A new fluorescent material and its application in sulfite and bisulfite bioimaging

Accepted Manuscript Title: A new fluorescent material and its application in sulfite and bisulfite bioimaging Authors: Caixia Yin, Xiaoqi Li, Yongkang Yue, Jianbin Chao, Yongbin Zhang, Fangjun Huo PII: DOI: Reference:

S0925-4005(17)30354-4 http://dx.doi.org/doi:10.1016/j.snb.2017.02.127 SNB 21858

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

28-10-2016 20-2-2017 21-2-2017

Please cite this article as: Caixia Yin, Xiaoqi Li, Yongkang Yue, Jianbin Chao, Yongbin Zhang, Fangjun Huo, A new fluorescent material and its application in sulfite and bisulfite bioimaging, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2017.02.127 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 new fluorescent material and its application in sulfite and bisulfite bioimaging

Caixia Yin,a,* Xiaoqi Li ,a Yongkang Yue, a Jianbin Chao,b Yongbin Zhang, b Fangjun Huo b,**

a

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of

Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China. b

Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China.

*Corresponding author: C.X. Yin, E-mail: [email protected]; F.J. Huo, E-mail:[email protected]

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GRAPHICAL ABSTRACT

A new fluorescent material 1 was designed and synthesized, which was able to sense sulfite and bisulfite through fluorescence “turn-on” manner. And 1 showed great selectivity toward sulfite or bisulfite and fast response time (within 4 min) as well as low detection limit (8.8 nM). We characterized the products through NMR and X-ray diffraction analysis. Finally, 1 had low cytotoxicity that could be applied in bioimaging in living cells.

2

Highlight 1. A new fluorescent material was synthesized and characterized through NMR and X-ray diffraction analysis. 2. The probe has low cytotoxicity that can be applied in bioimaging in living cells.

Abstract: In daily life, sulfite and bisulfite have been extensively used as antimicrobial

agent

and

antioxidant

for

food

products,

beverages

and

pharmaceutical products, but, SO 32-/HSO3 - are fantastically harmful to human health, so it is meaningful to develop an effective probe for these two anions. In this work, a new fluorescent material probe 1 was designed and constructed, which was able to sense sulfite and bisulfite through fluorescence “turn-on” manner. Compared with correlative fourteen other anions used, probe 1 showed great selectivity toward sulfite or bisulfite. Furthermore, the probe can detect SO3 2-/HSO3- with fast response (within 4 min) as well as low detection limit (8.8 nM) compared with the previous work. We characterized the adduct through NMR and X-ray diffraction analysis and further confirmed the detection mechanism of probe 1. Finally, fluorescence imaging of MCF-7 cells indicated that 1 could be used for monitoring SO 2 derivatives in living cells with low cytotoxicity.

Keywords: Sulfite/ bisulfite; Fluorescent; Detection; Turn-on; Bioimaging

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1. Introduction Sulfur dioxide (SO2) as one of the well-known primary air contaminant can be easily hydrated to generate sulfite (SO32-) and bisulfate (HSO3-) ions (3:1, M/M) in neutral liquids [1, 2]. In daily life, SO32-/HSO3- have been extensively used as antimicrobial agent, enzyme inhibitor and antioxidant for food products, beverages [3] and pharmaceutical products [4-6], and they also have been used in a wide range of industries [7]. However, a good deal of epidemiological studies have confirmed that the intake of excess amount of these anions would induce poisonous effects to cells and tissues in some individuals [8, 9], not only induce a large number of respiratory diseases [9], but is also connected to cardiovascular diseases, and many neurological disorders, such as lung cancer, asthmatic attacks, strokes, allergic reactions, migraine headaches, ischemic heart diseases, myocardial ischemia [10-12], and brain cancer [13-15]. And some people are extremely sensitive even to low doses of them [16]. Owing to these harmful effects for humans, the threshold levels of SO 32-/HSO3- in food, beverage and in the air have been strictly regulated in many countries. Therefore, to completely understand the biological activity of SO2 derivatives, it is highly needed to develop efficient analytical methods for monitoring cellular SO 32-/HSO3concentration, which is crucial for biological researches as well as clinical diagnoses. Up to now, to the best of our knowledge, there are several methods have been developed to detect SO32-/HSO3- in vitro or vivo, such as spectrophotometry [17, 18], capillary electrophoresis [19], chromatography [20], and fluorescent probes [21-25].

