Fluorescence turn-on detection of hypochlorous acid via HOCl-promoted dihydrofluorescein-ether oxidation and its application in vivo

Article abstract of DOI:10.1039/c2cc30265a

We report three new water-soluble dihydrofluorescein-ether probes FCN1, FCN2 and FCN3 for the detection of hypochlorous acid (HOCl), which were designed on the basis of a specific HOCl-promoted oxidation reaction. This work also provided a useful method t

Full text of DOI:10.1039/c2cc30265a

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Chemical Communications
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Volume 48 | Number 39 | 16 May 2012 | Pages 4641–4780
ISSN 1359-7345
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C. Yao et al.
Fluorescence turn-on detection of hypochlorous acid via HOCl-promoted
dihydrofluorescein-ether oxidation and its application in vivo
1359-7345(2012)48:39;1-2

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Fluorescence turn-on detection of hypochlorous acid via HOCl-promoted
dihydrofluorescein-ether oxidation and its application in vivow
Yi Zhou,a Ju-Ying Li,a Kai-Hui Chu,a Ke Liu,a Cheng Yao*a and Jing-Yun Lib
Received 12th January 2012, Accepted 15th February 2012
DOI: 10.1039/c2cc30265a
We report three new water-soluble dihydrofluorescein-ether probes
FCN1, FCN2 and FCN3 for the detection of hypochlorous acid
(HOCl), which were designed on the basis of a specific HOCl-
promoted oxidation reaction. This work also provided a useful
method to monitor the accumulated HOCl in specific organelles
using a zebrafish model.
Reactive oxygen species (ROS) and reactive nitrogen species
Scheme 1 Structures of probes FCN1–FCN3.
(RNS) form as natural byproducts of the normal metabolism
and play important roles in cell signaling and homeostasis.1 ROS
exhibit ideal properties in detecting HOCl quantitatively,
of particular interest is hypochlorous acid (HOCl, pKa = 7.46),
fluorescent probes that show single-wavelength fluorescence
which is approximately half dissociated into the hypochlorite
enhancement (turn-on) response and work well in 100% aqueous
anion (OClꢀ) under physiological conditions and is one of the
physiological media are still a little scarce. In addition, although
most powerful natural oxidants.2 Biologically, hypochlorous acid
several examples have been focused on the studies of the HOCl
is produced by myeloperoxidase (MPO)-mediated peroxidation
level in living cells or endogenous HOCl in stimulated macrophage
of chloride ions in activated neutrophils.3 Although HOCl
cells,7,8 the rapid and specific methods for studies of the
contributes to the destruction of bacteria in living organisms,
biological functions and deleterious effects of HOCl at the
the abnormal levels of HOCl caused by MPO have been impli-
organ level in vivo have not yet been reported.
cated in several human diseases including neuron degeneration,
Probes based on the fluorescein platform possess several
cardiovascular diseases, osteoarthritis and renal disease.4
favorable properties such as excitation and emission wavelengths
Moreover, a specified amount of HOCl is used to treat food
in the visible region as well as a high fluorescence quantum
preparation surfaces and water supplies in daily life.5 These
yield.13 Therefore, we designed three probes (FCN1–FCN3,
biologically relevant findings motivate us to develop sensitive
Scheme 1) with the dihydrofluorescein component that could
and specific probes for detecting HOCl in both water samples
selectively detect HOCl by a specific HOCl-promoted oxidation
and living systems.
reaction, resulting in the photoswitch of the non-fluorescent
Compared with other detection methods, fluorescent probes
deconjugated form to the strongly fluorescent conjugated form
have innate advantages including greater sensitivity, fast response
(Scheme 2). Moreover, we introduced a phenolic group and an
time and simplicity of implementation, offering application
ester group into FCN1 and FCN2 to increase the water
methods not only for in vitro assays but also for in vivo
solubility and modify the cell permeability. To the best of
imaging studies.6 Recently, the known reported fluorescent
our knowledge, FCN1–FCN3 are the first fluorescent probes
probes for HOCl were designed based on the strong oxidation
for HOCl based on dihydrofluorescein in 100% aqueous
property of HOCl, thus p-methoxyphenol,7 thiol,8 alkoxyaniline,2
media, and applied successfully in specific organelles using a
dibenzoylhydrazine,9 hydroxamic acid,10 oxim derivatives11 and
