Design and synthesis of a novel colorimetric fluorescent probe for the selective detection of sulfur dioxide in SH-SY5Y neuroblastoma cells and its applications in traditional Chinese medicines

Article abstract of DOI:10.1039/C8NJ06326E

Sulfur fumigation has attracted extensive attention as one of the important post-harvest processing methods for some traditional Chinese medicines (TCMs) in the last decade. However, sulfur fumigation has recently emerged as a controversial topic due to its potential detrimental effects on the safety and efficacy of TCMs as some sulfur-fumigated TCMs contain numerous sulfur dioxide derivatives; moreover, high levels of sulfur dioxide derivatives can cause some diseases and dangerous environmental pollution. In this study, a DTCC probe with a fast response time, low limit of detection and a high fluorescence quantum yield was designed and synthesized to detect SO₂ derivatives based on the coumarin-thiophene dye, which was fused with a coumarin moiety and 2-thiophenecarboxaldehyde. The probe DTCC exhibited a fast response time (less than 10 s), satisfactory selectivity for SO₂ derivatives in the presence of other ROS and excellent sensitivity for SO₂ derivatives with a low limit of detection (0.23 μM) as well as a wide linear range (0-100 μM). Furthermore, the probe DTCC was successfully applied in fluorescence imaging in SH-SY5Y neuroblastoma cells with excellent membrane permeability and stability. It was also employed in monitoring the total SO₂ derivatives in several real TCM samples. These results illustrate that the probe DTCC has an excellent capability for monitoring SO₂ derivatives in living cells and real TCM samples.

Full text of DOI:10.1039/C8NJ06326E

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Design and synthesis of a novel colorimetric fluorescent probe for
selective detection of sulfur dioxide in SH-SY5Y neuroblastoma
cells and its application in Traditional Chinese Medicines
Received 00th January 20xx,
Accepted 00th January 20xx
Lili Yanga,b, Mofan Liua,b, Kangjia Shenga,b, Xiaolu Lia,b, Junli Dua,b, Yaoyao Ninga,b, Xiaoqing Wangc,
DOI: 10.1039/x0xx00000x
Jianli Lic, Yongmin Zhanga,b,d and Shaoping Wua,b
*
www.rsc.org/
Sulfur fumigation has attracted more and more attention as one of the important post-harvest processing methods for
some Traditional Chinese Medicines (TCMs) in the last decade. However, sulfur-fumigated TCMs have recently emerged as
a controversial topic due to their potential detrimental effect on the safety and efficacy, as some sulfur-fumigated TCMs
contain lots of sulfur dioxide derivatives. Additionally, high levels of the sulfur dioxide derivatives could cause some
diseases and dangerous environmental pollutant. In this work, a fast response time, low limit of detection and high
fluorescence quantum yield probe DTCC was designed and synthesized to detect SO2 derivatives based on coumarin-
thiophene dye which was fused with a coumarin moiety and 2-thiophenecarboxaldehyde. Probe DTCC exhibited fast
response time (less than 10 s), satisfactory selectivity for SO2 derivatives in the presence of other ROS and excellent
sensitivity for SO2 derivatives with low limit of detection (0.23 µM) and widely linear range (0～100 µM). Furthermore,
probe DTCC was successfully applied in fluorescent imaging in SH-SY5Y neurobalstoma cells with excellent membrane
permeability and stability. It was also employed for monitoring the total SO2 derivatives in several real TCMs sample. These
results illustrated that probe DTCC has an excellent capability for monitoring SO2 derivatives in living cells and real TCMs
sample.
and its derivatives have received recently increasing attention
because they are involved in various physiology and pathological
1. Introduction
processed as a possible gasotransmitter2,3 following nitro oxide,4,5
carbon monoxide6,7 and hydrogen sulfide.8 On the other hand,
exogenous sulfur dioxide derivatives (SO32-/HSO32-) are widely used
as a kind of preservative in foods, beverages and pharmaceutical
products due to their capacity to inhibit the growth of
microorganisms and prevent mildew.9 However, high levels of the
sulfur dioxide derivatives could cause some diseases, such as
rheumatoid arthritis, Parkinson’s disease, Alzheimer’s disease10,11
and lung cancer.12,13
Reactive Sulfur Species (RSS) are associated with numerous
physiological and pathophysiological processes, which are produced
by various biochemical and physiological oxidative processes in the
body. RSS play a major role in the pathogenesis of various human
diseases. At low concentrations, RSS exhibit beneficial effects by
regulating intracellular signaling and homeostasis; at high levels,
however, RSS play a major role in the damage of proteins, lipids,
DNA and carcinogenesis.1 Among RSS, endogenous sulfur dioxide
For the sake of preserve moisture, colour and freshness, and to
prevent problems with insects and mold,14 sulfur fumigation has
attracted more and more attention as one of the important post-
harvest processing methods for some Traditional Chinese Medicines
(TCMs) in the last decade. However, sulfur-fumigated TCMs have
recently emerged as a controversial topic due to their potential
detrimental effect on the safety and efficacy.15 So residual sulfur
dioxide content is officially used as the exclusive indicator for the
a. School of Pharmacy; Key Laboratory of Resource Biology and Biotechnology in
Western China (Northwest University), Ministry of Education; Biomedicine Key
Laboratory of Shaanxi Province, Northwest University, Xi’an 710069, China.