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Compared with other techniques, fluorescence detection using small molecular probes [26-31], has regarded as a promising detection technique in solution and living cells due to its high sensitivity, selectivity, simple operation, and especially noninvasive characteristic [32-37] as well as real-time imaging [38-41]. Up to now, some fluorescent sensors have been developed for SO32-/HSO3-. For instance, Sun [42] constructed a coumarinhemicyanine dye which the probe’s detection limit was 3.8×10-7 M. Chen [43] synthesized a coumarin-based fluorescent probe with detection limit and response time were 5×10-6 M and 60 min respectively. In addition, our previous work [44] reported a coumarin-based fluorescent probe with 2×10-6 M detection limit and its reaction time was 10 min. Most of the reported probes have much of improving demands for practical applications in detection limit and response time. As a consequence, it is of great worth to construct an original fluorescent probe for sulfur dioxide derivative SO32-/HSO3- with high sensitivity, selectivity and rapid response in the point view of environmental protection and biological technology. Holding this in mind, we designed and synthesized a new fluorescent turn-on probe 1 for SO32-/HSO3- detection based on nucleophilic addition reaction (Scheme 1). The synthesis route was summarized in Scheme 2. This new probe not only could be readily prepared, but also showed excellent sensing properties for SO32-/HSO3- including highly selective and sensitive for SO32-/HSO3- over various common analytes. Also, probe 1 as a turn-on fluorescent for SO32-/HSO3- can response quickly (within 4 min) with a 33-fold enhancement in DMSO-HEPES buffer solution under mild conditions. Moreover, probe 1 can be used for SO32-/HSO3- bioimaging in living cells with low cytotoxicity.

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2. Experimental 2.1. Materials All chemicals were purchased from commercial suppliers and used without further purification. All solvents were of analytical grade also without further purification. Distilled water was used after passing through a water ultrapurification system. Deionized water was used to prepare all aqueous solutions. All metal ions salts and amino acids were purchased from Shanghai Experiment Reagent Co., Ltd (Shanhai,, China). 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Sigma-Aldrich (St. Louis, MO). 2.2. Instruments TLC analysis was performed using precoated silica plates. A pH meter (Mettler Toledo, Switzerland) was used to determine the pH. Ultraviolet-Visible (UV-Vis) spectra were recorded on an Agilent 8453 UV-Vis spectrophotometer. Hitachi F-7000 fluorescence spectrophotometer was employed to measure fluorescence spectra. Shanhai Huamei Experiment Instrument Plants, China provided a PO-120 quartz cuvette (10 mm). 1H-NMR and

13

C-NMR experiments were performed with a

BRUKER AVANCE III HD 600 MHz and 151 MHz NMR spectrometer, respectively (Bruker, Billerica, MA). Coupling constants (J values) are reported in hertz. ESI determinations were carried out on AB Triple TOF 5600 plus System (AB SCIEX, Framingham, USA). The red single crystal of probe was mounted on a glass fiber for data collection. Cell constants and an orientation matrix for data collection were

6

obtained by least-squares refinement of diffraction data from reflections within 2.428-27.505◦, using a Bruker SMART APEX CCD automatic diffractometer. Data were collected at 173 K using Mo Kα radiation (λ=0.71073 Å) and the ω-scan technique, and corrected for the Lorentz and polarization effects (SADABS) [45]. The structures were solved by direct methods (SHELX97) [46], and subsequent difference Fourier maps were inspected and then refined in F2 using a full-matrix leastsquares procedure and anisotropic displacement parameters. The ability of probe 1 reacting with sulfite in the living cells was also evaluated by a Leica DMi8 fluorescence inversion microscope system. 2.3. Preparation and characterization of probe and analytes 2.3.1. Preparation of compound 3 The synthesis of intermediate compound 3 is summarized in Scheme 2. Phosphorous oxychloride (POCl3) (10 mL) was slowely added to dimethylformamide (DMF) (10 mL) at 0-5 ℃ under the stirring about half an hour. Subsequently, 3-(N,N-diethyl amino) phenol (4.95 g, 20 mmol) dissolved in DMF (10 mL) was added into above system under the stirring and the reaction mixture was subsequently heated at 75 °C for 4 h. The reaction mixture was cooled to room temperature and then poured into ice cold water (60 mL). Then the reaction system was neutralised with sodium carbonate, brown colored solid was precipitated. Separated product was filtered and washed with cold water, dried and crystallised from ethanol to give compound 3.

