Novel NIR fluorescent probe with dual models for sensitively and selectively monitoring and imaging Cys in living cells and mice
Accepted Manuscript Title: Novel NIR fluorescent probe with dual models for sensitively and selectively monitoring and imaging Cys in living cells and...
Accepted Manuscript Title: Novel NIR fluorescent probe with dual models for sensitively and selectively monitoring and imaging Cys in living cells and mice Authors: Qianqian Wang, Hong Wang, Jinxin Huang, Nan Li, Yueqing Gu, Peng Wang PII: DOI: Reference:
Please cite this article as: Qianqian Wang, Hong Wang, Jinxin Huang, Nan Li, Yueqing Gu, Peng Wang, Novel NIR fluorescent probe with dual models for sensitively and selectively monitoring and imaging Cys in living cells and mice, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.166 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.
Novel NIR fluorescent probe with dual models for sensitively and selectively monitoring and imaging Cys in living cells and mice
Qianqian Wang,a,1 Hong Wang,b,1 Jinxin Huang,a Nan Li,a Yueqing Gu*a, Peng Wang*a
a
Department of Biomedical Engineering, School of Engineering, China
Pharmaceutical University, Nanjing 210009, China b
State Key Laboratory of Pathogen and Biosecurity, Department of Pharmacy, Beijing
Institute of Microbiology and Epidemiology, Beijing, 100071, China *
A novel NIR fluorescent probe with dual models for detection of Cys was designed and synthesized.
Fast response to Cys with obvious turn-on fluorescence signal at the low concentration range of Cys.
Providing a ratiometric fluorescence response to the high concentration range of Cys.
Detection limit of Cys in PBS was found to be 9.1 × 10-8 M.
Imaging Cys in living cells and mice with low cytotoxicity.
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Abstract Cys plays paramount roles in human physiologies and pathologies. Abnormal levels of Cys are linked to a variety of diseases. Near-infrared fluorescence probe capable of detecting intracellular Cys in vivo would be beneficial to the study of mechanisms of certain diseases. Herein, we reported a rational and novel dual-site fluorescence probe JC-2 responded to Cys, which enjoyed different modes of fluorescence signals towards different concentration ranges of Cys. The probe was cell- membranepermeable and could selectively detect Cys over other species. The potential biological utility of JC-2 was based on the fluorescence imaging of Cys in living cells and animals. The combined results elucidated the promising application of JC-2 in pathological analysis for diseases related to Cys.
1. Introduction Small molecular biological thiols like cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play crucial roles in a multitude of physiological and pathological processes, for instance, maintaining biological redox homeostasis and involvement in the intracellular signal transduction[1].However, much of the researches in biothiols have suggested that abnormal levels of these biothiols are associated with certain diseases[2]. Normal level of Cys in human metabolism is 30−200 μM, which acts as a source of protein synthesis and a precursor of GSH. A deficiency or excess amounts of Cys are linked to edema, liver damage, skin lesions, Parkinson’s disease, Alzheimer’s disease and slow growth[3, 4]; elevated Hcy in plasma gives rise to cardiovascular disease and osteoporosis[5]; and abnormal levels of GSH is a risk factor for HIV infection, leucocyte loss and cancer[6]. Thus, the paramount roles of these biological thiols have spurred strong interest in methods for determination of them. Over the past several decades, a substantial effort has been devoted to 2
developing an efficient strategy for the detection of biothiols, including UV–vis absorption spectrophotometry[7], mass spectrometry (MS)[8], high-performance liquid chromatography (HPLC)[9, 10], and fluorescent probe[11-16]. Among the available analytical methods, fluorescent probe is considered as a desirable tool for detection of biothiols owing to its non-invasiveness, real-time detection, sensitivity and simplicity. An ocean of fluorescent probes have been developed to distinguish biothiols from other amino acids, cations and anions, utilizing the nucleophilic addition and substitution reactions [17-25]. However, to our knowledge, it is still a significant challenge to simultaneously discriminate between Cys/Hcy and GSH due to their similar structure and reaction, let alone the detection of abnormal levels of these biothiols. Our group have successfully developed a novel fluorescence probe (JC-2) for selective and sensitive detection of Cys over Hcy and GSH. The rational design of JC-2 contained two reaction sites for the target of interest, which could show different modes of fluorescent signal towards the high and low concentration range of Cys. Furthermore, JC-2 was applicable for detection in living cells due to its good cell membrane permeability and low cytotoxicity. More importantly, JC-2 possessed the properties of near-infrared fluorescence which would have advantages in live animals imaging owing to the deep tissue penetration of light in this region.
