A fluorescent and colorimetric probe for imaging the mitochondrial sulfur dioxide in living cells

Accepted Manuscript Title: A Fluorescent and Colorimetric Probe for Imaging the Mitochondrial Sulfur Dioxide in Living Cells Authors: Haidong Li, Jiangli Fan, Saran Long, Jianjun Du, Jingyun Wang, Xiaojun Peng PII: DOI: Reference:

S0925-4005(18)31245-0 https://doi.org/10.1016/j.snb.2018.07.008 SNB 24977

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

2-3-2018 30-6-2018 2-7-2018

Please cite this article as: Li H, Fan J, Long S, Du J, Wang J, Peng X, A Fluorescent and Colorimetric Probe for Imaging the Mitochondrial Sulfur Dioxide in Living Cells, Sensors and Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.07.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A Fluorescent and Colorimetric Probe for Imaging the Mitochondrial Sulfur Dioxide in Living Cells

Key Laboratory of Fine Chemicals, Dalian University of Technology, 2 Linggong Road,

SC R

aState

IP T

Haidong Lia, Jiangli Fana, Saran Longa, Jianjun Dua, Jingyun Wangb, Xiaojun Peng*a

Dalian 116024, P.R. China.

of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong

U

bSchool

N

Road, Dalian 116024, P.R. China.

M

A

*Email: [email protected]. Tel/Fax: +86 -411- 84986306.



ED

Graphical Abstract

Highlights

A novel mitochondria-targeting probe CZ was designed and synthesized based on

PT

carbazole derivative.

Probe CZ was used to detect SO2 with high selectivity, sensitivity and rapid response.



The detection limits were calculated as low as 0.504 μM.



Probe CZ was successfully employed for the detection of SO2 in living cancer cells.

A

CC E



1

ABSTRACT Sulfur dioxide (SO2), one of active sulfur species (RSS), plays important roles in various physiologies and pathological processes. Hence, it is urgent to monitor the fluctuation of mitochondrial SO2 in vivo. In this work, a novel mitochondria-targeting probe CZ was

IP T

designed and synthesized based on carbazole derivative, which showed that probe was

SC R

employed to the detection of SO2 with high selectivity, rapid response as well as naked eyes. Besides, an excellent linear relationship (R2=0.9977) was observed and the detection limit was calculated as low as 0.504 μM (3σ/k). Surprisingly, with addition of SO2 derivative,

U

fluorescence intensity ratios (F475 nm/F607 nm) of probe CZ increased by 1104-fold ranging from

A

N

0.007 to 7.73. Owing to its biocompatibility, probe CZ was also successfully employed for

M

monitoring of mitochondrial SO2 by fluorescence confocal microscopy.

ED

Keywords: Mitochondria-targeted; Colorimetric; Imaging; Fluorescence; Environment detection; Sulfur dioxide

PT

1． Introduction

CC E

Sulfur dioxide (SO2), a well-known as environmental pollution gas[1-2], brings serious damage to agricultural production and urban construction owing to the effect of acid rain[3-4]. In recent years, there is increasing evidences that SO2 regarded as

A

signaling molecule in human body is involved in various physiological and pathological processes[5-7], such as regulating biological sulfur balance, relaxing blood vessels as well as maintaining redox equilibrium[8-10]. Through the catalysis of aspartate 2

aminotransferase-2, endogenous sulfur dioxide is mainly produced in mitochondria and cytosol[11]. On the other hand, it is very harmful to humans (e.g. cardiovascular diseases, neurological disorders and lung cancer) when excess SO2 is inhaled through

development of efficient methods is urgently needed in vivo.

IP T

the respiratory tract[7, 12-13]. Thus, in order to explore chemical biology of SO2, the

SC R

Until to now, there are some prominent detection techniques for the detection of SO2

in various sample, such as capillary electrophoresis[14], spectrophotometric[15],

U

coulometer[16] and electro-analytical methods[17]. Unfortunately, these methods are

N

time-consuming and complicated operation as well as biological tissue destructiveness,

A

which severely restrict their application in the level of vivo. On the contrary, owing to

M

high selectivity, excellent sensitivity, spatial and temporal resolution, especially for

ED

noninvasiveness, fluorescent probes have been attracted unprecedented attention in recent years[18-29]. So far, a class of smart fluorescent probes have been developed for

PT

specific the recognition of SO2 in vitro or vivo[30-40]. But, still existing shortcomings

CC E

including slow response time, no subcellular localization, no significant fluorescence changes, non-sensitivity and so on limit their further biological applications. Therefore,

A

developing a novel fluorescent probe with rapid response, mitochondria-targeted, biocompatibility and obvious fluorescence changes for the detection of SO2 appear to be particularly important.

3

Herein, a novel fluorescent probe CZ was designed and synthesized based on carbazole derivative. With addition of NaHSO3 (commonly the exogenous donor of SO2), fluorescence intensity ratios (F475 nm/F607 nm) of probe CZ increased by 1104-fold ranging from 0.007 to 7.73, accompanied with the color change of solution, which

IP T

showed that probe could be employed to sense SO2 through ratio detection in vitro and naked eyes. Nicely, only NaHSO3 triggered obvious the change of fluorescence

SC R

emission intensity of probe CZ in common ions, active molecules and amino acids.

Two good linear relationships were observed and limit detections (3σ/k) were

U

calculated as low as 0.504 and 0.194 μM, respectively. The Michael addition response

A

N

mechanism of probe CZ to SO2 was proved through high resolving mass spectrum

M

(ESI-HRMS) analysis. By introducing “dual” quaternary ammonium cation of benzyl, probe CZ more accurately located in the mitochondria with Pearson's correlation of

ED

0.98. In the consideration of its biocompatibility, the probe CZ was successfully

PT

employed to visualize the mitochondrial SO2 derivatives of living cancer cells.

