Biosensors and Bioelectronics 71 (2015) 68–74
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A colorimetric and fluorescent probe for detecting intracellular GSH Chunyang Chen, Wei Liu, Cong Xu, Weisheng Liu n Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 25 December 2014 Received in revised form 16 March 2015 Accepted 5 April 2015 Available online 8 April 2015
A new rapid and highly sensitive coumarin-based probe (probe 1) has been designed and synthesized for detecting intracellular GSH. Probe 1 was prepared from 4-methylumbelliferone using a 3-step procedure. It was converted into a latent fluorescence probe, which allowed it to achieve high sensitivity (LOD 122 nM) and fluorescence turn-on response (F/F0 415) toward GSH over other various natural amino acids in PBS buffer solution at pH 7.4. Owing to a specific Michael addition between thiols and the double bond of nitroolefin moiety, probe 1 displayed a high selectivity toward GSH. Afterwards, probe 1 was successfully used for fluorescence imaging of GSH in Hela and Hek-293a cells, and a rapid response was observed in the Hek-293a cells after incubating with probe 1 for 1 min. Simultaneously, a quantitative determination was achieved in Hela cells in the range of 8–48 μM. Specific fluorescence imaging of plants tissue was obtained for proving the permeability of probe 1. Finally, the viability was measured to be more than 80%, which shows probe 1 can serve as a rapid and biocompatible probe for GSH in cells. & 2015 Elsevier B.V. All rights reserved.
Keywords: Coumarin Fluorescent Glutathione Colorimetric Probe Bioimaging
1. Introduction Intracellular thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a pivotal role in many physiological processes, including redox homeostasis and cellular growth (Ball et al., 2006; Hong et al., 2006; Krauth-Siegel et al., 2005; Schulz et al., 2000). Alternations in the level of cellular thiols have been involved in the development of a number of diseases. A low level of GSH is a dangerous sign of various syndromes, including slow growth in children, hair depigmentation, lethargy, psoriasis, liver damage, substance abuse, muscle and fat loss, weakness and edema (Ibrahim, 2012; Njalsson and Norgren, 2005; Shahrokhian, 2001; Townsend et al., 2003; Uys et al., 2014). An elevated level of GSH in human plasma is also a risk factor for cardiovascular disease, Alzheimer’s disease, neural tube defects, inflammatory bowel disease, osteoporosis, cancer, and AIDS (Herzenberg et al., 1997; Lasierra-Cirujeda et al., 2013; Rossoni et al., 2010; Zhang et al., 2014; Zhao et al., 2006). Thus, a rapid and high-efficiency method for monitoring these diseases is essential. Consequently, the development of efficient methods for the recognition and quantification of special mercapto biomolecules in physiological media is of immense scientific interest and importance for those in the field of biological chemistry. To date, many strategies have been developed for detecting n
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http://dx.doi.org/10.1016/j.bios.2015.04.016 0956-5663/& 2015 Elsevier B.V. All rights reserved.
