Reversible Ratiometric Fluorescent Probe for Sensing Bisulfate/H2O2 and Its Application in Zebrafish

Article abstract of DOI:10.1021/acs.analchem.7b01580

Herein, a novel near-infrared fluorescent probe for ratiometric detection of bisulfate was designed and developed based on a conjugation of naphthopyran-benzothiazolium system. The sensor showed excellent selectivity, high sensitivity and a rapid response toward bisulfite in aqueous solution. Upon the addition of HSO₃^-, the sensor displayed 37-fold (I₅₂₀/I₆₃₀) fluorescence intensity enhancement, accompanied by an apparent color change from violet to colorless, suggesting that the sensor can be used to detect HSO₃^- with naked-eye . Notably, the addition product can be applied to the design of regenerative chemodosimeters based on the H₂O₂ promoted elimination of bisulfite and recovery of probe 1. Further cell and zebrafish imaging experiment demonstrated that the sensor could image the bisulfite/H₂O₂ redox cycle in biological system with ratiometric manners.

Full text of DOI:10.1021/acs.analchem.7b01580

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Article
Reversible ratiometric fluorescent probe for sensing
bisulphate/H2O2 and its application in zebrafish
weijie zhang, tao liu, Fangjun Huo, Peng Ning, Xiangming Meng, and Caixia Yin
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017
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Page 1 of 6
Analytical Chemistry
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Reversible ratiometric fluorescent probe for sensing
bisulphate/H O and its application in zebrafish
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†
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‡
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†,
§
Weijie Zhang, §Tao Liu, Fangjun Huo, Peng Ning, Xiangming Meng, Caixia Yin *
†
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for
Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030ꢀ006,
China.
‡
Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China
ǁ
Department of Chemistry, Anhui University, Hefei, 230601, China
*Corresponding author. * Eꢀmail: yincx@sxu.edu.cn. Tel/Fax: +86ꢀ351ꢀ7011022.
ABSTRACT: Herein, a novel nearꢀinfrared fluorescent probe for ratiometric detection of bisulphate was designed and developed
based on a conjugation of naphthopyranꢀbenzothiazolium system. The sensor showed excellent selectivity, high sensitivity and a
ꢀ
rapid response toward bisulfite in aqueous solution. Upon the addition of HSO , the sensor displayed 37ꢀfold (I520/I630) fluorescence
3
intensity enhancement, accompanied with an apparent color change from violet to colorless, suggesting that the sensor can be used
ꢀ
to detect HSO3 with “nakedꢀeye”. Notably, the addition product can be applied to the design of regenerative chemodosimeters
based on the H O promoted elimination of bisulfite and recovery of probe 1. Further cell and zebrafish imaging experiment
2
2
demonstrated that the sensor could image the bisulfite/H O redox cycle in biological system with ratiometric manners.
2
2
Recently, studies have suggested that bisulfite could be enꢀ
dogenously generated during the decomposition of sulfurꢀ
containing amino acids by reactive oxygen species (ROS),
which could induce oxidative stress, and diseases caused by
As a consequence, an ideal fluorescent probe for reversible
monitoring the intracellular bisulfite/ROS is badly needed. It
is worth pointing out that none ratiometric fluorescent probes
for imaging the bisulfite /ROS redox cycle in living body has
been reported.
1
ꢀ4
aging . H O , as one type of reactive oxygen species (ROS)
2
2
is an inevitable byproduct of cell metabolism and a common
To address the above issues, a novel fluorescent probe was
synthesized based on the naphthopyran and benzothiazolium
cation as described in the Scheme 1. Cyanine dyes usually
possess large Stokes shifts and excellent water solubility. And
the hemicyanine fluorophore was reported to be able to capꢀ
5
ꢀ7
marker and signal molecule of oxidative stress. Sulfur dioxꢀ
8
ide (SO ), as a toxic environment pollutant, is usually associꢀ
2
ated with the symptoms of neurological disorders, cardiovasꢀ
9
cular diseases, and lung cancer. The toxicity of SO is mainly
2
2ꢀ
23,24
attributed to its two reductive derivatives: sulfite (SO ) and
bisulfite (HSO ) (3 : 1 M/M, in neutral fluid). Therefore, it is
3
ture nucleophilic agents via 1,2ꢀ or 1,4ꢀaddition.