b. Joint International Laboratory of Glycobiology and Medicinal Chemistry,
Northwest University, Xi’an, Shaanxi 710069, China.
c. Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of
Ministry of Education, College of Chemistry & Materials Science, Northwest
University, Xi'an, Shaanxi 710127, P. R. China
d. Sorbonne Université, Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, 4
place Jussieu, 75005 Paris, France.
*
Tel.: +86 029 88304569; Fax: +86 029 88304569. E_mail:
wushaoping@nwu.edu.cn
Electronic Supplementary
DOI: 10.1039/x0xx00000x
†
Information
(ESI)
available.
See
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safety evaluation of sulfur-fumigated products worldwide, and the
residual content is expected to be controlled as low as possible.16
Therefore, it is of extremely emergency to develop a convenient,
efficient and reliable method for monitoring sulfur dioxide
derivatives in sulfur-fumigated TCMs. At present, several traditional
analytical methods have been used to detect SO2 and its
derivatives, such as spectrophotometry,17 high-performance liquid
chromatography (HPLC),18,19 electrochemistry,20,21 capillary
electrophoresis,22 flow injection analysis23,24 and so on. However,
these methods depend on high-cost instruments, complex pre-
treatment processes and are time-consuming, which limit their
practical application.
View Article Online
DOI: 10.1039/C8NJ06326E
Previous work：
O
OH
SO3
-
Ф = 0.43
LOD = 3.0 μM
Response time: 2 min
Linear rang: 0～0.3 μM
-
H
H
HSO3
N
O
O
N
O
O
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Present work：
O
-
HO
SO3
H
H
Ф = 0.65
LOD = 0.23 μM
Response time: < 10 s
Linear rang: 0～100 μM
S
S
2-
SO3
N
O
O
N
O
O
Scheme 1 Strategies to response of probe DTCC.
To solve these problems, fluorescent probes have been
extensively used as promising detection tools due to its non-
invasiveness, high sensitivity and selectivity, fast response, as well
as high temporal and spatial resolution.25,26 Accordingly, some
fluorescent and chromogenic chemosensors have been developed
for the monitor of sulfur dioxide derivatives based on familiar
mechanism contained nucleophilic reaction with aldehyde,27
Michael addition,28 dequenching of levulinate29 and coordinative
interactions.30 However, these works have several drawbacks, such
presence of other RSS and excellent sensitivity for SO2 derivatives
with low limit of detection (0.23 µM) and widely linear range (0～
100 µM). Furthermore, probe DTCC was successfully applied in
fluorescent imaging in SH-SY5Y neurobalstoma cells with excellent
membrane permeability and stability. For practical application,
probe DTCC was employed for monitoring the total SO2 derivatives
in several real TCM sample (Scheme 1).
as long detecting time (up to 10 h), poor water solubility and 2. Materials and methods
interference by hydrogen sulfide or biothiols.31 Therefore, it is
2.1. Materials
utmost important to design a fast response, widely linear range and
the sophisticated background sample application to detect SO2
All chemical reagents and solvents were purchased from
derivatives.
commercial sources with analytical grades, and used without
further purification. All the reactions were carried out in dried
glassware with magnetic stirring and the progress of reactions was
monitored by thin layer chromatography (TLC). TLC was performed
by using Merck F254 gel-60 plates. Silica gel (200 mesh) was used as
the solid phase for column chromatography. Stock solutions (1.0
Coumarin scaffold is frequently used as fluorescence dyes for the
detection of RSS due to their less toxicity, good water solubility and
convenience to be modified for sensing of different analytes.32 In
addition, aromatic heterocyclic compounds containing a five-
membered furan or thiophene unit have been extensively studied
in recent years due to their impressive optical properties and
excellent charge-transport properties.33,34 To the best of our
knowledge, there has been no report on the properties and
applications of thiophene modified coumarin derivatives as the
-
-
-
Mm) of Cys, GSH, S2O52-, S2O82-, NO2 , NO3 , F-, Cl-, I-, Br-, SO42-, HSO4 ,
CO32-, AcO- were prepared by direct dissolution of proper amounts
of sodium salts. The Hepes buffer solution was prepared from 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (2.3800 g) in 100
mLdeionized water and 20% NaOH solution. The deionized water
was prepared from a Millipore water purification system. All
glassware were cleaned with deionized water for three times and
dried before use.
