A highly sensitive and rapidly responding fluorescent probe based on a rhodol fluorophore for imaging endogenous hypochlorite in living mice

Article abstract of DOI:10.1039/c7tb02862h

Hypochlorous (HOCl) acid is generated as a defense tool in the immune system and plays a vital role in killing a wide range of pathogens. There is therefore great interest in developing fluorescent probes that can endogenously respond to the change in concentration of HOCl in vivo. To address this challenge, we here present a rapidly responding fluorescent probe RO610 to image endogenous HOCl in living mice. The development of RO610 was based on a novel water-soluble and pH-independent fluorescent xanthene dye, 2′-formylrhodol ROA, which exhibits highly selective and sensitive responses to HOCl/ClO^- over other reactive species. Moreover, adding a little more than 5 equiv. of ClO^- to the solution of RO610 resulted in a clearly observable fluorescence enhancement (48-fold) within 30 s. Based on these properties, RO610 was used to detect ClO^- in A549 cells without interference by other oxidants. It was applied for the imaging of endogenous HOCl in living nude mice with satisfactory results.

Full text of DOI:10.1039/c7tb02862h

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ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

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A highly sensitive and rapidly responding fluorescent probe
based on a rhodol fluorophore for imaging endogenous
hypochlorite in living mice
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
b
a
a
a
Yanhui Zhang , Lin Ma , Chunchao Tang , Shengnan Pan , Donglei Shi , Shaojing Wang ,
Minyong Li b,* and Yuan Guo a,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Hypochlorous (HOCl) acid is generated as a defense tool in the immune system and plays a vital role in killing a wide range
of pathogens. There is therefore great interest in developing fluorescent probes that can endogenously respond to the change
in concentration of HOCl in vivo. To address this challenge, we here present a rapidly responding fluorescent probe RO610
to image endogenous HOCl in living mice. The development of RO610 was based on a novel waterꢀsoluble and pHꢀ
independent fluorescent xanthene dye, 2'ꢀformylrhodol ROA, which exhibits highly selective and sensitive responses to
HOCl/ClOꢀ over other reactive species. Moreover, adding a little more than 5 equiv. of ClOꢀ to the solution of RO610
resulted in a clearly observable fluorescence enhancement (48ꢀfold) within 30 s. Based on these properties, RO610 was used
to detect ClOꢀ in A549 cells without interference by other oxidants. It was applied for the imaging of endogenous HOCl in
living nude mice with satisfactory results.
the availability of appropriate fluorescent molecules. Although a
number of fluorescent probes have been reported based on traditional
fluorophores, some drawbacks can be encountered, such as single
Introduction
Hypochlorous acid (HOCl)/hypochlorite (ClOꢀ) is known to be
one of the biologically important reactive oxygen species
(ROS).1 It is widely employed in our daily life as a cobleaching
agent and a disinfectant for potable water.2 Biologically, the
generation of endogenous hypochlorite is driven by hydrogen
peroxide and chloride ions in activated neutrophils catalyzed by
emission intensity, low quantum yield and short emission
wavelength.6 There is therefore a growing need to develop a
fluorescent probe which is able to overcome these limitations based
on novel and efficient fluorophores. In recent years, with the hybrid
structure of fluorescein and rhodamine, rhodol fluorophores and
derivatives thereof (also named “Rhodafluor”)7,8 have been found to
be excellent and interesting candidates for fluorescent probes since
they inherit all the superior photophysical properties from
fluorescein and rhodamine, such as high extinction coefficients,
quantum yield, photostability and solubility in a variety of solvents.
Based on the relationships between the structures of rhodol
derivatives and their excellent photophysical and photochemical
properties, we presumed that the reasonable modification of the
xanthene skeleton of rhodol would allow us to optimize the
photophysical properties of rhodol dyes for the desired applications.