CC E

2．Experimental section

2.1. General information and materials

A

All reagents used were obtained from commercial suppliers and were used without

further purification unless otherwise stated. Solvents used were purified via standard methods. Twice-distilled purified water used in all experiments was from Milli-Q systems. 1H-NMR and

13

C-NMR spectra were recorded on a Bruker Avance II 400 4

MHz spectrometer. Chemical shifts (δ) were reported as ppm (in DMSO-d6, with TMS as the internal standard). Fluorescence spectra were performed on a VAEIAN CARY Eclipse fluorescence spectrophotometer (Serial No.MY15210003) in 10×10 mm quartz cell. Excitation and emission slit widths were modified to adjust the fluorescence

IP T

intensity to a suitable range with the excitation at 340 nm. Absorption spectra were

measured on a Agilent Technologies CARY 60 UV-Vis spectrophotometer (Serial No.

SC R

MY1523004) in 10×10 mm quartz cell. Mass spectrometric data were achieved with

HP1100LC/MSD MS and an LC/Q-TOF-MS instruments. Mito-Tracker Green, Lyso-

U

Tracker Green and Hoechst 33342 were purchased from Life Technologies Co. (USA).

A

N

All pH measurements were performed using a Model PHS-3C meter. The fluorescence

M

quantum yields for compounds with Absolute PL Quantum Yield Spectrometer (HAMAMATSU C11347). Instruments used in cell imaging tests were carried out on

ED

FV1000-IX81confocal microscopy (Olympus, Japan). Slight pH variations in the

PT

solutions were achieved by adding the minimum volumes of HCl or NaOH (1 M). Flash column chromatography was performed using silica gel (200-300 mesh) obtained from

CC E

Qingdao Ocean Chemicals. All the interferential reagents were prepared based on

A

published literature[41-43].

2.2. Determination of the detection limit The test method was carried out according to our previous literature[42]. 2.3. Determination of the quantum yield 5

The fluorescence quantum yields for compounds with Absolute PL Quantum Yield Spectrometer (HAMAMATSU C11347). The PL Quantum Yield (Φ) is expressed as the ratio of the number of photons emitted from molecules (PNem) to that absorbed by

IP T

molecules (PNabs).

Φ= PNem/ PNabs

SC R

2.4. MTT assays

Measurement of cell viability was tested by reducing of MTT (3-(4, 5)-dimethylthiahiazo (-

U

2-yl)-3, 5-diphenytetrazoliumromide) to formazan crystals using mitochondrial

N

dehydrogenases. MCF-7 and Hela cells were seeded in 96-well microplates at a density of

M

A

1×105 cells/mL in 100 μL medium containing 10 % FBS. After 24 h of cell attachment, the plates were then washed with 100 μL/well PBS. The cells were then cultured in medium with

ED

0, 1.25, 2.50, 3.75 and 5.00 μM of probe CZ for 24 h and 40 h, respectively. Cells in culture

PT

medium without probe CZ were used as the control. Six replicate wells were used for each control and test concentration. 10 μL of MTT (5 mg/mL) prepared in PBS was added to each

CC E

well and the plates were incubated at 37 ºC for another 4 h in a 5% CO2 humidified incubator. The medium was then carefully removed, and the purple crystals were lysed in

A

200 μL DMSO. Optical density was determined on a microplate reader (Thermo Fisher Scientific) at 570 nm with subtraction of the absorbance of the cell-free blank volume at 630 nm. Cell viability was expressed as a percent of the control culture value, and it was calculated using the following equation [44]: 6

Cells viability (%) = (OD dye - ODK dye)/ (OD control - ODK control) × 100

2.5. Cell incubation Human breast cancer cells (MCF-7) and human cervical carcinoma cells (HeLa cells)

IP T

were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese Academy of Medical Sciences. Cells were cultured in Dulbecco’s modified Eagle’s

SC R

medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen).

The cells were seeded in confocal culture dishes and then incubated for 24 h at 37 ºC

U

under a humidified atmosphere containing 5% CO2.

N

2.6. Living cells imaging

A

MCF-7 cells and HeLa cells were seeded in glass-bottom culture dishes at

M

approximately concentration of 2×104 cells/mL and allowed to culture for 24 h at 37 ºC

ED

in a 5% CO2 humidified incubator. For the detection of exogenous SO2, MCF-7 cells

PT

and HeLa cells were incubated with probe CZ (1 µM) 37 oC for 1.5 h, followed by washing thrice with free DMEM and then NaHSO3 (100 μM) was added at 37 oC for

CC E

45 min. Under the confocal fluorescence microscope (Olympus FV1000-IX81) with a 60 × objective lens, probe CZ was excited at 515 nm, fluorescence emission at 575-

A

675 nm of red channel was gathered. Quantitative image analysis of the average fluorescence intensity of cells, determined from analysis of 7 regions of interest (ROIs) across cells.

