A novel rhodol-based colorimetric and ratiometric fluorescent probe for selective detection of sulfite in living cells

Article abstract of DOI:10.1007/s13738-018-1483-z

A new type of colorimetric and ratiometric fluorescent probe 1 for sulfite is developed based on a rhodol-benzothiazole platform. Ratiometric sensing of sulfite is achieved by utilizing the nucleophilic addition of sulfite to the vinyl bridge (?C=C?) to block the π-conjugated system of the probe, which results in significant blueshifts in the absorption and emission spectra of the sensing system (from 590 to 530?nm in the absorption spectra and from 650 to 555?nm in the emission spectra). This probe exhibits the desired selectivity for sulfite over other anions and biothiols. The fluorescence intensity ratio at 555?nm and 650?nm (I₅₅₅/I₆₅₀) increases linearly with the sulfite concentration in the range of 0.5?15?μM with a detection limit of 0.28?μM. A cytotoxicity assay indicates that probe 1 has low cytotoxicity and good cell membrane permeability and can be used for sulfite detection in practical samples and for ratiometric fluorescent imaging of sulfite in living HepG2 cells.

Full text of DOI:10.1007/s13738-018-1483-z

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-1483-z
ORIGINAL PAPER
A novel rhodol-based colorimetric and ratiometric fluorescent probe
for selective detection of sulfite in living cells
Hongmin Lv1,2,3 · Zhaoyu Wang1,2,3 · Jing Lv1,2,3 · Nan Gao1,2,3 · Xiangfeng Kong1,2,3 · Yingying Zhang1,2,3
Received: 18 April 2018 / Accepted: 27 August 2018
©
Iranian Chemical Society 2018
Abstract
A new type of colorimetric and ratiometric fluorescent probe 1 for sulfite is developed based on a rhodol-benzothiazole plat-
form. Ratiometric sensing of sulfite is achieved by utilizing the nucleophilic addition of sulfite to the vinyl bridge (‒C=C‒)
to block the π-conjugated system of the probe, which results in significant blueshifts in the absorption and emission spectra
of the sensing system (from 590 to 530 nm in the absorption spectra and from 650 to 555 nm in the emission spectra). This
probe exhibits the desired selectivity for sulfite over other anions and biothiols. The fluorescence intensity ratio at 555 nm
and 650 nm (I /I ) increases linearly with the sulfite concentration in the range of 0.5‒15 µM with a detection limit of
5
55 650
0
.28 µM. A cytotoxicity assay indicates that probe 1 has low cytotoxicity and good cell membrane permeability and can be
used for sulfite detection in practical samples and for ratiometric fluorescent imaging of sulfite in living HepG2 cells.
Keywords Ratiometric fluorescent probe · Sulfite · Rhodol · Nucleophilic addition reaction
Introduction
Consequently, many countries strictly control the content of
sulfite in food, wine and medicine. The FAO/WHO Expert
Committee stipulates that daily intake of sulfite should be
less than 0.7 mg/kg [7]. Therefore, sensitive and selective
detection techniques for sulfite in biological systems are
highly valuable.
Sulfur dioxide (SO ) is a stimulant odor and one of the
2
main contaminants in the atmosphere. In the physiological
2
−
−
environment, SO exists in the forms of SO and HSO .
2
3
3
Sulfite is often used for storing food, wine, and medicine
because it inhibits the decomposition of enzymes [1]. Toxi-
Fluorescent probes have attracted continuing attention
due to the simplicity and high sensitivity of fluorescence
detection. A number of fluorescent probes for the detec-
tion of sulfite have been developed, and their reaction
mechanisms can be divided into the following categories:
nucleophilic reaction with an aldehyde [8–16], selective
deprotection of a levulinate group [17–21], and coordina-
tive interactions [22, 23]. Nevertheless, these probes are sus-
ceptible to interference from reactive sulfur species, such
as cysteine (Cys), glutathione (GSH) and hydrogen sulfide.
Furthermore, their detection limits are not satisfactory, and
the response time is relatively long. Moreover, the detection
principle of these probes is based on a unique increase or
decrease in the emission intensity at a single wavelength,
which is easily affected by external environments. Thus,
these probes are not suitable for detecting sulfite in liv-
ing organisms. Guo et al. reported a coumarin‒hemicya-
nine dye as a ratiometric fluorescent probe for sulfite based
on a novel addition-rearrangement cascade reaction [24].
