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A colorimetric and fluorescent probe for detecting intracellular biothiols
Biosensors and Bioelectronics 85 (2016) 46–52
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A colorimetric and fluorescent probe for detecting intracellular biothiols 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 1 March 2016 Received in revised form 28 April 2016 Accepted 29 April 2016 Available online 30 April 2016
A new rapid and highly sensitive coumarin-based probe (probe 1) has been designed and synthesized for detecting intracellular thiols. Probe 1 was prepared by a 4-step procedure as a latent fluorescence probe to achieve high sensitivity and fluorescence turn-on response toward cysteine and homocysteine over GSH and other various natural amino acids under physiological conditions. Owing to specific cyclization between thiols and aldehyde group, probe 1 displayed a highly selectivity toward cysteine and homocysteine. Above all, probe 1 was successfully used for fluorescence imaging of biothiols in Hela cells, and quantitative determination had been achieved within a certain range. Then specific fluorescence imaging of mice organ tissues was obtained for proving the permeability of probe 1. Simultaneously, the viability was measured to be more than 80%, which shows probe 1 can be a rapid and biocompatible probe for biothiols in cells. Furthermore, the measurement of thiols detection in 5 kinds of animal serum showed that probe 1 can be used in determination of biothiols in blood. & 2016 Elsevier B.V. All rights reserved.
Keywords: Coumarin Fluorescent Thiols Colorimetric Probe Bioimaging
1. Introduction Compounds with thiols (–SH) functionality are very important as low molecular weight aliphatic thiols containing amino acids (cysteine and Homocysteine) and peptides (glutathione) play pivotal roles in biological systems. For example, cysteine (Cys) is the only amino acid with a thiols functional group that serves as a unique unit in protein construction, enzyme active sites and cofactors (Albers et al., 2013; Lipton et al., 2002; Marino and Gladyshev, 2010). On the other hand, glutathione (GSH) is critical in maintaining redox homeostasis in the intracellular environment, which is important for maintenance of cellular defense against reactive oxygen species and for a number of biological processes (Kanzok et al., 2000; Wu et al., 2004). Recently, much research interest has been paid to Homocysteine (Hcy) because of its special role as a biomarker in many diseases (Austin et al., 2004; Xiao et al., 2011). Generally, the levels of intracellular biothiols have been associated to toxic agents and diseases. A low level of biothiols would be a dangerous signal for various syndromes, such as slow growth in children, hair depigmentation, lethargy, psoriasis, liver damage, substance abuse, muscle and fat loss, edema and weakness. While an elevated level of biothiols in human plasma is a risk factor for cardiovascular, Alzheimer's disease, neural tube n
Corresponding author. E-mail address: [email protected] (W. Liu).
http://dx.doi.org/10.1016/j.bios.2016.04.098 0956-5663/& 2016 Elsevier B.V. All rights reserved.