2.7. Synthesis of probe CZ 7

2.7.1 Synthesis of 2, 3 and 5 2, 3 and 5 were synthesized by the previous literature reported[45-46]. 2.7.2 Synthesis of probe CZ 3 (12.5 mg, 0.5 mmol), 5 (382.8 mg, 1.2 mmol), and catalytic amounts of piperidine (0.05

IP T

mL) were dissolved in 10 mL absolute ethyl alcohol. The mixture was stirred at 85 oC for refluxing overnight with N2 protection. After cooling to room temperature and vacuum filter, the red residue was washed by absolute cool ethyl alcohol three times. Then probe CZ was

SC R

obtained as deep red solid. Yield: 221.8 mg (52%) 1H-NMR (400 MHz, DMSO-d6), δ: δ 9.11 (s, 2H), 8.54 (d, J = 15.5 Hz, 2H), 8.48 (d, J = 7.6 Hz, 2H), 8.37-8.25 (m, 4H), 8.18 (d, J = 8.2 Hz, 2H), 7.91 (d, J = 8.7 Hz, 2H), 7.85-7.71 (m, 4H), 7.50-7.29 (m, 10H), 6.40 (s, 4H), 4.59 (d, J =

U

7.0 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H). 13C-NMR (100 MHz, DMSO-d6): δ 173.28, 151.74, 143.45,

N

141.73, 134.51, 129.97, 129.60, 129.32, 128.75, 128.41, 127.46, 126.96, 124.98, 124.94,

A

123.50, 117.28, 111.56, 111.18, 51.79, 38.44, 14.45. TOF ESI-HRMS: m/z calcd for

A

CC E

PT

ED

3．Results and discussion

M

C46H38BrN3S2+ [M]+: 774.1614, found: 774.1607.

Scheme 1 Synthetic procedures of probe CZ and sensing mechanism 8

3.1. Design and Synthesis of fluorescent probe CZ Owing to the outstanding properties of carbazole compounds, such as good photostability, high quantum yield and biocompatibility[33, 47-48], this make them widely used in the fields of fluorescent probe. On the basis of our previous research work,

IP T

through introducing “dual” quaternary ammonium cation of benzyl for more accurate in locating the mitochondria, the probe CZ was prepared by condensation reaction

confirmed via 1H-NMR,

13

SC R

between compound 3 and 5, as described in Scheme 1. In addition, Probe CZ was C-NMR, ESI-HRMS and HPLC spectrometry in the

U

Supporting Information section (Figure S10-13). Furthermore, proposed response

A

N

mechanism of probe CZ for the detection of SO2 derivatives was also demonstrated in

M

Scheme 1.

A

CC E

PT

ED

3.2. Spectroscopic properties and response time

9

IP T SC R U N

A

Figure 1 a) Absorption and b) fluorescence spectra of probe CZ (10 μM) towards

M

various NaHSO3 concentrations (0-70 μM) in phosphate buffer-DMSO solution (10

ED

mM, pH 7.4, v/v, 1:1); c) fluorescence intensity ratio (F475 nm/F607 nm) changes upon the addition of different NaHSO3 concentrations; d) time response curve of probe CZ for

PT

detecting NaHSO3, λex=340 nm.

CC E

Firstly, fundamental spectroscopy properties of probe CZ (10 μM) were studied in

various solvents (Figure S1). With the increase of NaHSO3 in phosphate buffer-DMSO

A

solution (10 mM, pH 7.4, v/v, 1:1), the absorption band at 425 nm and 510 nm were obvious diminished (Figure 1a), accordingly, the color of probe solution from light red to colorless which demonstrated probe CZ had the ability to identify SO2 by naked eyes (Figure S2). Meanwhile, the emission intensity at 607 nm gradually decreased appeared 10

along with the enhancement of emission intensity at 475 nm (the quantum yield varies from 0.087 to 0.043) with the excitation at 340 nm (Figure 1b), clearly indicating the probe CZ could the detection of SO2 through ratio method. Besides, with the excitation at 510 nm, the fluorescence emission intensity of 607 nm declined sharply upon

IP T

addition of NaHSO3 ranging from 0 to 70 μM (Figure 3a). Based on above results, dual

mode of probe CZ could be applied to the recognition of SO2 in vitro, respectively. In

SC R

order to research the sensitivity of probe CZ (10 μM) to SO2, low concentration

fluorometric titration experiments were carried out. As shown in Figure 1c, an excellent

N

and various concentrations of NaHSO3. Furthermore, another good linearity

A

nm/F607 nm)

U

linear relationship (R2=0.9977) between the fluorescence emission intensity ratios (F475

M

(R2=0.9819) was observed when the fluorescent emission intensity of 607 nm were plotted against the concentration of NaHSO3 ranging from 0 to 18 μM (Figure S3b).

ED

Then, the detection limits were calculated as low as 0.504 μM and 0.194 μM on basis

PT

of 3σ/k principle, respectively, which verified the probe CZ possessed outstanding sensitivity for the detection SO2 in vitro. To further evaluate the response speed of probe

CC E

towards SO2, the time-dependent (0-800 s) fluorescence responses of probe CZ (10 μM) in the presence of 50 μM NaHSO3 was also performed. As is seen in Figure 1d,

A

the recognition process was almost completed within 5 min, showing the potential of probe CZ for the detection of SO2 in real-time.

3.3. Selectivity and competivity 11

IP T

Figure 2 a) Selectivity and b) competivity of probe CZ (10 μM) towards various

SC R

analytes (5 equiv) in phosphate buffer-DMSO solution (10 mM, pH 7.4, v/v, 1:1). Insert

1: blank; 2: Na+; 3:K+; 4:Fe2+; 5:NH4+; 6:Mn2+; 7:Br-; 8:I-; 9:HCO3-; 10:CO32-; 11:

N

A

Glutathione (GSH); 18: NaHSO3, λex=340 nm.

