactivity, and numerous electron donating ability of the 4-position substituent group based
biomacromolecules are vulnerable to oxidative damage from on the ‘push–pull’ internal charge transfer from the 4-position group
hydroxyl radicals, including carbohydrates, nucleic acids, lipids, to the electron withdrawing imide. Therefore, the 4-position is
3
and amino acids. Methods to detect hydroxyl radicals have been an important target in the design of fluorescent probes using
widely explored, such as electron spin resonance spectroscopy 1,8-naphthalimide derivatives. Similarly, the fluorescence spectra of
4
and fluorescent probes. The use of fluorescent probes is more 7-methyl-naphthyridine derivatives can be tuned by a 2-position
advantageous than other methods because they have high substituent group. Herein, we have designed a ratiometric fluorescent
sensitivity and accuracy, and can monitor hydroxyl radicals for probe 1, by connecting the 4-position of the naphthalimide group and
5
real-time visualization in living cells.
the 2-position of naphthyridine group using ethylenediamine
Over the past decade, several types of fluorescent probes (Scheme 1). The probe has a higher selectivity and sensitivity towards
have been developed for the detection of hydroxyl radicals. Spin hydroxyl radicals over other reactive oxygen species, and can be used
trapping hydroxyl radical probes, which can react with free for intracellular detection of hydroxyl radicals.
radicals, causing a significant fluorescence enhancement,
The details of synthesis and characteristics of 1 are provided in
6
–9
are the most common. Aromatic hydroxylation probes were the ESI.† The absorption spectrum of 1 (10 mM) shows two typical
1
0,11
3+
12
reported based on coumarin
and a special Tb complex.
2
absorption bands at 371 and 461 nm in H O–DMF (98 : 2, v/v)
In addition, rhodamine dyes have been applied in the design of (Fig. 1), which are characteristic of the naphthyridine and naphthal-
1
3
a hydroxyl radical probe by oxidative hydrogen abstraction.
imide moieties, respectively, by comparison to the contrasting
These probes show high sensitivity, but have low spatial
resolution and are easily affected by environmental factors.
Department of Chemistry & Innovation Center of Chemistry for Energy Materials,
Fudan University, 220 Handan Road, Shanghai 200433, P. R. China.
E-mail: yitao@fudan.edu.cn; Fax: +86-21-55664621
†
Electronic supplementary information (ESI) available: Synthesis details and
additional spectra. See DOI: 10.1039/c4cc00975d
Scheme 1 Chemical structure and synthetic routine of probe 1.
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Fig. 3 Changes in the (a) absorbance and (b) fluorescence spectra of
1
2
0 mM 1 in H O–DMF (98 : 2, v/v) with the addition of different concentra-
tions of hydroxyl radicals (from 0 to 10 eq.). The inset of (b) shows the ratio
of emission intensities (F418/F552) as a function of concentrations of
hydroxyl radicals.
2
Fig. 1 Absorption and fluorescence spectra of 1 (10 mM) in H O–DMF
(98 : 2, v/v).
ratio (F418/F552 o 0.5) were noted upon addition of H
2
O
2
, tert-
À
2 2
butylhydroperoxide, ONOO , O , O , F and Fe (Fig. 2b).
À
1
À
2+
The fluorescence images also display the sharp contrast response
between hydroxyl radicals and other ROS by 1 (Fig. 2c). Moreover,
both the fluorescence wavelength and intensity of 1 showed a very
tiny difference upon the change of pH value from 3.0 to 8.0
compounds S1 and S2 (Scheme S2 and Fig. S1 in the ESI†). The
fluorescence spectrum of 1 (10 mM) is characterized by a
maximal emission band centred at 552 nm with a fluorescence
lifetime of 7.12 ns (Fig. S2 in the ESI†) related to the naphthal-
imide moiety, with a much weaker emission band at 426 nm
related to the naphthyridine moiety when excited at 371 nm in
(Fig. S3 in the ESI†), which meant that pH had almost no
influence on the detection of hydroxyl radicals by probe 1.
To investigate the reactivity and spectral details of 1 (10 mM)
with hydroxyl radicals, spectroscopic titration was carried out
H O–DMF (98 : 2, v/v). The fluorescence quantum yield of 1 in
2
DMF was 0.74 using flavin mononucleotide (FMN) as the
standard (FFMN = 0.25).