Ratiometric sensing was realized via the interruption of
cological studies have found that SO and its derivatives
2
can change the mercaptan content in proteins, impede the
redox balance, destroy the nervous system, and even change
the characteristics of voltage-gated sodium and potassium
channels in rat hippocampal neurons [2–4]. Epidemiologi-
cal studies have found that excessive inhalation of SO can
2
not only cause respiratory diseases such as chronic bronchi-
tis, asthma, and emphysema, but also induce lung cancer,
cardiovascular disease and neurological disorders [5, 6].
*
Hongmin Lv
lhm_sdqd@hotmail.com
1
Institute of Oceanographic Instrumentation, Qilu University
of Technology (Shandong Academy of Sciences), Jinan,
China
2
3
Shandong Provincial Key Laboratory of Marine Monitoring
Instrument Equipment Technology, Qingdao, China
National Engineering and Technological Research Center
of Marine Monitoring Equipment, Qingdao 266061, China
Vol.:(0
1
123456789)
3

Journal of the Iranian Chemical Society
the π-conjugated system of the whole molecule through the
nucleophilic addition of sulfite to the conjugated bridge (‒
C=C‒). It is indeed a promising methodology, on which
several ratiometric fluorescent probes for selective detec-
tion of sulfite have further been based [25–31]. However,
these probes are mostly developed based on the coumarin
platform and are far from meeting the research demand due
to their smaller size and limited variety. Therefore, finding
a new active fluorophore instead of coumarin to extend the
π-conjugation system is necessary for designing a series of
ratiometric fluorescent probes for sulfite.
used throughout all experiments. Flash chromatography
was performed using Qingdao Haiyang silica gel (200–300
mesh). The inorganic salt and reactive sulfur species used
were Na SO , Na S O , Na SO , Na CO , NaOAc, NaNO ,
2
3
2
2
3
2
4
2
3
3
NaNO , NaSCN, NaF, NaCl, NaBr, KI, NaHS, Na S, NaClO,
2
2
NaCN, GSH, and Cys.
Instrumentation
The fluorescence spectra and relative fluorescence intensity
were measured with a Shimadzu RF-5301 spectrofluorim-
eter with a 10 mm quartz cuvette at the slits of 3.0/5.0 nm.
Absorption spectra were recorded using a Shimadzu
UV-2550 spectrophotometer. IR spectra were recorded on a
Bruker Vertex 70 FT-IR spectrometer using a diamond ATR
attachment. High-resolution mass spectrometry (HRMS)
data were collected using a Bruker micrOTOF-Q II mass
spectrometer (Bruker Daltonics Corp., USA) in electrospray
As a hybrid structure of fluorescein and rhodamine, the
rhodol fluorophore inherits excellent photophysical proper-
ties [32], such as a large extinction coefficient, high fluores-
cence quantum yield, considerable water solubility and low
pH dependence, and hence has received considerable atten-
tion from our group. To the best of our knowledge, a nucleo-
philic addition reaction based on the rhodol fluorophore has
not been used to develop a selective fluorescent probe for
sulfite. Thus, probe 1 is first synthesized by extending the
π-conjugated system through incorporation of a vinyl bridge
into rhodol. In this structure, the rhodol moiety acts as the
parent fluorophore, and the vinyl bridge acts as the receptor
1
13
ionization (ESI) mode. H NMR and C NMR spectra were
recorded at 400 and 100 MHz, respectively, on an INOVA-
400 spectrometer (Varian Unity) using tetramethylsilane
(TMS) as the internal standard. Cell images were acquired
with an Olympus BX 61 fluorescence microscope (Tokyo,
Japan) at 40× magnification. pH measurements were carried
out on a Sartorius PB-10 pH meter. All measurements were
performed at room temperature (25 °C).
(
Scheme 1). The nucleophilic attack of sulfite on the vinyl
bridge of probe 1 is envisaged to interrupt the π-conjugation
of the sensing system, which restores the fluorescence of the
−
rhodol moiety and leads to the 1-SO adduct. As a result, an
3
obvious blueshift in absorption and emission spectra before
Synthesis of compounds
and after adding sulfite can be observed due to the distinct
−
emission between 1 and the corresponding 1-SO adduct.
