A sensitive and selective fluorescence probe based fluorescein for detection of hypochlorous acid and its application for biological imaging

Article abstract of DOI:10.1016/j.dyepig.2014.03.012

A highly sensitive and selective fluorescein-based probe 1 for hypochlorite anion was synthesized. The probe 1 has favorable characteristics for biological imaging, including high water solubility, high fluorescence yield, pH-independent fluorescence, and biocompatibility. Results show that it has a detection limit of 40 nM to hypochlorite anion. In addition, confocal fluorescence microscopy imaging using RAW264.7 cells showed that the new probe 1 could be used as an effective fluorescent probe for detecting HOCl in living cells.

Full text of DOI:10.1016/j.dyepig.2014.03.012

Dyes and Pigments 107 (2014) 127e132
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A sensitive and selective fluorescence probe based fluorescein for
detection of hypochlorous acid and its application for biological
imaging
,
b
Wenzhu Yin a, Hongjun Zhu a , Ruiyong Wang
*
a Department of Applied Chemistry, College of Science, Nanjing Tech University, Nanjing 211816, PR China
b School of Life Science, Nanjing University, Han Kou Road 22, Nanjing, Jiangsu 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A highly sensitive and selective fluorescein-based probe 1 for hypochlorite anion was synthesized. The
probe 1 has favorable characteristics for biological imaging, including high water solubility, high fluo-
rescence yield, pH-independent fluorescence, and biocompatibility. Results show that it has a detection
limit of 40 nM to hypochlorite anion. In addition, confocal fluorescence microscopy imaging using
RAW264.7 cells showed that the new probe 1 could be used as an effective fluorescent probe for
detecting HOCl in living cells.
Received 11 February 2014
Received in revised form
9 March 2014
Accepted 10 March 2014
Available online 27 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescein
Hypochlorite anion
Biological imaging
Water solubility
Flourescent probe
Spirolactam
1. Introduction
desirable to develop new fluorescent probes that can overcome
these limitations in detecting HOCl/OClꢀ. The fluorescein frame-
Hypochlorite anion (ClOꢀ) is one of the biologically important
reactive oxygen species (ROS), and produces in organisms by the
reaction of H2O2 with Clꢀ ions under the catalysis of a heme
enzyme, myeloperoxidase [1,2]. Endogenous ClOꢀ is essential to life
and has important antibacterial properties. However, excessive or
misplaced production of ClOꢀ can lead to tissue damage and dis-
eases, such as atherosclerosis, arthritis, and cancers [3]. Therefore,
real-time monitoring and accurate detection of ClOꢀ anion are
attracting increasing attention due to its extremely important role
in health and environmental science. Fluorescent probes possess
some native advantages over the probes of other types because of
their high sensitivity, specificity, simplicity of implementation, and
fast response times, offering application methods for not only
in vitro assays but also in vivo imaging studies [4e8]. So far, a
number of small-molecule fluorescent probes for specific detection
of HOCl/OClꢀ have been reported [9e21]. However, some of them
still face some drawbacks, such as poor water-selectivity, low
sensitivity, and pH dependency [22]. Therefore, it is highly
work seems to be an ideal model to construct “turn-on” fluorescent
chemosensors due to its favorable properties such as excitation and
emission wavelengths in the visible region with a high fluorescence
quantum yield [23e26], easy synthesis and functionalization [27],
excellent biocompatibility and cellular membrane-penetrating ca-
pacity [28e31]. With these considerations in mind, we synthesized
a new fluorescein-based chemosensor bearing a catechol moiety
which responded to the amount of ClOꢀ for detection of hypo-
chlorite anion (Scheme 1). Being a closed spirolactam structure, the
probe itself was nearly nonfluorescent, while the strong green
fluorescence was restored after addition of HClO. It was ascribed to
the opening ring of spirolactam via oxidation of catechol moiety by
HClO and then hydrolysis of diacylhydrazine [32]. The probe
display highly sensitive and selective detection toward ClOꢀ over
other ROS or reactive nitrogen species (RNS).
