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A curcumin-based fluorescent and colorimetric probe for detecting cysteine in living cells and zebrafish Lanfang Pang, Yanmei Zhou, Wenli Gao, Junli Zhang, Haohan Song, Xiao Wang, Yong Wang, and Xiaojun Peng Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b02133 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 20, 2017

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Industrial & Engineering Chemistry Research

A curcumin-based fluorescent and colorimetric probe for detecting cysteine in living cells and zebrafish Lanfang Panga, Yanmei Zhoua,b,*, Wenli Gaoa, Junli Zhangc, Haohan Songa, Xiao Wanga, Yong Wanga, Xiaojun Pengb a

Institute of Environmental and Analytical Sciences, College of Chemistry and Chemical

Engineering, Henan University, Kaifeng, Henan 475004, PR China b

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024,

PR China c

Key Laboratory of Plant Stress Biology, Henan University, Kaifeng, Henan 475004, PR

China

*Corresponding author: Tel: +86-371-22868833-3422; Fax: +86-371-23881589 E-mail address: [email protected] (Y.M. Zhou)

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Abstract: We synthesized a new curcumin-based fluorescent and colorimetric probe (CAC) , and described its application for the specific detection of Cys, including in solution, in living cells, and in a living vertebrate organism. The probe CAC itself showed a strong fluorescence emission, the conjugate addition-cyclization reaction with Cys resulted in the fluorescence quenching at 490 nm and the absorbance decreasing at 400 nm, accompanied by the appearance of a new absorption band at 427 nm. The probe CAC exhibited higher properties such as acting as a “naked eye” probe, readily synthesized, highly selective for Cys, and a lower detection limit of 0.19 µM. In addition, the probe CAC can be successfully applied to detect Cys in living cells and zebrafish with good cell and organism permeable, indicating its significant potential in living biological systems.

Keywords: Fluorescent probe, Cysteine, Curcumin, Living cells imaging, Zebrafish imaging

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1. Introduction Detection of biological thiols (sulfhydryl-containing amino acids) in physiological system has become an active research area nowadays.1,2 Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are the most important intracellular thiols that play significant roles in maintaining biological systems with similar structures and reactivities but different physiological functions.3,4 Among them, Cys involved in catalysis, protein synthesis, metal binding, signal transduction, and metabolism.5,6,7 Cys insufficiency is associated with many human diseases such as retarded growth, edema, hair depigmentation, lethargy, loss of muscle and fat, hematopoiesis reduction and liver damage.8,9 On the other hand, excessive levels of Cys could induce cardiovascular complications, neurotoxicity, Parkinson's disease, Alzheimer's disease, adverse pregnancy outcomes and rheumatoid arthritis.10 Consequently, detecting the Cys in living systems is of considerable significance for better understanding its biological functions as well as accelerating the advancement of diagnosis and therapy means.11 Among the various detection techniques available,12,13 molecular imaging based on fluorescence might be the most attractive approach owing to its operational simplicity, high sensitivity and non-damaging detection of targets in living systems.14,15 To date, quite a few of fluorescent probes for biothiols have been developed that can distinguish biothiols from other amino acids by different sensing mechanisms.16-24 However, the design of a highly selective detection system that can discriminate Cys from Hcy and GSH is seriously hampered owing to their similar structures and reactivities.25,26 Despite this

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challenge, a few fluorescent probes based on organic molecules have been reported recently to be able to detect Cys with improved selectivity over Hcy and GSH. For example, the Yin's group designed an off-on fluorescent probe based on Michael addition reaction for specifically detecting cysteine in living cells.27 Chung and coworkers also designed a Mitochondria-Targeted fluorescent probe based on Michael addition and a subsequent cyclization reaction to improve the selectivity of Cys over Hcy and GSH.28 Moreover, Qin's group synthesized a BODIPY-based fluorescent probe based on reacting with the unsaturated aldehyde for the specific recognition of Cys.29 Nevertheless, most of those seminal probes need complicated synthesis30,31 and have the limitation of the applications in vivo studies.32,33 In addition, the colorimetric probes are widely developed because they have the capability to recognize analytes by naked-eye, without the aid of any advanced instruments.34,35 Therefore, we are interested in developing a Cys specific fluorescent and colorimetric probe with high sensitive, better living cells and organism imaging. In order to complete our design, we use curcumin as the mother molecule due to its eminent optical properties and biocompatibility.36,37 This new probe CAC contains two acrylate groups as the thiol reaction site (Scheme 1). Notably, the probe CAC shows excellent sensing properties such as highly sensitive and selective for detecting Cys over other relevant analytes,

including

Hcy

and

GSH.