The proposed ratiometric fluorescent probe 1 was produced
through a stepwise synthetic route of three steps shown in
Scheme 2.
3
Based on the above mechanism, dual-emission ratiometric
detection of sulfite can be achieved.
Synthesis of compound E
Experimental
Compound C was synthesized according to a reported proce-
dure [33]. Compound C (150 mg, 0.48 mmol) and 2,4-dihy-
droxybenzaldehyde (66 mg, 0.48 mmol) were dissolved in
methanesulfonic acid (5 mL) and stirred at 90 °C for 30 min.
After cooling to room temperature, the mixture was poured
into 50 mL of ice water, followed by the dropwise addition
Reagents and materials
All reagents and solvents were of analytical grade and were
used without further purification. Solvents were dried by
standard procedures before use. Double-distilled water was
Scheme 1 The sensing mecha-
2
−
nism of probe 1 for SO
3
1
3

Journal of the Iranian Chemical Society
Scheme 2 Synthesis of probe 1
of 1 M NH ·H O solution until the pH reached approxi-
(CH Cl :CH OH = 10:1 v:v) to afford probe 1 (45.8 mg,
2 2 3
3
2
1
mately 8.0. The solution was extracted with DCM and
washed with brine. The solvent was then removed under
reduced pressure, and the resulting residue was purified by
silica gel flash chromatography (CH Cl :CH OH = 100:1
34% yield). H NMR (CDCl , 400 MHz): δ ppm 8.34 (d,
3
J=8.0 Hz, 2H), 8.22 (d, J=8.4 Hz, 2H), 8.05 (t, J=6.8 Hz,
2H), 7.79 (m, 5H), 7.37 (s, 1H), 7.31(d, J=6.8 Hz, 1H), 6.50
(d, J=18 Hz, 3H), 4.19 (s, 3H), 1.10 (t, J=5.6 Hz, 6H) (Fig.
2
2
3
1
13
v:v) to afford compound E (39.85 mg, 20% yield). H NMR
S5); C NMR (CDCl , 100 MHz): 172.62, 168.93, 160.23,
3
(
(
CDCl , 400 MHz): δ ppm 11.19 (s, 1H), 9.57 (s, 1H), 8.05
154.59, 152.58, 151.57, 149.15, 144.62, 141.84, 135.67,
133.65, 130.11, 129.22, 128.56, 128.51, 128.11, 127.55,
127.44, 126.11, 124.78, 124.09, 124.01, 118.34, 116.71,
113.25, 111.95, 108.94, 104.53, 103.19, 97.00, 43.81, 36.24,
3
d, J=7.2 Hz, 1H), 7.68 (m, 2H), 7.22 (d, J=7.6 Hz, 1H),
7
1
.01 (s, 1H), 6.80 (s, 1H), 6.57 (d, J=8.8 Hz, 1H), 6.48 (s,
H), 6.39 (d, J=8.4 Hz, 1H), 3.37 (q, J=6.8 Hz, 4H), 1.18
1
3
+
(
t, J=6.8 Hz, 6H) (Fig. S1); C NMR (CDCl , 100 MHz):
12.36 (Fig. S6); ESI-HRMS: [M] m/z 561.1838, calcd for
3
+
−1
1
1
1
8
4
94.62, 169.28, 162.91, 157.83, 152.47, 152.19, 149.71,
35.49, 135.07, 129.83, 128.72, 126.94, 125.92, 125.07,
23.86, 117.80, 113.24, 108.86, 104.45, 104.37, 97.67,
C H N O S 561.1843 (Fig. S7); IR υ (cm ): 3397.55,
34 29 2 4
2927.44, 2862.05, 1719.51, 1578.99, 1464.98, 1393.39,
1340.52, 1279.93, 1234.70, 1188.48, 1078.02, 1017.82,
937.43, 758.17, 704.91 (Fig. S8).
+
2.99, 44.45, 12.38 (Fig. S2); ESI-HRMS: [M + H] m/z
16.1483, calcd for C H NO 416.1492 (Fig. S3). IR υ
2
5
22
5
−
1
(
cm ): 3672.28, 2978.58, 2902.54, 1750.47, 1625.98,
General procedure for the spectral measurements
1
9
521.23, 1405.66, 1343.70, 1282.61, 1237.12, 1071.37,
28.18, 875.84, 801.42, 697.41 (Fig. S4).