35
fluorescence probe. Herein, a fast response time, low limit of
detection and high fluorescence quantum yield probe DTCC was
designed and synthesized to detect SO2 derivatives based on
coumarin-thiophene dye which was fused with a coumarin moiety
and 2-thiophenecarboxaldehyde. Probe DTCC exhibited fast
response time (less than 10 s), satisfactory selectivity for SO2 in the
2.2. Instrumentation and methods
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The UV–vis absorption spectra were determined at room further purification (Yield: 70%, Rf = 0.2, PE:EtOAc =V1ie:w1)A.rticHle ONnMlinRe
temperature on a Shimadzu UV-2550 spectrophotometer in a 1 cm (600 MHz, DMSO-d6) δ 11.89 (s, 1H), 7.54D(Od,I:J1=09.1.003,91/CH8),N6J0.66532(6dE,
quartz. The fluorescence spectra were taken on a Hitachi F-7000 J=9.0, 2.5, 1H), 6.44 (s, 1H), 5.25 (s, 1H), 3.40 (q, J=7.0, 4H), 1.11 (s,
Fluorescence spectrophotometer using a 5.0 nm slit width in a 1×1 6H). HRMS (C13H15NO3): calcd. for [M+H]+ 234.1130; found: [M+H]+
cm quartz cell. The cell fluorescence imaging was obtained by laser 234.1134 (Fig. S3).
scanning confocal fluorescence microscopy Olympus FV1000
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2.5. Synthesis of 4-chloro-7-(diethylamino)-2-oxo-2H-chromene-3-
(Olympus Corporation, Tokyo, Japan). HRMS spectra were recorded
carbaldehyde (3)
on a micro TOF-QII mass spectrometer. 1H NMR and 13C NMR
spectra were obtained from a Varian Gemini 2000 DMX600 MHz FT
Fresh distilled DMF (2.6 mL) was added dropwise to POCl3 (2.6 mL)
NMR spectrometer using CDCl3/DMSO-d6 as solvent and TMS as
at room temperature and was stirred for 30 min to obtain a red
internal standard. The chemical shifts were represented by ppm.
solution under nitrogen. Then a portion of 7-(diethylamino)-4-
The quantum yield was measured at room temperature referenced
hydroxy-2H-chromen-2-one (2.0000 g, 8.6 mmol, 1.0 equiv.,
to fluorescein in aqueous solution of 0.1 M sodium hydroxide. The
dissolved in 9 mL DMF) was added dropwise to the above solution
structures of synthesized compounds were fully characterized by
and yield a scarlet suspension. After the mixture was stirred at 60
1
HRMS, H NMR, 13C NMR and the related spectra are shown in the
℃ for 12 h, it was poured into 100 mL of ice water. NaOH solution
Supporting Information.
(20%) was used to adjust the pH to obtain a large amount of
precipitate. The crude product was subjected to column
2.3. Synthesis of compound bis-(2, 4, 6-trichlorophenyl) malonate
chromatography (PE:EtOAc=1:4) to give 4-chloro-7-(diethylamino)-
(1)
2-oxo-2H –chromene-3-carbaldehyde as an orange solid (Yield:
74%, Rf = 0.75, PE:EtOAc=1:3). 1H NMR (600 MHz, CDCl3) δ 10.26 (s,
1H), 7.80 (d, J=9.0, 1H), 6.67 (d, J=9.0, 2.5, 1H), 6.41 (d, J=2.5, 1H),
3.45 (q, J=7.1, 4H), 1.25 (t, J=7.0, 6H). HRMS (C14H14ClNO3): calcd.
For [M+H]+ 280.0740; found: [M+H]+ 280.0784 (Fig. S4).
A mixture of commercial available malonic acid (0.5200 g, 5.0
mmol, 0.63 equiv.) and 2,4,6-trichlorophenol (1.5800 g, 8.0 mmol,
1.0 equiv.) in toluene (20 mL) and POCl3 (1 mL, 10.5 mmol, 1.31
equiv.) was heated at 100 ℃ for 4 h in oil bath. After cooling to
room temperature, the mixture was poured into ice water and
extracted with ethyl acetate (3×50 mL). The combined organic layer
was dried with MgSO4 and concentrated under reduced pressure to
give bis-(2, 4, 6-trichlorophenol) malonate 1 as a white solid, which
was used directly in the next step without further purification
2.6. Synthesis of 7-(diethylamino)-4-oxo-4H-thieno [3, 2-c]
chromene-2-carbaldehyde (DTCC)
A solution of Na2S•9H2O (0.0470 g, 0.2 mmol, 1.1 equiv.) was added
4-chloro-7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde
(0.0500 g, 0.2 mmol, 1.0 equiv.) in DMF (3 mL). The mixture was
stirred at 60 ℃ for 2 h. Then chloroacetaldehyde (0.013 mL, 0.2
mmol, 1.1 equiv.) was added rapidly and the reaction was stirred
during 3 h at 60 ℃, K2CO3 (0.0280 g, 0.2 mmol, 1.1 equiv.) was
dissolved in water (1.0 mL) and added to the reaction solution. The
mixture was stirred for 10 min at 60 ℃, cooled at room
1
(Yield: 43%, Rf = 0.64, PE: EtOAc = 5:1). H NMR (600 MHz, CDCl3) δ
7.40 (s, 4H), 4.05 (s, 2H). HRMS (C15H6Cl6O4): calcd. For [M+H]+
460.8475; found: [M+H]+ 460.8470 (Fig. S2).