Besides,

the

CAC

is

readily

synthesized,

visible-light-excitable, and the colorimetric probe can visual recognize Cys with the naked eye. More importantly, the probe CAC is successfully used for fluorescence imaging of Cys

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in living cells and zebrafish38, indicating its significant potential in living biological systems. 2. Experimental 2.1. Apparatus and Chemicals 1

H and

13

C NMR spectra were obtained on a Bruker DMX-300 spectrometer. The MS

spectra were measured on a Bruker ESQUIRE HPLC-MS AB 4000Q. The UV-Vis absorption spectra were obtained on a U-4100 spectrophotometer. An Edinburgh FS5 spectrofluorometer was used for ﬂuorescence measurements. The PC12 cells fluorescence imaging were record through an Olympus Zeiss 710 laser scanning confocal microscopy. An Olympus MVX10 fluorescence microscopy was used for fluorescence image of zebrafish. The pH value was measured by Jingke PHS-3D digital PH-meter. Curcumin, acryloyl chloride and Hcy were purchased from Energy Chemical. Cys, GSH and amino acids were purchased from Aladdin. The solvents were used as received without further purification. Distilled water was used throughout. 2.2. The synthesis of probe CAC Curcumin (0.184 g, 0.5 mmol), triethylamine (2 mmol) was dissolved in 40 mL of anhydrous dichloromethane. Then acryloyl chloride (2 mmol) was added dropwise at 0 °C. After stirring at this temperature 2 h, the mixture was warmed to room temperature and stirred overnight. The solution was washed with H2O (15 mL × 3) and dried over anhydrous MgSO4. The solvent was removed in rotavapor. The crude product was purified on a silica gel column using dichloromethane/ethanol (v/v, 20:1) as the eluent afforded a yellow solid (0.15g, yield

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63%). Mass spectrometry: m/z, calcd: 476.47, found: 475.6 ([M − H]−). 1H NMR (400 MHz, CDCl3) δ 15.89 (s, 1H), 7.65 (d, J = 15.8 Hz, 2H), 7.24-7.07 (m, 6H), 6.70-6.60 (m, 4H), 6.38 (dd, J = 17.3, 10.5 Hz, 2H), 6.07 (dd, J = 10.4, 1.3 Hz, 2H), 5.89 (s, 1H), 3.90 (s, 6H).

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C

NMR (100 MHz, CDCl3) δ 183.12, 163.88, 151.48, 141.15, 139.95, 134.04, 133.00, 127.38, 124.31, 123.31, 121.09, 111.55, 101.88, 76.85, 55.97. 2.3. General spectra measurements Stock solution (10 mM) of the probe CAC was prepared in C2H5OH. Stock solutions (10 mM) of the biologically relevant analytes were prepared in distilled water. For spectra measurements, test solutions were prepared by placing 10 µM probe stock solution into a solution of phosphate buffered (0.05 M, pH 7.4) aqueous C2H5OH solution (v/v, 1:1). The fluorescence spectra were record after the addition of analytes for 60 min at room temperature (λex = 408 nm, slit width: 3 nm/3 nm). 2.4. Cells and zebrafish imaging PC12 cells (rat adrenal pheochromocytoma cells) were seeded in glass bottom culture dishes and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5 % fetal bovine serum (FBS) and 15 % horse serum at 37 °C with 5 % CO2 atmosphere until harvesting for the experiment. When harvesting, the DMEM was drawn out from the culture dishes, and the dishes were rinsed three times with 10 mM phosphate buffer and then treated with 4 mL trypsinase solution which contain 0.25 % EDTA for 3 min in the incubator. After the samples had been cultured with the probe CAC and different concentrations of analytes in