zebrafish model.
oxazine conjugated nanoparticles12 have been utilized as the
components of HOCl-selective probes. Although those probes
a State Key Laboratory of Materials-Oriented Chemical Engineering
and College of Science, Nanjing University of Technology,
Nanjing 210009, P. R. China. E-mail: yaochengnjut@126.com;
Fax: +86-25-8358-7433
b Animal Model Research Center, Nanjing University,
Nanjing 210061, P. R. China
w Electronic supplementary information (ESI) available: Experimental
procedures, characterization data and fluorescence spectra. See DOI:
10.1039/c2cc30265a
Scheme 2 Turn-on sensing of HOCl via HOCl-promoted FCN2 oxidation
reaction (photos show fluorescence of FCN2 in 100% aqueous media).
c
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up to 1000 equiv. (Fig. 1d). Fluorescent titration experiments
demonstrated that the fluorescence intensities of FCN2 were
linearly proportional to the concentration of HOCl in the
range of 0.5–5 mM (Fig. 1c). The detection limit (S/N = 3) was
also estimated to be 6.68 nM (0.71 ppb) on the basis of the
signal-to-noise ratio (Fig. S3, ESIw), which is comparable to
that of the fluorescence turn-on type probe for HOCl (Table S3,
ESIw). Furthermore, the HOCl-promoted FCN2 oxidation
mechanism was supported by HPLC-MS in 50% CH3CN/H2O.
In the presence of 10 equiv. of HOCl, a new peak appeared at
361.1 (m/z) in the spectrum, suggesting that the transformation
from FCN2 to FCN-Et was induced by HOCl.
Since HOCl in living cells or organisms is synthesized
predominantly from hydrogen peroxide and chloride ions
catalyzed by the MPO enzymatic system,7 we employed
NIH3T3 cells (mouse embryonic fibroblast cells) to examine
the capacity of FCN2 to detect the production of HOCl in a
biological environment. Incubation of NIH3T3 cells with 5 mM
FCN2 in an MPO enzymatic system (MPO, 1.5 U per 100 mL;
NaCl, 250 mM) for 30 min at 37 1C yielded negligible
intracellular background fluorescence (Fig. 2b). Conversely,
addition of 10 mM H2O2 to probe-loaded cells for further
30 min incubation elicited a significant enhancement in intra-
cellular fluorescence (Fig. 2d). Also, FCN2 exhibited rapid
fluorescence response to HOCl in cells, and over 40% of the
total fluorescence intensity increase was observed after 5 min
(Fig. 2f). The overlay of fluorescence and bright-field images
revealed that the fluorescence signals were localized in the
whole cells (Fig. 2a and e). In addition, the MTT assays
suggested that FCN2 had almost no change in cell viability
even when the amount of probe is up to 100 mM (Fig. S13,
ESIw). Thus, all results indicated that FCN2 was cell
membrane permeable, nontoxic, and capable of detecting
HOCl produced by MPO-mediated peroxidation of Clꢀ.
Zebrafish has proved to be a highly valuable vertebrate
model for in vivo imaging due to its transparent nature during
all stages of embryonic development.14 Therefore, the external
HOCl in the embryo was tracked by FCN2 at various time
points during development. At 19 h post-fertilization (hpf),
Fig. 1 (a) The time-dependent fluorescence changes acquired from
reaction of FCN1–FCN3 (1.5 mM) with HOCl (5 mM) in 20 mM HEPES
buffer (pH 7.2) at room temperature. (b) Fluorescence spectra of FCN2
(1.5 mM) in HEPES buffer recorded 30 min after the addition of HOCl
(ranging from 0 to 5 mM). (c) The fluorescence enhancement at 485 nm as
a function of HOCl concentration. (d) Fluorescence responses of FCN2
(1.5 mM) with various ROS and RNS species (see ESIw for details):
H2O2 (50 mM), NaNO2 (200 mM), ONOOꢀ (50 mM), OHꢂ (1 mM),
ꢀ
ꢂ
O2 (50 mM), NO (2 mM), HNO (100 mM) and ROOꢂ (200 mM).
The fluorescence intensity of FCN1 and FCN2 respectively,
showed 6.5-fold and 2.4-fold enhancement upon exposure to
20 mM HEPES buffer in air for 24 h, indicating their auto-
oxidation characteristics (Fig. S2, ESIw). FCN3 did not show
any auto-oxidation over more than 48 h due to its stability
contributed by the benzenemethanol group. Upon interaction
with HOCl, the fluorescence intensity of FCN2 increased rapidly
(Fig. 1b) and an approximately 1643.4-fold fluorescence
enhancement after 30 min was observed (485 nm, Ff = 0.71),
which was extraordinarily sensitive compared to the previously
reported probes (Table S3, ESIw). Similar fluorescence
enhancements were observed for FCN1 (1232.8-fold) and FCN3
(31.6-fold) under the same conditions (Fig. 1a). Compared with
FCN1 and FCN2, FCN3 had much longer time and less response
increment to reach fluorescence intensity maximum. The auto-