Herein, we present a new rhodol dye, ROA (2'ꢀformylrhodol), which
readily dissolves in water in the form of a ringꢀopened zwitterion
structure. Moreover, it has high quantum yields (Φ = 0.94, measured
in ethanol) and displays strong tolerance to pH, being capable of
generating stable optical signals in the pH range of 5.5ꢀ9. More
interestingly, colorimetric and ratiometric sensing systems can be
established by modifying or using directly the active formyl,
carboxyl and hydroxyl of ROA.
a
hemeꢀcontaining enzyme myeloperoxidase (MPO).3
Nevertheless, abnormal levels of hypochlorite can contribute to
tissue damage and diseases such as nephropathy,
atherosclerosis, cardiovascular disease and cancers.4 In order to
comprehend the vital role hypochlorite plays in the biosystem,
it is important to develop techniques for realꢀtime monitoring
and accurate detection of ClOꢀ. In particular, fluorescent probes
have become a powerful tool for sensing trace amounts of species
in chemical and biological samples due to the high sensitivity,
specificity, simplicity of implementation and fast response of
fluorescence detection.5 Unfortunately, the feasibility of using
fluorescence methods for a particular application is often limited by
We also developed a novel “turnꢀon” fluorescence probe
RO610 via the condensation reaction between the formyl group
of ROA as a fluorophore and diaminomaleonitrile. It is known
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that C=N isomerization is the predominant decay process of A stock solution of RO610 (1.0 mM) was prepared in acetone.
excited states in compounds with an unfixed C=N structure.9 Stock solutions (10 mM) of various analytes were prepared by
Thus, as expected, RO610 itself was nearly nonꢀfluorescent due dissolving an appropriate amount of the test species in water.
•
1
ꢀ
to C=N isomerization, while the strong yellow fluorescence Various ROS and RNS including ClOꢀ, H2O2, OH, O2, O2 ,
was restored upon addition of ClOꢀ. The phenomenon occurred NO•,
by the formation of a formyl group which was caused by reports.11 All UVꢀvis and fluorescence titration experiments
oxidation and hydrolysis of C=N. Most notably, the were performed for min at room temperature using
t
ꢀBuOOH and TBO• were prepared according to previous
5
fluorescenceꢀon reaction showed high sensitivity (LOD, EtOH/phosphate buffer (20 mM, pH 7.4, 3:7) solution. Any
2.88×10−8 M), high selectivity and a rapid response time (30 s) changes in the fluorescence intensity were monitored using a
for ClOꢀ over other common oxidants and nucleophiles, which fluorescence spectrometer (λex = 535 nm, λem = 577 nm, slit: 5
makes it practical for detecting ClOꢀ in cells and mice.
nm/5 nm).
Measurement of fluorescence quantum yields
The fluorescence quantum yields were determined using
Rhodamine B as a reference with a known
EtOH.12 The sample and the reference were excited at the same
wavelength (λex 495 nm), maintaining nearly equal
Φ value of 0.89 in
=
absorbance (0.05). The quantum yield was estimated following
the equation (1):
2
2
ΦS
/
ΦR = (AS
/
AR) × (AbsR
/
AbsS) × (ŋS
/
ŋR
)
(1)
where ΦS and ΦR 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 absorbances of the sample
and the reference solution at the excitation wavelength of
excitation; ŋS and ŋR are the refractive indices of the sample and
the reference, respectively.
Cytotoxicity assay
The methyl thiazolyl tetrazolium (MTT) assay with A549 was
used to identify the cytotoxic potential of RO610. 90%
confluence A549 cells were seeded into a 96ꢀwell cellꢀculture
plate with density of 5000 cells per hole. After incubating for
24 h, different concentrations of RO610 (5, 10, 15, 20, 25, 30
µM) were added to the wells. The cells were treated and
incubated at 37 °C under 5% CO2 for another 24 h. 10 ꢁL MTT
was added to each well and incubated for 4 h in the same
conditions. The MTT solution was removed and yellow
precipitates (formazan) observed in the plates were dissolved in
200 ꢁL DMSO. The absorbance was recorded at 490 nm using
the microplate spectrophotometer system (Varioskan Flash).
The viability of the cells was calculated by the ratio of mean of
the absorbance value of the treatment group and the control
group.
Fig. 1 (A) Synthetic route of the ROA. (B) The crystal structure of
spirocyclized ROA. (C) Synthesis of the probe RO610
.
Materials and methods
Materials and instruments
Unless otherwise specified, all materials were obtained from
commercial suppliers and used without further purification. All
inorganic salts used were of analytical grade. Twiceꢀdistilled
water was used throughout the experiments. A Mettler Toledo
pH meter was used to determine the pH 1H NMR and 13C NMR
spectra were recorded on
a
VARIAN INOVAꢀ400
spectrometer, using TMS as an internal standard. Mass
spectrometry data were obtained with an AXIMAꢀCFR plus
MALDIꢀTOF Mass Spectrophotometer. UVꢀvisible spectra
were collected on a Shimadzu Uꢀ1700 spectrophotometer.