Therefore,
ꢀ
10
3
we speculate that the potential reaction sites may respond to
2ꢀ/
ꢀ
meaningful and valuable to develop efficient methods for
monitoring bisulfite/ROS redox cycle processes with different
fluorescence signals, which is vital for biological research as
well as clinical diagnoses.
SO3 HSO3 with ratiometric manner, and the original probe
could be regenerated with reversible fluorescent responses
towards H O .
2
2
Fluorescent probes for realꢀtime sensing and imaging are
indispensable tools in life science and materials science,
11
Scheme 1. Diagram of probe 1 for bisulphate/H
tion.
O detec-
2 2
providing an excellent detection technique that can identify
appropriate signals immediately and provide useful inforꢀ
mation in a short time. To date, lots of fluorescent probes for
2ꢀ/
ꢀ
SO3 HSO3 detection have been reported and the reaction
mechanisms can be sorted into two types, one is the nucleoꢀ
1
2ꢀ17
philic addition to aldehydes/ketones,
vantage as it may suffer from the interference of thiols,
and the other is the nucleophilic addition to double bonds,
which has a disadꢀ
18, 19
EXPERIMENTAL SECTION
20ꢀ21
especially the unsaturated compound addition reaction,
which can avoid the thiols interference. Along with the rapid
development of fluorescent probes for SO3 HSO , consideraꢀ
Materials and Chemicals. All chemicals were purchased
from commercial suppliers and used without further purifiꢀ
cation. All solvents were purified prior to use. Distilled
water was used after passing through a water ultraꢀ
purification system. TLC analysis was performed using
precoated silica plates. Hitachi Uꢀ3900 UVꢀvis spectrophoꢀ
2
ꢀ/
ꢀ
3
ble advances have been made in recent years. However, to the
best of our knowledge, only a few fluorescent probes could
22
realize reversible sensing for sulfur dioxide and peroxides.
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Analytical Chemistry
Page 2 of 6
tometer was employed to measure UVꢀvis spectra. Hitachi MHz, DMSO) δ 9.72 (s, 1H), 8.46 (s, 1H), 8.24 (d, J = 8.4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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F−7000 fluorescence spectrophotometer was employed to
measure fluorescence spectra. Shanhai Huamei Experiment
Instrument Plants, China provided a POꢀ120 quartz cuvette
Hz, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H),
7.65 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.19 (d, J
13
= 8.9 Hz, 1H), 5.06 (s, 2H). C NMR (151 MHz, DMSO)
δ 190.9 (s), 155.4 (s), 138.3 (s), 134.5 (s), 130.8 (s), 129.9
(s), 129.4 (s), 129.2 (s), 128.5 (s), 125.1 (s), 122.1 (s),
117.9 (s), 114.3 (s), 63.0 (s). ESI–MS: m/z Calcd 211.0754,
Found 211.0755 (Fig. S1).
1
13
(
10 mm). H NMR and C NMR experiments were perꢀ
formed with a BRUKER AVANCE III HD 600 MHz and
151 MHz NMR spectrometer, respectively (Bruker, Billeriꢀ
ca, MA). Coupling constants (J values) are reported in
hertz. ESI determinations were carried out on AB Triple
TOF 5600plus System (AB SCIEX, Framingham, USA).
The cell imaging experiments were measured by an Olymꢀ
pus FV1200 confocal laser scanning microscope. The
zebrafish imaging experiments were measured by an
Olympus FV1200 confocal laser scanning microscope.
Synthesis of (E)-2-(2-(3H-benzo[f]chromen-2-yl)vinyl)-3-
methylbenzo[d]thiazol-3-ium iodide (Probe 1). Compound 2
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
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0
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9
0
(0.105 g, 0.5 mmol) and 2,3ꢀdimethylbenzothiazolium iodid
(0.204 g, 0.7 mmol) were dissolved in 10 mL EtOH
with piperidine 30 µL. The reaction mixture was refluxed with
stirring for 12 h and then cooled to r.t., the precipitate was
filtered, washed with cold ethanol and dried in vacuum to
afford the desired product as a dark brown solid (0.2 g, 84%).