2. Experimental section 2.1 General All solvents and other reagents were of commercial quality and used without further purification. 1H-NMR and 13C-NMR spectra were taken on Bruker Advance 300-MHz spectrometer, δ values are in ppm relative to TMS. Mass data (ESI) were recorded by quadruple mass spectrometry. Microspectrophotometer (One drop, Nanjing, China) was used for the absorption measurements. PerkinElmer LS55 was utilized for fluorescence spectra detection. Laser confocal fluorescence microscopy (FluoViewTM, FV1000, Olympus, Japan) was used for cell imaging. Caliper IVIS Lumina II was used for the animal imaging. 3
2.2 Synthesis of JC-1 BBr3 (1.0 g) was added to a solution of compound (E)-2-(2-(1-chloro-6methoxy-3,4-dihydronaphthalen-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide (505 mg, 1.0 mmol) in dry dichloromethane (20 mL) under ice bath and stirred for 12 h at N2 atmosphere. The mixture was poured onto 100 g of crushed iced and extracted with dichloromethane (3 × 200 mL). The organic layers were collected and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel flash chromatography using CH2Cl2/MeOH (20:1) to give the compound JC-1 as a red solid (Yield 75%). 1H NMR (300 MHz, d6-DMSO) δ 10.49 (s, 1H), 8.46 (d, J = 15.8 Hz, 1H), 7.90-7.87 (m, 2H), 7.69 (d, J = 8.3 Hz, 1H), 7.64-7.61 (m, 2H), 7.16 (d, J = 15.8 Hz, 1H), 6.83 -6.80 (m, 2H), 4.09 (s, 3H), 2.91 (s, 4H), 1.75 (s, 6H). 13C NMR (75 MHz, d6-DMSO) δ 180.78, 161.19, 147.53, 143.15, 142.67, 141.88, 141.57, 129.37, 129.13, 129.00, 128.79, 123.30, 122.78, 115.01, 114.89, 114.45, 112.86, 51.69, 34.23, 26.61, 26.10, 23.35. MS (ESI) m/z = 364.1 [M]+. 2.3 Synthesis of JC-2 Triethylamine (101 mg, 1.0 mmol) was added to the solution of compound JC-1 (246 mg 0.5 mmol) in dry CH2Cl2 (20 mL). Then acryloyl chloride (108 mg, 0.6 mmol) was dropped to the mixture and stirred at room temperature for 4 h. The solvents were removed under rotary evaporator and the rude product was purified by silica gel column chromatography using CH2Cl2/MeOH (30:1) to afford compound JC-2 as red solid (Yield 83%). 1H NMR (300 MHz, d6-DMSO) δ 10.49 (s, 1H), 8.44 (d, J = 16.1 Hz, 1H), 7.96-7.86 (m, 3H), 7.68-7.65 (m, 2H), 7.34-7.24 (m, 3H), 6.606.39 (m, 2H), 6.20 (m, 1H), 4.15 (s, 3H), 2.97 (s, 4H), 1.77 (s, 6H). 13C NMR (75 MHz, d6-DMSO) δ 181.17, 152.15, 146.66, 143.42, 141.86, 140.43, 139.82, 134.11, 132.05, 129.63, 129.46, 129.08, 128.03, 127.31, 122.87, 121.14, 120.61, 115.44, 115.25, 52.06, 34.67, 26.09, 25.79, 23.26. MS (ESI) m/z = 418.2 [M]+. 2.4 Cytotoxic assay MCF-7 cells were seeded in a 96-well plate (1×104 cells/well). After cultivation for 24 h, JC-2 (DMSO dissolve first, then added it into the cell culture medium) of 4
different concentrations were added into the wells (n = 6) and incubated for 24 h. Then stock solution of MTT (20 μL; 5 mg/mL) was added into each well. After 4 h incubation at 37 oC, the MTT solution was replaced with 150 μL DMSO in each well. The absorbance in each well was measured at 570 nm with a multi-well plate reader. Cell viability was calculated using the following formula: Cell viability = (Mean absorbance of test wells - Mean absorbance of medium control wells) / (Mean absorbance of untreated wells - Mean absorbance of medium control well) × 100%. 