Fluorescence measurements were performed on a Hitachi Fꢀ
4500 fluorescence spectrophotometer with a xenon discharge
lamp, in a 1 cm quartz cell. HPLC spectra were collected on an
Agilent HP1100 high performance liquid chromatograph. The
ability of probes reacting to hypochlorite in the cells was also
evaluated by laser confocal fluorescence imaging using an
Olympus FV1000 laser scanning microscope.
Animals
All animal studies were approved by the Ethics Committee and
IACUC of Qilu Health Science Center, Shandong University,
and were conducted in compliance with European guidelines
for the care and use of laboratory animals. Imaging procedures
were conducted with adult mice (4ꢀ6 weeks old, 15ꢀ18 g,
Shandong University, Jinan, China) under general anesthesia by
injection of sodium pentobarbital (0.5 ml/0.03%).
Results and discussion
General UV-vis and fluorescence spectra measurements
Synthesis and characterization
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Synthesis of Compound 3. 1.00
g
(6.05 mmol) 3ꢀ Firstly, the photochemical properties of ROA in solvents with
diethylaminophenol and 1.07
g (7.26 mmol) oꢀphthalic different polarities were investigated by fluorescence
anhydride were gradient heated in 15 mL methylbenzene for 10 measurements (Fig. 2a). In polar solvent, the ROA was ringꢀ
h, 80 °C; 5 h, 90 °C; 2 h, 100 °C; 1 h, 110 °C. Then, the opened and exhibited a strong fluorescence intensity at 577nm,
reaction mixture was refluxed for 1 h. After the reaction was which was attributed to the πꢀπ* transitions of the ringꢀopened
complete, the mixture solution was cooled then filtered and xanthene conjugate. In order to verify the fluorescence
washed with methanol. The pink solid thus obtained was dried mechanism further, the fluorescence features were investigated
to give compound
3
in 25% yield. 1H NMR (400 MHz, DMSOꢀ by TDꢀDFT calculations. Based on the optimized geometry of
d6): δ ppm 13.11 (s, 1H), 12.59 (s, 1H), 7.97 (d, J = 7.2 Hz, ROA, it was found that the strongest fluorescence peak came
1H), 7.68 (t, J = 7.2 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.37 (d, J from the HOMOꢀLUMO transition, and partly from the
= 7.2 Hz, 1H), 6.80ꢀ6.78 (m, 1H), 6.17 (d, J = 9.2 Hz, 1H), 6.08 HOMOꢀ1ꢀLUMO transition. The three orbitals (HOMOꢀ1,
(s, 1H), 3.37 (q, J = 6.4 Hz, 4H), 1.08 (t, J = 6.2 Hz, 6H).
Synthesis of Compound ROA. A mixture of 300 mg (0.96 plane of ROA (shown in Fig. S10), corresponding to the πꢀπ*
mmol) and 132 mg (0.96 mmol) 2,4ꢀdihydroxybenzaldehyde transition rather than to ICT between diethylin and formyl
HOMO and LUMO) are π type distributed on the conjugate
3
was added in 5 mL of sulfuric acid and stirred at 90 °C groups. In contrast, ROA was spirocyclized without any
monitoring with TLC. After the reaction was complete, the fluorescence in aprotic solvent. These results indicated that
mixture solution was cooled to room temperature and then ROA is sensitive to the solvent. Furthermore, we obtained the
poured into iceꢀcold water (100 mL). After adjusting the pH crystal structure of spirocyclized ROA (CCDC number:
value of the mixture to 7ꢀ8 with ammonium hydroxide, the 1588170, Fig. 1, Fig. S3 and Table S1) by slowly evaporating
precipitate was filtered, washed with brine, and then dried its dichloromethane solution at room temperature which also
under vacuum to give the crude product, which was further proved that the spirolactone structure of ROA is stable in the
purified by chromatography on
a
silica gel column most aprotic organic solvents. Conversely, it showed a ringꢀ
(CH2Cl2:CH3OH = 300:1 v:v) to give a red product ROA in opened πꢀconjugate form in the protic solvent resulting in
1
30% yield. H NMR (400 MHz, CDCl3): δ ppm 11.19 (s, 1H), strong fluorescent emission. Therefore, the compound ROA
9.57 (s, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.68 (m, 2H), 7.22 (d, J = can be easily modified by chemical approaches because of its
7.6 Hz, 1H), 7.01 (s, 1H), 6.80 (s, 1H), 6.57 (d, J = 8.8 Hz, 1H), various modifiable sites of interconversion structures in
6.48 (s, 1H), 6.39 (d, J = 8.4 Hz, 1H), 3.37 (q, J = 6.8 Hz, 4H), different solvents, which is of paramount importance for the
1.18 (t, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3): 194.62, molecular design of the compound as a signaling fluorophore
169.28, 162.91, 157.83, 152.47, 152.19, 149.71, 135.49, for chemosensors and biological applications (Fig. S1). A
135.07, 129.83, 128.72, 126.94, 125.92, 125.07, 123.86, continuous pH titration of ROA in water revealed that there
117.80, 113.24, 108.86, 104.45, 104.37, 97.67, 82.99, 44.45, was enhanced emission at 577 nm from pH 1.5 to 12 (Fig. 2b).
