A novel colorimetric and ratiometric fluorescent probe for selective detection of bisulfite in real samples and living cells

Article abstract of DOI:10.1016/j.dyepig.2018.12.009

An abnormal level of bisulfite can induce toxicological effects associated with lung cancer, cardiovascular and neurological disorders. Therefore, it is of significance to develop an effective fluorescent probe to detect bisulfite in living cells. Herein, a novel fluorescent probe QPCT, based on a 1,4-addition mechanism, was constructed for the colorimetric and ratiometric detection of bisulfite. QPCT displayed high selectivity and anti-interference ability to bisulfite over other anions and biothiols. The probe renders a sensitive ratiometric response to bisulfite with a remarkable fluorescence blue shift from 590 to 537 nm and the fluorescence ratio was linear with bisulfite concentration over the range of 0–120 μM. More importantly, QPCT has been successfully applied to detect bisulfite in sugars and living A549 cells, which indicated that QPCT had a great capability for monitoring bisulfite in complex systems.

Full text of DOI:10.1016/j.dyepig.2018.12.009

Accepted Manuscript
A novel colorimetric and ratiometric fluorescent probe for selective detection of
bisulfite in real samples and living cells
Jin Li, Ying Gao, Haoran Guo, Xiaokang Li, Haoyang Tang, Jian Li, Yuan Guo
PII:
S0143-7208(18)32489-6
https://doi.org/10.1016/j.dyepig.2018.12.009
DYPI 7214
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 12 November 2018
Revised Date: 1 December 2018
Accepted Date: 6 December 2018
Please cite this article as: Li J, Gao Y, Guo H, Li X, Tang H, Li J, Guo Y, A novel colorimetric and
ratiometric fluorescent probe for selective detection of bisulfite in real samples and living cells, Dyes and
Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2018.12.009.
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ACCEPTED MANUSCRIPT
A novel colorimetric and ratiometric fluorescent probe for
selective detection of bisulfite in real samples and living cells
Graphical abstract
A novel fluorescent probe QPCT for the colorimetric and ratiometric detection
-
of HSO3 was constructed and successfully applied for sugar analysis as well as
imaging in living A549 cells.

ACCEPTED MANUSCRIPT
A novel colorimetric and ratiometric fluorescent probe for selective
detection of bisulfite in real samples and living cells
Jin Lia,#, Ying Gaoa,#, Haoran Guoa, Xiaokang Lib, Haoyang Tangc, Jian Lib, Yuan Guoa,*
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education,
National Demonstration Center for Experimental Chemistry Education, College of Chemistry and Materials
Science, Northwest University, Xi’an 710127, PR China
bSchool of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
cSchool of Automation, Xi’an University of Posts and Telecommunications, Xi’an 710121, PR China
#J. Li and Y. Gao contributed equally to this work.
E-mail address: guoyuan@nwu.edu.cn (Y. Guo).
Abstract
An abnormal level of bisulfite can induce toxicological effects associated with lung cancer, cardiovascular and
neurological disorders. Therefore, it is of significance to develop an effective fluorescent probe to detect
bisulfite in living cells. Herein, a novel fluorescent probe QPCT, based on a 1,4-addition mechanism, was
constructed for the colorimetric and ratiometric detection of bisulfite. QPCT displayed high selectivity and
anti-interference ability to bisulfite over other anions and biothiols. The probe renders a sensitive ratiometric
response to bisulfite with a remarkable fluorescence blue shift from 590 to 537 nm and the fluorescence ratio
was linear with bisulfite concentration over the range of 0-120 µM. More importantly, QPCT has been
successfully applied to detect bisulfite in sugars and living A549 cells, which indicated that QPCT had a great
capability for monitoring bisulfite in complex systems.