Preparation of Solutions of Probe 1 and Analytes. Stock
solution of probe 1 (2 mM) was prepared in DMSO. Stock
1
−
2−
H NMR (600 MHz, DMSO) δ 8.43 (d, J = 7.9 Hz, 1H), 8.32
solutions (2 mM) of Cys, Hcy, GSH, H S, CN , CO
,
2
3
2−
−
−
−
−
2−
−
−
−
−
(s, 1H), 8.23 (d, J = 8.5 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.06
(d, J = 15.8 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 7.9
Hz, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.79 (t, J = 7.5 Hz, 1H), 7.67
SO4 ,F , Cl , Br , I , HPO , H PO , NO , SCN , HCO ,
4
2
4
3
3
2−
−
−
2−
−
S2O3 , N , AcO , SO and HSO , were prepared by direct
3
3
3
dissolution of proper amounts of sodium salts in deionized
water. All chemicals used were of analytical grade.
(t, J = 7.7 Hz, 1H), 7.49 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 15.8
Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 5.38 (s, 2H), 4.31 (s, 3H).
General fluorescence spectra measurements. All the deꢀ
tection experiments were measured in DMSOꢀPBS buffer
solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25℃. The process
13
C NMR (151 MHz, DMSO) δ 170.77 (s), 153.89 (s), 144.86
(
1
s), 141.56 (s), 132.80 (s), 132.10 (s), 129.46 (s), 128.80 (s),
28.51 (s), 128.32 (s), 127.70 (s), 127.43 (d, J = 13.5 Hz),
127.17 (s), 124.15 (s), 123.66 (s), 120.91 (s), 116.81 (s),
16.15 (s), 114.73 (s), 111.87 (s), 63.65 (s), 35.61 (s). ESI–
was monitored by fluorescence spectrometer (λ = 400 nm,
ex
slit: 5 nm/10 nm).
1
Confocal Fluorescence Imaging. The MCFꢀ7 cells were
grown in Dulbecco’s Modified Eagle’s medium, supplemented
with 12% Fetal Bovine Serum and 1% antibiotics at 37 °C in
MS: m/z Calcd. For: m/z Calcd 356.1104, Found 356.1116
(Fig. S2).
humidified environment of 5% CO . Cells were plated on 6
RESULTS AND DISCUSSION
2
well plates and allowed to adhere for 24 h. Before the experiꢀ
ments, cells were washed with PBS 3 times. Firstly, we incuꢀ
bated probe 1 (10 ꢁM) with MCFꢀ7 cells for 30 min at 37℃ in
PBS as a control experiment. Secondly, probe 1, which located
MCFꢀ7 cells were treated with bisulfite (50 ꢁM) for another
Design of probe 1 and the proposed detection mecha-
nism. Probe 1 was synthesized by two steps with high yield
(Scheme 2). In our research, an ideal fluorescent probe should
satisfy the following properties: high selectivity; rapidly reꢀ
sponse; ratiometric detection; good permeability and biocomꢀ
patibility. Bearing these facts in mind, herein, a novel naphꢀ
thapyrones chromophore was efficiently synthesized. With the
aldehydeꢀfunctionalized chromophore in hand, a ratiometric
3
0 min. Finally, all cells were washed with PBS for 3 times
before imaging, and all images were carried out on a confocal
microscope, separately. For bioꢀimaging in vivo, 5ꢀdayꢀold
zebrafishes were prepared. Zebrafishes were fed with 10 ꢁM
of probe 1 in E3 embryo media (15 mM NaCl, 0.5 mM KCl, 1
2ꢀ
ꢀ
fluorescent probe for SO /HSO was developed based on the
3
3
fundamental notion of the nucleophilic addition reaction.