2. Experimental
2.1. Materials
Unless otherwise stated all chemicals were commercially ob-
tained and used without further purification. NaOCl was purchased
* Corresponding author. Tel.: þ86 25 83172358; fax: þ86 25 83587428.
E-mail addresses: zhuhj@njtech.edu.cn, zhuhjnjut@hotmail.com (H. Zhu).
http://dx.doi.org/10.1016/j.dyepig.2014.03.012
0143-7208/Ó 2014 Elsevier Ltd. All rights reserved.

128
W. Yin et al. / Dyes and Pigments 107 (2014) 127e132
d
(ppm): 7.77 (t, 1H, J ¼ 4.8 Hz), 7.49 (t, 2H, J ¼ 3.6 Hz), 6.98 (m, 1H),
6.59 (d, 2H, J ¼ 2.1 Hz), 6.42 (m, 4H), 4.38 (s, 2H).
2.3.3. Synthesis of compound 1
Compound 3 (346 mg, 1.0 mmol) was dissolved in absolute
methanol (20 mL). 3,4-dihydroxybenzalhyde (138 mg, 1.0 mmol)
was added and the mixture was heated at reflux for 4 h. The pre-
cipitate produced was filtered and washed with cold ethanol. The
crude product was purified by recrystallization from ethanol to
afford 1 as brown solid (420 mg, 90%). M.p.: >300 ꢁC (dec); 1H NMR
(300 MHz, DMSO-d6) d (ppm): 9.88 (s, 2H), 9.39 (s, 1H), 9.18 (s, 1H),
8.76 (s, 1H), 7.86 (dd, J ¼ 6.1, 2.2 Hz, 1H), 7.64e7.47 (m, 2H), 7.06 (dd,
J ¼ 5.9, 1.8 Hz, 1H), 6.87 (s, 1H), 6.70e6.58 (m, 4H), 6.45 (m, 4H). 13
C
Scheme 1. The routes of synthesis of compound 1.
NMR (75 MHz, DMSO-d6), d 163.32, 158.42, 152.03, 150.85, 150.70,
148.34,145.56, 133.60, 128.98, 128.90,127.95,125.71,123.53,122.96,
120.70, 115.46, 112.29, 112.25, 110.21, 102.45, 65.07. ESI-MS (-p) (m/
z): 465.75 ([MꢀH]ꢀ), 501.58 ([Mþ2H2OꢀH]ꢀ), 525.33
([MþCH3CO2HꢀH]ꢀ), 931.25 ([2MꢀH]ꢀ)
from Sinopharm Chemical Reagent Co., Ltd (China) and the con-
centration was determined by titration with Na2S2O3.
2.2. Measurements
2.4. Determination of quantum yield
The UVevis spectra were recorded on a Perkin Elmer Lambda 35
spectrophotometer. Fluorescence measurements were performed
at room temperature on a Perkin Elmer LS-55 spectrophotometer
or Hitachi Fluorescence spectrophotometer-F-4600, and Fluores-
cein was used as a standard for the determination of fluorescence
quantum yields. The biological imaging tests were carried out with
an Olympus FV-1000 and Leica TCS-SP5 laser scanning confocal
fluorescence microscopes. 1H NMR and 13C NMR were measured on
a Bruker AV-300 spectrometer with chemical shifts reported in
ppm (in DMSO-d6 or CDCl3, TMS as internal standard). Mass spectra
were recorded using a Thermo Finnigan LCQ Duo electrospray mass
spectrometer in positive ion mode by direct infusion, with spray
voltage 4.5 kV and capillary temperature of 200 ꢁC.
The fluorescence quantum yields were determined using fluo-
rescein as a reference with a known V value of 0.89 in EtOH [34].
The sample and the reference were excited at the same wavelength
(
lex ¼ 480 nm), maintaining nearly equal absorbance (0.06). The
quantum yield was calculated according to the following eqn (1):
ꢀ
.
