A ratiometric fluorescent probe for cysteine and its application in living cells

Sensors and Actuators B 207 (2015) 872–877

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A ratiometric fluorescent probe for cysteine and its application in living cells Xi Dai a,1 , Tao Zhang b,1 , Yi-Zhi Liu c , Tao Yan a , Yun Li a , Jun-Ying Miao b,∗ , Bao-Xiang Zhao a,∗∗ a

Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China c Key Laboratory of Colloid and Interface Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China b

a r t i c l e

i n f o

Article history: Received 17 August 2014 Received in revised form 15 October 2014 Accepted 18 October 2014 Available online 29 October 2014 Keywords: Coumarin Pyrazoline Ratiometric Fluorescence Cysteine Living cells

a b s t r a c t A ratiometric fluorescent probe DPCA based on coumarin and pyrazoline was designed and synthesized for sensing cysteine (Cys) in physiological pH. The probe can detect Cys by fluorescent spectrometry within 40 min with a detection limit of 5.08 ␮M. The mechanism of DPCA sensing Cys involved conjugate addition/cyclization which was confirmed by HRMS and fluorescence spectra analysis. Moreover, DPCA was able to be applied to complicated conditions with negligible interference from these species and successfully applied in cell imaging. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Cysteine (Cys), one of the most important small biothiols, is involved in multiples of vital cells process, such as posttranslational modifications, biocatalysis, and detoxification of xenobiotics and so on [1,2]. Moreover, Cys is an essential component of proteins and a precursor of CoA, glutathione (GSH) and taurine [3]. In human, clinically abnormal level of Cys is associated with edema, liver damage, Alzheimer’s disease, Parkinson’s disease, and so forth [4]. Therefore, it is quite necessary to effectively detect intracellular Cys. Fluorescence spectroscopy is the popular method for analyte detection in biological and environmental sciences because of its high sensitivity and selectivity, real-time monitoring, instrument manipulability, lower detection limit and live cell imaging [5–8]. To date, numerous fluorescent probes for sundry targets have been developed [9–11]. Certainly, many thiol probes are reported based on the inherent nucleophilicity of sulfydryl group which

∗ Corresponding author. ∗∗ Corresponding author. Tel.: +86 531 88366425; fax: +86 531 88564464. E-mail addresses: [email protected] (J.-Y. Miao), [email protected], [email protected] (B.-X. Zhao). 1 Equal contribution. http://dx.doi.org/10.1016/j.snb.2014.10.082 0925-4005/© 2014 Elsevier B.V. All rights reserved.

commonly consist in Cys, GSH and homocysteine (Hcy). The different mechanisms such as conjugate addition/cyclization reaction [12–14], aldehyde addition [15–17], cleavage reaction [18–20], and Michael addition [21–24] and so on [25–30] have also been proposed. However, it is seriously hampered to distinguish Cys from Hcy and GSH because of their similar structure. Recently, many of new strategies were used to design fluorescent probes [31–37]. A glutathione-protected silver nanocluster was first used for the detection of Cys [38]. Also, a rhodamine-based fluorescent probe masked with a para-hydroxybenzyl alcohol can discriminate Cys over Hcy/GSH in HEPES buffer through a series of reactions of Michael addition, intramolecular cyclization and the deprotection [39]. In addition, three coumarin fluorophore cationic probes were designed based on a response-assisted electrostatic attraction strategy, and enabled response to Cys in aqueous solution [40]. While, only the meta-position charge configuration probe can respond to Cys persistently, and when positive charge is at paraposition or ortho-position the fluorescence quenches. Compared to single wavelength response fluorescent probes, ratiometric fluorescent probes have two emission wavelengths to give a built-in correction for probe concentration and measured environment effects [41,42]. Although significant advances of fluorescent probes for Cys have been made [16,43–47], the ratiometric probes are still rare [17,21,38,48].