Benefited from the strong electron withdrawing moiety of
benzothiazole moiety, probe 1 featured ultrafast responses
mM MgSO , 1 mM CaCl , 0.15 mM KH PO , 0.05 mM
4
2
2
4
Na HPO , 0.7 mM NaHCO , 10ꢀ5% methylene blue; pH 7.5)
2
4
3
2ꢀ
ꢀ
at 28 °C for 20 min. Then incubated with Na SO (50 ꢁM) for
2
3
towards SO /HSO while maintained high sensitivity and
3 3
3
0 min, respectively, and finally incubated with H O (50 ꢁM)
2 2
selectivity properties. The sensing mechanism were characꢀ
for 30 min. All the fishes were terminally anaesthetized using
MS222, and images were carried out on a confocal microꢀ
scope.
1
terized by H NMR and electrospray ionization mass specꢀ
trometry (ESIꢀMS), which was consistent with the change
appeared in the absorption and fluorescence spectrum.
Synthesis of 3H-benzo[f]chromene-2-carbaldehyde
Scheme 2. Synthesis Route for Probe 1
(
Compound 2). 2ꢀHydroxyꢀ1ꢀnaphthaldehyde (0.516 g, 3.0
mmol) and acrolein (0.5 mL, 7.5 mmol) were dissoloved in
, 4ꢀdioxane (60 mL) with K CO (1.035g, 7.5 mmol). The
1
2
3
mixture was heated at 105 °C for 72 h. After the reaction
was completed, it was removed from the heating bath,
poured into 150 mL of ice water, The resulting mixture was
extracted three times with ether (40 ml). The combined
organic layer was washed with saturated NaCl twice (40
ml), then dried over anhydrous Na SO , and concentrated
2
-
-
Sensing Properties of Probe 1 towards SO /HSO .
3
3
Probe 1 was steady in a broad pH range (2.0ꢀ9.0). However,
upon the addition of SO /HSO , probe 1 displayed the best
response in the pH range of 7.0ꢀ9.0 (Figure. S3). Thus, to
study the spectroscopic response of probe 1 towards SO
HSO , pH 7.4 was selected for the following experiments.
Fluorescence titration experiments were carried out firstly. As
shown in Figure 1A, the free probe displayed a nearꢀinfrared
2ꢀ
ꢀ
3
3
2ꢀ
2
4
3
under vacuum. Compound 2 was isolated using a silica gel
chromatographic column eluted with EA/ PE (v/v, 1:5),
ꢀ
/
3
1
resulting a light yellow solid (0.126 g, 20%). H NMR (600
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Page 3 of 6
Analytical Chemistry
fluorescence emission at 630 nm. However, the addition of an
increasing amount of SO /HSO (0ꢀ30 µM) elicited a gradual
3 3
decrease of the fluorescence intensity at 630 nm, accompanied
with a distinct increase at 520 nm. These results indicated that
the πꢀconjugation of probe 1 was interrupted. Then, the correꢀ
sponding UVꢀvis spectral changes were also studied (Figure
2
ꢀ
ꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
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1
4
B). Probe 1 (50 µM) shows a strong absorption maximum at
2ꢀ
70 nm and a shoulder at 350 nm. Upon addition of SO3
ꢀ
/
HSO (0ꢀ150 µM) to the buffered solution of probe 1, the two
3
Figure. 2 (A) Fluorescent spectral changes upon addition of
H O (50 ꢀM) in DMSOꢀPBS buffer solution (10.0 mM, pH =
absorption bands gradually decreased, with an obvious color
2
2
change from violet to colorless, suggesting that probe 1 can be
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
2ꢀ
ꢀ
7.4, 1:9, v/v) at 25 °C, λex = 400 nm , slit: 5 nm/10 nm. (B)
used to detect SO /HSO with the “nakedꢀeye”.
3
3
ꢀ
UVꢀvis absorption spectra of probeꢀHSO3 in the presence
H O (200ꢁM) in DMSOꢀPBS buffer solution (10.0 mM, pH =
2
2
7.4, 1:9, v/v) at 25 °C.