12.38; ESIꢀHRMS: [M+H]+ m/z 416.1483, calcd for We also found that the response to changes in pH fitted a
C25H22NO5 416.1492; FTꢀIR υ (KBr, cmꢀ1): 3672.28, 1750.47, sigmoid curve in a pH range of 2ꢀ6 and 9ꢀ11.5 (see Fig. S2a).
1625.98, 1521.23, 1405.66, 875.84, 801.42, 697.41.
Synthesis of the Probe RO610. To a suspended solution of HendersonꢀHasselbachꢀtype mass action equation (see Fig.
compound ROA (83.05 mg, 0.2 mmol) in CH3OH:CH2Cl2 = S2b). Interestingly, ringꢀopened ROA showed stable
The pKa of 4.54 and 11.5 were calculated by using the
a
5:3 (4 mL) was added an excess of 2,3ꢀdiaminoꢀ2ꢀ fluorescence signal from pH 5.5 to 9, implying that the pH
butenedinitrile (32.43 mg, 0.3 mmol) and the solution was independent properties meet the requirements of fluorescent
refluxed and monitored with TLC. After the reaction was labels or tracers for biological applications.
complete, the mixture solution was cooled to room temperature.
The yellow solid formed was filtered and washed with a small
amount of methanol several times. 64.67 mg brown probe
RO610 was dried in vacuum and then obtained with a yield of
1
64%. H NMR (400 MHz, Acetoneꢀd6): δ ppm 8.46 (s, 1H),
7.97 (d, J = 7.2 Hz, 1H), 7.80 (t, J = 7.2 Hz, 1H), 7.73 (t, J = 7.6
Hz, 1H), 7.39 (s, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.13 (s, 2H),
6.79 (s, 1H), 6.57 (d, J = 9.6 Hz, 1H), 6.52ꢀ6.51 (m, 2H), 3.44
(q, J = 6.8 Hz, 4H), 1.17 (t, J = 6.8 Hz, 6H); 13C NMR (100
MHz, Acetoneꢀd6) δ 210.57, 173.79, 166.24, 164.64, 160.69,
157.79, 157.61, 154.96, 140.30, 139.57, 135.06, 133.98,
132.50, 130.39, 129.78, 129.24, 121.78, 119.14, 118.43,
117.93, 114.16, 110.55, 109.77, 108.52, 102.56, 49.30, 17.07;
HRMS (ESI): [M+H]+ m/z 506.1831, calcd for C29H24N5O4
506.1823; FTꢀIR υ (KBr, cmꢀ1): 3432, 2976, 2233, 2205, 1739,
1626, 1501, 1373, 1264, 1215, 1108, 874, 767, 692, 546.
Optical properties of ROA
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Fig. 3 (a) Kinetic analysis of RO610 towards ClOꢁ (0, 6, 20, 50 ꢂM).
(b) Fluorescence emission spectra of RO610 (10 ꢀM) upon the
addition of increasing concentrations of sodium hypochlorite (0–50
ꢀM). Inset: linear plot of emission changes at 577 nm against ClOꢁ
concentration (λex = 535 nm, λem = 577 nm, slit: 5 nm/5 nm).