Keywords: Colorimetric and ratiometric; Fluorescent probe; Bisulfite; Real samples; Live-cell imaging
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1. Introduction
Sulfur dioxide (SO2), produced by the combustion of coal and fossil fuels [1,2], is traditionally regarded
as an environmental pollutant [3,4]. SO2 can be easily inhaled by human beings and then turned into sulfite
2-
-
(SO3 ) and bisulfite (HSO3 ) salts in the physiological environment [5-7]. Epidemiological studies have
revealed that prolonged exposure to SO2 derivatives may cause cancer, asthma [8], cardiovascular and
2-
-
neurological disorders [9], which can be mainly attributed to sulfite (SO3 ) or bisulfite (HSO3 ) [10]. In addition,
2-
-
SO3 and HSO3 play a vital role in food, beverages, and pharmaceutical products to prevent oxidation,
browning, and microbial reactions during production and storage [11-16]. There is therefore a pressing need to
-
develop a convenient and rapid technique for detecting SO32- and HSO3 in the environment and in vivo.
-
Among the current detection techniques for HSO3 , fluorescent probes are the most attractive for both
environmental and bioimaging applications because of their high selectivity and sensitivity, non-invasiveness,
real-time detection and ease of operation [17-20]. In recent years, some fluorescent probes have been developed
-
for detecting HSO3 based on the nucleophilic reaction with aldehyde [21,22], selective deprotection of
levulinate [23], coordinative interactions [24] and Michael-type additions [25]. However, most of these probes
-
respond to HSO3 with changes only in fluorescence intensities, which can be influenced by many factors such
as probe concentration, environment effects and instrumental factors [26-28]. In contrast, ratiometric probes
can eliminate the aforementioned interferences by self-calibration of two emission bands [29,30]. Moreover, a
majority of fluorescent probes suffer from great interference by reactive sulfur such as H2S, Cys and Hcy
[19,31]. As a consequence, it is of vital importance to develop a ratiometric and high selectivity fluorescent
-
probe for detecting HSO3 .
-
To address the above issues, we developed a novel fluorescent probe QPCT for detecting HSO3 based on
-
a 1,4-addition mechanism (Scheme 1) [32]. Upon the addition of HSO3 , QPCT exhibited visible color and
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-
fluorescence changes to HSO3 with excellent selectivity. What is more, QPCT was not only employed for
monitoring the concentrations of bisulfite in several real samples but was also applied to visualize bisulfite in
living cells.
-
Scheme 1. Chemical structure of QPCT and the proposed mechanism of QPCT for HSO3 .
2. Materials and Methods
2.1 Materials and measurements
Unless otherwise noted, all chemical reagents and solvents were purchased from commercial suppliers and
1
used as received. Water used throughout the experiments was doubly distilled. H NMR and 13C NMR were
recorded on a Varian INOVA 400 spectrometer, using tetramethylsilane (TMS) as internal standard. Chemical
shifts were expressed in ppm and coupling constants (J) in Hz. High-resolution mass spectra (HRMS) were
obtained on a Bruker micro TOF-Q mass spectrometer. Fluorescence spectra were recorded on an F-2700
fluorescence spectrometer with a 10 mm quartz cuvette. UV-Vis absorption spectra were recorded on a
Shimadzu UV-1700 spectrophotometer. All pH measurements were carried out with an FE28-Standard pH
meter. Cell imaging experiments were performed under a confocal laser scanning microscope (Olympus FV
1000).
2.2 Synthesis
As demonstrated in Scheme 2, the target fluorescent probe (QPCT) was successfully synthesized through
four steps.
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Scheme 2. The synthesis procedures of QPCT.
2.2.1 Synthesis of compound 1
1
Compound 1 was prepared following the previously reported method [33]. Yield: 32%. H NMR (400
MHz, CDCl3) δ (ppm): 6.67 (d, J = 8.00 Hz, 1H), 6.08 (d, J = 8.00 Hz, 1H), 4.46 (s, 1H), 3.10 (d, J = 4.00 Hz,
4H), 2.72-2.64 (m, 4H), 2.00-1.97 (m, 4H) (Fig. S1).
