Lysozyme-targeted ratiometric fluorescent probe for SO2 in living cells

Article abstract of DOI:10.1016/j.dyepig.2020.108440

Sulfur dioxide (SO₂) as a common environmental pollutant, participates in a variety of physiology and pathological processes in vivo. Lysosomes are considered to be cleaners of living cells and play a vital role in the process of metabolism. Exposure to excess SO₂ will result in the increase risk of lysosomal dysfunction and induce multiple diseases. Herein a smart fluorescent probe LYSO-SO₂ is developed, which achieved the ratio (F_{561 nm}/F_{634 nm}) identification of SO₂ in vitro. LYSO-SO₂ showed high sensitivity (detection limit 15.8 nM), rapidly response (within 5 s), and excellent selectivity. Additionally, this probe LYSO-SO₂ has been successfully applied to the ratio monitoring SO₂ in lysosomes which may provide an effective quantitative method for studying the physiological and pathological functions of SO₂ in living cells.

Full text of DOI:10.1016/j.dyepig.2020.108440

Journal Pre-proof
Lysozyme-targeted ratiometric fluorescent probe for SO in living cells
2
Yang Lu, Haidong Li, Qichao Yao, Wen Sun, Jiangli Fan, Jianjun Du, Jingyun Wang,
Xiaojun Peng
PII:
S0143-7208(20)30772-5
DOI:
https://doi.org/10.1016/j.dyepig.2020.108440
DYPI 108440
Reference:
To appear in: Dyes and Pigments
Received Date: 26 March 2020
Revised Date: 9 April 2020
Accepted Date: 9 April 2020
Please cite this article as: Lu Y, Li H, Yao Q, Sun W, Fan J, Du J, Wang J, Peng X, Lysozyme-
targeted ratiometric fluorescent probe for SO in living cells, Dyes and Pigments (2020), doi: https://
2
doi.org/10.1016/j.dyepig.2020.108440.
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© 2020 Published by Elsevier Ltd.

Contributor Roles Taxonomy
Yang Lu: Data curation, Formal analysis, Software,Validation, Writing - Original Draft
Haidong Li: Conceptualization, Methodology,preparation, Writing - Review & Editing
Qichao Yao: Investigation, Data curation Wen Sun: Supervision Jiangli Fan:Resources
Jianjun Du: Project administration Jingyun Wang: Visualization Xiaojun Peng:Writing -
Review & Editing,Funding acquisition,Project administration

Lysozyme-targeted Ratiometric Fluorescent Probe for SO2 in
Living Cells
Yang Lua,#, Haidong Liab,#, Qichao Yaoa, Wen Sunac, Jiangli Fanac, Jianjun Duac, Jingyun Wangd,
Xiaojun Peng*ac
aState Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road,
Dalian 116024, P.R. China.
bDepartment of Chemistry and Nanoscience, Ewha Womans University, Seoul 03760, Korea.
cSchool of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road,
Dalian 116024, P.R. China.
dShenzhen Research Institute, Dalian University of Technology, Gaoxin South fourth Road,
Nanshan District, Shenzhen 518057, P. R. China.
These authors (#) contributed equally.
Email: pengxj@dlut.edu.cn.
1