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2.3.2. Preparation and characterization of compound 4 4-(diethylamino)-2-hydroxybenzaldehyde (1.93 g, 10 mmol), ethyl acetoacetate (1.95 g, 15 mmol), and 0.2 mL of piperidine were dissolved in 40 mL of absolute ethanol. After the mixture solution was refluxed for 10 h. Then the reaction mixture was cooled to room temperature, the solvent was removed under reduced pressure. The yellow solid was precipitated and collected, and the product was recrystallized from absolute ethanol to afford 4. 1H-NMR (DMSO-d6, 600 MHz): 8.49 (s, 1H), 7.66 (d, 1H, J = 9.0), 6.79 (dd, 1H, J = 9.0), 6.57 (d, 1H, J = 1.5), 3.49 (q, 4H, J = 6.9), 3.34 (s, 2H), 1.14 (t, 6H, J = 7.0);

13

C-NMR (DMSO-d6, 151 MHz): 191.1, 163.9,

154.3, 134.4, 111.7, 104.9, 96.4, 44.6, 12.9 (Fig. S1). 2.3.3. Preparation and characterization of probe 1 Phosphorous

oxychloride

(POCl3)

(10

mL)

was

slowely

added

to

dimethylformamide (DMF) (10 mL) at 0-5 ℃ under the stirring about half an hour. Then the intermediate compound 4 by dissolving it into DMF (10 mL) was added to this cooled system under the stirring for 3 h, and the reaction mixture was subsequently heated at 75 °C for 6 h. The reaction mixture was cooled to room temperature and then poured into ice cold water (60 mL). Then the reaction system was neutralised with sodium carbonate and subsequently was filtered and washed with cold water, dried and crystallised from ethanol to give brick-red solid probe 1. 1

H-NMR (DMSO-d6, 600 MHz): 10.15 (d, 1H, J=6.9), 8.66 (s, 1H), 7.72 (d, 1H, J =

9.0), 7.46 (d, 1 H, J = 6.9), 6.84 (d, 1H, J = 9.1), 6.62 (s, 1H), 3.51 (q, 4H, J = 7.0), 1.16 (t, 6H, J = 7.0 Hz); 13C-NMR (DMSO-d6, 151 MHz): 192.4, 158.4, 157.1, 153.4,

8

146.3, 146.0, 132.4, 125.0, 111.4, 110.9, 108.5, 96.2, 45.0, 12.8; Elemental analysis (calcd. %) for C16H16ClNO3: C, 62.85; H, 5.27; Cl, 11.59; N, 4.58; O, 15.70. ESI-MS m/z: [probe]+ Calcd for C16H16ClNO3 306.0891; Found 306.0891 (Fig. S3). Crystal data for C16H16ClNO3: crystal size: 0.34 × 0.26 × 0.13 mm3, space group P-1. a = 7.522 (3) Å, b = 9.588 (4) Å, c = 11.127 (4) Å, V = 702.1 (5) Å3, Z = 2, T = 173 K, θmax = 27.50°, 7313 reflections measured, 3202 unique (Rint = 0.0337) Final residual for 192 parameters and 3202 reflections with I > 2σ(I): R1= 0.0451, wR2= 0.1091 and GOF = 1.097 (Fig. 1, Fig. S4). 2.4. Preparation of Solutions of Probe 1 and Analytes. Stock solution of probe 1 (2 mM) was prepared in DMSO. Stock solutions (20 mM) of SO32-, HSO3-, S2O52-, F-, Cl-, Br-, I-, N3-, NO3-, H2PO4-, NO2-, S2O32-, AcO-, SO42-, HS-, HCO3- were prepared by direct dissolution of proper amounts of sodium salts in deionized water. Stock solution (20 mM) of sulfur-containing amino acid (GSH) was prepared by direct dissolution of proper amounts of GSH in deionized water. All chemicals used were of analytical grade. 2.5. General UV-vis and fluorescence spectra measurements All the detection experiments were measured in DMSO-HEPES buffer (pH 7.4, 1 : 1, v/v). The procedure was as follows: into a DMSO-HEPES buffer, containing 5 μM probe 1, a SO32- sample was gradually titrated. The process was monitored by fluorescence spectrometer (λex= 370 nm, slit: 2.5 nm/5 nm). 2.6. Cytotoxicity Experiments