thiols include mass spectrometry (MacCoss et al., 1999; Vellasco et al., 2002), high performance liquid chromatography (HPLC) (Ivanov et al., 2000; Michaelsen et al., 2009; Tcherkas and Denisenko, 2001), capillary electrophoresis (Chen et al., 2004; Inoue and Kirchhoff, 2002), optical assays (Bragin et al., 2010; Jung et al., 2013; Lim et al., 2013; Yin et al., 2013; Yoshida et al., 2014), and the electrochemical method (Wang et al., 2008). Most of these methods require complicated and expensive instruments and cumbersome preconditioning procedures such as the separation and purification of sample prior to the implementation of formal analysis. Furthermore, few of these methods can be applied in intracellular detection due to their limitations in vivo studies. Among the various strategies, optical assays based on synthetic colorimetric and fluorescent probes have received continuous attention since they are simple, sensitive, efficient, and can be used for detecting intracellular mercapto biomolecules. In the past few years, a variety of colorimetric and fluorescent thiol probes have been constructed based on different mechanisms. These probes exploit the high nucleophilic reactivity or transition metal-affinity of the thiol group, including thiol cleavage reaction (Lee et al., 2012; Long et al., 2011; Wang et al., 2014), cyclization reaction with aldehyde (Lim et al., 2010; Liu et al., 2012; Yuan et al., 2011), Michael addition (Chen et al., 2010a; Guo et al., 2009; Hong et al., 2009; Kwon et al., 2011; Matsumoto et al., 2007; Sreejith et al., 2008), metal complexes-displace coordination and others (Chen et al., 2010b; Hong et al., 2013; Jung et al., 2011; Liu et al., 2010). Among the various strategies, Michael addition
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probes have been most actively developed in recent years. A number of excellent Michael acceptors have been exploited such as maleimide (Guy et al., 2007), squaraine (Sreejith et al., 2008), quinone (Zeng et al., 2008), chromene (Chen et al., 2010a), acrylic acid (Lee et al., 2011), as well as α,β-unsaturated aldehyde (Yuan et al., 2011), ketone (Lim and Kim, 2011), diesters (Jung et al., 2013) and malonitrile (Hoyle et al., 2010; Kwon et al., 2011). Coumarin was selected as the fluorophore due to its desirable photophysical properties, such as a large Stokes’ shift, visible emission wavelengths, and high fluorescence quantum yields (Lim and Bruckner, 2004; Lim et al., 2005; Trenor et al., 2004). Nitroolefin was chosen among the various Michael acceptors due to its strong electron deficiency; it serves as not only an electrophile but also a quencher of the coumarin fluorophore. After adding GSH, the nitroolefin moiety acts as an electron acceptor for the photoinduced electron transfer (PET) process, leading to a low fluorescence of probe 1. After treatment with GSH, the double bond between the fluorophore and the quencher is broken, and blocking of the PET process can cause fluorescence recovery. Moreover, the reaction between GSH and nitroolefin inhibits the weak intramolecular charge transfer (ICT) from the 4-Methylumbelliferone group to the electron-poor nitroolefin moiety, producing a hypochromic shift in the fluorescence spectra of the probe upon addition of GSH (Scheme 1).
2. Experimental details 2.1. Materials and instruments All reagents and solvents were obtained commercially and used without further purification. 1H NMR and 13C NMR spectra were recorded on a JEOL ECS 400M spectrometer and referenced to the solvent signals. Mass spectra (ESI) were obtained on a LQC system (Finnegan MAT, USA). UV–visible spectra were gathered on a Varian Cary 100 spectrophotometer. The melting points were measured with an X-6 melting point apparatus without calibration (Beijing Fuka Keyi Science and Technology Co., Ltd.). Fluorescence spectra were performed on a Hitachi F-7000 luminescence spectrometer. 2.2. Preparation of amino acids solutions for fluorescent study Stock solutions (2 mM) of amino acids including GSH, Cys, Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln, Trp, Ser, Gly, Tyr, Asn and Asp were prepared in ultrapure water, and the stock solution for probe 1 was prepared in dimethyl sulfoxide. In a typical experiment, examination solutions were prepared by placing 2 μL of the probe stock solution into a solution of 2 mL phosphate buffer. Fluorescence spectra were measured after the addition of analytes for 10 min in a water bath at 37 °C. An excitation and emission slits with a width of 5.0 nm were used for the fluorescent measurements. A Phosphate Buffered Saline (PBS) buffer was prepared using the following method: to create solution A, dissolve 1.775 g of Dibasic Sodium Phosphate in ultrapure water, then dilute to 250 ml. To create solution B, dissolve 0.680 g of Potassium Phosphate Monobasic in ultrapure water, then dilute to 100 ml. Afterwards, solution B is added to solution A until the pH comes to 7.40, then the final PBS buffer (20 mM pH ¼ 7.40) is preserved at 4 °C. 2.3. Fluorescence microscope experiment Hela and Hek-293a cells were cultured in DMEM (Dulbecco Modified Eagle Medium) in an atmosphere of 5% CO2 and 20% O2 at 37 °C, then washed with PBS buffer three times. Then
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Scheme 1. The design concept of probe 1 for GSH.