2
upon addition of hydroxyl radicals (from 0 to 500 mM) in H O–
DMF (98 : 2, v/v) at room temperature (Fig. S4 in the ESI†). As
shown in Fig. 3, the absorbance of 1 at both 371 and 461 nm
increased upon the addition of less than 2 eq. of hydroxyl
radicals. Correspondingly, the intensity of the emission band at
552 nm of 1, which is characteristic of the naphthalimide moiety,
increased at first and then decreased. As the amount of hydroxyl
radicals increased to 10 eq., the absorbance at 461 nm, a char-
acteristic of the naphthalimide moiety, gradually weakened, while
the absorbance at 371 nm, a characteristic of the naphthyridine
moiety, enhanced and blue shifted to 355 nm. Simultaneously, the
intensity of the emerging fluorescence at 418 nm was drastically
enhanced during the titration process. In addition, at less than 10
eq. of hydroxyl radical, an excellent linear correlation between the
ratio of two emission intensities (F418/F552) and the hydroxyl radical
High-level selectivity is of paramount importance for an
excellent chemosensor. The selectivity of 1 was checked among
seven species related to ROS. Fluorescence changes of 1 upon
addition of hydroxyl radicals (50 eq.) and other ROS (50 eq. of
À
À
1
À
À
H O , tertbutyl-hydroperoxide, ClO , ONOO , O , O , F and
2
2
2
2
2+
Fe ) in H O–DMF (98 : 2, v/v) are shown in Fig. 2a. Upon treatment
2
with 50 eq. of hydroxyl radicals, a marked strong blue emission at
418 nm was observed in less than 2 min, which indicated that
hydroxyl radicals reacted with 1 rapidly at room temperature. In
addition to the fluorescence changes, a large ratiometric fluores-
cence response at 418 to 552 nm was observed (F418/F552 = 53.2)
upon addition of 50 eq. of hydroxyl radicals. By contrast,
hypochlorite only induced a very weak ratiometric response
with F418/F552 = 1.2, and negligible changes in the emission
2
concentration was obtained with R = 0.99946, which indicated that
the ratio of fluorescence intensity at 418 and 552 nm increased as a
linear function of the hydroxyl radical concentration. The detection
À7
limit of 1 to the hydroxyl radical is 2.0 Â 10 M, which is superior
À7
16,17
to that in previous reports (7.3 Â 10 M).
Thus, probe 1 has a
high selectivity, quick reaction time and low detection limit for
hydroxyl radicals, and can likely be used to detect intracellular
hydroxyl radicals.
The mechanism of the reaction between 1 and hydroxyl radicals
was studied. The main product with blue fluorescence emission was
isolated and purified by column chromatography as a yellow oil. The
1
H NMR and mass spectra suggest that the product is a hydroxyl
modified naphthyridine moiety without naphthalimide (Fig. S5 and
S6, ESI†). This means that probe 1 dissociates into a blue emissive
naphthyridine moiety and a nonfluorescence naphthalimide part
after the treatment of hydroxyl radicals.
Fig. 2 Fluorescence spectra (a) and images (c) of 1 (10 mM) upon addition
of 50 eq. of various ROS in H O–DMF (98 : 2, v/v, lex = 365 nm). (b) The
2
Cell experiments were performed by confocal laser micro-
scopy (CLMS) to evaluate the potential utility of probe 1 for
ratio (F418/F552) of emission intensities at 418 to 552 nm of 1 with 50 eq. of
various ROS.
4
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proliferation was determined by means of a MTT assay in
Hela cells. 1 showed no cellular toxicity in 36 h at a concentration
of 5 mM. Even at a higher concentration (20 mM), the cellular
viabilities were estimated to be greater than 85% in 36 h (Fig. S8,
ESI†). The low cytotoxicity and high biocompatibility make 1 a
suitable candidate for intracellular detection of hydroxyl radicals.
In summary, a novel naphthalimide–naphthyridine derivative
has been designed and synthesized for utility as a ratiometric
fluorescence probe for the detection of hydroxyl radicals. The
fluorescent probe exhibits high selectivity and can clearly distinguish
hydroxyl radicals from other reactive oxygen species. It also has
good sensitivity to hydroxyl radicals and responds rapidly in less
than 2 min in H O–DMF solution. In addition, the detection limit
2
À7
for hydroxyl radicals can reach as low as 2.0 Â 10 M. Moreover, the
fluorescent probe shows excellent photostability, low cytotoxicity and
high biocompatibility, and can be used for intracellular detection of
hydroxyl radicals, which is very important to follow the tracks of
hydroxyl radicals in physiological and pathological processes.
The authors are thankful for the financial support from 973
(2013CB733700), NNSFC (21125104, 51373039), PIRTU
Fig. 4 CLMS images of RAW264.7 cells (a) incubated with 1 (5 mM) for (IRT1117) and Shanghai Sci. Tech. Comm. (12XD1405900).
3
0 min and (b) followed by incubating with Fenton’s reagent (5 mM) for
1
h, (c) cells incubated with 1 (5 mM) for 30 min, and then with PMA
Notes and references
À1
(
50 ng mL ) for 1 h and (d) incubated with PMA for 1 h, TEMPOL (5 mM)
for another 1 h and finally with 1 for 30 min; 1–4 are bright field
images, fluorescence images in blue channel (420–450 nm), green
channel (520–570 nm) and overlay images, respectively.
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À1
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1
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2
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Chem. Commun., 2014, 50, 4843--4845 | 4845