2.4. Synthesis of 7-(diethylamino)-4-hydroxy-2H-chromen-2-one
(2)
3-Diethylamino- phenol (0.8150 g, 5 mmol, 1.0 equiv.) was added to temperature and quenched in water. The aqueous phase was
a solution of compound 1 (2.2840 g, 5 mmol, 1.0 equiv.) in dry extracted with EtOAc (3×50 mL). The organic layer was dried over
MgSO4 and concentrated under reduced pressure. The residue was
purified by column chromatography (PE:EtOAc=5:1) to obtain the
toluene (5 mL). The mixture was heated under reflux at 110 ℃ for 2
h. Then the reaction mixture was cooled to room temperature and
filtered. The resultant grey solid was washed with toluene and dried
under high vacum to generate 7-(diethylamino)-4-hydroxy-2H-
chromen-2-one 2, which was used directly in the next step without
1
desired compound DTCC (Yield: 40%, Rf = 0.83, PE:EtOAc=1:1). H
NMR (600 MHz, DMSO-d6) δ 9.96 (d, 1H), 8.39 (s, 1H), 7.69 (d, J =
8.9 Hz, 1H), 6.76 (d, J = 8.9 Hz, 1H), 6.62 (s, 1H), 3.46 (dd, J = 7.0 Hz,
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4H), 1.14 (t, J = 6.9 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 156.55, After the cells were incubated with probe DTCC at different
154.98, 154.30, 151.15, 139.08, 125.98, 120.62, 109.70, 103.81, concentrations (0, 5, 10, 15 and 20 µM) for D2O4Ih: 1,0C.1C0K3-98/C(180NJµ0L6)3w26aEs
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97.00, 67.38, 44.17, 12.30. HRMS (C16H15NO3S): calcd. For [M+Na]+
added to each well of the 96-well plate for 2 h at 37 ℃, the
absorbance was measured at 450 nm using a microplate reader.
Five replicates were done for each treatment group.
324.0773; found: [M+Na]+ 324.0664 (Fig. S5-S7).
9
2.7. Titration experiments of probe DTCC
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2.10. Cell imaging of probe DTCC
For UV-vis and fluorescence titrations, Sodium sulphite and sodium
bisulfite (Na2SO3/NaHSO3) solution was prepared by dissolving
Na2SO3/NaHSO3 in deionized water (5.0 mM). Probe stock solution
(1.0 mM) was prepared by dissolving 15.068 mg DTCC in 50 mL
absolute ethanol. Different portions of the stock solution were then
diluted to different concentrations for further usage. Generally, 25
µL of the DTCC solution was added into 5 mL of buffer solution (0.1
mM Hepes buffer, pH =7.4, 20% CH3CN) in a colorimetric tube. The
For intracellular imaging of SO32-, SH-SY5Y neuroblastoma cells were
placed on a 20 mm diameter glass bottom cell culture dish and
allowed to adhere for 24 h. Before confocal scanning imaging, these
cells were incubated with 10 µM of probe DTCC at 37 ℃ for 10 min
followed by washing three times with phosphate buffered saline
(PBS, pH = 7.4). Subsequently, the cells were washed with PBS for
three times to remove excess DTCC, then in situ treated with
different concentrations of Na2SO3 solution (0, 5, 10, 15, 20 µM,
respectively). Cells were then visualized with an FV1000 laser
scanning confocal microscope. The excitation wavelength was set to
405 nm and the emission collected in the range 450-550 nm.
-
-
2-
other ions (Cys, GSH, S2O52-, S2O82-, NO2 , NO3 , F-, Cl-, I-, Br-, SO4
,
HSO4 , CO32-, AcO-) was dissolved in water and added to the probe
solution under the same condition. The spectra of these solutions
were recorded by means of fluorescence method. Fluorescence
measurements were carried out with a slit width of 5 nm (λex = 385
nm) in 10 mm quartz cuvettes at room temperature and the scan
rate was 1200 nm/min.