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MCF-7 cells were seeded in 96-well plates and cultured at 37 °C (5% CO2) for 24 h. After washing with PBS, different concentrations of probe 1 (0, 1, 2.5, 5 and 10 μM) in culture medium (without serum) were added to the wells and incubated for 5 or 10 h. Subsequently, CCK-8 (10% in serum free culture medium) was added to each well which was washed with PBS two times, and the plate was incubated for another 1 h. Optical densities at 450 nm were then measured. 2.7. Cell culture and fluorescence imaging The MCF-7 cells were grown in Dulbecco’s Modified Eagle’s medium supplemented with 12% Fetal Bovine Serum and 1% antibiotics at 37 °C in humidified environment of 5% CO2. Some of the MCF-7 cells were pretreated with 10 μM of probe 1 in culture media for 30 min at 37 °C, and washed three times with phosphate-buffered saline (PBS). Meanwhile, another portion of MCF-7 cells were incubated with 10 μM of probe 1 in culture media for 30 min at 37 °C with 100 μM of SO32- added for the final 5 min, 10 min and 20 min respectively. Cell imaging was then carried out after washing cells with PBS buffer. 3. Results and Discussion 3.1. The selective response of probe to sulfite An important feature of probe is that it has superior selectivity for one analyte over other substances. In order to value its special recognition ability, we carried out the experiment by fluorescence spectrometer. Fig. 2 showed the fluorescence spectral changes that the 5 μM probe with the addition of various analytes (10 equiv.) in HEPES (10 mM, pH 7.4)/DMSO (v/v, 1:1) solution, including F -, Cl-,

10

Br-, I-, N3 -, NO3-, H2 PO4 -, NO2 -, S2 O3 2-, AcO-, SO4 2-, HS-, HCO3 - and biothiol (GSH). Delightfully, even 10 equiv. of other analytes did not induce any fluorescent changes (Fig. 2a) of the system except the introduction of SO 32-. At the same time, the competition experiments were carried out with the presence of other analytes. As shown in Fig. 2b, all the competing analytes did not interfere the detection of SO 32-. In addition, S 2O52- can also cause fluorescence enhancement (Fig. S5) which may be caused by their decomposition to produce sulfite under the above test conditions. This result displayed highly selectivity of probe 1 towards sulfite over other analytes mentioned above. 3.2. UV-Vis and fluorescence spectra of detecting sulfite A detailed spectral titration for sulfite was carried out. The sensing ability of probe 1 towards SO32- was measured in HEPES buffer (10 mM, pH 7.4)/DMSO (v/v, 1:1) at 25 °C. As shown in Fig. 3a, the absorption of probe 1 in the solution displayed a strong band centred at 471 nm. Along with the addition of SO 32-, the original absorption band decreased and a new band centred at 423 nm increased gradually and peaked when SO32- reached 140 μM. A well-defined isosbestic point occurred at 435 nm. Apparently, the color of the system changed from brilliant yellow to yellowish. Further experiment of probe 1 towards HSO3- in DMSO-HEPES buffer (pH 5.0, 1 : 1, v/v) displayed same UV-Vis spectral changes with the above system which demonstrated the reactable of probe 1 with HSO3- [44] (Fig. S5). For fluorescent responses, probe 1 itself had feeble fluorescent emission at 486 nm with the excitation