X. Dai et al. / Sensors and Actuators B 207 (2015) 872–877

Thus, we have designed and developed a ratiometric fluorescent probe, 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-2-oxo2H-chromen-7-yl acrylate (DPCA), containing two fluorophores and a acrylate for the reaction site. Probe DPCA not only distinguish Cys from other thiols, but also can be used to live cell imaging. 2. Experiment 2.1. Apparatus and chemicals Thin-layer chromatography (TLC) involved silica gel 60F254 plates (Merck KGaA) and column chromatography involved silica gel (mesh 200–300). 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were acquired on a Bruker Avance 400 spectrometer, with CDCl3 or DMSO used as a solvent and tetramethylsilane (TMS) as an internal standard. Melting points were determined with an XD4 digital micro-melting-point apparatus. IR spectra were recorded with the infra-red (IR) spectrophotometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were obtained on a Q-TOF6510 spectrograph (Agilent). UV–vis spectra were measured by use of a Hitachi U-4100 spectrophotometer. Fluorescent measurements were performed on a Perkin–Elmer LS-55 luminescence spectrophotometer. Quartz cuvettes with a 1-cm path length and 3-mL volume were used for all measurements. The pH was determined with a model PHS-3C pH meter. Unless otherwise stated, all reagents were purchased from Aladdin, J&K or Sinopharm Chemical Reagent Co. and used without further purification. Twice-distilled water was used throughout all experiments. The salts used in stock aqueous solutions of metal ions were KNO3 , Ca(NO3 )2 ·4H2 O, Mg(NO3 )2 ·6H2 O, Zn(NO3 )2 ·6H2 O, NaNO3 and Fe(NO3 )3 ·9H2 O. 2.2. Synthetic procedures 2.2.1. Synthesis of 3-cinnamoyl-7-hydroxy-2H-chromen-2-one (3) 3-Acetyl-7-hydroxy-2H-chromen-2-one (2) was prepared from 2,4-dihydroxybenzaldehyde according to the reported method [14]. Compound 2, benzaldehyde and piperidine (catalytic amount) were dissolved in absolute ethanol, and the mixture was heated at reflux for 4 h [49,50]. After cooling to room temperature, the mixture was poured into ice water and was adjusted to neutral with acetic acid. The ensuing yellow solid was filtered and recrystallized from ethanol to give compound 3. The purity is enough for the next step. 2.2.2. Synthesis of 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3yl)-7-hydroxy-2H-chromen-2-one (4) Compound 3 (2 mmol) and phenylhydrazine (6 mmol) were dissolved in hot EtOH (50 mL), and catalytic amount of acetic acid was added. The mixture was heated at reflux for 4 h, until TLC indicated the end of reaction. After cooling to room temperature, the mixture was poured into ice water. The ensuing solid was filtered and washed with EtOH. The crude product recrystallized from ethanol to give 4 in 32% yield. Red solid, m.p. 237–238 ◦ C. IR (KBr, cm−1 ): 3421 (O–H), 3152 (Ar–H), 2926 (–CH2 –), 1677 (C O), 1594 (C C), 1232 (C–O–C). 1 H NMR (400 MHz, DMSO-d6 ): ı = 3.18 (1H, dd, J = 18.0 and 6.3 Hz, pyrazole, 4-Ha), 3.96 (1H, dd, J = 12.5 and 18.0 Hz, pyrazole, 4-Hb), 5.47 (1H, dd, J = 6.3 and 12.5 Hz, pyrazole, 5-H), 6.71–7.67 (m, 13H, ArH), 8.41 (1H, s, C4 -H), 10.72 (1H, s, OH). 13 C NMR (100 MHz, DMSO-d6 ): 45.29, 63.45, 102.31, 112.18, 113.58 (2C), 114.18, 115.75, 119.35, 126.24 (2C), 127.87, 129.32 (2C), 129.45 (2C), 130.77, 140.01, 142.89, 144.30, 144.38, 155.58, 159.08, 162.17. HRMS: m/z [M+H]+ calcd for C24 H19 N2 O3 : 383.1396, found: 383.1382.