Stock solutions of probe 1 (1.0 mM) were prepared in
0
.5 mL of EtOH and diluted in a 25 mL volumetric flask
Synthesis of probe 1
with double-distilled water. Solutions of inorganic salts
10 mM) and biothiols (5.0 mM) were prepared from
(
2
,3-Dimethylbenzothiazole was synthesized according to a
Na SO , Na S O , Na SO , Na CO , NaOAc, NaNO ,
2 3 2 2 3 2 4 2 3 3
reported procedure [27]. Compound E (100 mg, 0.24 mmol)
and 2,3-dimethylbenzothiazole (70 mg, 0.24 mmol)
were dissolved in EtOH (5 mL) and stirred at 85 °C for
NaNO , NaSCN, NaF, NaCl, NaBr, KI, NaClO, NaCN,
2
GSH, and Cys. Test solutions were prepared by adding
50 µL of probe 1, 3 mL of DMSO, 1.0 mL of phosphate
buffer (0.2 M, pH 7.4), and appropriate aliquots of each ana-
lyte stock solution in a 10 mL volumetric flask and diluting
the solution to 10 mL with water. The resulting solution
6
h. After cooling to room temperature, the solvent was
then removed under reduced pressure, and the resulting
residue was purified by silica gel flash chromatography
1
3

Journal of the Iranian Chemical Society
was kept at room temperature (25 °C) for 15 min, and then
the absorption or fluorescence spectra were recorded. The
2.5
2.0
2
-
1
1
+SO
3
fluorescence ratio at I /I was measured with excitation
5
55 650
and emission wavelengths at 530/590 nm and 555/650 nm,
respectively.
1
1
.5
.0
Cytotoxicity assay
HepG2 cells were seeded in 96-well plates at a density of
5
1
× 10 per well and incubated with varied concentrations
0.5
.0
of probe 1 (0–25 µM) (n=5) in an atmosphere of 5% CO
2
and 95% air at 37 °C for 12 h. Then, 20 µL of MTT solu-
0
−
1
tion (5.0 mg mL ) was added to each well, followed by
incubation at 37 °C for 4 h. Next, 100 µL of the supernatant
was removed, and 150 µL of DMSO was added to each well
to dissolve the formed formazan. The plate was shaken for
4
6
8
10
12
pH
Fig. 1 The ratio (I /I ) of fluorescence intensity for probe 1
5
55 650
2
−
1
0 min, and then the absorbance of the solution was meas-
(5 µM) with absence and presence of SO (4 equiv) as a function of
3
pH in phosphate buffer (20 mM, 30% DMSO)
ured at 570 nm with a microplate reader (ELX 800 UV, BIO-
TEK Instruments Inc.). Cell viability (VR) is calculated by
the following equation: VR = A∕A × 100%, where A and
0
A are the absorbance of the experimental group and control
pH values ranging from 6.0 to 8.0. This observation can be
explained by the fact that the nucleophilic addition of sulfite
interrupts the π-conjugation of the sensing system and that
the rhodol fluorophore is subsequently restored. When the
pH is greater than 8.0, I /I decreases significantly, which
0
group, respectively.
Cell cultures and fluorescence imaging
5
55 650
5
−
HepG2 cells were seeded at a density of 2×10 per well in
might be due to the decomposition of the 1-SO adduct
3
6
-well culture plates containing sterile coverslips and were
under basic conditions. The above results indicate that a
good response of probe 1 to sulfite can be obtained in solu-
tions at physiological pH.