2.2.2 Synthesis of compound 2
1
Compound 2 was prepared following the previously reported method [33]. Yield: 63%. H NMR (400
MHz, CDCl3) δ (ppm): 11.80 (s, 1H), 9.36 (s, 1H), 6.83 (s, 1H), 3.29-3.25 (m, 4H), 2.65 (t, J = 6.00 Hz, 4H),
1.94-1.90 (m, 4H) (Fig. S2).
2.2.3 Synthesis of compound 3
Compound 2 (0.651 g, 3.0 mmol), 4-hydroxy-6-methyl-2H-pyran-2-one (0.450 g, 3.6 mmol) and a
catalytic amount of piperidine (one drop) were dissolved in 6.0 mL absolute ethyl alcohol. The mixture was
stirred at 80 ºC for 5 h with Ar protection. After cooling to room temperature, the precipitates were filtered and
washed by diethyl ether three times. Then, compound 3 was obtained as a red powder (0.48 g). Yield: 48%. 1H
NMR (400 MHz, CDCl3) δ (ppm): 16.31 (s, 1H), 8.35 (s, 1H), 6.95-6.93 (m, 2H), 3.32 (s, 4H), 2.85 (s, 2H), 2.74
(s, 2H), 2.19 (s, 3H), 1.95 (s, 4H) (Fig. S3). 13C NMR (100 MHz, CDCl3) δ (ppm): 196.20, 176.87, 159.87,
152.88, 148.53, 145.90, 127.22, 119.56, 105.64, 99.85, 50.39, 49.98, 27.50, 26.72, 21.20, 20.23, 20.13 (Fig.
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S4). HRMS m/z [M + Na]+ calcd for [C19H19NO4 + Na]+ 348.1206, found 348.1219 (Fig. S5). IR (KBr) ν: 2848,
1710, 1516, 1356, 1306, 1170, 810, 689, 550, 472, 445.
2.2.4 Synthesis of probe QPCT
Compound 3 (0.325 g, 1.0 mmol) and boron trifluoride ethyl etherate (123.5 µL, 1.0 mmol) were dissolved
in 4.5 mL acetic acid and 1.5 mL acetic anhydride, and the mixture was refluxed for 6 h. Then the red solid
precipitates were filtered and washed with methyl tert-butyl ether to afford the target fluorescent probe QPCT
(0.276 g). Yield: 70%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.64 (s, 1H), 7.42 (s, 1H), 7.28 (s, 1H), 3.46 (s,
4H), 2.74-2.67 (m, 4H), 2.33 (s, 3H), 1.91-1.88 (m, 4H) (Fig. S6). 13C NMR (150 MHz, DMSO-d6) δ (ppm):
189.16, 177.05, 159.15, 154.10, 152.01, 148.14, 129.73, 121.68, 109.84, 105.60, 104.54, 90.09, 50.94, 50.38,
27.17, 21.65, 21.46, 20.87, 19.82 (Fig. S7). HRMS m/z [M + H]+ calcd for [C19H18BF4NO3 + H]+: 396.1183,
found 396.1196 (Fig. S8). IR (KBr) ν: 2858, 1715, 1620, 1503, 1424, 1349, 1308, 1274, 1215, 1187, 1056, 694,
482, 431.
2.3 Determination of detection limit of QPCT
The detection limit was calculated based on the following formula (1):
DL = 3σ/k
(1)
where σ is the standard deviation of the blank solution and k is the slope of the calibration curve.
2.4 Spectroscopic measurements
A stock solution of the probe QPCT (1 mM) was dissolved in DMSO and stored at 4 ºC. Stock solutions of
2-
-
-
2-
2-
-
varieties of testing species (10 mM, including SO3 , HSO3 , ClO-, HCO3 , CO3 , SO4 , HSO4 , F-, Br-, I-, Cl-,
HPO4 , PO4 , NO2 , S2O3 , S2-, AcO-, Cys, Hcy, GSH) were prepared in doubly distilled water. Test solutions
3-
3-
-
2-
were prepared by placing 200 µL of QPCT stock solution and an appropriate aliquot of each substance stock
solution in a test tube and diluting the solution to DMSO/PBS buffer solution (7/3, v:v, pH = 7.4, 5 mM). Then
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3 mL of the resulting solution was taken out a quartz cuvette (path length = 1 cm) to measure UV-Vis absorption
and fluorescence spectra at room temperature.