2.5 Cell culture and confocal fluorescence imaging The human cell line MCF-7 (human breast adenocarcinoma cell line) was purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 μg/mL penicillin and 100 μg/mL streptomycin at 37 oC in a humidified atmosphere containing 5% CO2. One day before imaging, cells were seeded in laser scanning confocal microscope (LSCM) culture dishes with a density of 5×105 cells per well. The dishes were subsequently incubated at 37 oC in a humidified atmosphere containing 5% CO2. Then the cells were only incubated with 20 µM JC-2 for 20 min as control. For imaging different concentration ranges of Cys in living cells, the cells were incubated with Cys (500 µM) for 20 min, and then treated with the probe JC-2 for 20 min. For the thiol blocking experiment, the cells are incubated with N-ethylmaleimide (NEM, 2 mM) for 60 min, and then co-incubated with probe JC-2 for 20 min. The cells were washed three times with Dulbecco's PBS (pH 7.0) to remove free compound before analysis. Confocal luminescence images of MCF-7 cells were carried out on an Olympus FV1000 laser scanning confocal microscope. 2.6 Fluorescent imaging in living mice Kuming mice were purchased from Charles River Laboratories (Shanghai, China) for in vivo imaging investigation. All animal experiments were carried out in compliance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China (Document no. 55, 2001) and the guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University. The Kunming mice were divided into three groups. The first group was given an intraperitoneal 5
(i.p.) injection of saline (100 μL), followed by JC-2 (20 μM, in 20 μL DMSO). The second group was given an i.p. injection of N-ethylmaleimide (1 mM, in 100 μL saline), and followed by JC-2 (20 μM, in 20 μL DMSO) as the negative control experiment. The third group was given an intraperitoneal injection of Cys (100 μM, in 100 μL saline), and followed by JC-2 (20 μM, in 20 μL DMSO). Images were taken by using the Caliper IVIS Lumina II fluorescence imaging system. With an excitation filter of 570 nm and the orange and red channels are corresponding to the emission windows of 580-640 nm, and 650-750 nm, respectively.
3. Results and discussion 3.1 Synthesis of probe JC-2 The synthetic route of probe JC-2 is shown in Scheme 1. Compound JC-1 was easily achieved by previous reports [26]. Then JC-1 was treated with acryloyl chloride in the presence of triethanolamine and dichloromethane to afford probe JC-2. The chemical structure of probe JC-2 was identified by using NMR and mass spectrometry.
3.2 Spectral Properties of JC-2 The UV-vis properties of JC-2 was measured in the PBS (pH 7.4, 10 mM). Probe JC-2 displayed a wide absorption peak around 380 nm (Figure S1). After reacting with different concentrations of Cys (10 μM and 100 μM) for 30 min at 37 oC, the UV-vis spectrum of probe JC-2 had obvious changes. The maximum absorption peak increased prominently and was bathochromic shift to 540 nm. 3.3 Spectral response of JC-2 to low and high concentration ranges of Cys The response capacity of JC-2 to low concentration of Cys was as expected (Figure 1a). JC-2 enjoyed almost no fluorescence signals at 680nm while a gradual increase of intensity was observed with the Cys elevation. When the concentration reached 5 μM, the intensity was even 12 folds than that in the absence of Cys. A good 6
linear correlation was observed from Figure 1a, the limit of detection (LOD) was calculated to be 9.1 × 10-8 M (S/N = 3) according to the previous literatures. The regression equation was Y=22.026x + 6.7125 × [Cys] with a linear coefficient R2 of 0.9914. In the presence of high concentration of Cys (5-200 μM), a new emission band centered at 625nm was recorded (Figure 1b). The linear correlation was relatively favorable within the range of 5-50 μM. JC-2 depicted a turn-on mode of fluorescence signals upon the addition of low concentration of Cys and elicited a ratiometric mode response towards the high concentration range of Cys (Figure S2). These results were suggestive of the ability of JC-2 to be an efficient tool for rapid quantitative analysis of Cys.