ꢁ
FS
F
=
¼ ðAS A Þ ꢂ ðAbs Abs Þ ꢂ h2S h2R
;
(1)
=
=
R
R
R
S
where VS and VR are the fluorescence quantum yields of the
sample and the reference, respectively; AS and AR are the emission
areas of the sample and the reference, respectively; AbsS and AbsR
are the corresponding absorbance of the sample and the reference
solution at the wavelength of excitation; hS and hR are the refractive
indices of the sample and the reference, respectively.
2.3. Synthesis of compound 1 (probe 1)
2.5. Cell culture
As shown in Scheme 1, the probe 1 (compound 1) was readily
synthesized by treating fluorescein with methanol using H2SO4 as
catalyst, which was followed by hydrazine hydrate and 3,4-
dihyrdoxybenzaldehyde. The structure of probe 1 was confirmed
by 1H NMR, 13C NMR, ESI-MS. Compound 2 and 3 were synthesized
according to our previous procedures [33].
RAW264.7 macrophages were first incubated with LPS (1
and IFN-
(50 ng/mL) in culture medium for 4.5 h at 37 ꢁC, and then
were stimulated with PMA (10 nM) for 45 min at 37 ꢁC. Subse-
quently, part of the treated cells was incubated with -methionine
(300
M) for 40 min at 37 ꢁC. After washed with PBS buffer (0.10 M,
pH 7.4) for three times, all the cells were incubated with probe 1
(10
M) in culture medium for 30 min at 37 ꢁC. Before imaging, the
mg/mL)
g
L
m
2.3.1. Synthesis of compound 2
m
To fluorescein (1.0 g, 3.1 mmol) methanol solution (10 mL) in a
25 mL round-bottom flask, was added concentrated sulfuric acid
(98%) (1 mL). The solution was refluxed and stirred for 4 h. After
cooling, excess methanol was removed under reduced pressure and
excess water was added to the residue. The red solid formed was
washed with water several times and filtered in vacuum until
almost free from fluorescence. After dried in vacuum, 0.95 g red
solid fluorescein methyl ester 2 was obtained with a yield of 91%.
cells were washed again with PBS (0.10 M, pH 7.4) for three times.
3. Results and discussion
3.1. Selectivity studies
To evaluate whether probe 1 can selectively respond to OClꢀ
under simulated physiological conditions (pH ¼ 7.4), the fluores-
cence responses of probe 1 to other potentially competing ROS/
RNS, were also performed. As shown in the selectivity profiles
(Fig. 1), only OClꢀ incurs a dramatic fluorescence enhancement for
probe 1. Other ROS or RNS, including H2O2, t-BuOOH, NOꢀ2 , NO3ꢀ,
and S2O28ꢀ, exert no obvious spectral changes.
M.p.: 212e214 ꢁC; 1H NMR (CDCl3, 300 MHz)
d (ppm): 8.31 (d, 1H,
J ¼ 7.8 Hz), 7.79 (m, 2H), 7.34 (d, 1H, J ¼ 7.2 Hz), 7.28 (d, 2H,
J ¼ 3.6 Hz), 7.19 (d, 2H, J ¼ 9.0 Hz), 7.08 (dd, 2H, J1 ¼ 9.3 Hz,
J2 ¼ 2.1 Hz), 3.63 (s, 3H).
2.3.2. Synthesis of compound 3
Compound 2 (0.40 g) and hydrazine hydrate (0.24 g, 4.8 mmol)
were added to methanol (5 mL), refluxed and stirred for 6 h. After
collecting by filtration, the light brown precipitate was washed by a
small amount of methanol and water. 0.41 g straw yellow fluores-
cein hydrazone 3 was dried in vacuum and then obtained with a
yield of 98%. M.p.: 252e253 ꢁC; 1H NMR (DMSO-d6, 300 MHz)
In addition, probe 1 was treated with a wide variety of cations
and anions to examine the selectivity. As shown in Fig. 3, the
addition of ClOꢀ induced a significant redshift of the fluorescence
emission spectra. However, representative species such as Liþ, Naþ,
Kþ, Mg2þ, Ca2þ, Zn2þ, Cu2þ, Al3þ, Clꢀ, CH3COOꢀ, SO42ꢀ, CO23ꢀ, elicited
almost no changes in the fluorescence spectra. Furthermore, the

W. Yin et al. / Dyes and Pigments 107 (2014) 127e132
129
Fig. 3. Changes in absorption spectra of probe 1 (5
m
M) in a H2O/DMSO (v/v ¼ 20/1,
TriseHCl buffer, 0.05 M, pH 7.4) solution with various amount of OClꢀ (0e75
mM).