fluorescence imaging of Hela and Hek-293a cells was obtained with a Zeiss Leica DM 4000B microscope (40 objective lens). 2.4. Synthesis Initially, compound 2 was synthesized according to the previous report (Scheme 1) (Sun et al., 2011; Wang et al., 2013; Wu et al., 2012). A solution of compound 2 (0.612 g, 3 mmol), MeNO2 (0.244 g, 3.6 mmol), and piperidine (0.340 g, 3.6 mmol) in 15 ml EtOH was then heated to 40 °C and stirred for 8 h at this temperature. After cooling, the solvent was removed by vacuum, and chromatography on silica gel using CH2Cl2/CH3OH (v/v, 20:3) as the eluent afforded an aurantium solid (yield 52%). m.p. 152 °C. m/ z 246.0725 [M–H þ ]. 1H NMR (400 MHz, DMSO) δ: 12.13 (s, 1H), 8.42 (d, J ¼ 13.6 Hz, 1H), 8.22 (d, J ¼ 13.6 Hz, 1H), 7.79 (d, J ¼ 8.9 Hz, 1H), 7.00 (d, J ¼ 8.9 Hz, 1H), 6.27 (s, 1H), 2.40 (s, 3H). 13C NMR (101 MHz, DMSO) δ: 161.64, 158.94, 153.72, 153.64, 139.11, 129.90, 127.87, 112.57, 112.08, 110.54, 110.48, 104.81, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.89, 18.20.
3. Results and discussion 3.1. UV–vis absorption and fluorescence spectra of probe 1 towards GSH Thiol-containing biomolecule GSH plays a significant role in vivo, which was used in the spectral characteristics measurement of probe 1 in PBS buffer. Due to the influence of PET and ICT effect caused by nitroolefin moiety, probe 1 has a very weak fluorescence that can hardly be observed with the naked eye. Upon the addition of GSH (20 eq.), there was a significant decrease in the absorption peak of probe 1 (Fig. 1a). Meanwhile, the fluorescence intensity caught a 6-fold enhancement, and the maximum emission peak shifted from 442 nm to 453 nm (Fig. 1b). This causes the solution to make a remarkable transformation from yellow to a colorless, transparent hue, which can be clearly observed with the naked eye. Additionally, a variation in the solution from no fluorescence to a strongly blue fluorescence can be distinguished using a 365 nm UV lamp. These results indicate that GSH can be detected using both colorimetric and fluorescence methods with probe 1. To ensure a suitable reaction condition, time dependent modulations in the fluorescence spectra of probe 1 with GSH and Cys were monitored. Probe 1 (10 μM) was treated with 20 eq. of GSH and Cys. Then, the fluorescence signal at 453 nm was plotted as a function of time (Fig. 1c). A gradual enhancement of probe 1 fluorescence intensity with Cys was observed, which proves that Cys can also react slowly with probe 1. As a result, probe 1 has the capacity to separate GSH with Cys within 10 min. For a better understanding of the detection process, fluorescence spectra changes using different concentrations of GSH were measured in PBS buffer with probe 1 (2 μM). As shown in Fig. 1d, a progressively enhancive fluorescence intensity was observed,
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Fig. 1. (a) UV/vis absorption spectral changes of probe 1 (2 μM) upon treatment with GSH (20 eq.) 10 min later at 37 °C in PBS buffer. (b) Fluorescence spectral changes of probe 1 (2 μM) upon treatment with GSH (20 eq.) 10 min later at 37 °C in PBS buffer. (c) Time-dependent fluorescence intensity of probe 1 (2 μM) at 453 nm in the absence and presence of 20 eq. GSH or Cys in PBS buffer. (d) Fluorescence emission spectra of probe 1 (2 μM) in the presence of a gradually varied concentration of GSH (0, 1, 2, 4, 8, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μM) in PBS buffer. Inset: The change in the fluorescence intensity of probe 1 (2 μM) at 453 nm against varied concentrations of GSH from 0.5 to 5.0 eq. in PBS buffer, R2 ¼ 0.99107. Error bars represent the standard deviations of 3 trials. Ex ¼325 nm.
which corresponded to an incrementally increasing concentration of GSH in the mixture solution. In addition, the fluorescence intensity of the probe 1 solution also demonstrated a linear increase, corresponding to a growing concentration of GSH. These data prove probe 1 to be an ultrasensitive quantitative detection tool for GSH.