3.3 Kinetic data The time-dependent changes of the fluorescence intensity of JC-2 is shown in Figure 2. The detection process of probe JC-2 toward low concentration of Cys balanced within 10 minutes at 680nm. Meanwhile, the reaction of JC-2 with high concentration of Cys completed within 20 minutes at 625nm. The difference between the two reacting sites was indicative of the application of JC-2 in the detection of the concentration range of Cys.
3.4 pH - dependent fluorescence response to Cys To investigate the effects of pH on the fluorescent response of JC-2 to Cys, the fluorescence changes of JC-2 induced by Cys was measured from pH 4 to 10 (Figure S3).The fluorescence intensity of JC-2 had little changes at 625nm and 680nm, while upon the addition of Cys (JC-1), the emission at 680nm increased dramatically in neutral and slightly alkaline media. In other words, JC-2 is suitable for detecting Cys in the physiological pH region. 7
3.5 Selectivity experiments To evaluate the capability of selective detection of probe JC-2, other biothiols (Hcy and GSH), amino acids, metal ions, reactive nitrogen species and reactive oxygen species (ROS) were treated with JC-2 under simulated physical conditions (37 oC, pH 7.4). As shown in Figure 3, no obvious change in fluorescence intensity was observed with the addition of other species while a significant fluorescence turn on emission appeared within the addition of Cys, which showed the excellent selectivity of JC-2 towards Cys over other species.
3.6 Study on Reaction Mechanism Proposed detection mechanism of probe JC-2 to high and low concentration of Cys are shown in Scheme 2. The acrylate group in JC-2 was used as an efficient reaction site for the low concentration range of Cys. the mechanism was based on conjugated addition of Cys to terminal acrylate group, which formed the intermediate thioether. The intramolecular cyclization generated the desired lactam and compound JC-1[27]. The chloro group was chosen as the low-sensitivity site for its relative low sensitivity to biothiols[28]. It is universally acknowledged that once the chloro moiety of the cyanine substituted by the heteroatoms, the ratiometric mode in fluorescence would be induced[29]. The chloro group in JC-1 was first replaced by the sulfur group of Cys to form the 4-sulfhydryl intermediate. Subsequently, the intramolecular SNAr substitution of the sulfhydryl moiety produced the 4-amino product JC-1-Cys. The mass spectrometry analyses confirmed the formation of JC-1 and JC-1-Cys (Figure S4 and S5).
3.7 MTT assay The cytotoxicity of JC-2 was evaluated via a cell viability assay conventionally. 8
Different concentrations of JC-2 from 0 to 50 μM was incubated with the MCF-7 cells (Figure S6). The cells maintained a high viability (near 90%) even in the presence of 50 μM probe, demonstrating the great potential of JC-2 in terms of its low cytotoxicity. 3.8 Fluorescence imaging in living cells To investigate whether JC-2 can selectively detect Cys in living cells, cell-based experiments using confocal fluorescence microscope were performed. As shown in Figure 4, bright-field measurements manifested the viability of cells under the experimental environment. NEM as the thiol-blocking reagent acted to reduce the concentration of Cys in the control experiments. The cells treated only with the probe JC-2 exhibited strong fluorescence signals in red channel, when the cells incubated with NEM and then co-incubated with the probe, almost no fluorescence signal was observed. When MCF-7 cells were incubated with the probe and 5 μM Cys, orange fluorescence emission were observed inside the cells. A markedly higher fluorescence intensity increase took place when the cells were pretreated with 500 μM Cys. These results implied that the probe showed a turn on mode of fluorescence signals towards low concentration range of Cys and a ratiometric mode of fluorescence signals under the condition of high concentration of Cys. Thus, we harbor the idea that JC-2 will be a feasible probe for detection of abnormal levels of Cys.