-
3. Results and discussion
3.1. Synthesis of probe DTCC
2.8. Quantum calculations
The synthesis of probe DTCC was achieved in four steps as shown in
Scheme 2. Treatment of 2, 4, 6-trichlorophenol with malonic acid in
POCl3 at 100 ℃ gave the compound 1. The compound 1 reacted
with 3-diethylaminophenol in anhydrous toluene to give coumarin
derivative 2. The intermediate aldehyde 3 was prepared by the
Vilsmeier-Haack reaction, and it was further used to synthesize the
compound DTCC through condensation with sodium ulphide in DMF
in the presence of chloroacetaldehyde and K2CO3 at 60 ℃ without
purification.40,41 The detailed synthetic procedures were presented
in the Materials and methods section. All synthetic compounds
were carefully characterized by 1H NMR, 13C NMR and HRMS.
Geometries of probe DTCC in the ground state (S0) were optimized
by the density functional theory (DFT) using B3LYP with 6-311G(d)
basis set.36 The excited state geometries and related optical
properties of all molecules were calculated by TD-DFT at the same
level of theory. Vibrational analysis calculations were done in order
to confirm that optimized geometries corresponded to local minima
on the potential energy surfaces of the whole molecules. The
olarisable continuum model (PCM)37,38 using the integral equation
formalism variant (IEFPCM) was employed in DMF to describe the
solvent effects throughout theoretical calculations. All calculations
were performed using the Gaussian 09 package.39
3.2. Photophysical properties of probe DTCC
2.9. Cytotoxicity experiments
With probe DTCC in hand, the emission properties of probe DTCC
were investigated in 20% CH3CN solution (0.1 mM Hepes buffer, pH
7.4) at room temperature. As a new designed fluorescent probe,
DTCC was evaluated the spectroscopic properties in different
polarity solvents, such a DCM, DMF, EtOH, DMSO and so on.
Cell counting kit-8 (CCK-8) was used to detect the cytotoxicity of
probe DTCC. SH-SY5Y cells were purchased from the ATCC Cell
Bank. Cells were seeded in 96-well plates at a concentration of 1 ×
105 cells per well and cultured for 24 h in DMEM (supplemented
with 10% fetal bovine serum (FBS)) in an incubator (37 ℃, 5% CO2).
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OH
Cl
Cl
Cl
Cl
O
O
Cl
Cl
a
b
Cl
O
O
Cl
N
O
O
HO
Cl
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9
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2
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Cl
O
S
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c
d
H
N
O
O
N
O
O
3
Probe DTCC
Fig. 1 Fluorescence spectra of probe DTCC reaction with Na2SO3 and
NaHSO3 in a 20% CH3CN solution (10 mM Hepes buffer, pH 7.4) at
room temperature. λex= 385 nm, λem= 475 nm, slits: 5.0 nm / 5.0
nm.
Scheme 2 Reagents and conditions: a) Malonic acid, POCl3, 100 ℃,
reflux, 4 h, 43%; b) 3-Diethylaminophenol, toluene, 100 ℃, 70%; c)
DMF, POCl3, 74%; d) DMF, Na2S•9H2O, Chloroacetaldehyde, K2CO3,
40%.
2-
3.3. Fluorescence response of probe DTCC towards SO3
Meanwhile, the fluorescence quantum yield of DTCC-hydrogen
sulphite adduct was carefully measured using fluorescein in
aqueous solution of 0.1 M sodium hydroxide (Φf = 0.79) as the
standard reference compound. The photophysical data for
fluorophore DTCC were depicted in Table S2.
The fluorescence emission spectra of probe DTCC (10 µM) after
2-
addition of SO3 in a 20% CH3CN water solution (10 mM Hepes
buffer, pH 7.4) were shown in Fig. 2. Probe DTCC exhibited weak
fluorescence emission at 550 nm under the excitation wavelength
of 385 nm, since the fluorescence could be nearly completely
quenched in polar solvents when an aldehyde group was
introduced to coumarin. However, a significant enhancement of the
In the fluorescence emission study, only very weak fluorescence
was observed for DTCC in 550 nm when excited at 385 nm. A new
fluorescence emission peak at 475 nm could be readily observed
2-
emission intensity positioned at 475 nm when SO3 was added to
2-
after reaction with SO3 as shown in Fig. 1. At the same time, the
the probe solution. An excellent linear relationship (R2 = 0.9974)
was obtained with SO32- concentrations ranging from 0 to 100 µM
by plotting the corresponding fluorescence intensity ration and the
colour of solution was noticeable changed from earthy yellow to
blue under fluorescent lamp (Fig. 3a, inset). These results illustrated
that probe DTCC could be used as a sensitive probe for detection of
2-
SO3
.