U

HPO4-; 12:SO42-; 13:SCN-; 14: Ascorbic acid (AA); 15: HClO; 16: Cysteine (Cys); 17:

M

Selectivity of probe is an important index in practical application. Thus, we studied the

ED

fluorescent response of probe CZ towards various biological relevant species, including ions (Na+, K+, Fe2+, NH4+, Mn2+, Br-, I-, HCO3-, CO32-, HPO4-, SO42- and SCN-),

PT

oxidation and reducing substances (Ascorbic acid and HClO) and amino acids (Cys,

CC E

GSH, Ile, Ala, Pro, Arg, Thr, Met, Ser, Tyr, Glu, Trp, Phe and Asn). As is seen in Figure 2a and Figure S4a, only adding NaHSO3 triggered huge change of F475 nm/F607 nm ratio

A

in phosphate buffer-DMSO solution (10 mM, pH 7.4, v/v, 1:1) with excitation at 340 nm, which confirmed that the probe CZ possessed high selectivity to SO2. In addition, to further evaluate the availability of probe, competitive experiments were carried out. As is shown in Figure 2b and Figure S4b, other interferences did not affect the 12

specificity for SO2 of probe CZ, which successfully displayed that probe CZ could be the detection of SO2 in complex biological system with high selectivity.

U

SC R

IP T

3.4. Effect on pH and response mechanism

N

Figure 3 Effect on pH the F475 nm/F607 nm ratio of probe CZ (10 μM) towards NaHSO3

M

A

(5 equiv) in phosphate buffer-DMSO solution (10 mM, pH 2.14-10.02, v/v, 1:1),

ED

λex=340 nm.

Subsequently, we investigated the effect on probe CZ and its detecting process in

PT

broad range of pH (2.14-10.02). As shown in Figure 3, in absence of NaHSO3, no

CC E

changes of F467 nm/F607 nm ratio values of probe CZ (10 μM) were obtained in the range of pH from 2.14 to 9.05, which showed that probe CZ could be stable in physiological environment (Figure S5). On the contrary, when 5 equiv NaHSO3 was added into

A

solution, the F467 nm/F607 nm ratio values of probe CZ (10 μM) exhibited no obvious changes in alkaline solution environment (pH 7.08-10.02), which clearly indicated that probe CZ could be a promising tool for bio-imaging applications in vivo. It is known 13

that NaHSO3 reacts with some special double bonds via addition reactions, forming sulfo-group compounds [33, 49]. In order to verify response mechanism, the high resolving mass spectrum (ESI-HRMS) analysis was carried out. As is seen in Figure S6, one prominent peak centered at m/z 856.1663 (calcd. 856.1649 for C46H38N3O6S4-)

IP T

and another remarkable peak centered at m/z 878.1479 (calcd. 878.1468 for

SC R

C46H37N3NaO6S4-) corresponding to deprotonating product [M + HSO3+SO3]- and [M

+ NaSO3+SO3]-, respectively. Thus, two SO2 molecules added to the two double-bonds of CZ, forming two sulfo-groups, blocking the conjugation of CZ molecule, and

N

U

shortening the wavelengths (Scheme 1).

CC E

PT

ED

M

A

3.5. Cytotoxicity

Figure 4 Cytotoxicity of probe CZ in HeLa@24h and MCF-7@40h through MTT

A

assay. Error bar = RSD (n=6).

Before cell imaging, the cytotoxicity of probe CZ should be thoroughly studied.

Then, standard MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays were carried out in HeLa cells for 24 h and MCF-7 cells for 40 h with 0, 1.25, 14

2.50, 3.75 and 5.00 μM probe. As demonstrated in Figure 4, the cell survival rates of probe CZ (2.50 μM) were not affected in HeLa or MCF-7 cells compared to the control group, showing the good biocompatibility of probe CZ to living cells.

PT

ED

M

A

N

U

SC R

IP T

3.6. Cell imaging

CC E

Figure 5 Confocal fluorescence imaging in living cells. a-f) represent MCF-7 cells. hm) represent HeLa cells. a, d, h and k) represent bright imaging. b, e, i and l) represent

A

fluorescence imaging. c, f, j and m) represent merged imaging. g and n) represent emission intensities of MCF-7 and HeLa cells counted as averages of 7 regions of interest (ROIs) Error bar = RSD (n=7). λex=515 nm, λem=575-675 nm, scale bar = 20 μm.

15

To show the biological applicability of probe CZ for the detection of SO2 in vivo imaging assays, fluorescence confocal imaging experiments were carried out in living MCF-7 and HeLa cells. As shown in Figure 5d-f, MCF-7 cells were incubated with probe CZ (1 μM) at 37 ºC under a humidified atmosphere containing 5% CO2. After

IP T

removing medium, the MCF-7 cells were washed by free DMEM for three times.

Afterwards, prominent fluorescence signals (Figure 5e) at the red channel of 575-675

SC R

nm was collected upon excitation at 515 nm, as expected. On the contrary, when 100

μM exogenous NaHSO3 was added into confocal dish, fluorescence emission intensity

U

of red channel obviously decreased (Figure 5b). the specific quantitative values of 7

A

N

regions of interest were shown in Figure 5g, which indicated probe CZ had good cell

M

membrane permeable and could detect the exogenous SO2 in living cells. In addition, we also chose living HeLa cells as research model and these results (Figure 5h-n) were

ED

in agreement with the fluorescence intensity changes of living MCF-7 cells (Figure 5a-

PT

g).

A

CC E

3.7. Co-localization imaging

16

IP T SC R U N

A

Figure 6 Co-localization imaging in MCF-7 cells. Cells were pretreated probe CZ (1

M

μM, λex=515 nm, λem=575-675 nm) for 1.5 h and then treated Mito-Tracker Green (0.5

ED

μM, λex=488 nm, λem=500-560 nm), Hochest 33342 (0.5 μM, λex=405 nm, λem=410-480 nm) and Lyso-tracker Green DND-26 (0.5 μM, λex=488 nm, λem=500-560) for 30 min.

PT

a, e, and i) represent green channel of commercial dye; b, f, and j) represent red channel

CC E

of probe CZ; c, g, and k) represent merged imaging between commercial dye and probe CZ; d, h, and l) represent Pearson’s correlation, scale bar = 20 μm.