2.5 Cell culture and fluorescence imaging
A549 cells were obtained from Procell Life Science&Technology Co., Ltd (Wuhan, China). A549 cells
were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 1%
penicillin and streptomycin in an atmosphere of 5% CO2 and 95% air at 37 ºC in the incubator. For confocal
microscopy imaging, cells were adjusted to the density of 1×106 cells per mL in a 35 mm confocal dish (15 mm
glass bottom well) and incubated for 24 h. Subsequently, the first group was incubated with QPCT (10 µM) for
30 min; the other groups were incubated with NaHSO3 (5, 10, 15, 20, 25 µM) for 30 min after the QPCT
cultured A549 cells. After being washed three times with PBS buffer, cells were imaged under a confocal laser
scanning microscope (Olympus FV1000).
2.6 Cytotoxicity assay
The cytotoxicity of QPCT was evaluated in A549 cells by the methyl thiazolyl tetrazolium (MTT) assay
[34]. 90% Confluence A549 cells were seeded into a 96-well cell-culture plate at 37 ºC and 5% CO2 atmosphere
overnight followed by incubation for 24 h. After incubating for 24 h, different concentrations of QPCT (0, 5, 10,
15, 20 and 25 µM) were added to the wells. The cells were treated and incubated in the same conditions for 24 h.
MTT solution (10 µL) was then added to each well and kept for another 4 h. The cells were washed with PBS
buffer and were dissolved in 1 mL DMSO. Absorbance at 490 nm was measured using the microplate
spectrophotometer system (Varioskan Flash).
3. Results and Discussion
-
3.1 Spectral properties of QPCT to HSO3
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-
We first investigated the effect of pH on the response of QPCT and QPCT to HSO3 because nucleophilic
-
addition is sensitive to pH [35]. As depicted in Fig. S9, in the absence of HSO3 , the fluorescence intensity ratio
(I537/I590) of the QPCT showed no obvious change in the pH range of 4.0-9.0, demonstrating that probe
-
QPCT was stable at physiological pH conditions. Upon treatment with HSO3 , QPCT displayed the best
response in the pH range of 7.0-9.0, indicating that QPCT can be applied to the physiological pH range. Thus,
the following experiments were conducted at pH = 7.4 to study the spectroscopic response of QPCT toward
-
HSO3 .
-
Then, we evaluated the sensing ability of QPCT to HSO3 in DMSO/PBS buffer solution (7/3, v:v, pH =
7.4, 5 mM). As shown in Fig. 1a, QPCT exhibited two major absorption peaks at 325 nm and 530 nm. Upon
-
treatment with increasing concentrations of HSO3 , it was observed that the absorbance gradually decreased at
530 nm and increased at 325 nm with a well-defined isosbestic point at 360 nm. Meanwhile, the color of the
QPCT solution changed from pink to yellow, which could be easily observed by the naked eye (inset of Fig. 1a).
Fluorescence titration experiments were also carried out. As shown in Fig. 1b, the addition of an increasing
-
amount of HSO3 (0-120 µM) triggered an obvious decrease in the intrinsic emission peak at 590 nm and a
remarkably blue-shifted peak at 537 nm, showing a noticeable fluorescence color change from orange to green.