Fluorescence Reversibility and LOD of Probe 1. In a furꢀ
ther experiment, we carried out the reversibility study of probe
2
ꢀ
ꢀ
1 towards SO /HSO upon addition of H O . As shown in
3
3
2
2
2ꢀ
ꢀ
Fig. 3A, three times supplement of SO /HSO and H O
3
3
2
2
caused negligible intensity attenuation, which provided a
2ꢀ
ꢀ
unique approach for monitoring SO /HSO and H O in vitro
3
3
2
2
Figure 1. (A) Fluorescent spectral changes of probe 1 (10
and in vivo. Next, we performed fluorescence titration experiꢀ
2
ꢀ
ꢀ
2ꢀ ꢀ
ꢀ
M) upon addition of SO /HSO (0 ꢀ 30 ꢀM) in DMSOꢀPBS
ment of probe 1 for SO
with various concentrations of SO
fluorescence ratio of I520/I presented an excellent linear
/HSO . Probe 1 (10 µM) was treated
3 3
2ꢀ ꢀ
3
3
buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C. (B)
/HSO (0ꢀ30 µM), and the
3 3
UVꢀvis absorption spectra of Probe 1 (50.0 ꢁM) in the presꢀ
630
2
2ꢀ
ꢀ
2
ꢀ
ꢀ
relationship (R = 0.9934) towards increased SO /HSO
ence of different amounts of SO /HSO (0 ꢀ 150 ꢁM) in
3
3
3
3
concentration (Fig. 3B). Moreover, the detection limit for
DMSOꢀPBS buffer solution (10.0 mM, pH = 7.4, 1:9, v/v) at
2ꢀ
ꢀ
SO /HSO was calculated to be 95 nM based on IUPAC
2
5 °C. λ = 400 nm , slit: 5 nm/10 nm.
3
3
ex
25ꢀ27
(
CDL=3 Sb/m)
.
Sensing Properties of the addition towards H O . After
2
2
the demonstration of probe 1 as a suitable chemosensor for the
detection of SO derivatives, we further investigated some
2
possible applications such as the reversibility of the nucleoꢀ
2ꢀ
ꢀ
philic addition product induced by SO /HSO . In the course
3
3
of our research, it was found that hydrogen peroxide could
effectively restore the fluorescence intensity. As show in Figꢀ
ure 2A, an addition of 50 ꢁM H O to the solution could inꢀ
2
2
duce big fluorescence variation. A dramatic fluorescence
enhancement was observed at 630 nm and the emission peak
at 520 nm decreased simultaneously. The relative fluorescent
Figure. 3 (A) Reversibility study of probe 1 (10 µM) toꢀ
2ꢀ
ꢀ
intensity (I 20/I630) was calculated as 0.65, indicating that the
wards SO /HSO (30 µM) upon addition of H O (50 µM).
3 3 2 2
5
2ꢀ
ꢀ
SO /HSO added system of probe 1 effectively restored by
3
3
(B) Relative emission intensity at I520/I630 of probe 1 (10 ꢁM)
the addition of H O . Fig. 2B shows the change of the UVꢀ
2ꢀ
ꢀ
2
2
with SO /HSO (6ꢀ24 ꢁM) in DMSOꢀPBS buffer solution
3
3
Visible spectrum upon addition of H O . The absorption band
2
2
(10.0 mM, pH = 7.4, 1:9, v/v) at 25 °C. λex = 400 nm , slit: 5
at 470 nm gradually increased, which accompanied by a clear
visual change in the color of the experimental solution from
colorless to violet. In other words, when oxidized by H O , the
nm/10 nm.
Kinetic Study and Selectivity of Probe 1. The kinetic
2
2
2ꢀ
ꢀ
analysis of probe 1 towards 10 equivs of SO /HSO disꢀ
color of the addition product can be changed almost the same
3
3
ꢀ
played that the reaction was completed within 40 s (Fig. S6).