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2.2.3. Synthesis of 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3yl)-2-oxo-2H-chromen-7-yl acrylate (5, probe DPCA) A solution of acryloyl chloride (1.8 mmol) in dichloromethane (10 mL) was added dropwise to a solution of compound 4 (0.6 mmol) and triethylamine (0.5 mL) in dichloromethane (40 mL) in an ice-bath. After stirring for 2.5 h at room temperature, water (50 mL) was added to the reaction mixture. The organic phase was washed with water, then dried over Na2 SO4 . After the solvent was evaporated under reduced pressure, the crude solid recrystallized from 30 mL ethanol to give product 5 in 85% yield. Red solid, m.p. 228–230 ◦ C. IR (KBr, cm−1 ): 3059 (Ar–H), 2917 ( CH), 1729 (COO), 1597 (C=O), 1499 (C C), 1155 (C–O–C). 1 H NMR (400 MHz, CDCl3 ): ı = 3.38 (1H, dd, J = 18.3 and 7.3 Hz, pyrazole, 4-Ha), 4.09 (1H, dd, J = 12.8 and 18.3 Hz, pyrazole, 4-Hb), 5.34 (1H, dd, J = 7.3 and 12.8 Hz, pyrazole, 5-H), 6.09 (1H, dd, J = 0.94 and 10.44 Hz), 6.34 (1H, dd, J = 10.44 and 17.26 Hz), 6.66 (1H, dd, J = 0.94 and 17.26 Hz), 6.81–7.61 (m, 13H, ArH), 8.40 (1H, s, C4 -H). 13 C NMR (100 MHz, CDCl3 ): 45.17, 64.92, 109.96, 113.68, 118.55, 119.89, 125.51 (2C), 125.76 (2C), 127.65, 128.51 (2C), 128.83 (2C), 129.03, 129.11 (2C), 133.63, 137.38, 139.86, 142.02, 144.37, 152.80, 154.07, 159.56, 163.83. HRMS: m/z [M+H]+ calcd for C27 H21 N2 O4 : 437.1501, found: 437.1564. 2.3. Absorption and fluorescence spectroscopy Probe DPCA was dissolved in acetonitrile for a stock solution (1 mM). The amino acids (Cys, Hcy, GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, proline, threonine, tryptophan and tyrosine), cationic (K+ , Ca2+ , Na+ , Mg2+ , Zn2+ and Fe3+ ), H2 O2 and glucose stocks were all in deionized water at 10−2 M for absorption and fluorescence spectrum analysis. Test solutions were prepared by displacing 100 ␮L of the stock solution and an appropriate aliquot of each testing species solution into a 10 mL volumetric flask, and the solution was diluted to 10 mL in a mixture of acetonitrile and water (1:1, v/v) buffered at pH 7.4 (PBS buffer, 1 mM CTAB). The resulting solution was shaken well and recorded after 40 min at room temperature. All measurements of fluorescence spectra were performed at excitation wavelength 430 nm, slit 10.0/4.0 nm and scan speed 900 nm min−1 . Fluorescence quantum yield was determined at room temperature with fluorescein (˚s = 0.95 in 0.1 M NaOH) as standard, and it was calculated by Eq. (1) [51,52]: ˚ = ˚s

 IA   2  s Is A

2s

(1)

where A is the absorbance, I is the integrated fluorescence intensity, and  is the refractive index of the solvent. 2.4. Cell culture and cell imaging A549 cells were cultured in DMEM. The probe was dissolved in DMSO at a storage concentration of 10 mM. Cells were adherentcultured in 24-well culture plates for 12 h. A549 cells were washed from the culture medium, incubated with 0.1, 0.3, 0.5, 1.0 ␮M probe solution at 37 ◦ C, respectively. Then washed 3 times with phosphate buffered saline (PBS) and underwent imaging measurement by ultraviolet light with a Nikon TE2000-E fluorescent microscope. Imaging analysis involved the use of ImageJ. 2.5. Cytotoxicity assay The cytotoxicity in vitro of the probe to A549 cells was measured by a standard sulforhodamine B (SRB) assay. Briefly, A549 cells were loaded in 96-well culture plates at 4 × 104 cells per well.

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Scheme 1. Synthesis of compound 5 (Probe DPCA).