-
This result may be attributed to the 1,4-addition reaction between HSO3 and QPCT, leading to the shortening
of the π-conjugation of QPCT. It can be seen from Fig. 1c that the fluorescence intensity ratios at 590 and 537
nm (I537/I590) increased from 0.21 to 3.6, which is an 18-fold enhancement. The fluorescence ratio of I537/I590
-
also displayed an excellent linear relationship (R2 = 0.9929) to increased HSO3 concentration. This quantitative
-
analysis gave a low detection limit for HSO3 at 0.44 µM. Moreover, the time-dependent fluorescence response
-
of QPCT (20 µM) towards HSO3 (120 µM) were also examined. Upon the addition of HSO3-, the
fluorescence intensity at 590 nm gradually decreased, accompanied by a distinct increase at 537 nm, which
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reached stability after 7 min (Fig. 1d).
-
Fig. 1. (a) UV-vis spectra of QPCT (20 µM) upon addition of HSO3 (0-120 µM) in DMSO/PBS buffer (7/3, v:v,
pH = 7.4, 5 mM) at 25 ºC. The inset photo shows the colorimetric changes of QPCT (20 µM) upon the addition
-
of HSO3 in DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC. (b) Ratiometric fluorescence spectra of
-
QPCT (20 µM) upon addition of HSO3 (1-120 µM) in DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC;
λex = 490 nm, slit: 2.5 nm/2.5 nm. The inset photo shows the fluorescence color changes of QPCT (20 µM)
-
upon the addition of HSO3 in DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC. (c)The linear curve of the
-
fluorescence intensity ratio (I537/I590) of QPCT (10 µM) versus the concentrations of HSO3 (1-120 µM) in
DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC. (d) Time-dependent fluorescence intensity of QPCT
-
(20 µM) in the presence of HSO3 (120 µM) in DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC.
Selectivity and competition are two crucial parameters of a probe for biological applications [24]. We
therefore measured the fluorescence spectra of QPCT in the presence of various interference species (including
-
2-
-
-
2-
HSO3 , Cl-, Br-, I-, SO4 , NO3 , NO2 , S2-, SCN-, ClO-, AcO-, S2O3 , CN-, CO32- Cys, Hcy, GSH). As shown in
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-
Fig. 2a, upon the addition of various species to the probe QPCT solutions, only HSO3 caused an obvious blue
shift of the fluorescence band, while other interfering species including H2S, Cys and Hcy did not trigger any
detectable fluorescence changes. Moreover, the competitive experiments confirmed that the fluorescence
-
response of QPCT to HSO3 in the presence of other competitive species is nearly consistent with that in the
absence of these species (Fig. 2b). The above results suggested that QPCT showed excellent selectivity to
-
HSO3 and anti-interference ability.
-
Fig. 2. (a) Fluorescence spectra of QPCT (20 µM) in the presence of various ions (120 µM for HSO3 , Cl-, Br-,
2-
-
-
2-
2-
I-, SO4 , NO3 , NO2 , S2-, SCN-, ClO-, AcO-, S2O3 , CN- and CO3 ) and other biological thiols (120 µM for Cys,
Hcy and GSH in DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC; (b) Fluorescence spectra of QPCT (20
-
-
2-
-
-
µM) to HSO3 (120 µM) in the presence of various ions (120 µM for HSO3 , Cl-, Br-, I-, SO4 , NO3 , NO2 , S2-,
2-
2-
SCN-, ClO-, AcO-, S2O3 , CN- and CO3 ) and other biological thiols (120 µM for Cys, Hcy and GSH) in
DMSO/PBS buffer (7/3, v:v, pH = 7.4, 5 mM) at 25 ºC.
-
3.2 The sensing mechanism of QPCT to HSO3
-
A well-known sensing process takes place between QPCT and HSO3 with a 1,4-addition reaction [36,37].
In order to further demonstrate this sensing mechanism, 1H NMR analysis was performed. As shown in Fig. 3,
-
upon reaction with HSO3 , the characteristic proton (Ha) signal of QPCT dramatically shifted from 8.64 to 4.84
ppm, implying that the π-conjugation of QPCT was shortened. The results indicated that the sensing
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-
mechanism of QPCT to HSO3 was based on the 1,4-addition (Scheme 1).