defect, inflammatory bowel disease, osteoporosis, cancer, and AIDS (Refsum et al., 1998; Seshadri et al., 2002; Shahrokhian, 2001; Townsend et al., 2003). Therefore, the determination and quantification of cellular biothiols is of great importance and has attracted much attention. Of all the traditional detection methods for biothiols, one of the most common analytical techniques is liquid chromatography (LC) (Ivanov et al., 2000; Michaelsen et al., 2009; Refsum et al., 2004; Tcherkas and Denisenko, 2001). Although this method offers high accuracy, it typically requires staff with a certain skill level, cumbersome preconditioning procedures and expensive instruments for the analysis, which makes it unsuitable for on-site trials and household testing. Among the available techniques to detect and quantify thiols, fluorescent methods have attracted increasing interest due to their high sensitivity, inexpensiveness, selectivity, easy operation and nondestructive (Chen et al., 2010; Kaur et al., 2012; Moragues et al., 2011; Wang et al., 2014). Accordingly, during the past decade, considerable efforts have been devoted to developing fluorescent probes for thiols (Jung et al., 2013; Kim et al., 2008; Yin et al., 2013). Among these probes, there are two main mechanisms used to design fluorescent probes for thiols. One is based on cleavage reaction mechanism (Bouffard et al., 2008; Ji et al., 2009; Pires and Chmielewski, 2008; Tang et al., 2007; Yuan et al., 2012), the other one is based on nucleophilic reaction mechanism (Jung et al., 2013; Yin et al., 2013). By comparing the two kinds of mechanisms, nucleophilic reaction mechanism are more actively developed because of the diversity of
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the electrophiles. Especially in recent years, the selective reaction of aldehydes with thiols to form thiazolidines was applied to the detection of Cys and Hcy, as sensors with an aldehyde functionality can form a rapid ring with 1, 3- or 1, 2-aminothiols, while other biothiols, like GSH, cannot (Rusin et al., 2004; Wang et al., 2005; Yang et al., 2011). However, it has been known that the selective detection of Cys, Hcy and GSH is still a challenge due to their reactivity of thiols and similarity in structure. Based on the abovementioned consideration and our earlier work (Chen et al., 2015; Shi et al., 2014; Yang et al., 2014), we report a new type fluorescent probe (probe 1) containing coumarin fluorophore and aldehydes moiety. Coumarin was selected as the fluorophore by reason of 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). Simultaneously, aldehydes was chosen because it serves as not only an electrophile but also a quencher of the coumarin fluorophore. The aldehydes moiety plays an electron acceptor for the photoinduced electron transfer (PET) process, leading to a low fluorescence of probe 1. After treatment with thiols, ring between fluorophore and thiols are formed, and the blocking of PET process can cause recovery of fluorescence (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 Cys, Hcy, GSH, Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln, Trp, Ser, Gly, Tyr, Asn and Asp in ultrapure water were prepared, and stock solution of probe 1 was prepared in 10 mL of DMSO. In a typical experiment, examine solutions were prepared by placing 10 μL of the probe stock solution into the solution of 2 mL phosphate buffer. Fluorescence spectra were measured after addition of analytes for 10 min at 20 °C. An excitation and emission slit widths of 5.0 nm were used for the fluorescent measurements. Phosphate Buffered Saline (PBS) buffer was prepared with the following method. Dissolve 1.775 g of Dibasic Sodium Phosphate in ultrapure water, and dilute to 250 mL, which is the solution A.
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Then dissolve 0.680 g of Potassium Phosphate Monobasic in ultrapure water, and dilute to 100 mL, which is the solution B. Afterwards, solution B was added to solution A until the pH comes to 7.40, then the final PBS buffer was preserver at 4 °C. 2.3. Cell and tissue culture Hela cells were obtained from the school of life science of Lanzhou University (Gansu, China). The cells were propagated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 μg/mL), and streptomycin (100 μg/mL). Cells were maintained under a humidified atmosphere of 5% CO2 and at 37 °C incubator. The cytotoxic effect of compound probe was determined by an MTT assay following the manufacturer instruction (Sigma-Aldrich, MO). Hela cells were initially propagated in a 25 cm2 tissue culture flask in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 μg/mL), and streptomycin (100 μg/mL) in a CO2 incubator. For cytotoxicity assay, cells were seeded into 96-well plates (approximately 104 cells per well), and various concentrations of compound probe (20, 40, 60, 80, 100 and 120 μM) made in DMEM were added to the cells and incubated for 24 h. Solvent control samples (cells treated with DMSO alone) was also included in parallel sets. Following incubation, the growth media was removed, and fresh DMEM containing MTT solution 5 mg/mL was added. The plate was incubated for 2–3 h at 37 °C. Subsequently, the supernatant was removed, the insoluble colored formazan product was solubilized in DMSO, and its absorbance was measured in a microtiter plate reader (Infinite M200, TECAN, Switzerland) at 480 nm. The assay was performed in five sets for each concentration of compound probe complex. Data analysis and calculation of standard deviation was performed with Microsoft Excel 2010 (Microsoft Corporation). For statistical analysis, a one way analysis of variance (ANOVA) was performed using Sigma plot. 2.4. Fluorescence microscope experiment Fluorescence images of dye labeled cells and tissues were obtained by Olympus FV1000 laser confocal microscope IΧ81 with 40 objective (cells) and 10 objective (tissues), numerical aperture (NA) ¼0.65. The images signals at 400–500 nm range were collected by internal PMTs in a 12 bit unsigned 1024*1024 pixels at 40 Hz scan speed. 2.5. Synthesis Initially, compound 3 was synthesized according to the previous report (Scheme S1) (Bochkov et al., 2013; Liu et al., 2014). Then a solution of compound 3 (1.165 g, 5 mmol), phosphorus oxychloride (0.7 mL, 6.75 mmol) in 5 mL N, N-Dimethylformamide (DMF) and stirred at room temperature for 24 h. Then the mixture was poured into aqueous sodium acetate (5 g in 100 mL) and
Scheme 1. Design concept of probe 1 for thiols.