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Fig. 2 (a) Fluorescence spectra responses of probe DTCC (10 µM) in the presence of different concentrations of SO32- (0 ~ 100 µM) in a 20%
CH3CN solution (10 mM Hepes buffer, pH 7.4) at room temperature; Error bars are ± SD, n = 3; (b) Linear relationships between
fluorescence response at 475 nm and concentrations of SO32-. λex= 385 nm, λem= 475 nm, slits: 5.0 nm / 5.0 nm.
2-
Fig. 3 (a) Fluorescence spectra of probe DTCC (10 μM) upon addition of 100 μM SO3 and 19 kinds of other ions in a 20% CH3CN solution
(10 mM Hepes buffer, pH 7.4). (b) The pillars in the front row represent fluorescence response of the probe to the varying ions of interest.
2-
The pillars in the back row represent the subsequent addition 100 μM SO3 to the solution containing probe and the various anions 100
μM, respectively. The detection medium was in a 20% CH3CN solution (10 mM Hepes buffer, pH 7.4). λex= 385 nm, λem= 475 nm, slits: 5.0
nm / 5.0 nm.
2-
concentration of SO32-. The detection limit of DTCC for SO3 was no obvious change of the fluorescence signal was detected upon
calculated to be 0.23 µM according to signal to noise ratio (S/N = 3). addition of other analytes even in considerable concentrations.
These results illustrated that the probe DTCC possesses excellent
Then the anti-interference ability of DTCC to other analytes has
2-
capability for quantitative determination of SO3 with high
been examined as shown in Fig. 3b, all the competing analytes did
sensitivity in 20% CH3CN aqueous media.
not interfere the detection of SO32-, these results demonstrated
2-
3.4. Selectivity experiment
that probe DTCC has excellent selectivity for SO3 over a variety of
interfering species that could be present in cells and TCM sample,
Various biologically relevant species were investigated including
ROS, RSS, reactive nitrogen species (RNS), anions and cations for
evaluating the selectivity of probe DTCC toward SO32-. As shown in
2-
which enable it to detect SO3 in complex biological system and
other sophisticated background sample.
Fig. 3a, the fluorescence intensity ration of the probe DTCC 3.5. Influences of time and pH-dependent of probe DTCC
significantly increased after the addition of SO32-. On the contrary,
The time-dependent fluorescence changes of probe DTCC toward
2-
SO3 were investigated using kinetic study. As displayed in Fig. 4a,
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the fluorescence intensity at 475 nm increased dramatically and from 3.0 to 11.0. Probe DTCC itself was stable in the pVHiewraAnrtgiceleoOfn3lin.0e
DOI: 10.1039/C8NJ06326E
2-
reached a plateau within 10 s. In addition, the fluorescence to 11.0. After addition of SO3 to the DTCC solution, fluorescence
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intensity of probe DTCC almost kept unchanged after the reaction. intensity increased significantly in the pH range from 4.0 to 10.0,
The time-dependent absorption changes were in consistent with these results indicated that probe DTCC had a wide range of
the time-dependent fluorescence changes (Fig. S1). The results tolerance to pH change in sophisticated sample.
9
2-
illustrate that probe DTCC could fast monitor SO3 change in
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3.6. Proposed recognition mechanism and theoretical
biological environment and TCM sample.
computation.
For SO32- detection, pH change was an important factor in various
The proposed mechanism for the detection of SO32- was interpreted
environmental. The different pH range of the free probe DTCC and
in Scheme 3. The probe itself has a conjugated system that showed
2-
probe DTCC with SO3 was studied in 20% CH3CN mixed solution
weak fluorescence activity, ICT effect was open. When the probe
with addition of buffers prepared in the relevant pH range from 3.0
coexists with SO32- that aldehyde-hydrogen sulphite adduct
to 11.0, respectively (Fig. 4b). The fluorescence intensity of free
probe DTCC could not undergo any notable changes in the pH range
Fig. 4 (a) Time-dependent fluorescence intensity of free probe (10 μM) and probe (10 μM) with SO32-(100 μM) in Hepes buffer (10 mM, pH
7.4, containing 20% CH3CN, 25℃) solution. (b) pH-dependent changes of fluorescence intensity of probe (10 μM) in the absence and
2-
presence of SO3 (100 μM) in Hepes buffer (10 mM, pH 7.4, containing 20% CH3CN, 25℃) solution. Different pH values were adjusted by
NaOH
and
HCl
aqueous
solution.
λex
=
385
nm,
λem=
475
nm,
slit=5.0nm.
fluorescence was remarkably enhanced by the nucleophilic addition appropriately and subjected to mass spectroscopy, where an
reaction of sulfhydryl toward the α, β-unsaturated aldehyde in expected peak at m/z 382.0417, corresponding to aldehyde-
-
probe DTCC. To prove the above proposed sensing mechanism, we hydrogen sulphite adduct (C16H16NO6S2 , Exact Mass: [M]- 382.0425)
initially attempted to isolate the aldehyde-hydrogen sulphite was observed (Fig. S8).
adduct from the mixture of probe DTCC with sodium sulphite.