ABSTRACT
Sulfur dioxide (SO2) as a common environmental pollutant, participates in a variety of
physiology and pathological processes in vivo. Lysosomes are considered to be cleaners of living
cells and play a vital role in the process of metabolism. Exposure to excess SO2 will result in the
increase risk of lysosomal dysfunction and induce multiple diseases. Herein a smart fluorescent
probe LYSO-SO2 is developed, which achieved the ratio (F561 nm/F634 nm) identification of SO2 in
vitro. LYSO-SO2 showed high sensitivity (detection limit 15.8 nM), rapidly response (within 5
s), and excellent selectivity. Additionally, this probe LYSO-SO2 has been successfully applied to
the ratio monitoring SO2 in lysosomes which may provide an effective quantitative method for
studying the physiological and pathological functions of SO2 in living cells.
KEYWORDS: Ratiometric; Fluorescent probe; SO2; Environmental pollutant; Lysosomes; Cell
imaging;
1．Introduction
2-
Sulfur dioxide (SO2), is one of the main factors of air pollution, which exists in forms of SO3
and HSO3- in aqueous environments [1]. However, SO2 derivatives are widely applied in various
fields, such as antimicrobial agents [2], enzyme inhibitors [3], pharmaceuticals as well as food
additive [4]. Many research evidences have suggested that SO2 is related to many physiology and
pathological processes, including regulation of the organisms antioxidant activity and
cardiovascular functions [5]. In a biological environment, the endogenous sulfur dioxide is
mainly generated by the oxidation of H2S or decomposition sulfur-containing amino acid
accompanied with a catalysis of aspartate aminotransferase-2 (AAT-2) [6]. Lysosome, as the
digestive organelles in the cell, plays the vital role in regulating of cell [7, 8]. Usually, the
2

abnormal SO2 exposure could affect the amount and activity of lysosomal enzymes in organisms,
which will lead to some diseases such as rheumatoid arthritis, neurodegerative disease, and
cancer [9-11]. Thus, the development of efficient methods for the SO2 imaging of lysosome is of
highly value. Compared with previous analytical methods, such as coulometer, electrochemistry,
and capillary electrophoresis, fluorescence probes have been got widespread attention due to
high sensitivity, excellent selectivity, spatial and temporal resolution, and non-invasive features
[12-18]. Up to now, some elegant fluorescent probes were designed to the detection of SO2 in
vitro or vivo [19-23]. Unfortunately, most of fluorescent probes based on change in fluorescence
intensity, usually influenced by probe concentration, environment or laser instruments , which
considerably limited their application in vivo [24-29]. By comparison, ratiometric fluorescent
probes could solve the problem easily, because the fluorescence intensity ratio of two different
wavelengths is used to detect signals [30-33]. Meanwhile, it could also achieve trace detection
precisely by the self-correction without interference. Therefore, it has drawn increasingly notice
in biological and medical research [34-37].
Based on our group’s previous research work, we found that the slight structural differences
between 7-dimethylamino-1,4-benzoxazin-2-one (compound 5) and coumarin made the latter
exhibit a much longer emission wavelength [38]. Thus, malononitrile as strong electron-
withdrawing group was equipped to the fluorophore to design a ratiometric fluorescent probe
LYSO-SO2 for efficiently imaging SO2 derivatives in lysosomes. When the NaHSO3 was added
into the solution, nucleophile HSO-3 would attack the double bond carbon atom, resulting in a
blocked π-conjugation system, which leaded to the blue shift in the emission wavelength from
634 nm to 561 nm. The probe also showed many advantages, such as high selectivity, fast
response (within 5 s), and low detection limit (15.8 nM). In addition, after the probe enters the
3

cell, it mainly accumulated in lysosomes, which indicated that probe LYSO-SO2 has the
potential application value for imaging SO2 derivatives in cells.
Scheme 1 Proposed sensing mechanism for SO2 in cells.
2．Experimental section
2.1. General information and materials
All chemicals were purchased from commercial suppliers and used directly unless
additionally mentioned. All the other solvents were analytical purity. Ultrapure water used in the
1
experiments was obtained from Milli-Q systems. H NMR and 13C NMR spectra were measured
on a Bruker 500 MHz spectrometer. Absorption and fluorescence spectra were recorded on the
Agilent Technologies CARY 60 UV-Vis spectrophotometer and VAEIAN CARY Eclipse
fluorescence spectrophotometer, respectively. The width of the excitation and emission slits has
been modified, which is useful for a suitable the fluorescence intensity range. Mass
spectrometric data were measured on the HP1100LC/MSD MS instruments. The pH
measurements in this work were performed on a Model PHS-3C meter. Confocal laser scanning
microscope (CLSM) images were carried out on Olympus FV 3000 confocal microscopy
4