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stirred for another 1 h. The precipitate formed was filtered off and washed with water. Then 20% HCl was added and the mixture further heated for 30 min at 60 °C, then cooled and extracted with dichloromethane. The organic solvent was removed by vacuum, and chromatography on silica gel using EA/PE (v/v, 1:2) as the eluent afford red solid (yield 60%). m.p. 135 °C. m/z 260.1135 [M – H þ ]. 1H NMR (400 MHz, Chloroform-d) δ 10.31 (s, 1H), 7.85 (d, J ¼9.3 Hz, 1H), 6.69 (d, J ¼9.4 Hz, 1H), 6.44 (s, 1H), 3.49 (q, J ¼7.4 Hz, 4H), 1.26 (t, J ¼7.4 Hz, 6H). 13C NMR (101 MHz, Chloroform-d) δ 187.07, 160.00, 156.50, 154.16, 153.74, 129.34, 110.98, 110.66, 107.72, 96.65, 45.43, 12.53.
3. Results and discussion 3.1. UV–vis absorption and fluorescence spectra The absorption and fluorescence emission spectra of probe 1 (10 μM) in PBS buffer (20 mM, pH 7.4) are shown in Fig. 1(a). Owing to the influence of PET effect caused by aldehydes moiety, probe 1 has a very weak fluorescence that can be hardly observed with the naked eye. After treated with thiols (20 eq.) for 10 min at room temperature (20 °C), probe 1 exhibited an absorption peak shift from 457 nm to 384 nm, and meanwhile, the fluorescence
intensity at 480 nm caught a 5-fold enhancement. To acquire a better understanding of the reaction of probe 1, time dependent modulations in the fluorescence spectra of probe 1 with thiols were monitored. Probe 1 (10 μM) was treated with 20 eq. of Cys, Hcy and GSH, respectively. Then, the fluorescence signal at 480 nm was plotted as a function of time (Fig. 1(c)). As a result, we cannot observe an obvious enhancement of the fluorescence signal of GSH within 10 min. Therefore, probe 1 has the capacity to separate Cys and Hcy with GSH. The fluorescence spectra clearly show an increase in the emission intensity of probe 1 in PBS buffer as the concentration of Cys increases (Fig. 1(d)). The fluorescence intensity at 480 nm were plotted as a function of Cys concentration, and the plots show a proportional relationship between emission intensities at 480 nm and the corresponding concentrations in Fig. 1(d). Meanwhile, The detection limit of the probe for Cys was determined to be 0.874 μM. All these data attest to that probe 1 can be a sensitive quantitative detection for Cys and Hcy. 3.2. Selectivity studies To examine the selectivity toward thiols, probe 1 was treated with Cys, Hcy, GSH and 19 kinds of other common amino acids including Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln,
Fig. 1. (a) UV/vis absorption spectral changes of probe 1 (10 μM) upon treatment with Cys, Hcy and GSH (20 eq.) 10 min later at 20 °C in PBS buffer. (b) Fluorescence spectra of probe 1 (10 μM) upon treated with Cys, Hcy and GSH for 10 min at 20 °C in PBS buffer. (c) Time-dependent fluorescence intensity of probe 1 (10 μM) at 480 nm in the presence of 20 eq. Cys, Hcy or GSH in PBS buffer. (d) Fluorescence emission spectra of probe 1 (10 μM) in the presence of gradually varied concentration of Cys (0–70 eq.) in PBS buffer. Inset: The change in the fluorescence intensity of probe 1 (10 μM) at 480 nm against varied concentrations of Cys from 0 to 20 eq. in PBS buffer. Ex¼ 384 nm. (1 eq.¼ 10 μM).