We then implemented the titration experiments with
addition of ClOꢀ in the RO610 solution (10 µM) to evaluate the
quantitative detection capacity and detection limit. As shown in
Fig. 3b, RO610 displays weak fluorescence (fluorescence
quantum yield
Φ
= 0.04). When ClOꢀ was titrated from 0 to 50
µM, the mixture of RO610 exhibited a 48ꢀfold enhancement of
fluorescence intensity over that of blank (fluorescence quantum
Fig. 2 (a) Emission spectra of ROA (10 ꢀM) in different solvents. (b)
in different pH solutions. Inset: Fluorescent intensity of ROA at 577
nm under different pH conditions. (λex = 515 nm, λem = 577 nm, slit: 5
nm/5 nm).
yield
Φ
=0.35), and the phenomenon of brightness
enhancement could be observed by the naked eye under a 365
nm UV lamp. This can be attributed to the formation of ROA
by the reaction of RO610 with ClOꢀ, which was confirmed by
1H NMR and HRMS. Additionally, the UVꢀvis spectra of
RO610 exhibited maximum absorption at 572 nm. Upon
addition of ClOꢀ, the absorption at 572 nm decreased, whereas a
new absorption peak appeared at 542 nm which changed the
color of the solution from purple to pink, indicating the
colorimetric detection of ClOꢀ (Fig. S4). The absorption and
emission spectra showed that the changes reached a plateau
when the concentration of ClOꢀ increased to 50 ꢁM. What is
more, the fluorescence intensity of RO610 has a good linear
relationship with the ClOꢀ concentration ranging from 0 to 20
µM, suggesting that the probe is potentially useful for
quantitatively detecting ClOꢀ. Accordingly, the detection limit
(3σ/slope) was as low as 2.88×10ꢀ8 M.
Kinetic studies of RO610 towards ClO- and titration
experiments
To test the sensing ability of the probe RO610 for ClOꢀ, we
studied its optical spectrum in phosphate buffer:EtOH = 7: 3
(V/V, 20 mM, pH = 7.4) at room temperature (25 °C). Fig. 3a
shows the response time chart for the chemosensor upon
contact with ClOꢀ under these conditions. The data indicate that
the fluorescent intensity increased dramatically in 30 s and then
leveled off, while the fluorescence background of the detection
system in the absence of ClOꢀ remained unchanged at the same
time. To obtain a high sensitivity and reproducible results, a 5
min reaction time was selected in the following experiment.
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probe RO610 (10 ꢀM) upon the addition of several analytes and inset:
visual fluorescence color (under a handheld 365 nm UV lamp) changes (1:
ꢁ
Blank; 2: Fꢁ; 3: Clꢁ; 4: Brꢁ; 5: Iꢁ; 6: S2O32ꢁ; 7: AcOꢁ; 8: SO42ꢁ; 9: NO2 ; 10:
ꢁ
ꢁ
NO3 ; 11: CNꢁ; 12: HSꢁ; 13: SCNꢁ; 14: PO43ꢁ; 15: H2PO4 ; 16: HPO42ꢁ; 17:
ꢁ
ꢁ
ꢁ
CO32ꢁ; 18: HCO3 ; 19: HSO4 ; 20: HSO3 ; 21: SO32ꢁ; 22: Cys; 23: Hcy; 24:
GSH; 25: ClOꢁ) (λex = 535 nm, λem = 577 nm, slit: 5 nm/5 nm).
Selectivity of RO610 towards ClO-
As achieving a high selectivity toward the target molecule is a
major issue in the field of molecular sensing, we tested the
sensing performance of the probe RO610 towards various
reactive oxygen species (ROS) and reactive nitrogen species
(RNS), including sodium hypochlorite (NaOCl), hydrogen
peroxide (H2O2), hydroxyl radicals (•OH), superoxide (O2 ),
ꢀ
singlet oxygen (1O2), nitric oxide (NO•), tertꢀbutyl hydroꢀ
peroxide (TBHP) and tertꢀbutoxy radical (TBO•) in a phosphate
buffer solution. Strong yellow fluorescence was only observed
upon the addition of ClOꢀ to a solution containing RO610; the
other ROS and RNS produced no obvious change in
fluorescence (Fig. 4). The same result was obtained by the UVꢀ
vis spectrum (Fig. S6). Considering that the nucleophilic
addition of common anions and biothiols might act on the
position of the dicyanovinyl groups present in the chemosensor
and thus break the conjugation, which ultimately offsets the
effect of C=N isomerization and leads to fluorescence
enhancement, the fluorescent response of RO610 towards
common anions and biothiols was explored (Fig. 5). When 200
Fig. 4 (a) Fluorescence spectra of probe RO610 (10 ꢀM) in the
ꢁ
presence of 200 ꢀM analytes (H2O2, •OH, O2 , 1O2, NO•, TBHP, TBO•)
and 50 ꢀM ClOꢁ. (b) Optical density graph of the emission at 577 nm
of probe RO610 (10 ꢀM) upon the addition of several analytes for 5
min and inset: visual fluorescence color (under a handheld 365 nm
UV lamp) changes. (λex = 535 nm, λem = 577 nm, slit: 5 nm/5 nm).