After culture for 12 h, cells were incubated with fresh 1640 containing 1 ␮M probe for 4 h. Then cells were fixed with 4% TCA for 1 h at 4 ◦ C, then washed with deionized water 5 times; 50 ␮L SRB was added to each well, and after sufficient reaction with cells, the remaining SRB was removed by washing each well with 1% acetic acid solution, and 100 ␮L Tris–HCl was used to dissolve the SRB. Absorbance at 540 nm was measured in a 96-well multiwell-plate reader (TECAN). 3. Results and discussion 3.1. Synthesis We selected coumarin and pyrazoline as the fluorophores by virtue of their strong fluorescence and high quantum yield. Usually, the excitation wavelength of pyrazoline is less than 400 nm [53–55], and that of coumarin is between 400 and 450 nm [56–58]. We combined pyrazoline and 7-hydroxy coumarin to obtain a large conjugated compound, which will give a red shift. Pyrazoline was widely used to the fluorescent probe, but rarely in ratiometric sensors. That was the first time, coumarin and pyrazoline together applied to the fluorescent probe. Compound 4 (3-(1,5-diphenyl-4,5-dihydro1H-pyrazol-3-yl)-7-hydroxy-2H-chromen-2-one) was simply synthesized from 3-cinnamoyl-7-hydroxy-2H-chromen-2-one 3 and phenylhydrazine. Probe DPCA (3-(1,5-diphenyl-4,5-dihydro-1Hpyrazol-3-yl)-2-oxo-2H-chromen-7-yl acrylate) was conveniently synthesized from acylation of compound 4 with acryloyl chloride (Scheme 1) [59]. The structure of DPCA was confirmed by IR, NMR and HRMS. 3.2. Measurement studies of probe DPCA Probe DPCA alone exhibited two absorption peaks at 294 and 439 nm (see Figure S1 in Supporting Information). Upon addition of Cys to DPCA, absorbance at 439 nm increased and that at 294 nm decreased. Then, we added various analytes (Cys, Hcy, GSH, arginine, aspartic acid, glutamic acid, glycine, histidine, lysine, proline, threonine, tryptophan, tyrosine, K+ , Ca2+ , Na+ , Mg2+ , Zn2+ , Fe3+ , H2 O2 and glucose) to PBS-buffered solution of DPCA. Originally, probe DPCA alone had a weak fluorescent peak at 600 nm (Fig. 1). The addition of Cys caused the decrease of DPCA concentration and the increase of compound 4 concentration due to the reaction of

DPCA with Cys. An obvious fluorescence enhancement at 560 was observed because compound 4 showed the maximum emission wavelength at 560 nm (Figure S2). Therefore, the maximum emission wavelength of the sensing system afforded a hypochromatic shift from 600 nm to 560 nm ( = 40 nm). In order to clarify the specific process, various concentrations of Cys were added to the buffer solution. Obviously, with increasing Cys concentration, the fluorescence intensity enhanced gradually at 560 nm until Cys reached approximately 9.0 equiv. (Fig. 2). The intensity ratio of I560 /I460 decreased before 1.0 equiv. (from 4.4 to 3.1) and increased after 1.0 equiv. (from 3.1 to 5.0), that is, the ratiometric responses (I560 /I460 ) decreased with increasing Cys in low concentrations while increased in high concentrations. It is interesting that probe DPCA has two completely different responding trends to Cys. With the addition of Cys, the amount of probe DPCA decreased and that of compound 4 increased. However, besides the fluorescent intensity of the system was mainly depend on the effects of the probe and compound 4, it might be influenced by other weak interactions between many compounds such as the probe, compound 4, intermediate and Cys, because they could exist in the system and their composition change during the titration process. Therefore, the overall effects result in two completely different trends for the probe. Compared to probe ACA that we reported [13], DPCA shows ratio responses to Cys that has self-correction shielding environment interference. Moreover, DPCA with the large conjugated structure has longer emission wavelength and large storks shift (120 nm). Therefore, DPCA exhibits more advantage and figures a potential application. The linear correlation (k = −1.4275, R2 = 0.990) between intensity ratio and concentrations of Cys from 0 to 0.8 equiv. gave the detection limit 5.08 ␮M (Figure S3). Also, we observed GSH and Hcy induce slight change (Fig. 1). Therefore, the interference of these thiols, metal ions and other related amino acids was explored. The results revealed that probe DPCA was able to be applied to complicated conditions with negligible interference from these species (Figure S4). DPCA displayed good selectivity toward Cys, which outweighs numerous reported probes [60–62].

3.3. Reaction time and pH effect We examined the time-dependent response of DPCA in the presence of 10.0 equiv. of Cys (Figure S5). Time-dependent ratiometric response (I560 /I460 ) reached maximum at 40 min, and steadied for

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Fig. 1. Fluorescence spectra of DPCA (1 × 10−5 M) with GSH (3 mM) or other analytes (0.1 mM) in PBS buffer solution (CH3 CN/PBS = 1:1, pH 7.4, ex = 430 nm, slit: 10.0 nm/4.0 nm).