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Fig. 2. Up: natural amino acids selectivity and competitiveness of probe 1 (2 μM) to 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. (Gray lines mean the fluorescence intensity of probe 1 and various amino acids; red lines mean the competitiveness which react with probe 1 between Cys and various amino acids; green bold line means the fluorescence intensity of probe 1 in PBS; other bold lines mean the fluorescence intensity of probe 1 treated with Cys, Hcy or GSH) Down: color changes and fluorescence changes excited by a UV lamp (365 nm) in probe 1 upon addition of Cys, Hcy, GSH and various amino acids. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Trp, Ser, Gly, Tyr, Asn and Asp (20 eq.) in PBS buffer (pH 7.4) solution under the same conditions. Then, the fluorescence emission of samples were measured. As might be expected, a significant increase in fluorescence intensity was observed only the addition of Cys or Hcy. The other 19 kinds of amino acids and GSH exhibited no noticeable fluorescence change (Fig. 2). These results clearly demonstrated probe 1 to be a highly selective probe towards sulfydryl than other nucleophiles such as amino and hydroxyl. Furthermore, digital photograph of probe 1 with different amino acids under ambient light and 365 nm UV lamp was caught out (Fig. 2). As a consequence, a dramatic color change from yellow to colorless can be observed by naked eyes, and an obvious transform form weak green to strong blue fluorescent was obtained. These results indicated thiols can be detected through both colorimetric and fluorescence methods by probe 1. 3.3. Reaction kinetics and mechanism To confirm the formation of 1-Cys, probe 1 was treated with Cys, and the reaction product was isolated. The partial 1H NMR spectra of 1 and the isolated 1-Cys are shown in Fig. S11. The resonance signal corresponding to the aldehyde proton at 10.09 ppm disappeared; however, concomitantly, a new peak at 4.99 ppm assigned to the methine proton of the thiazolidine diastereomer emerged, consistent with the previous reports (Hu et al., 2011; Son et al., 2014; Wang et al., 2005). Besides, the formation of 1-Cys can
be verified by the mass spectra as shown in Fig. S15. 3.4. Suitable pH To test the capability of probe 1 to detect thiols, fluorescence spectra of probe 1 with 20 eq. Cys was obtained at different pH values (from 2 to 13, prepared with NaOH and HCl). After that, the relationship between fluorescence intensity at 480 nm and the pH value of examine solution was rendered in Fig. S4-left. The results shown that probe 1 exhibit an obvious response in slightly alkaline solution, because the deprotonated thiolate amino is more nucleophilic than its neutral form (Song et al., 2013). Since a preferable effect of the detection, a further measurement was taken in PBS buffer (Fig. S4-right, 5.8–8.0 20 mM). For detecting thiols in vivo, the PBS buffer of pH 7.4 was selected for the general measurement conditions, which is physiological condition. Thus, within the biologically relevant pH range (5.8–8.0), probe 1 could be used to detect intracellular thiols without interference. 3.5. Cytotoxicity In addition, the cell viability of 1 to Hela (Fig. S7) was evaluated using MTT assay. After treating with 120 μM of 1 at 37 °C, the viability was more than 80%, which indicates the low cytotoxicity and good biocompatibility of probe 1. Consequently, the highly selective probe 1 can rapidly detect Cys in living cells under the
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Fig. 3. Fluorescence images and bright field images of Hela cells. Up: Hela cells treated with different concentration of probe 1 for 10 min. Down: Hela cells treated with probe 1 (30 μM) for different times.