(Olympus, Japan). Lyso-Tracker Green, Mito-Tracker Green and Hoechst 33342 were ordered
from Life Technologies Co. (USA). Column chromatography was conducted over silica gel
(200-300 mesh), which was purchased from the Qingdao Ocean Chemicals.
2.2. Detection limit
The detection limit (DL) of the probe LYSO-SO2 (5 μM) was calculated through the
method of fluorescence titration. The ratio of the fluorescence intensity of the probe LYSO-SO2
and standard deviation of the blank measurements were obtained. The following formula was
used to calculate detection limit.
Detection limit = 3σ/k
Where σ is the standard deviation of the blank measurements, k is the slope between the
fluorescence ratio intensity (F561 nm/F643 nm) and various concentrations of NaHSO3.
2.3. MTT assays
The cytotoxicity studies of probe LYSO-SO2 was measured using the MTT (3-(4, 5)-
dimethylthiahiazo (-2-yl)-3, 5-diphenytetrazoliumromide) method. The detailed steps were as
follows:
100 μL of MCF-7 cells suspension were added into 96-well cell plate at a concentration of 1 ×
105 cells mL-1. After 12 h, the cells were growth, the cells was cultured in medium containing the
probe LYSO-SO2 at different concentrations (0, 1, 2, 5, and 10 μM) in cell incubator for 24 h. 10
μL of freshly prepared MTT (5 mg mL-1) solution in medium was added into each well. The
medium was replaced with the DMSO (100 μL) each well to dissolve blue precipitates after the
plates were stored at 37 ºC for another 4 h under 5% CO2 humidified incubator. Finally, the
5

absorbance of 570 nm and subtracted the absorbance of the cell-free blank volume at 630 nm
were measured via a microplate reader (Thermo Fisher Scientific) . Cell viability was expressed
as a percent of the control culture value, and the relative cell viability was calculated as follows:
Cells viability (%) = (ODdye - ODK dye)/ (ODcontrol - ODK control) × 100
2.4. Cell incubation
Human breast cancer cells (MCF-7) was provided by Institute of Basic Medical Sciences
(IBMS) of the Chinese Academy of Medical Sciences. Cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM, Invitrogen) mixed with 10 % fetal bovine serum (Invitrogen)
as well as 1% antibiotics (penicillin-streptomycin) and incubated at 37 ºC in a humidified
atmosphere with 5% CO2 for 24 h.
2.5. Cells imaging
MCF-7 cells were seeded into glass bottom culture dishes at density of 2×104 cells mL-1 and
allowed to be cultured at 37 ºC for 24 h in humidified atmosphere accompanied with 5% CO2
.The fresh culture with probe LYSO-SO2 (5 µM) was added into dishes and pre-incubated for 30
min. The dishes were washed three times carefully using DMEM, then cells were treated with
o
the fresh culture contained NaHSO3 (10 equiv) and stored at 37 C for another 30 min. Then,
cells were washed by DMEM three times again, and used for imaging (Olympus FV3000-IX81).
Cell imaging was observed through the confocal fluorescence microscope under 60 × objective
lens, probe LYSO-SO2 were excited at 488 nm and 561 nm respectively. afterwards,
fluorescence were harvest by emission at 625-725 nm of red channel and fluorescence emission
at 500-600 nm of green channel, respectively.
6