2-
To further investigate the optical property upon addition of SO3
Unfortunately, it was observed that the intermediate is unstable in
aqueous solution, and TLC analysis shows that it has been in probe DTCC solution, DFT calculations were performed using
recovered to its starting material gradually, indicating this addition
reaction could be reversible.42
f
f
o
T
n
O
HO
-
o
In order to verify our hypothesis, the reaction of DTCC （100 µM
）and SO32- （150 µM）was performed at room temperature for 3
min in MeOH solution, then the reaction solution was diluted
SO3
T
C
I
H
C
I
H
S
S
2-
SO3
N
O
O
N
O
O
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2-
Scheme 3 Proposed mechanism of the reaction of DTCC with SO3
.
laser confocal microscope. When the SH-SY5Y cells were incubated
View Article Online
with probe DTCC (10 µM) for 5 min, no fluoDreOsIc: e10n.c10e3w9/aCs8dNiJs0p6la3y2e6dE
at the blue channel (λex= 405 nm). On the contrary, certainly strong
fluorescence intensity was clearly observed in the cytoplasm after
adding SO32- (5 µM) and then incubating for another 5 min at 37 ℃,
indicating that probe DTCC could penetrate the cell membranes and
react with exogenous SO32- in the cellular environment at extremely
low concentrations. As shown in Fig. 6, the fluorescence intensity of
the cells was gradually increased when the concentration of probe
DTCC changed from 5 µM to 20 µM. At the same time, the
fluorescence intensity of the cells was basically stable and barely
any fluorescence quenching was observed until 30 min. The above
results proved that probe DTCC has the ability to visualize and
quantitatively detect SO32- in SH-SY5Y neuroblastoma cells.
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9
B3LYP/6-311+G(d) level of Gaussian 09 program. The
2-
optimized structures of DTCC and the adduct DTCC-SO3 were
shown in Fig. S9. The highest occupied molecular orbital (HOMO)
and the lowest unoccupied molecular orbital (LUMO) in DTCC were
shown in Fig. 5. Obviously, there was no ICT process occurred in
the adduct DTCC-SO32-, in that a sp3 hybridized carbon atom
existed in quaternary carbon moiety of thiophene ring. The
interruption of the ICT process also resulted in the increased
HOMO-LUMO energy gap of the adduct DTCC-SO32- (3.60 eV) in
comparison with that of the DTCC (3.12 eV), which brought
about a blue-shift in the emission spectra observed. Therefore,
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2-
these DFT calculations verified that SO3 was bound to the
aldehyde group of probe DTCC and are in good agreement
with the experimental results.
3.8. Practical application of probe DTCC in Traditional Chinese
Medicine
There was an excellent linear relationship between the
fluorescence intensity and the concentration of SO32- from 0 to 100
µM. In order to further explore the practical application of probe
DTCC in realistic sample, positive sample was collected and
detected the concentration of SO32- in Traditional Chinese Medicine
according to the excellent properties of probe DTCC. The
fluorescent titration spectra of DTCC with different concentrations
2-
of SO3 showed an increase at 475 nm in real TCM sample. The
detection results illustrate that probe DTCC was able to detect the
Fig. 5 HOMO-LUMO energy levels and frontier molecular orbital of
2-
probe DTCC and the adduct DTCC-SO3
.
3.7. Cytotoxicity and fluorescence imaging for living cells
CCK-8 was used to evaluate cytotoxicity of probe DTCC before
intracellular fluorescence imaging for living cells. As shown in Fig.
S10, after 24 h of incubation with 5 μM probe, more than 95% of
the SH-SY5Y neuroblastoma cells remained viable. In addition, the
cell viability was as high as 85% when 20 μM probes were incubated
for 24 h. These data indicated that probe DTCC exhibits low
cytotoxicity and good biocompatibility at concentrations of 0 ~ 20
μM in SH-SY5Y neuroblastoma cells.
With these preliminary data in hand, the potential application of
the probe DTCC for imaging SO32- in living cells was explored using a
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Table 2 Comparison of analysis results for sulfite in TCM sample by
DOI: 10.1039/C8NJ06326E
fluorescent probe DTCC with a traditional method. (n=5)
View Article Online
5
6
7
8
Sample
Batch No. Pharmacopoeia Fluorescenc
e method
method
(mg/kg)
(mg/kg)
9
201701001a
170301
386
361
390
5
361
372
363
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Dioscotea
opposite
Thunb
201701001b
161001
Angelica
sinensis
(Oliv.)