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phosphate buffer (pH 7.4), the fluorescence images were visualized under the Olympus Zeiss 710 laser scanning confocal microscopy. To test the practical applicability of the probe CAC in a real organism, the wild type Tuebingen (TU) strain of zebrafish (4 day old) were kept at 28 °C and maintained at optimal breeding conditions. The wild type zebrafish larvae were divided into five groups, and then fed with the probe CAC and different concentrations of analytes in phosphate buffer (pH 7.4). After washed with phosphate buffer for three times to remove free probe CAC, the zebrafish imaging were record through the Olympus MVX10 fluorescence microscopy. 2.5. Cytotoxicity assay The methyl thiazolyl tetrazolium (MTT) assay was used to measure the cytotoxicity of probe CAC in PC12 cells. PC12 cells were seeded into a 96-well cell-culture plate. Various concentrations (0, 10, 20, 40, 80 µM) of probe CAC were added to the wells. The cells were incubated at 37 °C under 5% CO2 for 1 h. 10 µL MTT (5mg mL-1) was added to each well and incubated at 37 °C under 5% CO2 for 4 h. Then the culture medium was removed and the cell layer was dissolved in DMSO (100 µL). Thermo Multiskan Ascent microplate reader was used to measure the absorbance at 570 nm for each well. 3. Results and discussion 3.1. Condition experimient During the initial attempt, the CAC probe exhibited high selectivity to Cys. We firstly investigated the influence of the fractions of water on the interaction of CAC probe (10 µM)

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with Cys. Among various fractions of water (0-100%), the combination of phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1) proved to be ideal in the sensing process (Fig. S1). In pure organic media or higher percentages of water, the probe CAC almost no response to Cys. Therefore, we chose phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1) as our test system. Then, we tested the effect of pH (3.0-11.0) on the fluorescence emission intensity of the probe CAC in the absence and presence of Cys. As shown in Fig. S2, the probe CAC exhibited an obvious response to Cys at physiological pH 7.4, which indicated that the probe CAC was highly suitable for biological applications. To acquire a better understanding of the reaction, the time dependent experiment was established under optimal conditions (phosphate buffer-C2H5OH, pH 7.4, v/v, 1:1). Fig. S3 showed the fluorescence intensity at 490 nm was completely quenched at about 10 min, indicating that the probe CAC reacted rapidly with Cys and could be used to detect Cys in real time. 3.2. Selectivity studies of CAC to Cys The selectivity of probe CAC to Cys in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1) solution was tested. The responses of the probe CAC to other relevant analytes including GSH, Hcy, threonine (Thr), Serine (Ser), glutamic acid (Glu), lysine (Lys), phenylalanine (Phe), histidine (His), glycine (Gly), alanine (Ala), valine (Val), tyrosine (Tyr) and hydrogen sulfide (H2S) were carefully tested at the same conditions as Cys. In Fig. 1, the probe CAC (10 µM) itself showed a strong fluorescence emission (ΦF = 0.20). When the addition of Cys (60 µM), the green fluorescence was greatly quenched (ΦF = 0.02) and the fluorescence

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emission peak exhibited a red shift from 490 nm to 555 nm. However, other relevant analytes including Hcy and GSH showed no apparent fluorescence quenching effect. The results indicated that the probe CAC high selectively responded to Cys. Furthermore, the UV-vis absorption spectra also showed the response of the probe CAC to Cys (Fig. 2). The probe CAC (10 µM) in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1) solution showed a strong absorption at 400 nm, whereas the absorption spectra didn’t changed significantly in the presence of different analytes except Cys. Upon the addition of Cys (60 µM), the absorption peak at 400 nm decreased and a new peak appeared at 427 nm. The red shift of the absorption wavelength was also reflected in a change in the colour of the solution from colourless to yellow, implying the probe CAC can be served as a “naked eye” probe toward Cys. As it can be seen in Fig. S4, when increasing concentration of Cys (0-60 µM) was added, the absorption intensity at 400 nm was gradually decreased, and the new absorption peak at 427 nm was gradually increased, which indicated a possible structural change of the probe CAC. 3.3. Linearity The fluorescence titration experiment was further investigated in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1) solution. As shown in Fig. 3, when increasing concentration of Cys (0-20 µM) was added, the fluorescence intensity of CAC dispersed in solution decreased gradually. And the fluorescent quantum yield decreased from 0.20 to 0.02 (See Supplementary Materials). As envisioned, a good linear correlation between the fluorescence