3.9 Imaging of JC-2 to different concentration ranges of Cys in live animals JC-2 featured a prominent NIR photophysical properties and high sensitivity and selectivity to Cys with dual sites. These favorable characteristics encouraged us to further study the utility of JC-2 in the imaging of different concentrations of Cys in living animals. Three groups of mice were chosen to carry on the experiments. Saline and JC-2 (20 μM, in 20 μL DMSO) were injected into the peritoneal cavity of one group. The second group were given an i.p. injection of NME and JC-2 as the negative group. The third group were injected with Cys and probe JC-2 in the similar 9
way. As shown in Figure 5, the fluorescence (pseudo-color) of the mice treated only with the probe was higher than that in the addition of NME in red channel. In addition, the fluorescence (pseudo-color) of the mice treated only with the probe was less than that in the presence of Cys in the orange channel. Consequently, JC-2 enjoyed a turn on mode of fluorescence signals towards the low levels of Cys (Figure 5d, 5e and 5g) and a ratiometric mode of fluorescence signal towards the high levels of Cys, which was consistent with the results discussed ahead. Thus, we take the attitude that this novel probe JC-2 will have a pleasurable application in the imaging of abnormal levels of Cys in the living animals.
4. Conclusion In this study, we have successfully developed a novel fluorescence probe JC-2, which could undergo remarkably selective detections of Cys in controlled aqueous solutions or living cells. The probe intrinsically beared two reaction sites with ratiometric fluorescent responses, which enabled the probe to monitor the alternation of Cys. Cellular experiments and imaging in living animals demonstrated that the probe would have a promising prospect in biological and clinical investigations.
Author Contributions 1
Q. Wang and H. Wang contributed equally.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NSFC 81501529 and 81220108012), the 973 Key Project (2015CB755504), and National Found for Fostering Talents of Basic Science (NFFTBS, No. J1030830 and J1310032). 10
Biographies Qianqian Wang She is studying for bachelor degree in School of Pharmacy at China Pharmaceutical University. Hong Wang She obtained her Doctor Degree in Pharmacology from Beijing Institute of Microbiology and Epidemiology in 2010. Now she is a researcher in department of Pharmacy at Beijing Institute of Microbiology and Epidemiology. Jinxin Huang He obtained his bachelor degree in biology from Zhejiang University of Technology in 2015. Now he is studying for master in School of Engineering at China Pharmaceutical University. Her current research is fluorescent probe. Nan Li She is studying for bachelor degree in School of Pharmacy at China Pharmaceutical University. Yueqing Gu She obtained her Doctor Degree in physics from Nanjing University of Aeronautics and Astronautics. Now she is a professor in School of Engineering at China Pharmaceutical University major in biomedical engineering. Her current research interests are molecular imaging and nanomedicine. Peng Wang He obtained his Doctor Degree in biomedical engineering from Southeast University in 2014. Now he is a lecturer in School of Engineering at China Pharmaceutical University. His current research interests are biosensor and molecular imaging.