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at 370 nm in the detection system. As shown in Fig. 3b, incremental changes of SO 32(0-140 μM) induced a notable enhancement in the intensity of the fluorescent band at 486 nm which peaked with a 33-fold enhancement. The optical responses of 1 to SO32- exhibited that probe 1 could be used as a visual probe for colorimetric and fluorescent detection of sulfite. It should be noted that a visual probe could offer great convenience, especially in cases where there was no instrument available. In order to exam the sensitivity of probe 1 to SO32-, the working curve was further measured upon treatment of 5 μM probe 1 with various concentrations of SO32- (0-80 μM) in DMSO-HEPES buffer. As shown in Fig. 3b, a linear calibration graph of the responses of the relative fluorescent intensity at 486 nm to the SO32- concentrations from 0 to 80 μM could be observed, which means that probe 1 could be potentially employed to detect SO32- quantitatively. The detection limit, based on the definition by IUPAC (CDL = 3 Sb/m), was found to be 8.8 nM. This result demonstrated probe 1 to be a highly sensitive probe for SO32-. 3.3. pH dependence The pH value of system is often considered as a significant influence factor on interactions. Hence, in order to investigate pH effect and find a suitable pH range, fluorescent spectra of probe 1 and probe 1-SO32- were evaluated. Fig. 4 showed the fluorescent responses of probe 1 at 486 nm in the presence of SO32- at different pH values. The results showed that probe 1 was pH independence in the range of pH 2~ 12. However, the fluorescence intensity of probe 1-SO32- reached a maximum in the

12

pH 7 system. It can be induced that probe 1 works well for SO32- detection under pH 7. Considering the potential biological application, the pH value in our detection system was set at 7.4 to fit the natural physiological requirements. 3.4. Time-dependence in the detection process of sulfite The kinetic analysis in the fluorescence spectra of probe 1 were monitored in the presence of 5 eq. of sulfite. It (Fig. 5) showed that the fluorescence intensity of 1 with addition of SO32- reached a plateau in about 4 min, which indicated that the reaction was completed within 4 min. These fast and distinct responses both in fluorescent emission and color changes promoted probe 1 to be used as a predominant fluorescent probe for sulfite. As indicated in Table 1, probe 1 exhibited excellent analytical performance compared with other recently reported fluorescent probes for SO32-/HSO3- in the aspects of detection medium, emission wavelength, detection limit, response time and cell imaging. It can be found that probe 1 has lower detection limit (8.8× 10-9 M) and fast response (4 min). In addition, the biological experiments demonstrated the application of probe 1 monitoring SO2 derivatives in living cells in this work. These traits suggested that probe 1 might be an excellent candidate for practical SO2 derivatives analysis. 3.5. Proposed mechanism

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To confirm the reaction mechanism, 1H-NMR and MS titration experiments were carried out through adding excess SO32- to a solution of probe 1 in DMSO-d6.

In the

adduct 1H-NMR spectra, the signal of original aldehyde proton at around δ 10.15 ppm disappeared and a new signal at 4.92 ppm appeared (Fig. 6). This result indicated that the aldehyde group reacted with sulfite ion. In addition, as showed in Fig. 6, other proton peaks all moved to high field. Among them, proton 4 and 5 were greatly influenced by opened aldehyde group with big shifts to high field. The mixture after probe 1 reacting with SO32- was characterized by HRMS spectrum. After the addition of SO32-, a peak at about 388.0613 appeared corresponding to the adduct species [1+SO32-+H]+ (m /zcalcd = 388.0616) (Fig. S3). The above data suggested that the probe’s formyl group hydrogen changed into alkyl hydrogen. The sensing mechanism of probe 1 towards SO32- was based on the nucleophilic addition as shown in Scheme 1. 3.6. Cytotoxicity experiments and cellular imaging Cytotoxicity experiment for 1 showed that the probe with a concentration range of 0-10 µM has only low cytotoxicity to cultured cells after 5 or 10 h (Fig. 7), and thus the probe at 10 µM was selected for further cell experiment. As shown in Fig. 8, MCF-7 cells incubated with 10 µM of probe for 30 min at 37 ℃ shown no fluorescence. In a further experiment it was found that MCF-7 cells displayed gradually enhanced cyan fluorescence when the cells were first incubated with 10 µM of probe for 30 min at 37 ℃ and then incubated with 100 µM sulfite for 5 min, 10