In the selectivity test, the probe was treated with various bioꢀ
logical relevant anions and small molecules (10 equals). Upon
as the original probe. Moreover, the probe 1 with HSO addiꢀ
3
tion product showed a negligible response to some reactive
.
oxygen species (HClO, OH , TBHP, KO ) and some metal
2
2ꢀ
ꢀ
2+
2+
+
+
2+
2+
2+
2+
2+
3+
excitation at 400 nm, Only SO /HSO leading to a big relaꢀ
ions (Cu , Zn , Na , K , Mg , Ca , Hg , Ba , Mn , Al )
even at the concentrations up to 1 mM. By contrast, noticeable
fluorescence ratio change was obtained in the presence of
H O (Fig. S4). Meanwhile, it was also found that, after treatꢀ
3
3
tive fluorescent intensity change at 520 nm and 630 nm
(I520/I630≈37), while other selected anions showed negligible
change in the emission spectra of probe 1 (Fig. S7).
2
2
ment with 50 µM of H O , the fluorescence ratio of I630/I
2
2
520
Proposed Mechanism. The sensing mechanism of probe
2
ꢀ
ꢀ
increased steadily and nearly reached a plateau within 60 min,
which was resulted from the red fluorescence enhancement
and the green fluorescence decline. (Fig. S5).
1
for SO /HSO was carefully examined by the NMR titraꢀ
3 3
tion experiment. Clearly, the proton signal at δ 4.32 (a ) and δ
1
8
.33 (d ) shifted forward (a , d , at δ 4.05 and δ 7.35 ppm)
1 2 2
2ꢀ ꢀ
after reaction with SO /HSO , and the characteristic proton
3
3
signal of the double bond in probe 1 (b , c , at δ 7.40, 7.38,
1
1
7
.26, 7.24 ppm) shifted to (b , c at δ 5.00, 4.98, 4.87, 4.84
2 2
ppm), respectively (Fig. S8). Furthermore, the mixture of
2ꢀ
ꢀ
SO /HSO and probe 1 was characterized by mass spectromꢀ
3
3
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Page 4 of 6
2
ꢀ
ꢀ
etry. As shown in Fig. S9, two dominant peaks at m/z 356.11
exogenous SO /HSO , accompanied with decreased red
3
3
+
ꢀ +
1
2
3
4
5
6
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and 438.08 were attributed to [probe 1] and [probe 1ꢀ HSO ] ,
3
fluorescence in the red channel. What is more, when the
aboveꢀmentioned zebrafish preꢀtreated with H O , green
fluorescence changes disappeared and the red fluorescence
restored. These imaging findings demonstrate that probe 1 can
effectively enable redox cycle process in living body.
respectively. Thus, the sensing mechanism of probe 1 towards
2
2
2ꢀ
ꢀ
SO /HSO was based on the nucleophilic addition, as shown
3
3
in Scheme 1.
Cellular Imaging. Inspired by the excellent properties of
2
ꢀ
ꢀ
probe 1 for reversible sensing SO /HSO and H O in vitro,
3
3
2
2
the ability of probe 1 for cell imaging were evaluated with
MCFꢀ7 cells. Firstly, cytotoxicity of probe 1 was conducted by
MTT assay (Fig. S10), and the result showed that probe 1 was
suitable for living cells below 10.0 ꢁM. Hence, the following
experiment was conducted with probe 1 at the concentration of
0
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1
0 ꢁM. In Fig. 4, MCFꢀ7 cells were incubated with probe 1
Figure 5. Confocal images of Probe 1 response to
(10 ꢁM) for 30 min at 37℃ in PBS buffer. On the excitation
2ꢀ
ꢀ
exogenous SO /HSO in zebrafish. (a1ꢀd1): Zebrafish was
3
3
of 488 nm, probe 1 exhibited red fluorescence in the red chanꢀ
nel, but negligible fluorescence in green channel. These data
was consistent with fluorescence spectra results of probe 1. In
incubated with Probe 1 (10 ꢁM) for 30 min; (a2ꢀd2): Zebrafish
was incubated with Probe 1 (10 ꢁM) for 30 min, then
2ꢀ
ꢀ
2ꢀ
ꢀ
incubated with SO /HSO (50 ꢁM) for 30 min. Probe 1
3 3
a control experiment, upon incubation SO /HSO to probe 1ꢀ
3
3
loaded Zebrafish incubated with 50 ꢁM of NaHSO for 30 min,
3
loaded MCFꢀ7 cells 0.5 h, the cells displayed a stronger green
fluorescence enhancement and a weaker red fluorescence.