A

To further evaluate where probe CZ existed in living cells, subcellular localization

experiments were carried out through co-staining living MCF-7 or HeLa cells with Mito-Tracker Green (a commercially targeting fluorescent dye of mitochondria) and probe CZ. As is seen in Figure 6a-c, the green channel signals of Mito-Tracker Green 17

(Figure 6a) and the red channel of probe CZ overlapped well (Figure 6c). Moreover, a higher Pearson’s correlation of 0.98 (co-localization coefficient) rather than in the nucleus (P=0.02, Figure 6e-h) and lysosomes (P=0.60, Figure 6i-l) were obtained, which demonstrated that probe CZ had the ability of mitochondria-targeting in living

IP T

MCF-7 cells. In the same way, mitochondria co-localization experiment was performed

in living HeLa cells to further examine targeting ability of probe CZ. As depicted in

SC R

Figure S7, similar phenomena were observed with a higher Pearson’s correlation of

0.96 (Figure S7a-d) and two intensity profiles marked ROI 1 and 2 crossing HeLa cells

U

vary in close synchrony (Figure S7e and S7f). Based on above results, probe CZ

A

N

possessed an excellent mitochondria-targeting ability.

M

4. Conclusions

In summary, based on carbazole derivative, a novel fluorescent probe CZ was

ED

designed and synthesized through condensation reaction, which displayed rapid

PT

response, high selectivity and sensitivity for the detection of SO2. The probe CZ had been proved good biocompatibility and precise localization in the mitochondria

CC E

(Pearson’s correlation 0.98). In addition, probe CZ had successfully applied to detect the exogenous SO2 in living MCF-7 and HeLa cells. Thus, we expect that this probe

A

will be used in environmental monitoring and biochemical study. Acknowledgments This work was supported by National Science Foundation of China (project 21421005, 21422601, 21576037, 21376039 and U1608222). 18

Supporting Information Available Biographies Haidong Li received his B.S. degree from Lanzhou University (China) in 2013. Currently, he is pursuing his Ph.D. degree under the supervision of Prof. Xiaojun Peng at Dalian University

IP T

of Technology. His research interests focused on the design and application of fluorescence

SC R

probes.

Jiangli Fan obtained her Ph.D. from Dalian University of Technology (China) in 2005. In 2010 she joined the University of South Carolina (America) as a visiting scholar. Currently,

N

U

she is a professor in State Key Laboratory of Fine Chemicals, Dalian University of

A

Technology (China). Her research is focused on fluorescent probes and nano materials.

M

Saran Long received her Ph.D. from Institute of Chemistry, Chinese Academy of Sciences (China) in 2015. Now, she is a lecture in State Key Laboratory of Fine Chemicals, Dalian

ED

University of Technology (China). Her current research interest is ultrafast photonchemistry.

PT

Jianjun Du received his Ph.D. degree from Dalian University of Technology (China) in

CC E

2010. He worked as postdoctoral fellowship in Nanyang Technological University (Singapore) from 2010 to 2013. Now, he is an associate professor at the State Key Laboratory of Fine Chemicals at Dalian University of Technology (China). His research interest is

A

focused on functional nanoparticles materials. Jingyun Wang obtained her Ph.D. from Dalian University of Technology (China) in 2001. She worked as postdoctoral fellowship in Northwestern University (America) from 2002 to 2004. Currently, she is a professor in School of Life Science and Biotechnology, Dalian 19

University of Technology (China). Her research is focused on biological nano materials and fluorescence probes. Xiaojun Peng received his Ph.D. degree from Dalian University of Technology (China) in 1990. He worked as postdoctoral fellowship in Nankai University (China) from 1990 to 1992.

IP T

In 2001 and 2002 he was a visiting scholar at Stockholm University (Sweden) and

SC R

Northwestern University (USA). Currently, he is a professor and the director of the State Key Laboratory of Fine Chemicals at Dalian University of Technology (China). His research interests digital printing/recording, fluorescence probes, and photodynamic therapy.

U

Absorbance, emission spectra, 1H-NMR, 13C-NMR, HRMS and HPLC spectra of probe CZ are

N

available in Supporting Information. This material is available free of charge.

A

References

M

[1] D.G. Streets and S.T. Waldhoff, Present and future emissions of air pollutants in China:

ED

SO2, NOx, and CO, Atmos. Environ. 34 (2000) 363-374. [2] A.S. Heagle, D.E. Body and W.W. Heck, An open-top field chamber to assess the impact

PT

of air pollution on plants, J. Environ. Qual. 2 (1973) 365-368.

CC E

[3] P.M. Irving and J.E. Miller, Productivity of Field-Grown Soybeans Exposed to Acid Rain and Sulfur Dioxide Alone Iand in Combination, J. Environ. Qual. 10 (1981) 473-478.

A

[4] J.S. Gaffney, G.E. Streit, W.D. Spall and J.H. Hall, Beyond acid rain. Do soluble oxidants

and organic toxinsinteract with SO2 and NOx to increase ecosystem effects? Environ. Sci. Technol. 21 (1987) 519-524. [5] L.A. Sayavedra-Soto and M.W. Montgomery, Inhibition of Polyphenoloxidase by Sulfite, 20

J. Food Sci. 51 (1986) 1531-1536. [6] Y. Huang, C. Tang, J. Du and H. Jin, Endogenous Sulfur Dioxide: A New Member of Gasotransmitter Family in the Cardiovascular System, Oxid. Med. Cell Longev. 2016 (2016) 1-9.

IP T

[7] X. Wang, H. Jin, C. Tang and J. Du, The biological effect of endogenous sulfur dioxide in

SC R

the cardiovascular system, Eur. J. Pharmacol. 1 (2011) 1-6.