2.6. Synthesis of probe LYSO-SO2
2.6.1 Synthesis of 2, 3, 4, and 5
2, 3, 4, and 5 were synthesized by the previous literature reported [38].
2.6.2 Synthesis of LYSO-SO2
5 (218 mg, 1 mmol), malononitrile (132 mg, 2 mmol), and piperidine (0.2 mL) were added into
o
15 ml ethyl alcohol. The mixture was stirred at 85 C for overnight with N2 protection. After
cooling to room temperature and vacuum filter, the red residue was purified by the column
chromatography. Then probe LYSO-SO2 was obtained as light red solid. Yield: 93 mg (35 %)
1H NMR (500 MHz, CDCl3): δ 8.05 (s, 1H), 7.68 (d, J = 9.3 Hz, 1H), 6.80 (dd, J = 9.3, 2.6 Hz,
1H), 6.45 (d, J = 2.6 Hz, 1H), 3.25 (s, 6H). 13C NMR (125 MHz, CDCl3): δ 155.72, 153.13,
151.23, 148.54, 133.08, 133.00, 125.19, 114.41, 112.71, 112.21, 97.32, 83.19, 40.87. ESI-HRMS:
m/z calcd for C14H11N4O2 [M+H]+: 267.0877, found: 267.0877.
+
Scheme 2 Synthetic procedures of probe LYSO-SO2
7

3．Results and discussions
3.1. Design and synthesis of fluorescent probe LYSO-SO2
Coumarin derivatives are a large family of compounds, which have been widely adopted in the
development of novel fluorescent probes owing to them strong and stable fluorescence emission,
good biocompatibility, and structural plasticity [39]. When 4-C atom is replaced by N atom, the
fluorescence emission of aza-coumarin will be red-shifted greatly. Unfortunately, few
fluorescence probes based on aza-coumarin platform have been reported, to the best of our
knowledge. Therefore, a ratio-type fluorescent probe LYSO-SO2 is designed to fill the gap with
SO2 as the identification target. Probe LYSO-SO2 could be successfully synthesized by five
steps (Scheme 2), which was characterized by nuclear magnetic resonance (1H and 13C NMR)
(Figure S5 and Figure S6), and high resolution mass spectrum (ESI-HRMS) (Figure S7) in the
Supporting Information section.
3.2. Spectroscopic characteristics
8

Figure 1 The spectroscopic properties of probe LYSO-SO2 (10 μM) in PBS solution (0.01 M,
pH 7.4, 30% DMSO). a) UV-Vis spectra of probe LYSO-SO2 towards different concentrations
of NaHSO3 and b) Fluorescence titration experiments towards different concentrations of
NaHSO3. λex = 488 nm, slit: 5/5 nm c) Fluorescence titration experiments towards different
concentrations of NaHSO3. λex = 598 nm, slit: 10/10 nm. d) Linearity of log(F561 nm/F634 nm
)
towards the concentration of NaHSO3. d) Fluorescence intensity ratio (F561 nm/F634 nm) changes
upon the addition of different NaHSO3 concentrations. The experiments were repeated three
times and the data were shown as mean (±S.D.).
The fundamental photo-physical properties of probe LYSO-SO2 were obtained in the aqueous
solution. In order to verify the optical response of probe LYSO-SO2 to SO2, we tested the
spectral properties of LYSO-SO2 in the presence and absence of NaHSO3. NaHSO3 (commonly
the exogenous donor of SO2) was dissolved in PBS buffer solution (0.01 M, pH 7.4, 30%
DMSO), the original absorption peak at 600 nm diminished gradually and the corresponding
absorption peak appeared at 450 nm (Figure 1a). At meanwhile, a clearly color change from blue
to yellow can be observed in (Figure S1). As for fluorescence spectra, with a series of
concentrations of NaHSO3 were added, the fluorescence emission band at 560 nm gradually
enhanced with excitation at 440 nm and the fluorescence with the excitation of 590 nm
decreased at 638 nm (Figure 1b and Figure 1c). The large change in emission made it helpful to
use probe LYSO-SO2 in ratiometric imaging. And, an outstanding linear relationship(R2 =
0.9950) was acquired when the fluorescence intensity ratios were plotted against the
concentration of NaHSO3 (Figure 1d). Detection limit (3σ/k) was calculated as low as 15.8 nM,
which confirmed that probe LYSO-SO2 is extremely high sensitivity to NaHSO3 in vitro.
9