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intensity and the concentration of Cys within the range of 0-20 µM (Fig. S5). The regression equation is Y = 844303 + 32132.8 X (R = 0.9856), which can be used for quantitative determination of Cys. And, we obtained a lower detection limit of 0.19 µM based on 3 × δblank/k (where δblank is the standard deviation of the blank solution and k is the slope of the calibration plot). 3.4. Tolerance of the CAC to Cys over other interferents The competition experiment was carried out to analyze the influence of other analytes on the reaction of probe CAC with Cys. As shown in Fig. 4, the changes of fluorescence emission intensity caused by Cys with various species together such as GSH, Hcy, Thr, Ser, Glu, Lys, Phe, His, Gly, Ala, Val, Tyr and H2S was similar to that caused by Cys alone. The results indicated that the selectivity of probe CAC to Cys was hardly affected in the presence of the other relevant analytes. 3.5. Reaction mechanism study The proposed mechanism of probe CAC with Cys was shown in Scheme 2. In the presence of the Cys, the probe CAC underwent conjugate addition and subsequently followed by cyclization to release the curcumin molecule and generate seven membered-ring cyclic amide.39-42 To further confirm the reaction mechanism, we performed ESI mass spectrometry in the reaction mixture of probe CAC with Cys. As shown in Fig. S6, a peak at m/z 367.3 corresponded to reaction product curcumin ([M − H]− calcd m/z 367.38) was predominantly observed. The peaks at m/z 174.1 corresponded to the seven membered-ring cyclic amide ([M

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− H]− calcd m/z 174.21) were clearly observed. Additionally, the UV-vis absorption spectra (Fig. S7) and fluorescence spectra (Fig. S8) of the reaction product were completely match with that of curcumin in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1). These results strongly indicated that the reaction shown in Scheme 2 took place. 3.6. Applications of the probe CAC The selective sensing and the low detection limit of Cys by probe CAC motivated us to explore the potential biological application of CAC for detecting Cys in PC12 cells. We investigated the cytotoxicity of probe CAC by MTT assay with PC12 cells (Fig. S12, Supporting information). When added various concentrations (10, 20, 40 µM) of probe CAC to the wells, compared with the control group (0 µM), the cell viability didn't declined and had no significant difference after 1 h. However, when added 80 µM of probe CAC to the wells, compared with the control group (0 µM), the cell viability declined and had significant difference. This results indicated that probe CAC (10 µM) we used in this work has low cytotoxicity. As shown in Fig. 5A, the PC12 cells themselves showed no fluorescence. However, when the PC12 cells were incubated with probe CAC (10 µM) for 60 min at 37 °C, a green fluorescence was exhibited inside the cells (Fig. 5B), which indicated the cell permeability of the probe CAC (transduction the cellular membrane). In another control group, the PC12 cells were pretreated with 0.5 mM of N-ethylmaleimide (NEM) for 30 min, which consumed all the free thiols within the cells. It was then incubated with probe CAC (10 µM) for 60 min, the cells exhibited a brighter green fluorescence signal (Fig. 5C) compared with

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Fig. 5B. This proved that the changes of the fluorescence intensity was dependent on the changes in intracellular Cys levels. In another set of experiment, the PC12 cells were pretreated with 100 µM Cys for 30 min followed by incubation with 10 µM probe CAC for another 60 min, the PC12 cells exhibited almost no fluorescence signal in Fig. 5D. These results demonstrated that the probe CAC could detect the Cys in living cells. And the bright-field images indicated that the PC12 cells were viable throughout the confocal microscopic imaging experimental conditions. In order to enlarge the applied range, a further exploratory effort was made to determine if the probe CAC could be used for detecting Cys in live zebrafish. As shown in Fig. 6, the 4-day-old zebrafish themselves showed no fluorescence (Fig. 6A). When the zebrafish were incubated with probe CAC (10 µM) for 60 min at 28 °C and imaged through fluorescence microscopy, the zebrafish gave a green fluorescence (Fig. 6B). Moreover, when the CAC-loaded zebrafish were fed with an increasing concentration of Cys (40 µM, 60 µM, and 80 µM) for 30 min, the fluorescence intensity was gradually decreased (Fig. 6C-6E). These results provided a way to detect the Cys in zebrafish. 4. Conclusion In summary, we have reported a new curcumin-based fluorescent and colorimetric probe CAC for highly selective and sensitive detection of Cys over other relevant analytes including Hcy and GSH. The sensing mechanism was carefully examined and proposed to proceed by the Michael addition and a subsequent cyclization reaction with Cys. The detection limit of