ꢀ
ꢀ
µM anions (Fꢀ, Clꢀ, Brꢀ, Iꢀ, S2O32ꢀ, AcOꢀ, SO42ꢀ, NO2 , NO3 , CNꢀ,
ꢀ
ꢀ
ꢀ
ꢀ
HSꢀ, SCNꢀ, PO43ꢀ, H2PO4 , HPO42ꢀ, CO32ꢀ, HCO3 , HSO4 , HSO3 ,
SO32ꢀ) and 200 µM biothiols (Cys, Hcy, GSH) were added to 10
µM RO610 in phosphate buffer, none of the ions produced a
noticeable change in the fluorescence intensity, indicating no
response toward the RO610 probe. Moreover, other analytes
showed negligible changes in absorbance spectra under the
same conditions. Thus, these results suggest that RO610
possesses
a a potential
higher selectivity for ClOꢀ and
application for detecting ClOꢀ in complex biological
environments.
Fig. 6 ESIꢁMS spectra of mixture of RO610 (10 ꢀM) with 5 equiv. of NaOCl
in methanol.
Fig. 5 (a) Fluorescence spectra of probe RO610 (10 ꢀM) in phosphate
buffer : EtOH = 7 : 3 (V/V, 20 mM, pH = 7.4) in the presence of 200 ꢀM of
various analytes; (b) Optical density graph of the emission at 577 nm of
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Fig. 7 1H NMR spectrum of probe RO610 and the product of the
complete reaction mixture of the probe RO610 with 5 equiv. ClOꢁ in
Actoneꢁd6.
N
O
O
Cl
N
NH2
CN
NC
HN
CN
HOOC
OCl CN
NH2
H2
O
Cl
ClO
O
N
O
Cl
N
NH2
CN
N
O
O
OCl
OH
HOOC
CN
HOOC
Fig. 8 Confocal fluorescence images of A549 cells. Fluorescence
image (aꢁd) and merge (eꢁi) of A549 cells pretreated with 5 ꢀM probe
RO610 for 10 min and then followed by incubation with 0, 5, 15, 25
ꢀM of ClOꢁ for 10 min, respectively. The cells were excited with a light
laser at 543 nm, and emission was collected at 555ꢁ655 nm. Scale
bar: 20 ꢀm.
HClO
H2O
NaClO
Cl
N
O
O
N
O
O
NH2
O
N
CN
COOH
COOH
CN
Bioimaging and cytotoxicity studies
ROA
RO610
NC
CN
Encouraged by the desirable optical response in aqueous
solution, we then explored the capability of RO610 to monitor
ClOꢀ in a cellular context using confocal fluorescence imaging.
Prior to that, an MTT assay was conducted to evaluate the
cytotoxicity of RO610. The cellular viability was estimated to
be greater than 80% after 24 h, indicating that the probe (<30
ꢁM) has low cytotoxicity (Fig. S8). The A549 cells incubated
with only RO610 (5 ꢁM, 10 min) displayed very dim
fluorescence (Fig. 8). However, after the treatment with 5 ꢁM,
15 ꢁM and 25 ꢁM ClOꢀ, a significant enhancement of the
fluorescence was observed in the yellow channel and it was
clearly seen that the fluorescence intensities of the cells rose
along with an increase in ClOꢀ concentration. These data
indicate that probe RO610 is cell membrane permeable and
capable of imaging of ClOꢀ in cells.
H2N
NH2
Scheme 1 The proposed recognition mechanism of RO610 towards
hypochlorite.