These results demonstrated that the probe was capable of
and then incubated with 50 ꢁM of H O for another 30 min.
2
2
From left to right: Green channel: λem = 520±20 nm (λ = 405
ex
2
ꢀ
ꢀ
nm), Red channel: λem = 650±20 nm (λex = 488 nm), Bright
field, Merge. Scale bar: 400 ꢁm.
detecting exogenous SO /HSO in living cells. Besides,
3
3
when H O was supplemented with the above stained MCFꢀ7
2
2
cells for another 30 min, the initial red fluorescence enhanced
and the green fluorescence faded, indicating that probe 1 unꢀ
derwent a redox activity in living cells, and it can be regenerꢀ
ated by hydrogen peroxide indeed.
CONCLUSIONS
In summary, a reversible and ratiometric fluorescent probe
was explored to sense bisulfite and hydrogen peroxide in
vitro and in vivo. The ratiometric fluorescence response was
based on the πꢀconjugation interruption along with the canꢀ
celation of an ICT process. In the reversibility process of the
2ꢀ
ꢀ
SO /HSO ꢀprobe 1 system induced by H O in vitro, we
3
3
2
2
successfully demonstrated the reversible redox properties of
2ꢀ
ꢀ
probe 1 for SO /HSO and H O . Besides, further cells and
3
3
2
2
zebrafish experiments clearly revealed that probe 1 could be
used for monitoring redox process in biological system. Moreꢀ
over, this will prompt us to explore the reversible process of
other bioactive molecules in the next step.
ASSOCIATED CONTENT
Supporting Information
Structure characterizations of probe 1, pH dependent spectra,
kinetic analysis of probe 1, and data for investigation of the sensꢀ
ing mechanism. The material is available and free of charge on
the ACS Publications website.
Figure 4. Confocal images of probe 1 responds to
2ꢀ
ꢀ
exogenous SO /HSO and H O in MCFꢀ7 cells. (a1ꢀd1):
3
3
2
2
MCFꢀ7 cells were incubated with probe 1 (10 ꢁM) for 30 min;
a2ꢀd2): MCFꢀ7 cells were incubated with probe 1 (10 ꢁM) for
0 min, then incubated with NaHSO (50 ꢁM) for 30 min; (a3ꢀ
(
AUTHOR INFORMATION
3
3
Corresponding Author
d3): Probe 1 loaded MCFꢀ7 cells incubated with 50 ꢁM of
ꢀ
* Eꢀmail: yincx@sxu.edu.cn.
HSO for 30 min, and then incubated with 50 ꢁM of H O for
3
2
2
another 30 min. From left to right: Green channel: λem
=
ORCID
5
20±20 nm (λ = 405 nm), Red channel: λ = 650±20 nm (λex
ex em
Caixia Yin: 0000ꢀ0001ꢀ5548ꢀ6333
=
488 nm), Bright field, Merge. Scale bar: 20 ꢁm.
Zebrafish Imaging. To further display the reversibility
Author Contributions
2
ꢀ
ꢀ
advantage of probe 1ꢀSO /HSO ꢀH O system, zebrafish
3
3
2
2
This manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script.
imaging experiment was performed to examine the redox
cycle processes in vivo. As is shown in Fig. 5, when zebrafish
was incubated with the probe 1 for 30 min, an obviously red
fluorescence signals can be achieved while none fluorescence
signals could obtained in the green channel. By contrast, the
green fluorescence increased obviously with the treatment of
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENT
Analytical Chemistry
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This work was financially supported by the National Natural
Science Foundation of China (No. 21472118), talents Support
Program of Shanxi Province (No. 2014401) and Scientific Instruꢀ
ment Center of Shanxi University (No. 2015).
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