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Reference [1] Z.A. Wood, E. Schroder, J. Robin Harris, L.B. Poole, Structure, mechanism and regulation of peroxiredoxins, Trends Biochem Sci, 28(2003) 32-40. [2] L.A. Herzenberg, S.C. De Rosa, J.G. Dubs, M. Roederer, M.T. Anderson, S.W. Ela, et al., Glutathione deficiency is associated with impaired survival in HIV disease, Proc Natl Acad Sci USA, 94(1997) 1967-72. [3] H.S. Jung, J.H. Han, T. Pradhan, S. Kim, S.W. Lee, J.L. Sessler, et al., A cysteineselective fluorescent probe for the cellular detection of cysteine, Biomaterials, 33(2012) 945-53. [4] H.S. Jung, T. Pradhan, J.H. Han, K.J. Heo, J.H. Lee, C. Kang, et al., Molecular modulated cysteine-selective fluorescent probe, Biomaterials, 33(2012) 8495-502. [5] Y.L. Li, S.Z. Wu, Relationship between Serum Hcy Levels and Arterial Stiffness in Elderly Patients with Hypertension in the High Altitude, J Am Geriatr Soc, 64(2016) S351. [6] F.P. Kong, Z.Y. Liang, D.R. Luan, X.J. Liu, K.H. Xu, B. Tang, A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging, Anal Chem, 88(2016) 6450-6. [7] L.S. Hu, S.Q. Hu, L.Y. Guo, T. Tang, M.H. Yang, Optical and electrochemical detection of biothiols based on aggregation of silver nanoparticles, Anal Methods, 8(2016) 4903-7. [8] B. Seiwert, U. Karst, Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides, Anal Chem, 79(2007) 7131-8. [9] O. Nekrassova, N.S. Lawrence, R.G. Compton, Analytical determination of homocysteine: a review, Talanta, 60(2003) 1085-95. [10] Y.V. Tcherkas, L.A. Kartsova, I.N. Krasnova, Analysis of amino acids in human serum by isocratic reverse-phase high-performance liquid chromatography with electrochemical detection, J Chromatogr A, 913(2001) 303-8. [11] Q.H. Hu, C.M. Yu, X.T. Xia, F. Zeng, S.Z. Wu, A fluorescent probe for 12
simultaneous discrimination of GSH and Cys/Hcy in human serum samples via distinctly-separated emissions with independent excitations, Biosens Bioelectron, 81(2016) 341-8. [12] J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, et al., Cyanine-based fluorescent probe for highly selective detection of glutathione in cell cultures and live mouse tissues, J Am Chem Soc, 136(2014) 5351-8. [13] Y. Yue, F. Huo, P. Ning, Y. Zhang, J. Chao, X. Meng, et al., Dual-Site Fluorescent Probe for Visualizing the Metabolism of Cys in Living Cells, J Am Chem Soc, (2017). [14] H.T. Zhang, R.C. Liu, J. Liu, L. Li, P. Wang, S.Q. Yao, et al., A minimalist fluorescent probe for differentiating Cys, Hcy and GSH in live cells, Chem Sci, 7(2016) 256-60. [15] P. Wang, Q.Q. Wang, J.X. Huang, N. Li, Y.Q. Gu, A dual-site fluorescent probe for direct and highly selective detection of cysteine and its application in living cells, Biosens Bioelectron, 92(2017) 583-8. [16] P. Wang, Y. Wang, N. Li, J.X. Huang, Q.Q. Wang, Y.Q. Gu, A novel DCM-NBD conjugate fluorescent probe for discrimination of Cys/Hcy from GSH and its bioimaging applications in living cells and animals, Sensor Actuat B-Chem, 245(2017) 297-304. [17] X. Chen, Y. Zhou, X. Peng, J. Yoon, Fluorescent and colorimetric probes for detection of thiols, Chem Soc Rev, 39(2010) 2120-35. [18] H.S. Jung, X. Chen, J.S. Kim, J. Yoon, Recent progress in luminescent and colorimetric chemosensors for detection of thiols, Chem Soc Rev, 42(2013) 6019-31. [19] S.H. Xue, S.S. Ding, Q.S. Zhai, H.Y. Zhang, G.Q. Feng, A readily available colorimetric and near-infrared fluorescent turn-on probe for rapid and selective detection of cysteine in living cells, Biosens Bioelectron, 68(2015) 316-21. [20] S.S. Ding, G.Q. Feng, Smart probe for rapid and simultaneous detection and discrimination of hydrogen sulfide, cysteine/homocysteine, and glutathione, Sensor Actuat B-Chem, 235(2016) 691-7. [21] Q. Zhang, D.H. Yu, S.S. Ding, G.Q. Feng, A low dose, highly selective and sensitive colorimetric and fluorescent probe for biothiols and its application in 13