14

min and 20 min respectively. Therefore, all these data demonstrated good cell-membrane permeability of probe and it can thus be used to detect sulfite in living cells. 4. Conclusions In summary, we developed a new turn-on fluorescent material for rapid, highly selective and sensitive detection of sulfite through colorimetric and fluorescent responses. The nucleophilic addition of SO32-/HSO3- with probe 1 in DMSO-HEPES buffer would display significant fluorescence enhancement (up to 33-fold) at 486 nm. Furthermore, with low detection limit (as low as 8.8 nM), good cellular permeability and low cytotoxicity, the probe was successfully applied in SO 32-/HSO3- imaging in living cells. All these results demonstrated that this probe can be further developed for extensive applications in biological sensing. Acknowledgments The work was supported by the National Natural Science Foundation of China (No. 21472118, 21672131), talents Support Program of Shanxi Province (2014401), Shanxi province Outstanding Youth Fund (2014021002).

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Fluorescent Probe for Imaging of Cellular Viscosity in Live Cells, Chem. Eur. J. 20 (2014) 4691-4696. [37] J. Du, M. Hu, J. Fan, X. Peng, Fluorescent chemodosimeters using “mild” chemical events for the detection of small anions and cations in biological and environmental media, Chem. Soc. Rev. 41 (2012) 4511-4535. [38] J. S. Kim, D. T. Quang, Calixarene-derived fluorescent probes, Chem. Rev.107 (2007) 3780-3799. [39] T. Q. Duong, J. S. Kim, Fluoro- and chromogenic chemodosimeters for heavy metal ion detection in solution and biospecimens, Chem. Rev. 110 (2010) 6280-6301. [40] M. S. T. Goncalves, Fluorescent labeling of biomolecules with ̧ organic probes, Chem. Rev. 109 (2009) 190-212. [41] M. Fernandez-Suarez, A. Y. Ting, Fluorescent probes for superresolution imaging in living cells, Nat. Rev. Mol. Cell Biol. 9 (2008) 929-943. [42]

Y. Q. Sun, J. Liu, J. Y. Zhang, T. Yang, W. Guo, Fluorescent probe for biological gas SO2 derivatives bisulfite and sulfite, Chem. Commun. 49 (2013) 2637-2639.

[43]

S. Chen, P. Hou, J. X. Wang, X. Z. Song, A highly sulfite-selective ratiometric fluorescent probe based on ESIPT, RSC Adv. 2 (2012) 10869-10873.

[44]

Y. T. Yang, F. J. Huo, J. J. Zhang, Z. H. Xie, J. B. Chao, C. X. Yin, H. B. Tong, D. S. Liu, S. Jin, F. Q. Cheng, X. X. Yan, A novel coumarin-based

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fluorescent probe for selective detection of bissulfite anions in water and sugar samples, Sensor. Actuat. B-Chem. 166-167 (2012) 665-670. [45]

G. M. Sheldrick, SADABS, Germany University of Gottingen, 1997.

[46]

G. M. Sheldrick, Program for the Refinement of Crystal Structure, University of Goettingen, Germany, 1997.

[47]

X. Ma, C. X. Liu, Q. L. Shan, G. H. Wei, D. B. Wei, Y. G. Du, A fluorescein-based probe with high selectivity and sensitivity for sulfite detection in aqueous solution, Sensor. Actuat. B-Chem. 188 (2013) 1196-1200.

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Biographies

Caixia Yin She obtained her Doctor Degree is in chemistry for Shanxi University in 2005. Now she is a Professor in Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science at Shanxi University major in inorganic chemistry. She current research interests are molecular recognition, sensors chemistry.

Xiaoqi Li She obtained her BSC in Chemistry for Datong University in 2015. Now she is studying for master in Institute of Molecular Science at Shanxi University. Her current research is molecular chemistry.

Yongkang Yue He obtained his BSC in Chemistry for Shanxi University in 2013. Now he is studying for doctor in Institute of Molecular Science at Shanxi University. His current research is molecular chemistry.

Jianbin Chao He obtained his master degree is in chemistry for Shanxi University in 2000. Now he is an Assoiate Professor in Research Institure of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are supramolecular chemistry.

Yongbin Zhang He obtained his master degree is in chemistry for Shanxi University in 2006. Now he is is a University Lecturer in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are molecular recognition, sensors chemistry.