-
Fig. 3. 1H NMR spectra of (a) QPCT in DMSO-d6 and (b) QPCT upon addition of HSO3 in DMSO-d6/D2O
(v/v = 4:1).
3.3 Detection of bisulfite in real samples
-
Table 1. Determination of HSO3 in real samples.
Bisulfite content/
Added/
Found/
(µM)
5.21
Recovery/
(%)
Sample
(µM)
(µM)
5
white
sugar
crystal
sugar
brown
sugar
98.46
100.66
96.66
95.41
98.50
97.51
2.13
1.65
4.86
10
5
12.05
6.88
10
5
12.21
10.01
15.24
10
Encouraged by the excellent optical properties of QPCT, the concentrations of the bisulfite in white sugar,
brown sugar and crystal sugar were examined by a standard addition method. The concentrations of all three
kinds of sugar solution were 0.2 g/mL, which was prepared by dissolving white sugar, brown sugar and crystal
sugar (2.0 g) in water (10 mL), respectively. As shown in Table 1, the total bisulfite contents in white sugar,
brown sugar and crystal sugar were determined to be 2.13 µM, 1.65 µM and 4.86 µM, respectively. Meanwhile,
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good recovery in Table 1 indicated that QPCT can be applied for the quantitative detection of bisulfite in the
environment.
3.4 Bioimaging properties of QPCT in A549 cells
Fig. 4. Confocal fluorescence imaging of QPCT (10 µM) in A549 cells incubated with different concentrations
-
of (0, 5, 10, 15, 20 and 25 µM) HSO3 . Excitation wavelength: 488 nm. Green green channel: 500-550 nm. Red
channel: 570-630 nm. Scale bar: 30 µm.
-
Inspired by the excellent properties of QPCT for HSO3 in vitro, the ability of QPCT for living cell
imaging was evaluated with A549 cells. We first examined the potential toxicity of QPCT against A549 cells.
A standard MTT assay indicated that more than 80% of cells remained alive when probe concentration was up
to 25 µM, demonstrating the acceptable cytotoxicity of QPCT (Fig. S10). Hence, QPCT could be used for
imaging in A549 cells at a working concentration below 25 µM. Subsequently, cell imaging experiments were
performed by using confocal fluorescence microscopy. It can be seen from Fig. 4 that A549 cells, incubated
with QPCT (10 µM) in the culture medium for 30 min at 37 ºC, displayed intense intracellular fluorescence in
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the red channel and inconspicuous intracellular fluorescence in the green channel. Further incubated with
-
different concentrations (5, 10, 15, 20, and 25 µM) of HSO3 for 30 min, a distinct enhancement in green
emission and decrement in red emission were observed. These results indicated that QPCT could be used for
ratiometric fluorescence imaging in living cells.
4. Conclusion
In summary, we have designed and synthesized a fluorescent probe QPCT that can achieve the
colorimetric and ratiometric detection of bisulfite ions based on a 1,4-addition reaction. Both the visible
colormetric change from pink to colorless and fluorescent color change from red to green (under 365 nm
-
hand-held UV lamp) can be easily observed by naked eye. This probe has a high selectivity to HSO3 with no
interference from common interfering agents such as H2S, Cys and Hcy. QPCT can be successfully applied for
-
the quantitative detection of HSO3 in real samples. Furthermore, preliminary biological experiments indicated
-
its potential to explore the role of HSO3 in biology.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 21472148 and
21072158), Open Funding Project of the State Key Laboratory of Bioreactor Engineering, Academic Backbone
of Northwest University Outstanding Youth Support Program, Technology Plan Project of Xi'an [No.
201805040YD18CG24(1)] and the Top-Rated Discipline Construction Scheme of Shaanxi Higher Education.
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Highlights
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1. QPCT displays a colorimetric and ratiometric response towards HSO3 .
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2. QPCT exhibits good selectivity to HSO3 among various anions and biothiols.
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3. QPCT has been successfully applied to detect HSO3 in real samples and living cells.