Fig. 1. Fluorescence changes of probe 1 (2
various ROS/RNS (20
M) in a H2O/DMSO (v/v ¼ 20/1, TriseHCl buffer, 0.05 M, pH 7.4)
solution. The excitation wavelength was 490 nm. Inset: Fluorescence response of probe
1 (2
mM) in response to NaOCl (20 mM) and
m
observed with an isosbestic point at 440 nm, which indicated for-
mation of a new unique compound. At this stage, the color of so-
lution became yellowish green from colorless and the absorbance
at 496 nm reached max when the addition of 15 equiv of NaClO.
These results indicated that probe 1 can be used as a “naked-eye
monitor” for detection of HClO.
m
M) to 10 equiv of various ROS/RNS in a H2O/DMSO (v/v ¼ 20/1, TriseHCl buffer,
0.05 M, pH 7.4) solution.
visual fluorescence response of probe 1 to various species (Fig. 2)
demonstrates that the probe can be used conveniently for hypo-
chlorite detection by simple visual detection.
3.3. Fluorescent response of probe 1 to OClꢀ
3.2. UV absorption response of probe 1 to OClꢀ
In order to evaluate the sensitivity of probe 1 to OClꢀ in a Trise
HCl buffer solution (pH ¼ 7.4), fluorescence titration were carried
out. Addition of NaOCl (aq) to the solution of the probe 1 caused an
immediate, strong change in fluorescence intensity with a new
band centered at 523 nm formed under 490 nm excitation wave-
length (Fig. 4). The emission intensity reached its maximum after
the addition of 15 equiv of HOCl. The quantum yield of the probe 1
The absorption spectra of probe 1 in the absence and presence of
different amounts of OClꢀ are shown in Fig. 3. Probe 1 only
exhibited an absorption peak centered at 338 nm, which was
ascribed to the absorption of xanthene moiety. Meanwhile, there
was almost no typical ring-opened spirolactam absorption in the
496 nm region. Upon addition of increasing concentrations of OClꢀ,
absorption peak at 496 nm enhanced significantly, indicating that
the OClꢀ promoted ring opening of spirolactam in the fluorescein
moiety, while the absorption of xanthene moiety at 338 nm red-
shifts to 368 nm with the peak intensity decreasing gradually.
During the titration a beautiful UVevisible spectral pattern was
with NaClO was
F
¼ 0.87, which is 1240-fold higher than that of 1,
at
F
¼ 0.0007. Notably, there was a good linear correlation between
the fluorescence intensity and the concentration of NaOCl (0e
1.0
m
M, R2 ¼ 0.9989) (seeing Supporting information, Fig. S1).
Furthermore, it was found that 1 has a detection limit of 40 nM, on
the basis of the signal-to-noise ratio (S/N ¼ 3), which makes it
sufficiently sensitive for application in living systems. Thus, probe 1
Fig. 2. The fluorescence spectra of 1 (10 mM) upon addition of 5 equiv of various metal
ions including of Liþ, Naþ, Kþ, Mg2þ, Ca2þ, Zn2þ, Cd2þ, Hg2þ, Pb2þ, Mn2þ, Cu2þ, Agþ
,
Fe2þ, Fe3þ, Co2þ, and Ni2þ in a H2O/DMSO (v/v ¼ 20/1, TriseHCl buffer, 0.05 M, pH 7.4)
Fig. 4. Fluorescence changes of probe 1 (5 mM) in the presence of various equivalents
solution. Inset: Fluorescence response of 1 (10
mM) to 5 equiv of various metal ions in a
of NaOCl (0e15 equiv) in a H2O/DMSO (v/v ¼ 20/1, TriseHCl buffer, 0.05 M, pH 7.4)
H2O/DMSO (v/v ¼ 20/1, TriseHCl buffer, 0.05 M, pH 7.4) solution.
solution. The excitation wavelength was 490 nm.