bioimaging, Chem Commun, 50(2014) 14002-5. [22] X.F. Yang, Y.X. Guo, R.M. Strongin, Conjugate Addition/Cyclization Sequence Enables Selective and Simultaneous Fluorescence Detection of Cysteine and Homocysteine, Angew Chem Int Edit, 50(2011) 10690-3. [23] Z.Q. Guo, S. Nam, S. Park, J. Yoon, A highly selective ratiometric near-infrared fluorescent cyanine sensor for cysteine with remarkable shift and its application in bioimaging, Chem Sci, 3(2012) 2760-5. [24] Y. Liu, D.H. Yu, S.S. Ding, Q. Xiao, J. Guo, G.Q. Feng, Rapid and Ratiometric Fluorescent Detection of Cysteine with High Selectivity and Sensitivity by a Simple and Readily Available Probe, ACS Appl Mater Interfaces, 6(2014) 17543-50. [25] H.Y. Zhang, W.Y. Feng, G.Q. Feng, A simple and readily available fluorescent turn-on probe for cysteine detection and bioimaging in living cells, Dyes Pigm, 139(2017) 73-8. [26] H. Chen, Y.H. Tang, M.G. Ren, W.Y. Lin, Single near-infrared fluorescent probe with high- and low-sensitivity sites for sensing different concentration ranges of biological thiols with distinct modes of fluorescence signals, Chem Sci, 7(2016) 1896-903. [27] G.L. Khatik, R. Kumar, A.K. Chakraborti, Catalyst-free conjugated addition of thiols to alpha,beta-unsaturated carbonyl compounds in water, Org Lett, 8(2006) 2432-6. [28] L.Y. Niu, Y.S. Guan, Y.Z. Chen, L.Z. Wu, C.H. Tung, Q.Z. Yang, BODIPY-Based Ratiometric Fluorescent Sensor for Highly Selective Detection of Glutathione over Cysteine and Homocysteine, J Am Chem Soc, 134(2012) 18928-31. [29] X.J. Peng, F.L. Song, E. Lu, Y.N. Wang, W. Zhou, J.L. Fan, et al., Heptamethine cyanine dyes with a large stokes shift and strong fluorescence: A paradigm for excited-state intramolecular charge transfer, J Am Chem Soc, 127(2005) 4170-1.
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Figure 1. (a) Fluorescence response of JC-2 upon addition of low concentration of Cys (0-5 μM). Each spectrum was recorded upon the excitation at 540 nm. Insert: Working curve of JC-2 to detect Cys obtained by addition of various concentrations of Cys (0-5 μM). (b) Fluorescence response of JC-2 upon addition of low concentration of Cys (5-200 μM). Each spectrum was recorded upon the excitation at 540 nm. Insert: Working curve of JC-2 to detect Cys obtained by addition of various concentrations of Cys (5-200 μM)
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Figure 2. (a) Time-dependent fluorescent emission of JC-2 (5 μM) and Cys (5 μM) at 680 nm. (b) Time-dependent fluorescent emission of JC-2 (5 μM) and Cys (200 μM) at 625 nm.
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Figure 3. Fluorescence intensity of JC-2 (5 μM) as determined in aqueous solution after the addition of various amino acids, metals, ROS, reducing agent, glucose, and Cys (200 μM). The spectra were recorded after incubated with JC-2 for 30 min. Emission at 680 nm (a) and 625 nm (b).
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Figure 4. Confocal microscope images of MCF-7 cells stained with JC-2. (a–d) bright-field and fluorescence images of the cells incubated only with the probe. (e-h) bright-field and fluorescence images of cells incubated with 5 μM Cys for 20 min, and then treated with the probe for 20 min. (i–l) bright-field and fluorescence images of cells incubated with 500 μM Cys for 20 min, and then treated with the probe for 20 min. (m-p) bright-field and fluorescence images of cells incubated with Nethylmaleimide, and then co-incubated with the probe for 20 min. The orange and red channels are corresponding to the emission windows of 580–640 nm, and 650–750 nm respectively.
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Figure 5. Fluorescent images (pseudo-color) of Cys in the mice: (a) and (d) the mice were given an i.p. injection of NME and JC-2; (b) and (e) only the probe was injected in the peritoneal cavity of the mice; (c) and (f) the mice were given an i.p. injection of Cys and JC-2. (g) the fluorescence emission intensity in the abdominal area of the each mouse; (h) average Forange/Fred intensity ratios of the only probe group and probe + Cys group. The mice were imaged with an excitation filter of 570 nm and the orange and red channels are corresponding to the emission windows of 580–640 nm and 650–750 nm respectively.
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Scheme 1. Synthetic route of probe JC-2
Scheme 2. Proposed detection mechanism of probe JC-2 to high and low concentration of Cys