[8] Z. Meng, Y. Li and J. Li, Vasodilatation of sulfur dioxide derivatives and signal transduction, Arch. Biochem. Biophys. 2 (2007) 291-296.

N

U

[9] Z. Meng, Z. Yang, J. Li and Q. Zhang, The vasorelaxant effect and its mechanisms of

A

sodium bisulfite as a sulfur dioxide donor, Chemosphere 5 (2012) 579-584.

M

[10] D. Liu, D. She, S.E. Yu, G. Shao and D. Chen, Predicted infiltration for sodic/saline soils from reclaimed coastal areas: sensitivity to model parameters, Sci. World J. 2014 (2014) 1-12.

ED

[11] N.D. Mathew, D.I. Schlipalius and P.R. Ebert, Sulfurous Gases As Biological

PT

Messengers and Toxins: Comparative Genetics of Their Metabolism in Model Organisms, J.

CC E

Toxicol. 2011 (2011) 1-14.

[12] Y. Yun, H. Li, G. Li and N. Sang, SO2 inhalation modulates the expression of apoptosisrelated genes in rat hippocampus via its derivatives in vivo, Inhal. Toxicol 22 (2010) 919-929.

A

[13] W.L. Beeson, D.E. Abbey and S.F. Knutsen, Long-term concentrations of ambient air pollutants and incident lung cancer in california adults: results from the AHSMOG study, Environ. Health Persp. 102 (1998) 813-822. [14] Z. Daunoravicius and A. Padarauskas, Capillary electrophoretic determination of 21

thiosulfate, sulfide and sulfite using in-capillary derivatization with iodine, Electrophoresis 15 (2002) 2439-2444. [15] O. Fatibello-Filho and I.D.C. Vieira, Flow injection spectrophotometric determination of sulfite using a crude extract of sweet potato root (Ipomoea batatas (L.) Lam.) as a source of

IP T

polyphenol oxidase, Anal. Chim. Acta 354 (1997) 51-57.

dechlorination of water, Anal. Chim. Acta 332 (1996) 155-160.

SC R

[16] N. Ekkad and C.O. Huber, A coulometer for determination of residual sulfite fol

[17] A. Isaac, J. Davis, C. Livingstone, A.J. Wain and R.G. Compton, Electroanalytical

N

U

methods for the determination of sulfite in food and beverages, TrAC Trends in Anal. Chem.

A

6 (2006) 589-598.

M

[18] D. Zhang, N. Xu, L. Xian, H. Ge, J. Fan, J. Du and X. Peng, A BODIPY-based fluorescent probe for thiophenol, Chin. J. Chem. 36 (2018) 119-123.

ED

[19] H. Li, J. Fan and X. Peng, Fluorescent probes for the recognition of hypochlorous acid,

PT

Prog. Chem. 29 (2017) 17-35.

CC E

[20] C. Liu, T. Xiao, F. Wang and X. Chen, Synthesis of binol-based fluorescence probe and recognition of arginine, Chin. J. Appl. Chem. 35 (2018) 40-45. [21] Z. Zhou, F. Wang, G. Yang, C. Lu, J. Nie, Z. Chen, J. Ren, Q. Sun, C. Zhao and W. Zhu,

A

A ratiometric fluorescent probe for monitoring leucine aminopeptidase in living cells and zebrafish model, Anal. Chem. 89 (2017) 11576-11582. [22] Q. Yao, D. Wu, R. Ma, M. Xia, Study on the structure-property relationship in a series of novel BF2 chelates with multicolor fluorescence, J. Organomet. Chem. 743 (2013) 1-9. 22

[23] Y. Tang. A. Shao, J. Cao, H. Li, Q. Li, M. Zeng, M. Liu, Y. Cheng and W. Zhu, cNGRbased synergistic-targeted NIR fluorescent probe for tracing and bioimaging of pancreatic ductal adenocarcinoma, Sci. China Chem. 61 (2018) 184-191. [24] D. Wu, J. Ryu, Y.W. Chung, D. Lee, J. Ryu, J.H. Yoon and J. Yoon, A far-red- emitting

IP T

fluorescence probe for sensitive and selective detection of peroxynitrite in live cells and

SC R

tissues, Anal. Chem. 89 (2017) 10924-10931.

[25] X. Chen, F. Wang, J.Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin and J. Yoon, Recent progress in the development of fluorescent, luminescent and colorimetric probes for detection

N

U

of reactive oxygen and nitrogen species, Chem. Soc. Rev. 45 (2016) 2976-3016.

A

[26] W.S. Shin, M. Lee, P. Verwilst, J.H. Lee, S. Chi and J.S. Kim, Mitochondria-targeted

Sci. 9 (2016) 6050-6059.

M

aggregation induced emission theranostics: crucial importance of in situ activation, Chem.

ED

[27] S. Wang, C. Li, J. Li, B. Chen, and Y. Guo, Novel coumarin-based fluorescent probes for

PT

detecting fluoride ions in living cells, Acta Chim. Sinica 75 (2017) 383-390. [28] K. Dou, Q. Fu, G. Chen, F. Yu, Y. Liu, Z. Cao, G. Li, X. Zhao, L. Xia, L. Chen, H.

CC E

Wang and J. You, A novel dual-ratiometric-response fluorescent probe for SO2 /ClO− detection in cells and in vivo and its application in exploring the dichotomous role of SO2

A

under the ClO− induced oxidative stress, Biomaterials 133 (2017) 82-93. [29] L. Zeng, S. Chen, T. Xia, W. Hu, C. Li and Z. Liu, Two-photon fluorescent probe for detection of exogenous and endogenous hydrogen persulfide and polysulfide in living organisms, Anal. Chem. 5 (2015) 3004-3010. 23

[30] W. Wu, H. Ma, M. Huang, J. Miao and B. Zhao, Mitochondria-targeted ratiometric fluorescent probe based on FRET for bisulfite, Sens. Actuators B 241 (2017) 239-244. [31] J. Yang, K. Li, J. Hou, L. Li, C. Lu, Y. Xie, X. Wang and X. Yu, Novel tumor-specific and mitochondria-targeted near-infrared emission fluorescent probe for SO2 derivatives in

IP T

living cells, ACS Sensors 2 (2016) 166-172.