170301
5
4
160801
3
6
Diels
20150416
17042601
170403
51
23
36
59
21
34
Lycium
barbarum
L.
a, b: The two samples with the same batch No. come from the
different regions.
Fig. 6 Confocal fluorescence microscopic images of SH-SY5Y cells
incubated by probe DTCC (10 µM) and observed under bright field
(a), blue channel (b), overlay (c), then further incubation with
Na2SO3 (5、10、15、20 µM) for 5 min at 37℃ and observed under
bright field, blue channel, overlay.
2-
concentration of SO3 in Traditional Chinese Medicine with good
recovery (Table 1) and excellent consistent with the traditional
experimental values according to the working curve, respectively
(Table 2). More importantly, the fluorescent probe DTCC methods
for quantitative analysis of SO32- in TCM involve short analysis time,
simple sample preparations and easy operation.
Table 1 Determination of sulfite in negative TCM sample.
Sample
Added (µM) Found (µM)
Recovery (%)
4. Conclusions
0
1.01
40.70
82.86
1.05
-
Dioscotea
opposite
Thunb
40.00
80.00
0
101.75
103.58
-
In summary, a novel colorimetric fluorescent probe was successfully
designed and synthesized based on coumarin scaffold for highly
2-
selective and sensitive detection of SO3 with fast response time,
low limit of detection and wide pH range in applications. Moreover,
Angelica
sinensis
(Oliv.) Diels
40.00
80.00
0
41.56
84.02
1.10
103.9
105.02
-
2-
the probe could be applied to sense exogenous imaging of SO3 in
SH-SY5Y neuroblastoma cells with negligible cytotoxicity, excellent
cell membrane permeability and good biocompatibility.
Additionally, the probe could be acted as an effective tool for the
Lycium
barbarum
L.
40.00
80.00
38.14
77.89
95.35
97.36
2-
detection of SO3 in Traditional Chinese Medicine and other
sophisticated sample. Furthermore, we envision that such a novel
probe DTCC could be applied to further reveal essential information
about SO32- in extensive biological systems and in real samples.
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The Directorate for the Quality of Medicines & VHieewaAltrthicCleaOrenlionef
DOI: 10.1039/C8NJ06326E
Conflicts of interest
There are no conflicts to declare.
the Council of Europe. (b) Pharmacopoeia of People’s
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Republic of China (2010). Vol. 1. Beijing: China Medical
Science Press. (c) United States Pharmacopoeia 38 (2014).
Rockville: United States Pharmacopeial Convention Inc.
17 P. West and G. Gaeke, Anal. Chem., 1956, 28, 1816–1819.
18 Y. Zhao, W. Qiu, C.-Y. Yang and J.-N. Wang, Energy Fuels,
2017, 31, 693–698.
Acknowledgements
This work was supported by the Program for Changjiang Scholars
and Innovative Research Team in University (No. IRT_15R55),
Scientific Research Program Funded by Shaanxi Provincial Education
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Department (No. 18JK0774), the International Science
&
Technology Cooperation Program of Shaanxi Province (No.
2016KW-003) and Program of Innovation for Undergraduates of
Northwest university (No. 2019230).
19 Z. Zhong, G. Li, B. Zhu, Z. Luo, L. Huang and X. Wu, Food
Chem., 2012, 131, 1044–1050.
20 U. T. Yilmaz and G. Somer, Anal. Chim. Acta., 2007, 603, 30–
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DOI: 10.1039/C8NJ06326E
Design and synthesis of a novel colorimetric fluorescent probe for
selective detection of sulfur dioxide in SH-SY5Y neuroblastoma cells
and its application in Traditional Chinese Medicines
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Lili Yanga,b, Mofan Liua,b, Kangjia Shenga,b, Xiaolu Lia,b, Junli Dua,b, Yaoyao Ninga,b,
Xiaoqing Wangc, Jianli Lic, Yongmin Zhanga,b,d and Shaoping Wua,b*
a School of Pharmacy; Key Laboratory of Resource Biology and Biotechnology in Western China
(Northwest University), Ministry of Education; Biomedicine Key Laboratory of Shaanxi Province,
Northwest University, Xi’an 710069, China
b Joint International Laboratory of Glycobiology and Medicinal Chemistry, Northwest University,
Xi’an, Shaanxi 710069, China
c
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of
Education, College of Chemistry & Materials Science, Northwest University, Xi'an, Shaanxi
710127, P. R. China
d Sorbonne Université, Institut Parisien de Chimie Moléculaire, CNRS UMR 8232, 4 place Jussieu,
75005 Paris, France
* Tel.: +86 029 88304569; Fax: +86 029 88304569. E_mail: wushaoping@nwu.edu.cn
Graphical Abstract:
Keywords: Coumarin scaffold, Fluorescent probe, Sulfur dioxide, Fast responsive,
Bioimaging, Traditional Chinese Medicines