130
W. Yin et al. / Dyes and Pigments 107 (2014) 127e132
properties of 1 was investigated in a series of TriseHCl buffers with
different pH values ranging from 5 to 9 (Fig. 5).
The probe in TriseHCl buffer (pH 5e9.5) exhibits almost no
emission when excited at 490 nm and no distinct variations are
observed after 24 h, suggesting that probe 1 is stable under the
measurement condition. Fig. 5 indicted that the probe 1 is capable
of selectively detecting hypochlorite without interference by other
biological ROS in pH variations (5e9.5). Probe 1 itself were essen-
tially pH-insensitive across a wide range of pH values. However, the
fluorescent responses to the probe 1 towards NaOCl was pH-
dependent; the maximal sensing responses of probe 1 upon
treatment with 2 equiv of NaOCl was observed at pH 6.0e8.0 (pKa
of HOCl is 7.6), suggesting that the assay is compatible with most
biological applications (pH ¼ 7.4).
3.5. Biological applications
Probe 1 is well-suited to fluorescence imaging in living cells. As
determined by laser scanning confocal microscopy imaging exper-
Fig. 5. Fluorescence intensity of probe 1 (10 mM) at various pH values in a H2O/DMSO
(v/v ¼ 20/1, TriseHCl buffer, 0.05 M, pH 7.4) solution in the absence and presence of
ClOꢀ (2 equiv) (excitation at 490 nm, emission at 517 nm, emission slit 5 nm).
iments (Fig. 6), staining RAW264.7 macrophages cells with 10 mM
probe 1 at 37 ꢁC for 30 min exhibited faint fluorescence in the optical
window 490e550 nm with 488 nm exited wavelength (Fig. 6(a)).
appears to be highly sensitive and selective for the detection of
HOCl. Moreover, it is worth noting that probe 1 can rapidly respond
to NaOCl within seconds, making it a good candidate for real-time
detection of hypochlorite generation.
Treating the cells with 10 mM NaOCl for 10 min led to remarkable
fluorescence enhancement (Fig. 6(b)), indicating that probe 1 is
extremely sensitive for detecting exogenous OClꢀ in living cells.
These cells kept well their morphologies, and suggested that probe 1
do not exhibit marked cytotoxicity in our experimental conditions.
The images of cells were obtained using a confocal fluorescence
microscope. As shown in Fig. 6(a), when RAW264.7 cells were
3.4. The effect of pH on the fluorescence
incubated with probe 1 (10 mM), no fluorescence was observed in
To essay the practical application of probe 1 in the detection of
HOCl (pKa ¼ 7.6), the pH dependence of the photophysical
the RAW264.7 cells (Fig. 6(b)). After the treatment with NaOCl,
yellow-green fluorescence was observed in the RAW264.7 cells
Fig. 6. Fluorescence images of RAW264.7 cells. (Left) fluorescence image; (Middle) Bright-field image and (Right) overlay image. (a) The cells incubated with probe 1 (10
30 min. (b) Subsequent treatment of the cells with NaOCl (10 M) for 10 min. (c) The cells treated with stimulant PMA (25 ng/mL) for 2 h in the presence of for 30 min.
lex ¼ 488 nm, lem ¼ 490e550 nm).
mM) for
m
(

W. Yin et al. / Dyes and Pigments 107 (2014) 127e132
131
Scheme 2. Turn-on sensing of HOCl via HOCl-promoted probe 1 oxidation reaction (photos show fluorescence of probe 1 in a H2O/DMSO (v/v ¼ 20/1, TriseHCl buffer, 0.05 M, pH
7.4) solution).
Scheme 3. Proposed mechanism for the reaction of probe 1 with HOCl.