SC R

[32] D. Li, Z. Wang, X. Cao, J. Cui, X. Wang, H. Cui, J. Miao and B. Zhao, A mitochondriatargeted fluorescent probe for ratiometric detection of endogenous sulfur dioxide derivatives in cancer cells, Chem. Commun. 13 (2016) 2760-2763.

N

U

[33] H. Li, X. Zhou, J. Fan, S. Long, J. Du, J. Wang and X. Peng, Fluorescence imaging of

A

SO2 derivatives in daphnia magna with a mitochondria-targeted two-photon ratiometric

M

fluorescent probe, Sens. Actuators B 254 (2018) 709-718. [34] Q. Sun, W. Zhang and J. Qian, A ratiometric fluorescence probe for selective detection

ED

of sulfite and its application in realistic samples, Talanta 162 (2017) 107-113.

PT

[35] M. Gómez, E.G. Perez, V. Arancibia, C. Iribarren, C. Bravo-Díaz, O. García-Beltrán and

CC E

M.E. Aliaga, New fluorescent turn-off probes for highly sensitive and selective detection of SO2 derivatives in a micellar media, Sens. Actuators B 238 (2017) 578-587. [36] J. Wang, Y. Hao, H. Wang, S. Yang, H. Tian, B. Sun and Y. Liu, Rapidly responsive and

A

highly selective fluorescent probe for bisulfite detection in food, J. Agr. Food Chem. 65 (2017) 2883-2887. [37] M. Wu, K. Li, C. Li, J. Hou and X. Yu, A water-soluble near-infrared probe for colorimetric and ratiometric sensing of SO2 derivatives in living cells, Chem. Commun. 50 24

(2014) 183-185. [38] Q. Wang, W. Wang, S. Li, J. Jiang, D. Li, Y. Feng, H. Sheng, X. Meng, M. Zhu and X. Wang, A mitochondria-targeted colorimetric and two-photon fluorescent probe for biological SO2 derivatives in living cells, Dyes Pigments 165 (2016) 297-305.

IP T

[39] H. Yu, Y. Wu, Y. Hu, X. Gao, Q. Liang, J. Xu and S. Shao, Dual-functional fluorescent

SC R

probe responds to hypochlorous acid and SO2 derivatives with different fluorescence signals, Talanta 165 (2017) 625-631.

[40] L. Zeng, A novel colorimetric and ratiometric fluorescent probe for visualizing SO2

N

U

derivatives in environment and living cells, Talanta 176 (2018) 389-396.

A

[41] H. Li, Q. Yao, J. Fan, J. Du, J. Wang and X. Peng, A two-photon NIR-to-NIR

M

fluorescent probe for imaging hydrogen peroxide in living cells, Biosens. Bioelectron. 94 (2017) 536-543.

ED

[42] H. Li, Q. Yao, J. Fan, J. Du, J. Wang and X. Peng, An NIR fluorescent probe of uric

PT

HSA for renal diseases warning, Dyes Pigments 133 (2016) 79-85.

CC E

[43] H. Li, Q. Yao, J. Fan, N. Jiang, J. Wang, J. Xia and X. Peng, A fluorescent probe for H2S in vivo with fast response and high sensitivity, Chem. Commun. 90 (2015) 16225-16228. [44] H. Zhang, J. Fan, J. Wang, S. Zhang, B. Dou and X. Peng, An off–on COX-2-Specific

A

fluorescent probe: targeting the Golgi apparatus of cancer cells, J. Am. Chem. Soc. 135 (2013) 11663-111669. [45] F. Liu, T. Wu, J. Cao, S. Cui, Z. Yang, X. Qiang, S. Sun, F. Song, J. Fan, J. Wang and X. Peng, Ratiometric detection of viscosity using a two-photon fluorescent sensor, Chem. -Eur. 25

J. 19 (2013) 1548-1553. [46] N. Jiang, J. Fan, F. Xu, X. Peng, H. Mu, J. Wang and X. Xiong, Ratiometric fluorescence imaging of cellular polarity: decrease in mitochondrial polarity in cancer cells, Angew. Chem. Int. Ed. 127 (2015) 2540-2544.

IP T

[47] F. Meng, Y. Liu, X. Yu and W. Lin, A dual-site two-photon fluorescent probe for

SC R

visualizing mitochondrial aminothiols in living cells and mouse liver tissues, New J. Chem. 40 (2016) 7399-7406.

[48] D. Li, Y. Feng, J. Lin, M. Chen, S. Wang, X. Wang, H. Sheng, Z. Shao, M. Zhu and X.

N

U

Meng, A mitochondria-targeted two-photon fluorescent probe for highly selective and rapid

A

detection of hypochlorite and its bio-imaging in living cells, Sens. Actuators B 222 (2016)

M

483-491.

[49] H. Li, Q. Yao, J. Fan, C. Hu, F. Xu, J. Du, J. Wang and X. Peng, A fluorescent probe for

PT

(2016) 1477-1483.