Detection limit =
3σbi m
where sbi is the standard deviation of blank measurements, and m is the slope between fluorescence intensity versus sample concentration. The detection limit of the probe for GSH was determined to be 122 nM. This is better than many probes based on same reactive group (Wang et al., 2013; Yuan et al., 2011).
3.2. Detection limit The detection limit was calculated based on the fluorescence titration (Joshi et al., 2010). To determine the S/N ratio, the emission intensity of probe 1 without GSH was measured times 30, and the standard deviation of blank measurements was determined. Three independent duplication measurements of emission intensity were performed in the presence of GSH and each average intensity value was plotted as a concentration of GSH for determining the slope (Fig. 1c). The detection limit was then calculated using the following equation.
3.3. Selectivity studies To investigate its selectivity, probe 1 (2 μM) was treated with natural amino acids in PBS buffer solution and monitored using fluorescent spectroscopy. The 20 kinds of amino acids include Cys, Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln, Trp, Ser, Gly, Tyr, Asn and Asp. In the presence of GSH, probe 1 displayed remarkable fluorescence enhancement (Fig. 2a). However, no obvious fluorescence spectra changes were observed upon the addition of other analytes under the same conditions.
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also provided evidence for the reaction of GSH with nitroolefin (Figs. 3 and S7). With addition of GSH to probe 1 in DMSO-d6, the alkene protons at 8.4 ppm (Ha) and 8.2 ppm (Hb) of probe 1 dramatically disappear while new peaks at 5.2, 5.3 and 4.5 ppm were observed. These peaks should be attributed to the saturated CH2 protons adjacent to the nitro group (Hbʹ and Hbʺ) and the original CH (Haʹ). In conclusion, all the measurements proved the occurrence of Michael addition between probe 1 and GSH. 3.5. Suitable pH To test the capability of probe 1 to detect GSH, the fluorescence spectra of probe 1 with 20 eq. GSH was obtained at different pH values (from 2 to 13, prepared with NaOH and HCl). The relationship between the fluorescence intensity at 453 nm and the pH value of the examination solution was then rendered in Fig. S3. The results showed that probe 1 exhibit an obvious response in the range of 7–11. For a preferable effect of the detection, a further measurement was taken in PBS buffer (Fig. S4, 5.8–8.0, 20 mM). For detecting GSH in vivo, the PBS buffer of pH 7.4 was selected as the general measurement condition. Thus, within the biologically relevant pH range (5.8–8.0), probe 1 could be used to detect intracellular GSH without interference. 3.6. Cell imaging and cytotoxicity
Fig. 2. (a) Natural amino acids selectivity and the competitiveness of probe 1 (2 μM) with various analytes, GSH, Cys, Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln, Trp, Ser, Gly, Tyr, Asn and Asp (20 eq.) for 10 min in PBS buffer. Ex ¼325 nm. (b) Color changes and fluorescence changes excited by a UV lamp (365 nm) in probe 1 upon the addition of various amino acids. [Probe 1] ¼40 μM, [amino acids] ¼ 80 μM.