oxidation did not affect the HOCl detection because of the fast
response time and the large fluorescence enhancement. In addition,
the pseudo-first-order rate constants of FCN1–FCN3 with HOCl
were determined as k0 = 5.50 ꢁ 10ꢀ3 sꢀ1, 2.54 ꢁ 10ꢀ3 sꢀ1, and
5.94 ꢁ 10ꢀ4 sꢀ1, respectively. Upon varying the concentrations
of analyte, the second-order rate constant for the reaction of
HOCl with FCN2 (2.14 ꢁ 103 Mꢀ1
s
ꢀ1) was greater than that
for the reaction of HOCl with FCN1 (1.51 ꢁ 103 Mꢀ1
s
ꢀ1).
Thus, among the three dihydrofluorescein-based probes,
FCN2 showed the best sensitivity for the detection of HOCl
(order: FCN2 > FCN1 c FCN3; Table S2, ESIw).
To further evaluate the specific nature of FCN2 for HOCl,
we examined the fluorescence enhancement of FCN2 incubated
with other biologically rꢀelevant ROS and RNS. Upon inter-
Fig. 2 DIC (a) and confocal fluorescence (b) images of NIH3T3 cells
preincubated with NaCl (250 mM) and 5 mM FCN2 in MPO (1.5 U per
100 mL); fluorescence images in the course of the production of HOCl
stimulated with H2O2: incubated with H2O2 (10 mM) for 5 min (c) and
30 min (d); (e) overlay image of (a) and (d); (f) the average emission
intensities of cells were analyzed by using IPP software.
ꢀ
action with H2O2, NO2 , ONOO , OHꢂ, O2 ꢀ, NO, HNO
and ROOꢂ, the resulting solution only showed negligible
fluorescent enhancement even when the amount of analyte is
ꢂ
c
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Fig. 3 Fluorescence microscopy images of AB/Tubingen larvae zebrafish
incubated with 10 mM HOCl (E3 embryo media, 28.5 1C) during the
development. 1-Phenyl-2-thiourea (PTU, 0.003%) was added to depress
the development of pigment after 8 h of incubation. Fluorescence and
DIC images of (a, b) 18 h-old, (c, d, e) 28 h-old, (f, g) 54 h-old, and
(h, i) 78 h-old zebrafish further incubated with 5 mM FCN2 for 1 h.
Fig. 4 (a) Adult zebrafish were exposed to 100 mM HOCl in E3
embryo media for 24 h at 28.5 1C and then incubated with 5 mM FCN2
for 30 min. (b) The average emission intensities of isolated organs were
analyzed by using IPP software.
the embryo displayed nonspecific faint green fluorescence,
conceivably because the protective chorion resisted FCN2
from penetrating (Fig. 3a). During development, the fluores-
cence signal was brighter and a few bright green dots distri-
buted in two zygomorphic areas around its yolk extension after
25 h of incubation (Fig. 3c and e). It was notable that the
bright green dots moved to the top of the yolk sac at 54 hpf
(Fig. 3f), which may be attributed to the accumulated HOCl
by excess ingestion of some cells around yolk sac and yolk
extension. At 78 h hpf, the bright green dots were no longer
observed (Fig. 3h), and a significant green area was distributed in
the yolk sac. There were no evident developmental defects in fish
exposed to 10 mM HOCl (E3 embryo media) during fluores-
cence imaging.
in living cells, but also estimate the accumulated HOCl level in
zebrafish organs. This methodology may also be used as an
alternative tool for assessing the deleterious effects of HOCl.
Notes and references
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with the decrease of sensitivity to airborne pathogens and
pollutants, and an increased ability to modulate infections
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difficult to assess the deleterious effects of HOCl. We explored
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lated HOCl. An adult zebrafish (3 months old with identifiable
organs) was exposed to 100 mM HOCl in E3 embryo media for
24 h for accumulating HOCl in organs, and then incubated
with 5 mM FCN2 for 30 min. Intriguingly, the fluorescence
signals were detected in the eye, eggs, intestine, and liver of the
zebrafish (the highest intensity was observed in the gall bladder),
but not in the brain and heart after a 24 h accumulation period
(Fig. 4a). These preliminary studies are informative for the
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In summary, we have developed three water-soluble
dihydrofluorescein-ether probes FCN1–FCN3 on the basis of
a specific HOCl-promoted oxidation reaction. Significantly,
FCN2 showed a fast response time (B20 min), meanwhile it
displayed the larger fluorescence enhancement (1643.4-fold)
and an extraordinarily lower detection limit (0.71 ppb) compared
to the previously reported probes. FCN2 can not only visualize
HOCl produced by MPO-mediated peroxidation of chloride ions
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 4677–4679 4679