ED

ratiometric imaging of SO2 derivatives in mitochondria of living cells, Ind. Eng. Chem. Res. 6

Table of Contents

CC E

Scheme 1 Synthetic procedures of probe CZ and sensing mechanism. Figure 1 a) Absorption and b) fluorescence spectra of probe CZ (10 μM) towards

A

various NaHSO3 concentrations (0-70 μM) in phosphate buffer-DMSO solution (10 mM, pH 7.4, v/v, 1:1); c) fluorescence intensity ratio (F475 nm/F607 nm) changes upon the addition of different NaHSO3 concentrations; d) time response curve of probe CZ for detecting NaHSO3, λex=340 nm. 26

Figure 2 a) Selectivity and b) competivity of probe CZ (10 μM) towards various analytes (5 equiv) in phosphate buffer-DMSO solution (10 mM, pH 7.4, v/v, 1:1). Insert 1: blank; 2: Na+; 3:K+; 4:Fe2+; 5:NH4+; 6:Mn2+; 7:Br-; 8:I-; 9:HCO3-; 10:CO32-; 11: HPO4-; 12:SO42-; 13:SCN-; 14: Ascorbic acid (AA); 15: HClO; 16: Cysteine (Cys); 17:

IP T

Glutathione (GSH); 18: NaHSO3, λex=340 nm.

Figure 3 Effect on pH the F475 nm/F607 nm ratio of probe CZ (10 μM) towards NaHSO3

SC R

(5 equiv) in phosphate buffer-DMSO solution (10 mM, pH 2.14-10.02, v/v, 1:1), λex=340 nm.

U

Figure 4 Cytotoxicity of probe CZ in HeLa@24h and MCF-7@40h through MTT

A

N

assay. Error bar = RSD (n=6).

M

Figure 5 Confocal fluorescence imaging in living cells. a-f) represent MCF-7 cells. hm) represent HeLa cells. a, d, h and k) represent bright imaging. b, e, i and l) represent

ED

fluorescence imaging. c, f, j and m) represent merged imaging. g and n) represent

PT

emission intensities of MCF-7 and HeLa cells counted as averages of 7 regions of interest (ROIs) Error bar = RSD (n=7). λex=515 nm, λem=575-675 nm, scale bar = 20 μm.

CC E

Figure 6 Co-localization imaging in MCF-7 cells. Cells were pretreated probe CZ (1 μM, λex=515 nm, λem=575-675 nm) for 1 h and then treated Mito-Tracker Green (0.5

A

μM, λex=488 nm, λem=500-560 nm), Hochest 33342 (0.5 μM, λex=405 nm, λem=410-480 nm) and Lyso-tracker Green DND-26 (0.5 μM, λex=488 nm, λem=500-560) for 30 min. a, e, and i) represent green channel of commercial dye; b, f, and j) represent red channel

27

of probe CZ; c, g, and k) represent merged imaging between commercial dye and probe

U

SC R

IP T

CZ; d, h, and l) represent Pearson’s correlation, scale bar = 20 μm.

A

CC E

PT

ED

M

A

N

Scheme 1 Synthetic procedures of probe CZ and sensing mechanism.

28

Figure 1 a) Absorption and b) fluorescence spectra of probe CZ (10 μM) towards various NaHSO3 concentrations (0-70 μM) in phosphate buffer-DMSO solution (10 mM, pH 7.4, v/v, 1:1); c) fluorescence intensity ratio (F475 nm/F607 nm) changes upon the addition of different NaHSO3 concentrations; d) time response curve of probe CZ for

A

N

U

SC R

IP T

detecting NaHSO3, λex=340 nm.

M

Figure 2 a) Selectivity and b) competivity of probe CZ (10 μM) towards various analytes (5 equiv) in phosphate buffer-DMSO solution (10 mM, pH 7.4, v/v, 1:1). Insert

ED

1: blank; 2: Na+; 3:K+; 4:Fe2+; 5:NH4+; 6:Mn2+; 7:Br-; 8:I-; 9:HCO3-; 10:CO32-; 11:

PT

HPO4-; 12:SO42-; 13:SCN-; 14: Ascorbic acid (AA); 15: HClO; 16: Cysteine (Cys); 17:

A

CC E

Glutathione (GSH); 18: NaHSO3, λex=340 nm.

29

Figure 3 Effect on pH the F475 nm/F607 nm ratio of probe CZ (10 μM) towards NaHSO3 (5 equiv) in phosphate buffer-DMSO solution (10 mM, pH 2.14-10.02, v/v, 1:1),

SC R

IP T

λex=340 nm.

U

Figure 4 Cytotoxicity of probe CZ in HeLa@24h and MCF-7@40h through MTT

A

CC E

PT

ED

M

A

N

assay. Error bar = RSD (n=6).

30

Figure 5 Confocal fluorescence imaging in living cells. a-f) represent MCF-7 cells. hm) represent HeLa cells. a, d, h and k) represent bright imaging. b, e, i and l) represent fluorescence imaging. c, f, j and m) represent merged imaging. g and n) represent emission intensities of MCF-7 and HeLa cells counted as averages of 7 regions of

IP T

interest (ROIs) Error bar = RSD (n=7). λex=515 nm, λem=575-675 nm, scale bar = 20

CC E

PT

ED

M

A

N

U

SC R

μm.

Figure 6 Co-localization imaging in MCF-7 cells. Cells were pretreated probe CZ (1

A

μM, λex=515 nm, λem=575-675 nm) for 1 h and then treated Mito-Tracker Green (0.5 μM, λex=488 nm, λem=500-560 nm), Hochest 33342 (0.5 μM, λex=405 nm, λem=410480 nm) and Lyso-tracker Green DND-26 (0.5 μM, λex=488 nm, λem=500-560) for 30 min. a, e, and i) represent green channel of commercial dye; b, f, and j) represent red 31

channel of probe CZ; c, g, and k) represent merged imaging between commercial dye

A

CC E

PT

ED

M

A

N

U

SC R

IP T

and probe CZ; d, h, and l) represent Pearson’s correlation, scale bar = 20 μm.

32