3.3. Response speed and pH influence
Figure 2 The time-dependent fluorescence responses curve of probe LYSO-SO2 (10 μM)
towards 5 equiv NaHSO3 in PBS solution (0.01 M, pH 7.4, 30% DMSO). a) λex = 488 nm, and b)
λex = 598 nm.
The response speed of the probe LYSO-SO2 toward SO2 was evaluated. With the accompany
of NaHSO3 (5equiv), the time-dependent fluorescence responses of probe LYSO-SO2 (10 μM).
As shown in Figure 2a) and b), the fluorescence intensity reached a stable within 5 s, which
indicated that probe had ability for real-time monitoring SO2 in solutions. Besides, the different
pH conditions effect on the probe LYSO-SO2 (10 μM) was also investigated in the range of pH
3.95-10.10 (Figure S2). These results show that there are no remarkable changes in emission of
the probe LYSO-SO2, which suggest probe could act as an outstanding molecular tool that not
be disturbed by biological pH micro-environment.
10

3.4. Selectivity of probe LYSO-SO2
Figure 3 Probe LYSO-SO2 (10 μM) were displayed towards different analytes. Insert 1: blank; 2:
+
-
2-
Na+; 3:K+; 4:Fe2+; 5: NH4 ; 6: Mn2+; 7: Ca2+; 8: F-; 9: Cl-; 10: Br-; 11: I-; 12: HCO3 ; 13: CO3 ;
2-
-
2-
-
-
14: HPO4 ; 15: H2PO4 ; 16: SO4 ; 17: NO3 ; 18: NO2 ; 19: SCN-; 20: Ascorbic acid (AA); 21:
HClO (100 μM); 22: Cysteine (Cys); 23: Glutathione (GSH); 24: NaHSO3 (50 μM). The others
concentration of interfering substances was 400 μM. The experiments were repeated three times
and the data were shown as mean (±S.D.).
Selectivity of probes is a crucial factors in complex cellular environments. Thus, the selectivity
of probe LYSO-SO2 towards NaHSO3 (50 μM) and other biologically compounds, including
+
-
2-
2-
-
ions (Na+, K+, Fe2+, NH4 , Mn2+, and Ca2+), anions (F-, Cl-, Br-, I-, HCO3 , CO3 , HPO4 , H2PO4 ,
2-
-
-
SO4 , NO3 , NO2 , and SCN-), amino acids (Cysteine and Glutathione), and reductive substances
(Ascorbic acid, AA), oxidizing substance (HClO) (100 μM) were investigated. The others
concentration of interfering substances was 400 μM. As illustrated in Figure 3, only addition of
NaHSO3 a significant change of fluorescence intensity ratio (F561 nm/F634 nm) was evoked, which
clearly confirmed that the probe LYSO-SO2 could be used to detect SO2 with high selectivity.
11

3.5. Recognition mechanism and MTT assays
For understanding the recognition mechanism of probe LYSO-SO2 for NaHSO3 clearer ,the
date of High Resolving Mass Spectrum (HRMS) analysis was displayed in Figure 4 where one
distinct peak centered at m/z 347.0466 (calcd. 347.0455 for C14H11N4O5S-) corresponds to
deprotonation product [M + SO3]-. According to above results, the sensing mechanism was
described accurately in Scheme 1. Besides, cytotoxic assay of the probe LYSO-SO2 in living
MCF-7 cells was also performed with 0, 2, 5, and 10 μM probe for 24 hours, respectively. As
demonstrated in Figure 5, the results exhibited extremely low cytotoxicity of probe LYSO-SO2
to cells under the experimental conditions.
Figure 4 HRMS of probe LYSO-SO2 and NaHSO3 adduct.
12