cultured in RPMI 1640 medium supplemented with 10%
−
1
(
v/v) fetal bovine serum (FBS), penicillin (100 U mL ),
−1
and streptomycin (100 µg mL ) at 37 °C in a 95% humidity
atmosphere under 5% CO for 2 days. Before imaging, the
Kinetic studies
2
cells were washed with PBS three times and then incubated
with probe 1 (10 µM) in a DMSO/PBS (5: 95, v/v) solution
at 37 °C for 30 min. Next, the samples were rinsed with PBS
three times to remove any remaining probe. Fluorescence
images were acquired with an Olympus BX 61 fluorescence
microscope. Experiments to assess the sulfite uptake were
performed in the same medium supplemented with Na SO
The time dependence of the emission ratio (I /I ) of
5
55 650
probe 1 in the absence and presence of sulfite was meas-
ured in phosphate buffer (pH 7.4, 20 mM, 30% DMSO)
(Fig. 2). Upon the addition of sulfite (10 equiv) to the solu-
tion of probe 1, the emission ratio (I /I ) of the sensing
5
55 650
system increases significantly and reaches a plateau after
2
3
(
100 µM) for 30 min at 37 °C. Fluorescence images were
15 min, whereas the emission ratio (I /I ) of the free
5
55 650
acquired after the cells had been rinsed with PBS.
probe (5 µM) remains almost unchanged under the same
conditions. Therefore, an assay time of 15 min is chosen in
subsequent evaluations of the selectivity and sensitivity of
probe 1 for sulfite.
Results and discussion
Effect of pH
Sensing of sulfite by probe 1
The effect of pH on the fluorescence properties of the sens-
ing system before and after the addition of sulfite in the pH
range of 3‒11 was investigated. As shown in Fig. 1, the
fluorescence intensity ratio (I /I ) of probe 1 (5 µM) is
The sensing behavior of probe 1 toward sulfite was investi-
gated by absorption and fluorescence spectroscopy in phos-
phate buffer (pH 7.4, 20 mM, 30% DMSO). As shown in
Fig. 3a, the free probe displays a strong absorption peak
at approximately 590 nm. However, upon the addition of
sulfite (0‒60 µM), the absorption of 1 at 590 nm decreases
gradually, and a new absorption peak at 530 nm increases
5
55 650
almost invariable within the pH interval of 3‒11, implying
that the probe is stable over a wide range of pH values. Upon
the addition of sulfite (4 equiv), I /I increases sharply for
555 650
1
3

Journal of the Iranian Chemical Society
2.5
2.0
1.5
1.0
0.5
0.0
2
5
2
-
4
3
2
1
0
0
0
0
0
1+SO
20
3
1
1
5
0
5
0
1
only
0
10
20
30
40
50
60
)
Sulfite concentration
(
µ
M
Y = 0.14208X - 0.0006
r = 0.9958
0
2
4
6
8
10
12
14
16
0
2
4
6
8
10
12
14
16
Time (min)
Sulfite concentration (µM)
Fig. 2 The time course of the emission ratio (I /I ) of probe 1
5
55 650
Fig. 4 Fluorescence intensity ratio (I /I ) of probe 1 (5 µM) as a
555 650
2
−
(
5 µM) in absence and presence of SO (10 equiv) in phosphate
3
function of sulfite concentration in phosphate buffer (pH 7.4, 20 mM,
0% DMSO). The inset shows the changes to the fluorescent intensity
ratio (I /I ) on increasing the concentration of Na SO (0‒60 µM)
buffer (pH 7.4, 20 mM, 30% DMSO)
3
5
55 650
2
3
progressively. Meanwhile, a well-defined isosbestic point is
observed at 545 nm, which might be contributed by a new
species formed during the sensing process.
ratio with respect to that of the free probe. The I /I value
5
55 650
is proportional to the sulfite concentration in the range from
0.5 to 15 µM with a detection limit of 0.28 µM, indicating
that probe 1 can detect sulfite quantitatively.