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Fangjun Huo He obtained his Doctor Degree in chemistry for Shanxi University in 2007. Now he is an Assoiate Professor in Research Institute of Applied Chemistry at Shanxi University major in organic chemistry. His current research interests are sensors, supramolecular chemistry.

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Figure captions Fig. 1 The crystal structure of probe 1. Fig. 2 (a) Fluorescent responses of probe 1 (5 μM) towards various anions (140 μM for SO32− and 1400 μM for other anions) in DMSO-HEPES buffer. (b) Competing responses of probe 1 (5 μM) towards various analytes (140 μM for SO32− and 1400 μM for other analytes) in DMSO-HEPES buffer. λex= 370 nm, slit: 2.5nm/5 nm. Each spectrum was recorded after 5 min. (Other analytes including 2: F-, 3: Cl-, 4: Br-, 5: I-, 6: N3-, 7: NO3-, 8: H2PO4-, 9: NO2-, 10: S2O32-, 11: AcO-, 12: SO42-, 13: HS-, 14: HCO3- and 15: GSH.) Fig. 3 (a) UV-Vis spectra of 1 (5 μM) upon addition of SO32- (140 μM) in DMSO-HEPES buffer at 25 °C. Each spectrum was collected 5 min after adding of SO32-. (Insert: the colorimetric responses of 1 towards SO32-). (b) Fluorescent spectral changes of 1 (5 μM) upon addition of SO32- (140 μM) in DMSO-HEPES buffer at 25 °C. (Insert: the color of fluorescent responses of 1 towards SO32-; Working curve of 1 (5 μM) at 486 nm with SO32- (80 μM) in DMSO-HEPES buffer at 25 °C. λex= 370 nm, slit: 2.5 nm/5 nm.) Fig. 4 Effect of pH on the emission intensity of the probe 1. Sulfite concentration: (a) 0 and (b)140 μM. Fig. 5 Kinetic analysis of probe 1 towards SO32- (5 μM 1 with 5 equiv. of SO32-). Fig. 6 1H-NMR comparison of the probe with the product upon addition of SO32- in DMSO-d6. Fig. 7 Percentage of viable MCF-7 cells after treatment with indicated concentrations

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of probe 1 after 5 hours and 10 hours. Fig. 8 Fluorescent imaging of MCF-7 cells: (a1) Bright field image of MCF-7 cells after been treated with probe 1 (10 µM) for 30 min and (a2) its fluorescence image; (a3) The overlay of (a1) and (a2); (b1, c1, d1) Bright field image of MCF-7 cells preincubated with 10 µM probe 1 and further incubated with 100 µM SO32- for 5 min, 10 min and 20 min; (b2, c2, d2) Corresponding fluorescence image of (b1, c1, d1); (b3, c3, d3) The overlay of (b1, c1, d1) and (b2, c2, d2).

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Figure 1

Figure 2(a)

Figure 2(b)

27

Figure 3(a)

Figure 3(b)

28

Figure 4

29

Figure 5

Figure 6

30

Figure 7

Figure 8

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Scheme 1. Proposed response mechanism of probe 1 towards SO32−/HSO3−. Scheme 2. The synthesis of probe 1.

Scheme 1

Scheme 2

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Table 1 Comparison of fluorescent probes for SO2 derivatives. Table 1 Reference Detection medium

Ref.[42]

HEPES buffer

λex/λem

Detection

(nm)

limit(M)

445/478

3.8× 10−7

5 min

HeLa cell

310/468

5 × 10−6

60 min

-

330/395

2 × 10−6

10 min

Time

Cell imaging

(pH 7.4) containing 30% DMF Ref. [43]

HEPES buffer (pH 7.4) containing 50% of CH3CN

Ref. [44]

Na2HPO4- citric acid Buffer (pH 5.0) containing

-

30% DMF Ref. [47]

HEPES buffer

490/520

10 × 10−6

25 min

-

370/486

8.8× 10−9

4 min

MCF-7

(pH 7.4) containing 1% of CH3CN This

HEPES buffer

work

(pH 7.4)/DMSO(v/v, 1:1)

cell

33