a long time. As we know, the pKa of Cys (8.30) is lower than that of Hcy (8.87) and GSH (9.20) [63] and Cys has a lower pI value (Cys 5.02, Hcy 5.27 and GSH 5.93) [64]. Under physiological conditions (pH 7.4), the sulfydryl group of Cys is probably changed to a better nucleophile – thiolate – in Michael addition reaction. Also, Cys has less steric hindered than that of Hcy and GSH. Namely, the above factors make Cys slightly more reactive than Hcy/GSH. Therefore, we should confirm the appropriate pH range for the probe. Ratiometric responses (I560 /I460 ) of probe DPCA with or without Cys were measured in buffer solution with various pH values (Figure S6). The probe is stable in weak alkaline condition, and the response of DPCA toward Cys is slight increased with increasing pH from 6.5 to 10.0. Because weak

alkaline conditions promoted the reaction, some probes were reported using higher pH (selected pH ≥ 9.0) [65–67], which should be not suitable for detecting biothiols in the physiological pH condition. In this work, probe DPCA can function over a wide range of pH, therefore, we chose physiological pH (pH 7.4) as experimental conditions. 3.4. Mechanism of DPCA in sensing Cys Acrylate group has been used as the thiols reaction site to detect thiols since the first reported by Strongin et al. [12,68]. Based on the reported conjugate addition/cyclization sensing mechanism, the mechanism of DPCA responding to Cys involved the next two

Fig. 2. Fluorescence responses of DPCA (10 ␮M) toward different concentrations of Cys (final concentrations: 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120 and 130 ␮M) in PBS buffer solution (CH3 CN/PBS = 1:1, pH 7.4, ex = 430 nm, slit: 10.0 nm/4.0 nm). Inset is the plot of ratiometric responses (I560 /I460 ) of DPCA vs equivalents of Cys.

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Fig. 3. Fluorescence images of DPCA in A549 cells at different times and concentrations.

steps: conjugate addition of Cys to an ␣,␤-unsaturated carbonyl moiety generated thioether (6), then intramolecular cyclization gave the desired compounds 4 and 7 (Scheme S1). In addition, the reaction products of DPCA with Cys underwent HRMS analysis, and compounds 4 ([M+H]+ calcd m/z 383.13957) and 7 (calcd m/z 174.02249) were observed according to m/z 383.13462, 174.01881 (Figure S7). Meanwhile, the intermediate thioether (6, [M+H]+ calcd m/z 558.16988) was found at the peak m/z 558.16462, which verified the proposed mechanism and indicated that DPCA cannot transform fully to compound 4. On the other hand, we explored the fluorescence sensing behavior of precursor (4), DPCA and DPCA with Cys in PBS buffer solution (Figure S2). Probe DPCA was non-fluorescent with a low fluorescence quantum yield (Ф = 0.0046), but the addition of Cys induced a fluorescence enhancement (fluorescence quantum yield Ф = 0.0235). Compared to them, fluorescence quantum yield of compound 4 is 0.0288.

3.5. Application of probe DPCA We applied probe DPCA for the fluorescence imaging in living cells. Living A549 cells were incubated with DPCA at different concentrations and times, which showed a bright and rapid change (Fig. 3). Obviously, weak orange fluorescence represented at living cells with 0.1 ␮M DPCA after 10 min, then increased with increasing time and concentration of DPCA. Relative to 1 ␮M DPCA, bright orange fluorescence always showed, even 10 min, and the same bright orange fluorescence showed with 0.5 ␮M DPCA after 30 min. The phenomena present DPCA has longer emission wavelength and good membrane permeability than that of ACA [14]. To confirm the biothiol specific of the probe, A549 cells were preincubated with N-methylmaleimide (NEM, a thiol-blocking agent), then incubated with 1 ␮M probe for 30 min. We observed the obvious fluorescent quench and quantified the fluorescent intensity to give a visual chart (Figure S8). Meanwhile, DPCA was added to the solution with Cys and NEM to evidence the probe specificity in vitro. Obviously, DPCA had no response because of the deficiency of Cys caused by NEM (Figure S9). Moreover, we used sulforhodamine B assays of probe DPCA (1 ␮M) in living A549 cells for 4 h to research the cytotoxicity (Figure S10). The cellular viability showed no significant change, which indicated probe DPCA is available in biological systems.