Furthermore, the fluorescent response of 1 to sulfite was
investigated under the same conditions (Fig. 3b). Upon exci-
tation at 590 nm, probe 1 itself exhibits an emission peak
centered at 650 nm. However, the fluorescence intensity at
Selectivity studies
6
50 nm decreases gradually with increasing sulfite concen-
tration, which indicates that the large π-conjugated system
is interrupted by the addition of sulfite to the vinyl bridge of
probe 1. Moreover, the progressive increase in a new emis-
sion band at 555 nm most likely results from the recovery of
rhodol fluorescence. A blueshift of 95 nm is observed during
the above sensing process, making probe 1 favorable for the
dual-emission ratiometric sensing of sulfite. The fluores-
cence intensity ratio I /I is plotted as a function of sulfite
The selectivity of probe 1 for sulfite over other anions
2
−
2−
2−
2−
−
−
−
(SO , S O , SO , CO , CH COO , NO , NO ,
3
2
3
4
3
3
3
2
−
−
−
−
−
−
−
−
2−
SCN , F , Cl , Br , I , ClO , CN , HS , S ) and biothi-
ols (GSH, Cys) was determined in phosphate buffer (pH 7.4,
20 mM, 30% DMSO). As shown in Fig. 5a, sulfite induces a
dramatic decrease in the absorption band at 590 nm and the
appearance of a new blueshifted absorption peak centered at
530 nm. Meanwhile, an absorption color change from blue to
pink is directly observed with the naked eye. In contrast, the
other anions and biothiols afford no significant changes in
the absorption spectra. In good accordance with the absorp-
tion spectra, only the introduction of sulfite to the solution of
555 650
concentration in Fig. 4, and a typical calibration graph is
also obtained. During treatment with 12 equiv of sulfite, the
I
/I increases from 0.034 to 23.25, which corresponds
55 650
5
to a ca. 672-fold enhancement in the fluorescence intensity
Fig. 3 The absorption (a) and
(
b) 600
00
400
00
200
(
a) 0
λ
= 530 nm
λ
= 590 nm
ex
[
sulfite] /µM
ex
fluorescence spectra (b) of the
probe 1 (5 µM) in phosphate
buffer (pH 7.4, 20 mM, 30%
DMSO) upon addition of
.4
.3
0
2
4
6
8
1
1
5
0
0
5
amounts of Na SO (0‒60 µM).
2
3
3
Each spectrum was recorded
0.2
20
2
3
3
4
5
6
5
0
5
0
0
0
after 15 min
0
0
.1
.0
1
00
0
4
00
500
600
700
540
560
580
630 660 690 720 750
Wavelength (nm)
Wavelength (nm)
1
3

Journal of the Iranian Chemical Society
Fig. 5 The absorption spectrum
(
b)600
500
(
a)0
(
(
a) and fluorescence spectrum
b) of probe 1 (5 µM) upon
.4
.3
Blank and others
analyte tested
Blank and others
analyte tested
addition of various species
in phosphate buffer (pH 7.4,
0
4
00
SO32-
2
0 mM, 30%DMSO) including
2− 2− 2− 2−
SO , S O , SO , CO ,
3
0.2
300
200
3
2
3
4
−
−
−
−
−
HCO , AcO , NO , NO ,
2
SO32-
3
3
2
SO
3
-
−
−
−
−
−
Br , Cl , SCN , F , I , ClO ,
0
0
.1
.0
−
CN (10 equiv each); GSH, Cys
1
00
−
2−
2−
(
0.5 mM); HS , S , SO (4
3
0
equiv)
4
00
500
600
700
540
560
580
630 660 690 720 750
Wavelength (nm)
Wavelength (nm)
Fig. 6 Images of probe 1 in the
presence of various anion and
biothiols under visible (left) and
UV lamp at 365 nm (right)
that probe 1 possesses a high selectivity for sulfite over other
3
2
2
1
1
0
0
.0
.5
.0
.5
.0
.5
.0
anions and biothiols.
Mechanism studies
To prove the sensing mechanism proposed above, the com-
pound rhodol was initially prepared by a procedure reported
previously [32]. Then, the absorption and emission spectra
of rhodol, probe 1 and probe 1 titrated with sulfite were
−
compared. As shown in Fig. 8, the 1-SO adduct affords
3
none of the spectroscopic characteristics of probe 1 but dis-
A B C D E F G H I J K L M N O P Q R
plays absorption and emission spectra (λ
em,max
= 531 nm;
abs,max
λ
= 556 nm) that are similar to those of rhodol, con-
Fig. 7 Fluorescencent ratio (I /I ) of 1 (5 µM) in phosphate buffer
5
55 650
firming the disruption of the π-conjugation in probe 1 caused
(
pH 7.4, 20 mM, 30%DMSO) to various species. (A: probe 1, B:
2− − − − − − − −
SO , C: F , D: Cl , E: Br , F: I , G: NO , H: NO , I: AcO , J:
by nucleophilic addition. This similarity can be explained
3
2
3
2−
2−
−
2−
−
S O , K: SO , L: SCN , M: CO , N: CN (10 equiv each), O:
−
2
3
4
3
by the fact that the 1-SO adduct and rhodol have almost
−
2−
3
Cys (0.5 mM), P: HS (4 equiv), Q: GSH (0.5 mM), R: S (4 equiv)
the same fluorophore.