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this probe for Cys was as low as 0.19 µM and bioimaging of Cys by this probe was successfully applied in living cells and zebrafish, indicating that this probe holds great potential for biological applications. Acknowledgments The authors are grateful for the State Key Laboratory of Fine Chemicals (KF1514), National Natural Science Foundation of China (21576071). Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. effect of fraction of water, effects of pH, time courses, additional spectroscopic, the linearity, MS spectra, 1H NMR and 13C NMR spectra, cytotoxicity assay. References 1. Yang, C.; Wang, X.; Shen, L.; Deng, W.; Liu, H.; Ge, S.; Yan, M.; Song, X. An aldehyde group-based P-acid probe for selective fluorescence turn-on sensing of cysteine and homocysteine. Biosens. Bioelectron. 2016, 80, 17. 2. Zhang, H.; Liu, R.; Liu, J.; Li, L.; Wang, P.; Yao, S. Q.; Xu, Z.; Sun, H. A minimalist fluorescent probe for differentiating Cys, Hcy and GSH in live cells. Chem. Sci. 2016, 7, 256. 3. Jung, H. S.; Han, J. H.; Pradhan, T.; Kim, S.; Lee, S. W.; Sessler, J. L.; Kim, T. W.; Kang, C.; Kim, J. S. A cysteine-selective fluorescent probe for the cellular detection of cysteine. Biomaterials 2012, 33, 945.

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31. Hu, Q.; Yu, C.; Xia, X.; Zeng, F.; Wu, S. A fluorescent probe for simultaneous discrimination of GSH and Cys/Hcy in human serum samples via distinctly-separated emissions with independent excitations. Biosens. Bioelectron. 2016, 81, 341. 32. Zhang, J.; Wang, J.; Liu, J.; Ning, L.; Zhu, X.; Yu, B.; Liu, X.; Yao, X.; Zhang, H. Near-infrared and naked-eye fluorescence probe for direct and highly selective detection of cysteine and its application in living cells. Anal. Chem. 2015, 87, 4856. 33. Kong, F.; Liu, R.; Chu, R.; Wang, X.; Xu, K.; Tang, B. A highly sensitive near-infrared fluorescent probe for cysteine and homocysteine in living cells. Chem. Commun. 2013, 49, 9176. 34. Shu, W.; Wang, Y.; Wu, L.; Wang, Z.; Duan, Q.; Gao, Y.; Liu, C.; Zhu, B.; Yan, L. Novel Carbonothioate-Based Colorimetric and Fluorescent Probe for Selective Detection of Mercury Ions. Ind. Eng. Chem. Res. 2016, 55, 8713. 35. Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Fluorescent and colorimetric probes for detection of thiols. Chem. Soc. Rev., 2010, 39, 2120. 36. Yue, Y.; Yin, C.; Huo, F.; Chao, J.; Zhang, Y. The application of natural drug-curcumin in the detection hypochlorous acid of real sample and its bioimaging. Sens. Actuators, B 2014, 202, 551. 37. Rohanizadeh, R.; Deng, Y.; Verron, E. Therapeutic actions of curcumin in bone disorders. Bonekey Rep. 2016, 5, 793.

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Abstract graphic

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Figures

Scheme 1. The design concept of probe CAC for Cys.

Scheme 2. Proposed response mechanism of CAC to Cys.

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Fig. 1. Fluorescence spectra of probe CAC (10 µM, λex = 408 nm) in the presence of different analytes (60 µM) in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1).

Fig. 2. UV-vis absorption spectral changes of probe CAC (10 µM) upon addition of various analytes (60 µM) in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1).

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Fig. 3. Fluorescence spectra of probe CAC (10 µM, λex = 408 nm) upon increased Cys concentrations (0-60 µM) in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1).

Fig. 4. Fluorescence intensities of probe CAC (10 µM, λex = 408 nm) in phosphate buffer-C2H5OH (pH 7.4, v/v, 1:1) solution upon addition of Cys (60 µM) in the presence of various species (60 µM). (Green bar: probe only. Gray bar: probe + Cys + various species.)

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Fig. 5. Confocal ﬂuorescence images of PC12 cells. (A) PC12 cells without incubation with probe CAC. (B) PC12 cells incubated with probe CAC (10 µM) for 60 min. (C) PC12 cells incubated with NEM (0.5 mM) for 30 min, then incubated with probe CAC (10 µM) for 60 min. (D) PC12 cells incubated with Cys (100 µM) for 30 min, then incubated with probe CAC (10 µM) for another 60 min.

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Fig. 6. Fluorescent images of Cys in 4-day-old zebrafish. (A) zebrafish only in bright field and dark field. (B-E) Dark filed of zebrafish fed with various concentrations of Cys (0 µM, 40 µM, 60 µM, and 80 µM), and then with probe CAC (10 µM) for 60 min. All scale bars = 200 µm.

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A curcumin-based fluorescent and colorimetric ... - ACS Publications

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