(Fig. 6(e)). An overlay of fluorescence and bright-field images
(Fig. 6(f)) show that the fluorescence signals are localized in the
intracellular area, indicating a subcellular distribution of HOCl and
the good cell-membrane permeability of probe 1.
Due to oxidation of sodium hypochlorite depends on pH value of
the solution, catechol moiety reacted with sodium hypochlorite to
form a higher concentration of chlorinated derivatives in lower or
higher pH [35], which did not lead to open ring of spirolactam. It is
agreement with Fig. 1. In the absence of ClOꢀ, probe 1 is colorless
and nonfluorescent due to the closed spirolactam ring. In the
presence of ClOꢀ (pH 6e8), the catechol moiety of probe 1 was
oxidized to form open-ring intermediate M, and then the M further
hydrolyzed to yellowish and strong fluorescent fluorescein in the
presence of water (Scheme 3).
Encouraged by these results as demonstrated above, we further
evaluated whether the probe 1 could be used to monitor endoge-
nous hypochlorite in living cells. Macrophages and other phago-
cytic cells are known to produce low micromolar levels of H2O2 or
other ROS during phagocytosis, or when stimulated by agents such
as phorbol myristate acetate (PMA) [35e37]. RAW264.7 macro-
phages were treated with PMA (25 ng/mL) for 2 h, after which the
cells were incubated with probe 1. After 30 min, the confocal
microscopic images of RAW264.7 macrophages exhibited intense
yellow-green fluorescence (Fig. 6(i)). The results suggested that
probe 1 could be utilized to investigate endogenous hypochlorite in
living cells.
4. Conclusion
In summary, a new fluorescein-based fluorescence probe 1 for
ClOꢀ has been synthesized and structurally characterized. Results
show that it has a detection limit of 40 nM to hypochlorite anion.
Moreover, most of background metal ions and anions showed small
or no interference with the detection of ClOꢀ. In addition, it has
favorable characteristics for biological imaging, including high
water solubility, high fluorescence yield and biocompatibility.
Confocal fluorescence microscopy imaging of RAW264.7 cells
showed that the new probe 1 could be used as an effective fluo-
rescent probe for detecting HOCl in living cells. We consider this
sensor may prove to be a useful tool for investigating the roles of
HOCl in biological and pathological processes.
3.6. Proposed mechanism for the reaction of probe 1 with HOCl
Zhang et al. reported [38] a rhodamine 6G hydrazide fluorescent
chemosensor for detecting HOCl. The hydrazide moiety of the
fluorescent probe might be oxidized by HOCl via an N-chlorination
and subsequent HCl elimination pathway to release the fluo-
rophore of rhodamine in the nucleophilic solvent.
To explorethe sensing mechanism of 1 toClOꢀ and determinethe
produced species, the reaction of 1 with 10 equiv. ClOꢀ was carried
out in H2O-DMSOsolution (v/v ¼ 20/1), which was carried out by TLC
and ESI-MS analysis. The formation of fluorescein as product was
confirmed by comparison of Rf value with that of authentic fluo-
rescein. The ESI mass spectra of the probe 1 solution before and after
the addition of HOCl were illustrated in Figs. S4 and S5 (seeing ESI).
The experimental results revealed that, with the presence of HOCl,
the peak of probe 1 (m/z 429.1 [MþH]þ) disappeared, while three
major new peaks emerged at m/z 621.58, 379.00 and 363.58,
respectively. The three peaks represented the formation of inter-
mediate oxide (I) and fluorescein (F) (m/z 621.58 [Iþ2DMSOþH]þ,
379.00 [FþC2H5OHþH]þ, 363.58 [FþCH3OHþH]þ). Based on the ESI-
MS results of the reaction products and TLC analyst, we proposed
that the fluorescence offeon reaction process might proceed as the
way depicted in Scheme 2.
Acknowledgments
We are grateful to financial support from the Jiangsu Key Lab-
oratory of Industrial Water Conservation & Emission Reduction and
the National Basic Research Program of China (973 Program, No.
2014CB846004) for financial support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2014.03.012.

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W. Yin et al. / Dyes and Pigments 107 (2014) 127e132
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