−
Next, efforts were made to isolate the 1-SO adduct
3
1
induces a marked decrease in the fluorescence at 650 nm
from the mixture of probe 1 with sodium sulfite. Unfortu-
−
and an increase in the fluorescence peak centered at 555 nm,
accompanied by an obvious emission color change from red
to orange-yellow (Fig. 5b). Figure 6 shows that other com-
petitive species do not induce significant color changes in
the sensing system under visible or UV light. The histogram
in Fig. 7 shows intuitively that the fluorescence intensity
ratio I /I is highly enhanced by sulfite, whereas bio-
nately, the 1-SO adduct was unstable in aqueous solution,
3
and TLC analysis showed that it gradually returned to the
starting material. Therefore, a pure product for NMR spec-
troscopic characterization could not be obtained. Then, the
reaction mechanism between 1 and sulfite was further exam-
ined by HRMS. The peak of probe 1 is at 561.1837 (Fig.
S7). Upon the addition of sodium sulfite to probe 1 solution,
the peak at 561.1837 disappears, and the two sets of new
peaks that concomitantly appear at 659.1481 and 681.1300
should be assigned to the hydrated sulfoacid anion (calcd for
C H N O S 659.1527) and sodium sulfonate (calcd for
5
55 650
thiols (GSH, Cys) possessing nucleophilic ability induce a
negligible change in the emission ratio because electrostatic
repulsion and steric hindrance effects suppress the nucleo-
philicity of the thiol groups [34]. The above results indicate
3
4
31
2
8 2
1
3

Journal of the Iranian Chemical Society
Fig. 8 Normalized absorption
1
1
(
a) 1.0
Probe 1
(b) 1.0
(
(
a) and fluorescence spectra
b) of rhodol and the solution
-
1-SO3-
Probe 1-SO
Rhodol
-
3
1-SO3
0.8
-
0
.8
1
1-SO
3
of probe 1 (5 µM) in absence
and presence of sulfite (20 µM)
in phosphate buffer (pH 7.4,
Rhodol
Rhodol
Rhodol
0.6
0
.6
.4
2
0 mM, 30% DMSO)
0.4
0
-
1
-SO3
650
0.2
0.2
0.0
0.0
3
00
400
500
600
700
540
560
580
700
750
Wavelength (nm)
Wavelength (nm)
Table 1 The application in the
Sample
Determined
sulfite (µM)
Added
Combined
Measured (µM)
Recovery (%)
practical samples
sulfite (µM) sulfite (µM)
Granulated sugar
Rock sugar
0.58
3
3.58
5.58
8.58
5.35
7.35
10.35
3.45
5.52
8.48
5.28
7.32
10.34
95.7
98.8
98.8
97.7
99.4
99.9
5
8
3
5
8
2.35
C H N NaO S 681.1341), respectively (Fig. S9), indicat-
3
4
30
2
8 2
1
00
ing that the nucleophilic attack of sulfite on probe 1 indeed
occurs at the vinyl bridge, which is consistent with existing
results [24, 29]. These data are sufficient to prove the pro-
posed sensing mechanism shown in Scheme 1.
8
0
6
0
Real sample analysis
40
2
0
To investigate the applicability of probe 1 to real samples,
the content of sulfite in granulated sugar and rock sugar was
examined. Sample solutions were prepared by dissolving
0
0
5
10
15
20
25
1
0.0 g of sugar in water and diluting to 50 mL. Test solu-
Concentration (µM)
tions were prepared by adding 50 µL of probe 1 solution
1.0 mM) and 1.0 mL of phosphate buffer (0.2 M, pH 7.4,
(
Fig. 9 Cytotoxicity assay of probe 1 at different concentrations for the
3
0% DMSO) into a 10 mL volumetric flask and then diluting
HepG2 cell
the solution to 10 mL with water. The fluorescence intensity
ratio I /I of the test solutions was recorded. According
5
55 650
to Fig. 4, the amount of sulfite in granulated sugar and rock
sugar were estimated to be 0.58 and 2.35 µM, respectively.
