Biosensors and Bioelectronics 55 (2014) 72–75

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A highly sensitive ratiometric fluorescent probe with a large emission shift for imaging endogenous cysteine in living cells Baocun Zhu n, Bingpeng Guo, Yunzhou Zhao, Bing Zhang, Bin Du School of Resources and Environment, University of Jinan, Shandong Provincial Engineering Technology Research Center for Ecological Carbon Sink and Capture Utilization, Jinan 250022, China

art ic l e i nf o

a b s t r a c t

Article history: Received 8 October 2013 Accepted 25 November 2013 Available online 3 December 2013

A new design strategy for the construction of ratiometric fluorescent probe with a large emission shift was developed. Based on this strategy, a highly selective and sensitive colorimetric and ratiometic fluorescent probe for cysteine (Cys) with a 117 nm red-shifted emission was synthesized and applied to the ratiometric imaging of endogenous Cys in living cells. & 2013 Elsevier B.V. All rights reserved.

Keywords: Ratiometric fluorescent probe Hydroxynaphthalimide Large emission shift Cysteine Bioimaging

1. Introduction Fluorescence imaging employing fluorescent probe is an ideal tool for the in situ visualization of biologically important and toxic species in vivo because of its advantages of high selectivity and sensitivity, non-invasiveness, and good compatibility for various biosamples (Wang et al., 2013; Yang et al., 2013; Li et al., 2013; Guo et al., 2013). Recently, ratiometric fluorescence imaging catches increasing attention as it can eliminate numerous ambiguities resulting from the localization of the probe, changes of environment around the probe (pH, polarity, temperature, and so on), excitation and emission efficiency (Fan et al., 2013; Zhu et al., 2011a; Yuan et al., 2011). Until now, two mechanisms are adopted to construct ratiometric fluorescent probe: internal charge transfer (ICT) and resonance energy transfer (RET) or electronic energy transfer (EET) (Fan et al., 2013). Comparatively, ICT-based ratiometric fluorescent probes are widely developed due to their simple structure and synthesis. But, most of the newly developed ratiometric fluorescent probes lose their capacity in the ratiometric fluorescence imaging for their small emission shift (under 80 nm) (Lin et al., 2008; Xuan et al., 2012; Chen et al., 2012; Zhu et al., 2013). Therefore, novel strategies for the construction of ratometric fluorescent probe with large emission shift (above 80 nm) are in high demand. Recently, we developed a series of carbamate protecting 4-aminonaphthalimide ratiometric fluorescent probes, but these

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Corresponding author. Tel.: +86 531 82769235; fax: +86 531 82765969. E-mail address: [email protected] (B. Zhu).

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probes possess common defect of small emission shift (under 60 nm) (Zhu et al., 2010a, 2010b; Zeng et al., 2011). To achieve ratiometric fluorescent probe with a large emission shift, we for the first time designed a 4-hydroxynaphthalimide-based ratiometric fluorescent probe with a 73 nm emission shift (Zhu et al., 2011b). Based on both design strategies, we reasonably hypothesize that the carbonyl protecting 4-hydroxynaphthalimide should show a larger emission shift than carbamate protecting 4-aminonaphthalimide and alkyl protecting 4-hydroxynaphthalimide did (Scheme 1). Intracellular biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play a crucial role in maintaining the biological systems owing to their participation in reversible redox reactions and important cellular functions, and abnormal levels of these compounds are associated with many diseases (Jung et al., 2013; Zhou and Yoon, 2012; Chen et al., 2010). For example, a decreased level of Cys is associated with slowed growth, hair depigmentation, edema, lethargy, liver damage, muscle and fat loss, skin lesions, and weakness (Jung et al., 2013; Zhou and Yoon, 2012; Chen et al., 2010). So, the determination of biothiols is of great importance for various biochemical investigations as well as the diagnosis of related diseases. Despite advances in the development of fluorescent thiol probes, only one example for the ratiometric detection of Cys with a large (above 80 nm) emission shift has been reported so far, but this probe cannot be used for the ratiometric imaging of Cys in living cells (Guo et al., 2012). Based on the above considerations, an ICT-based ratiometric fluorescent probe with a new design platform of carbonyl protecting 4-hydroxynaphthalimide was designed and synthesized to selectively detect Cys. Cysteine-mediated cleavage of the acrylic acid moiety releases a long-wavelength absorption and fluorescence of

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paper were obtained from commercial suppliers and used without further purification. Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co.) was used for column chromatography. 1H.-NMR was recorded on a Bruker AV-400 spectrometer with chemical shifts reported as ppm (in CDCl3, TMS as internal standard). Electrospray ionization (ESI) mass spectra were measured with an LC–MS 2010A (Shimadzu) instrument. Absorption spectra were recorded on UV-3101PC spectrophotometer. Fluorescence emission spectra were measured on Perkin-Elmer Model LS-55. All pH measurements were made with a Sartorius basic pH-meter PB-10.