4. Conclusions In summary, we developed a novel ratiometric fluorescent probe DPCA for Cys based on coumarin-pyrazoline fluorophore with an acrylate group as a reaction site. The detection mechanism involving conjugate addition/cyclization was confirmed by HRMS and fluorescence spectrum analysis. Upon addition of Cys to PBS buffer solution (CH3 CN/PBS = 1:1, CTAB 10 mM), DPCA was able to respond to Cys in ratiometric manner within 40 min with a low detection limit of 5.08 ␮M in a wide range of pH. Meanwhile, DPCA exhibited high sensitivity and selectivity toward Cys over other analytes at physiological condition. Finally, DPCA was successfully applied in cell imaging. Acknowledgments This study was supported by the National Basic Research Program of China (2010CB933504) and the National Natural Science Foundation of China (91313303). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.10.082. References [1] E. Weerapana, C. Wang, G.M. Simon, F. Richter, S. Khare, M.B. Dillon, D.A. Bachovchin, K. Mowen, D. Baker, B.F. Cravatt, Nature 468 (2010) 790–795. [2] Z. Sun, C. Han, M. Song, L. Wen, D. Tian, H. Li, L. Jiang, Adv. Mater. 26 (2014) 455–460. [3] L.Y. Niu, Y.S. Guan, Y.Z. Chen, L.Z. Wu, C.H. Tung, Q.Z. Yang, Chem. Commun. 49 (2013) 1294–1946. [4] L.M. Lopez-Sanchez, C. Lopez-Pedrera, A. Rodriguez-Ariza, Mass Spectrom. Rev. 33 (2014) 7–20. [5] X. Li, X. Gao, W. Shi, H. Ma, Chem. Rev. 114 (2014) 590–659. [6] H. Wang, G. Zhou, X. Chen, Sensor. Actuat. B 176 (2013) 698–703. [7] D. Kand, T. Saha, P. Talukdar, Sensor. Actuat. B 196 (2014) 440–449. [8] C. Liu, H. Wu, Z. Wang, C. Shao, B. Zhu, X. Zhang, Chem. Commun. 50 (2014) 6013–6016. [9] L. Yuan, W. Lin, K. Zheng, L. He, W. Huang, Chem. Soc. Rev. 42 (2013) 622–661. [10] M. Lan, J. Wu, W. Liu, H. Zhang, W. Zhang, X. Zhuang, P. Wang, Sensor. Actuat. B 156 (2011) 332–337. [11] J. Fan, Z. Wang, H. Zhu, N. Fu, Sensor. Actuat. B 188 (2013) 886–893. [12] X. Yang, Y. Guo, R.M. Strongin, Angew. Chem. Int. Ed. Engl. 50 (2011) 10690–10693. [13] H. Wang, G. Zhou, H. Gai, X. Chen, Chem. Commun. 48 (2012) 8341–8343.

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Biographies Xi Dai received her B.Sc. in chemistry from Qufu Normal University in 2011. At present, she is a PhD student in Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University. Her current research interest involves the synthesis and application of fluorescent sensors. Tao Zhang is studying assiduously for his M.Sc. at present in Institute of Developmental Biology, School of Life Science, Shandong University. His current research interest involves the chemosensor applied in living cells. Yi-Zhi Liu received his B.Sc. in chemistry from Shandong University in 2014. At present, he is studying assiduously for his M.Sc. at present in Key Laboratory of Colloid and Interface Chemistry, School of Chemistry and Chemical Engineering, Shandong University. His research focuses on self-assemble of imidazolium-type zwitterions. Tao Yan is studying assiduously for his B.Sc. at present in School of Chemistry and Chemical Engineering, Shandong University. His current research interest involves the synthesis of chemosensor. Yun Li is studying assiduously for her B.Sc. at present in School of Chemistry and Chemical Engineering, Shandong University. Her current research interest involves the synthesis of chemosensor. Bao-Xiang Zhao received his PhD (1998) in chemistry from the Nagoya University, Japan. Then, he became Associate Professor at Shandong University. After postdoctoral fellowship with Professor S. Blechert at Technology University Berlin, he spent his career at the Shandong University where he has been Professor of Organic Chemistry since 2000. His main scientific interests are the design and synthesis of small molecule with structural diversity for chemical biology research. One of his current research interests is synthesis of fluorescent probe for detecting bithiols in water and in living cells. Jun-Ying Miao received her PhD (1997) in Cell Biology from the Nagoya University, Japan. She spent her career at the Shandong University where she has been Professor of Cell Biology since 1999. Her main scientific interests are the research on differentiation, autophagy, apoptosis and modulation of cell fate by small molecules with structural diversity.