Simultaneously, the effects of Cys on monitoring GSH in the presence of probe 1 were investigated. The primary experimental results showed that the interference of Cys may be ignored. Generally speaking, in a mixed solvent of buffer solution and organic solvent, chemodosimeter shows a similar reaction speed to GSH and Cys. Certain methods were used to overcome interference from GSH in the articles that chemodosimeters were prepared for detecting Cys or Hcy over GSH. For example, Cetrimonium Bromide (CTAB) was used to reduce the reaction speed of the probe with macromolecular GSH (Yang et al., 2011). The effects of GSH on monitoring Cys may be ignored in the presence of an excess probe (Wang et al., 2012). In our case, a low concentration and high solubility will lead to a reaction speed for probe 1 similar to that of GSH and Cys. As we know, GSH own more amino and carboxyl, which makes the sodium salt of GSH reflect better solubility than Cys in slightly alkaline solutions. Furthermore, a photo was taken of probe 1 (40 μM) with different amino acids (80 μM) in 5 ml PBS buffer under ambient light and a 365 nm UV lamp (Fig. 2b). As a consequence, a dramatic color change from yellow to colorless can be observed by the naked eye, and an obvious blue fluorescence enhancement was obtained. 3.4. Reaction mechanism In order to investigate the mechanism, the stoichiometry of a binding event between GSH and probe 1 was first determined. The results obtained from the Job’s plot show a 1: 1 stoichiometry between probe 1 and GSH (Fig. S5). NMR spectroscopic analysis
To further demonstrate the ability of probe 1 to rapidly detect intracellular thiols, fluorescence microscopy experiments were carried out in Hela and Hek-293a. As shown in Fig. 4, Hek-293a exhibits no intracellular background fluorescence. After incubated with probe 1 (40 μM) for 1 min at 37 °C, strong blue fluorescence could be observed, and the fluorescence did not show distinct enhancement with the increase of time (Fig. S9). Hela was used in order to prove the generality of 1; a gradual increase of blue fluorescence was observed after incubated with 40 μM probe 1 for 5–60 min at 37 °C (Fig. S10). In contrast, when Hela and Hek-293a cells were pre-treated with 2 mM N-ethylmaleimide (NEM, a thiolblocking reagent) for 1 h to remove the endogenous biothiols, then incubated with 40 μM of probe 1 for 10 min under the same condition, no fluorescence was observed inside the cells. This further confirmed that the fluorescence turn-on in Figs. S9 and S10 was caused by the reaction of probe 1 and biothiols. These cell experiments revealed that probe 1 can rapidly penetrate cell membranes and could be an effective GSH imaging agent in living cells. We simultaneously studied the quantitative determination property of probe 1 to GSH in Hela. A distinct blue fluorescence could be observed when the concentration of 1 increased to 4 μM, and an enhancement of brightness occurred with the addition of more probe after treated with 1 for 20 min (Fig. 5a). In every fluorescent photo, 3 common cells were chosen and the average brightness was calculated using software. Predictably, the brightness of cells demonstrated a linear increase with a growing concentration of 1 in the range of 8–48 μM (Fig. 5b), which proved that probe 1 could also detect GSH quantitatively in vivo. Furthermore, because GSH also plays an important role in the metabolic system of plants (Frendo et al., 2013), we tested the absorptive capacity of beans for 1 and then obtained fluorescence imaging (Fig. S11). After soaking in the mixture solution (water: DMSO ¼2:1) of probe 1 (2 mM) for 3 days, distinct sample fluorescence could be observed, which proves the probe may enter into plant tissue and reflect its function. In addition, the cell viability of 1 to Hela (Fig. S12) and Hek293a (Fig. S13) was evaluated using Resazurin assay. After treating with 40 μM of 1 at 37 °C, the viability was more than 80%, which indicates the low cytotoxicity and good biocompatibility of probe
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Fig. 3. Michael addition of probe 1 to form probe 1 – GSH and partial 1H NMR spectra of 1 upon addition of GSH in DMSO-d6. (a) Only probe 1, and (b) probe 1 þ GSH.
Fig. 4. Fluorescence images of Hek-293a. Fluorescence images before (b) and after (d) treating the cells with 40 μM of probe 1 for 1 min and their bright field images (a and c), respectively.
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Fig. 5. (a) Fluorescence images (right, exposure time: 3 s) and bright field images (left, exposure time: 30.7 ms) of Hela treated with a different concentration of Probe 1. (b) The linear relationship between average brightness and the concentration of probe 1 (8–48 μM). Error bars represent the standard deviations of 3 trials.
1. Consequently, the highly selective probe 1 can rapidly detect GSH in living cells under the condition of low cytotoxicity.
4. Conclusion A colorimetric and turn-on fluorescent probe based on a coumarin unit for rapid and highly selective GSH detection in PBS buffer solution (pH 7.4) was designed and synthesized. Moreover, fluorescence imaging of Hela and Hek-293a cells was carried out successfully, which indicates the cell permeability and biocompatibility of probe 1. We plan to modify probe 1 to detect polypeptide and protein containing GSH. Following this, some living mice will be used to test the physiological toxicity of probe 1.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant nos. 21431002 and 91122007) and the Specialized Research Fund for the Doctoral Program of Higher Education (Grant no. 20110211130002).
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