2.2. Synthesis of probe 1 Acrylic acid (720.6 mg, 10 mmol) was dissolved in 15 mL anhydrous dichloromethane. Then thionyl chloride (1.1897 g, 10 mmol) was added to this solution while stirring under nitrogen atmosphere. After the mixture was refluxed for 6 h, the resulting mixture was cooled to room temperature. The solution of 4-hydroxy-1,8-naphthalimide (664.6 mg, 2 mmol) in 20 mL CH3CN was added to the above resulting mixture, and subsequently triethylamine (1.0119 g, 10 mmol) was added. And then, the resulting mixture was allowed to stir at room temperature for 18 h. After removal of solvent, the residues were purified by silica gel column chromatography using dichloromethane as eluent to afford pure product. 1H.-NMR (600 MHz, CDCl3) δ (  10  6): 0.984 (t, J ¼7.5 Hz, 3H), 1.434–1.472(m, 2H), 1.692–1.731(m, 2H), 4.181 (t, J¼7.8 Hz, 2H), 6.206(d, J¼ 10.8 Hz, 1H), 6.504(dd, J¼ 10.2 Hz, 10.8 Hz,1H), 6.788(d, J ¼17.4 Hz, 1H), 7.597(d, J ¼8.4 Hz, 1H), 7.764 (t, J¼ 7.8 Hz, 1H), 8.236(d, J ¼7.8 Hz, 1H), 8.605–8.624(m, 2H). ESI-MS calcd for C19H18NO4 [MþH] þ 324.1, found 324.1.

Scheme 1. Our previous work ((a) Zhu et al., 2010b and (b) Zhu et al., 2011b) and new design strategy for the construction of ratiometric fluorescent probe with a large emission shift (c) and a paradigm of Cys-selective probe employing this design strategy (d).

4-hydroxy-1,8-naphthalimide owing to the stronger electron-donor ability of oxygen anions (Jung et al., 2013; Zhou and Yoon, 2012; Chen et al., 2010). Thus, a colorimetric and ratiometric fluorescent probe for Cys with a large emission shift could be achieved.

2. Experimental 2.1. Materials and instrumentations 4-Hydroxy-1,8-naphthalimide was prepared according our previous work (Zhu et al., 2011b). All other chemicals used in this

Fig. 1. Fluorescence responses of 1(5 mM) toward different concentrations of Cys in PBS (20 mM, pH 7.4) solution (ethanol/water¼1:9, v/v). (a) Fluorescence spectra of 1 in the presence of increasing concentrations of Cys (final concentration: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 35 mM), (b) Fluorescence intensity ratio (F547/F430) of 1 versus increasing concentrations of Cys. Each spectrum was obtained after Cys addition at 25 1C for 20 min.

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3. Results and discussion 3.1. Characteristic spectrum Previous studies disclosed that the proper balance between hydrophilicity and lipophilicity of probe is favorable for both cell permeability and intracellular fluorescent imaging (Zhu et al., 2011a). So, the recognition of Cys with probe 1 was investigated under a mixture of ethanol and water (1:9, v/v) solution containing phosphate buffered saline (PBS) (20 mM, pH 7.4). The solution of free probe 1 shows one major absorption peak at around 350 nm and fluorescence emission peak at around 430 nm (Figs. S1 and 1). When Cys (50 μM) was added to the solution of probe 1, the maximum absorption peak showed about a 100 nm red shift and the color of the solution turned from colorless to yellow (Fig. S1), and the maximum emission peak underwent a red shift to 547 nm (Fig. 1). So, compound 1 can serve as a “naked-eye” probe for Cys. The remarkable changes of absorption and fluorescence spectra might be attributed to the cleavage of the opposite electron-withdrawing carbonyl group of acrylic acid moiety and the coinstantaneous production of stronger electron-push capacity of oxygen anion. These spectra of the

reaction solution are in good agreement with ones of 4-hydroxy1,8-naphthalimide reported by us (Zhu et al., 2011b). The results implied that the cleavage of acrylic acid moiety was induced by Cys. To further confirm the interaction mechanism of 1 with Cys, the reaction of probe 1 with Cys was conducted under the same conditions as described above. The green fluorescent reaction product was obtained and characterized to be compound 2 by 1 H -NMR and ESI-MS. Therefore, combined with the previous conclusions by other groups (Jung et al., 2013; Guo et al., 2012; Yang et al., 2011, 2012; Wang et al., 2012), a possible mechanism was proposed as shown in Scheme 1. 3.2. Effects of reaction time on sensing Cys Response time is a fundamental parameter for most reactionbased indicators. Then, the time required for the reaction of probe 1 and Cys at 25 1C was investigated. As shown in Fig. S2, the fluorescence intensity at 430 nm decreases with reaction time, and synchronously the fluorescence intensity at 547 nm increases with reaction time. The ratio of fluorescence intensities (F430/F547) decreases with reaction time and then levels off at reaction time