The proposed ClO- sensing mechanism of RO610
In order to investigate the mechanism of the reaction of probe
RO610 with ClOꢀ, a series of experiments was performed. The
proposed mechanism is shown in Scheme 1. According to the
coincident peak at normalized emission and absorbance spectra
(Fig. S7), we assumed that the product of this probe oxidized
by ClOꢀ was ROA, which was confirmed by the ESIꢀMS
spectra of the reaction mixture of the probe with 5 equiv. ClOꢀ.
The ESIꢀMS showed the presence of a dominant peak at m/z =
416.1485, which corresponded to ROA (m/z = 416.1492
[ROA+H]+) (Fig. 6). 1H NMR spectra also confirmed the
1
hypochlorite mediated conversion of RO610 to ROA. The H
NMR titrating experiment revealed that the presence of ClOꢀ in
the RO610 solution caused the disappearance of the signal at
8.46 ppm (ꢀCH=N proton of RO610) and the appearance of a
new signal at 9.85 ppm (ꢀCHO proton of ROA) (Fig. 7).
However, as shown in Fig. 7, the attribution of some peaks
could not be specified. We suspected that there could be byꢀ
products generated, combined with the above result of no clear
isosbestic point in the UVꢀvis spectrum (Fig. S4). An HPLC
experiment was therefore carried out to confirm the conversion
yield of ROA. The results showed that ROA was still the main
product and that the yield of ROA calculated by the
normalization method was 89% (Fig. S11).
Fig. 9 Representative fluorescence images (pseudocolor) of a mouse
given a skinꢁpop injection of RO610 (25 ꢀL, 1 mM, right abdomen) and a
subsequent (10 min later) skinꢁpop injection of ClOꢁ (5.0 equiv.) in the
same region. Images were taken after incubation for 0, 10 and 20 min,
respectively. Images were taken using an excitation laser at 530 nm and a
600 nm emission filter.
The suitability of the probe for visualizing ClOꢀ in living
was also evaluated. All these images were obtained from
normal mice without unhairing. The mouse was given an s.p.
(skinꢀpop) injection of RO610 (1 mM, 25 µL DMSO) on the
right abdomen, and 10 minutes later, the mouse was given an
6 | J. Name., 2012, 00, 1ꢀ3
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s.p. injection of 5.0 equiv. of ClOꢀ in the same region. The worthwhile tool for investigating the roles of ClOꢀ in biological
mouse was imaged by using an IVIS Spectrum small animal in and pathological processes.
vivo imaging system with a 530 nm excitation laser and a 600
nm emission filter. Fig. 9 shows representative fluorescence
images of a mouse at different times after the injection of ClOꢀ.
It was demonstrated that the fluorescence intensity was
enhanced gradually within 20 min, proving that RO610 can
detect ClOꢀ in vivo without the interference of background
signals.
Conflicts of interest
The authors have no conflicts of interest to declare.
Acknowledgements
This work was supported by the National Natural Scientific
Foundation of China (No. 21472148 and 21072158), Overseas
Students Science and Technology Activities Project Merit
Funding of Shaanxi Province (No. 20151190) and Academic
Backbone of Northwest University Outstanding Youth Support
Program.
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Fig.10 (Left) Representative fluorescence images of a nude mouse. a,
The mouse was given an intraperitoneal injection of saline (1 mL) for
5 h, finally a intraperitoneal injection of RO610 (0.2 mL, 1 mM). b,
The mouse was given an intraperitoneal injection of zymosan (1 mL,
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In summary, a new rhodol dye ROA with an aldehyde group
has been successfully developed, which exhibits high extinction
coefficients, quantum yield, and good photostability. Based on
the model platform, RO610 was synthesized and it displayed a
high sensitivity, selectivity and short response time for ClOꢀ over
other related species. A549 cells and living mice imaging
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fluorescent probe for detecting ClOꢀ in biological systems.
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Journal of Materials Chemistry B
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DOI: 10.1039/C7TB02862H
A highly sensitive and rapidly responding fluorescent probe based
on a rhodol fluorophore for imaging endogenous hypochlorite in
living mice
Yanhui Zhang a, Lin Ma b, Chunchao Tang b, Shengnan Pan a, Donglei Shi a, Shaojing Wang a, Minyong
Li b, * and Yuan Guo a,
*
Graphical abstract
A novel fluorescent probe RO610 was applied for the in vivo detection of endogenous ClO- using
living mice.