The determined recoveries are listed in Table 1, indicating
the feasibility of probe 1 for detecting sulfite in real samples.
cytotoxicity at concentrations below 25 µM. Thus, 10 µM
probe 1 was selected for cell imaging. The fluorescence
image of HepG2 cells incubated with probe 1 (10 µM) for
30 min in the red channel is very strong (Fig. 10a), whereas
that in the orange channel is rather dim (Fig. 10b), which
confirms the cell permeability of probe 1. In the control
experiment, the cells were pretreated with probe 1 (10 µM)
and further incubated with Na SO (100 µM). Marked fluo-
Intracellular sulfite imaging
Before examining the capability of probe 1 for ratiomet-
ric imaging applications in living cells, the cytotoxicity
was evaluated by MTT assays, and the result is illustrated
in Fig. 9. The cellular viability is approximately 80% even
with 25 µM probe 1, indicating that this probe has low
2
3
rescence quenching in the red channel and a fluorescence
increase in the orange channel are observed in Fig. 10c, d,
respectively. These results are in agreement with the sulfite-
induced ratiometric fluorescence response.
1
3

Journal of the Iranian Chemical Society
Fig. 10 Fluorescence images
of HepG2 cells. a Fluorescence
image of HepG2 cells incu-
bated with probe 1 (10 µM)
for 30 min in the red channel,
b fluorescence image in the
orange channel, c fluorescence
image of HepG2 cells incubated
with probe 1 for 30 min and
further incubated with sulfite
(
100 µM) for 30 min in the red
channel, d fluorescence image
in the orange channel
8
9
.
.
G.J. Mohr, Chem. Commun. 2646 (2002)
K.Y. Chen, Y. Guo, Z.H. Lu, B.Q. Yang, Z. Shi, Chin. J. Chem.
Conclusion
2
8, 55 (2010)
In summary, a new type of colorimetric and ratiometric fluo-
rescent probe 1 for the selective detection of sulfite has been
developed. Ratiometric sensing is realized via the interrup-
tion of the π-conjugated system in probe 1 through the nucle-
ophilic addition of sulfite to the vinyl bridge moiety. The
probe displays excellent selectivity for sulfite over other ref-
erence anions and biothiols. The fluorescence intensity ratio
1
1
1
0. X.F. Yang, M.L. Zhao, G. Wang, Sens. Actuators B Chem. 152,
8 (2011)
1. C.M. Yu, M. Luo, F. Zeng, S.Z. Wu, Anal. Methods 4, 2638
(
2012)
2. Y.Q. Sun, P. Wang, J. Liu, J. Zhang, W. Guo, Analyst 137, 3430
(
2012)
13. Y.T. Yang, F.J. Huo, J.J. Zhang, Z.H. Xie, J.B. Chao, C.X. Yin,
H.B. Tong, D.S. Liu, S. Jin, F.Q. Cheng, X.X. Yan, Sens. Actua-
tors B Chem. 166, 665 (2012)
(
I
/I ) is proportional to the sulfite concentration in the
555 650
1
4. X.H. Cheng, H.Z. Jia, J.G. Feng, Z. Li, J. Mater. Chem. B 1, 4110
range from 0.5 to 15 µM with a detection limit of 0.28 µM.
Biological experiments indicate that probe 1 has low cyto-
toxicity and high cell permeability and can be employed for
the ratiometric fluorescent imaging of sulfite in living cells.
(
2013)
15. A.K. Liu, R.X. Ji, S.L. Shen, X.Q. Cao, Y.Q. Ge, New J. Chem.
4
1, 10096 (2017)
1
1
1
6. L.J. Zhang, Z.Y. Wang, X.J. Cao, J.T. Liu, B.X. Zhao, Sens. Actu-
ators B Chem. 236, 741 (2016)
7. M.G. Choi, J. Hwang, S. Eor, S.K. Chang, Org. Lett. 12, 5624
Acknowledgements This project is supported by National Key
Research and Development Program of China (No. 2016YFC1400800)
and the National Natural Science Foundation of China (Grant nos.
(
2010)
8. X.F. Gu, C.H. Liu, Y.C. Zhu, Y.Z. Zhu, J. Agric. Food Chem. 59,
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