Fig. 2. Confocal fluorescence images of live RAW 264.7 macrophage cells: the cells incubated with probe 1 (10 μM) for 20 min: (a) bright-field transmission image and (b) ratio image generated from green channel and blue channel; the cells incubated with probe 1 (10 μM) for another 20 min after preincubation with 500 μM NEM for 1 h: (c) bright-field transmission image and (d) ratio image generated from green channel and blue channel. Incubation was performed at 37 1C under a humidified atmosphere containing 5% CO2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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greater than about 10 min. The result showed that the rate of the reaction of probe 1 with Cys is close to those reported by acrylic acid-based fluorescent probes (Jung et al., 2013; Guo et al., 2012; Yang et al., 2011, 2012; Wang et al., 2012). That is to say, our proposed probe could provide a rapid analytical method for the detection of Cys. 3.3. Quantification of Cys The subsequent addition of Cys to the solution of probe 1 resulted in a gradual decrease of fluorescence peak centered at around 430 nm and a progressive increase of fluorescence peak centered at around 547 nm (Fig. 1). In addition, a well-defined isoemission point at 512 nm is also observed (Fig. 1), which implicated that a new species came into being. Moreover, there was a good linearity between the fluorescence intensity ratio (F547/ F430) and the concentrations of sulfite in the range of 0–35 mM with a detection limit of 0.08 mM (Fig. 1). These results demonstrated that probe 1 could detect cysteine qualitatively and quantitatively by ratiometric fluorescence method with excellent sensitivity. 3.4. Selectivity to Cys Then the selectivity of probe 1 toward Cys under the same conditions was evaluated. As expected, nearly no fluorescence intensity changes were observed in the presence of non-thiol amino acids, and negligible fluorescence intensity changes were obtained in the presence of Hcy and GSH (Fig. S3), which is ascribed to the differences of the kinetics of intramolecular adduct/cyclizations of acrylic ester and Cys (Jung et al., 2013; Guo et al., 2012; Yang et al., 2011, 2012; Wang et al., 2012). In addition, the effects of interference of the above-mentioned amino acids on monitoring Cys were investigated (Fig. S3). These results demonstrated that probe 1 possesses high selectivity toward Cys when present with other amino acids. 3.5. Bioimaging and cytotoxicity investigation Next, to further demonstrate the practical application of probe 1, we carried out experiments in living RAW 264.7 macrophage cells. The obtained results revealed that probe 1 can be used for the ratiometric fluorescent imaging of endogenous Cys in living cells (Fig. 2). Moreover, to evaluate cytotoxicity of probe 1, we performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in RAW 264.7 macrophage cells with 5, 10, 25 and 50 mM probe 1 for 1 h, successively. The experiment results exhibited that the absorbances at 490 nm were almost invariable, which were in good agreement with the control experiment. This result implied that our proposed probe was of low toxicity to cultured cells under the experimental conditions at the concentration of 10 mM for 20 min (Fig. S4). 4. Conclusions In conclusion, we have presented the synthesis and properties of an ICT-based ratiometric fluorescent probe 1 for Cys with a new design platform of carbonyl protecting 4-hydroxynaphthalimide. Probe 1 exhibits high Cys-selectivity over various amino acids,

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which is ascribed to the differences of the kinetics of intramolecular adduct/cyclizations. Additionally, probe 1 displays about a 100 nm red-shift of absorption spectra and the color changes from colorless to yellow upon addition of Cys, and thus can serve as a “naked-eye” probe for Cys. Importantly, probe 1 can detect Cys quantitatively by ratiometric fluorescence method with a 117 nm red-shifted emission with excellent sensitivity. We highlight the simplicity of the design and synthesis, yet its combined properties, such as high specificity and sensitivity, visual and ratiometric fluorescent determination with a large red-shifted emission and ratiometric bioimaging in living cells, anticipate that this probe would be of great benefit to biological researchers for investigating the function of Cys in living systems. Our proposed strategy by modulation of the carbonyl protecting 4-hydroxynaphthalimide provides a promising methodology for the design of ratiometric fluorescent probe with a large emission shift.

Acknowledgments We thank the National Natural Science Foundation of China (No. 21107029), National Major Projects on Water Pollution Control and Management Technology (2008ZX07422), and National Science and Technology Major Project (2009ZX07212-003) for financial support.

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A highly sensitive ratiometric fluorescent probe with a large emission shift for imaging endogenous cysteine in living cells.

A new design strategy for the construction of ratiometric fluorescent probe with a large emission shift was developed. Based on this strategy, a highl...
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