Figure 5 Cytotoxicity studies of probe LYSO-SO2 in living MCF-7 cells for 24 h. Error bar =
RSD (n=6).
3.6. Confocal fluorescence imaging of probe LYSO-SO2 in living cells.
On the basis of the photophysical properties and response, we continued to verify the
capabilities of probe to image SO2 in the living cells. MCF-7 cells were incubated with probe
o
LYSO-SO2 for 20 min at 37 C. Then, a strong fluorescence signals could be observed in red
channel (Figure 6c) and almost no fluorescence signals in green channel (Figure 6b). Further
treatment with 10 equiv exogenous NaHSO3, the fluorescent intensity of red channel disappeared
gradually (Figure 6g). On the contrary, a gradually increasing ratiometric fluorescence signals
could be observed under the green channel (Figure 6f), which prove that probe LYSO-SO2 could
be applied to monitoring SO2 by the ratiometric biological imaging in living cells.
Figure 6 Confocal fluorescence imaging of living MCF-7 cells. a and e) Represent bright
imaging. b and f) Represent green channel. λex = 488 nm, λem = 500-600 nm. c and g) Represent
red channel. λex = 561 nm, λem = 625-725 nm. d and h) Represent merged imaging. Scale bar =
20 μm.
13

3.7. Lysosome-Localization of probe LYSO-SO2 in living cells.
In order to ensure which subcellular localization for probe LYSO-SO2 in living MCF-7 cells,
the co-localization imaging was conducted. MCF-7 was incubated in medium with the probe
LYSO-SO2 (10 μM) for 30 min at 37 oC, and then treated with 0.5 μM Lyso-Tracker Green for
another 30 min, under the same conditions. As displayed in Figure 7, the merged image show
that probe LYSO-SO2 (Figure 7c) and Lyso-Tracker Green (Figure 7b) are similarly overlap
(Figure 7d), proving that the probe LYSO-SO2 is predominantly localized in the the lysosome of
living cells with a co-localization coefficient of 0.919 (Figure 7e). By contrast, there was no
desirable overlap in the mitochondria (Figure S3) or nucleus (Figure S4). Herein, co-localization
imaging indicates that probe LYSO-SO2 could be used to detect SO2 in lysosome.
Figure 7 Subcellular-localization fluorescence imaging of MCF-7 cells. a) Bright imaging and b)
confocal imaging from Lyso-Tracker Green on green channel. c) Confocal imaging from probe
14

LYSO-SO2 (10 μM) on red channel. d) Merged imaging of a), b), and c). e) Correlation plot of
the intensities of probe LYSO-SO2 and Lyso-Tracker Green (Rr = 0.919). Scale bar = 20 μm.
4．Conclusions
In summary, we have designed and synthesized a novel lysosomes-targeted ratiometric
fluorescence probe LYSO-SO2, which exhibited ultrafast response (within 5 s), outstanding
sensitivity, high selectivity and significant ratiometric fluorescent response toward SO2. The
recognition mechanism of the probe LYSO-SO2 to SO2 was verified through HRMS. More
importantly, probe LYSO-SO2 possesses an excellently bio-compatibility, which is achieving the
exogenous SO2 imaging in cells. Relying on above merits, the probe LYSO-SO2 can be applied
as an effective tool in environmental detection and molecular biological research.
Acknowledgments
This work was financially supported by National Natural Science Foundation of China
(21421005, and 21576037), and NSFC-Liaoning United cFund (U1608222 and U1908202).
Supporting Information Available
Additional documentation available in Supporting Information. This material is available free of
charge.
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20

Hightlights
‹ A ratiometric fluorescent probe LYSO-SO2 was proposed, which achieved the
ratio (F561 nm/F634 nm) identification of SO2;
‹ Probe LYSO-SO2 showed excellent sensitivity (15.8 nM), high selectivity and
rapid response (within 5 s);
‹ Probe LYSO-SO2 was successfully applied to the detection of lysosomes SO2 in
living MCF-7 cells;

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.