condition of low cytotoxicity. 3.6. Cell imaging and possible detection of intracellular thiols The suitable amphipathicity and insignificant cytotoxicity of probe 1 offered the possibility of using this reagent for in vivo fluorescence imaging in living cells and tissue. To test the capability of probe 1 to image Cys in living cells, we further applied probe 1 to cells. Hela was used and a gradual increase of blue fluorescence over time was observed after incubated with 30 μM probe 1 for 5–60 min at 37 °C, the strongest intracellular fluorescence can be cached in 30 min. However, when cells were not incubated with probe 1, there was no intracellular background fluorescence under the same bioimaging conditions. The result demonstrated that probe 1 can easily penetrate cell membranes
and make fast fluorescent labeling. As another control experiment, when Hela cell were pre-treated with N-ethylmaleimide (NEM, a thiol-blocking reagent) for 30 min to remove the endogenous cellular thiols and then incubated with 30 μM of probe 1 for 10 min under the same condition, intracellular fluorescence cannot be observed, which further confirmed that the fluorescence turn-on in Fig. 3-down was caused by reacting probe 1 with the cellular thiols (Fig. 4). We simultaneously studied the quantitative determination property of probe 1 to thiols in Hela. A distinct blue fluorescence could be observed when the concentration of 1 increased to 30 μM, and an enhancement of brightness occurred with the addition of more probe after treated with 1 for 10 min (Fig. 3-up). Predictably, the brightness of cells demonstrated a linear increase with a growing concentration of 1 in the range of 0–50 μM (Fig.
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Fig. 4. Fluorescence images and bright field images of mice liver tissue treated with difference methods.
showed fluorescence due to the reaction between thiols and probe 1. Next, the fluorescence intensity did not become strong when the tissue slice was pre-treated with 20 mM of GSH, which proved that even high concentration of GSH will not interfere with the determination of Cys/Hcy. Furthermore, when tissue slice were pre-treated with 20 mM of Cys and further incubated with the probe, much stronger fluorescence was observed. These results indicate that the blue fluorescence emission in tissue is due to the formation of the 1-Cys adduct. 3.8. Thiols detection in animal serum
Fig. 5. The fluorescence intensity at 480 nm of different kind of animal (Chicken, Horse, Sheep, Cattle and Goat) serums, the number in the figure is the calculated concentration of thiols.
S6), which proved that probe 1 could also detect thiols quantitatively in vivo.
To ascertain the practical applicability of probe 1, thiols content in the different kinds of animal serum samples were studied. Estimation the concentration of thiols in blood serum is crucial for understanding its role in the pathogenesis of cardiovascular diseases. The serum samples were diluted 10 times with PBS, then make titration curve in these solution. Upon addition of probe 1 (10 μM) to the animal serum solution, we observed obviously enhancement of fluorescence signal at 480 nm, and the concentration calculated with titrating equation is much close to the normal concentration of thiol compounds in animal serum (Fig. 5) (Yang et al., 2014). This result indicates that probe 1 can be potentially employed to detect thiols quantitatively in serum samples.
3.7. Tissue imaging 4. Conclusion We also investigated the utility of this probe to image the process in mice organ tissue. After injected with 100 μL of the probe solution (1 mM/L; DMSO: normal saline ¼1:1), the mice was dissected and frozen, then the photo of different mice organ slices was obtained (Fig. S8). These photos show similar fluorescence intensity, which revealed that our probe has a good permeability in vivo. Meanwhile, the organ slices of liver was incubated with 50 μM probe 1 for 10 min at 37 °C, the dye-stained tissue slice
A colorimetric and turn-on fluorescent probe (probe 1) for rapid detecting biothiols was synthesized. Probe 1 exhibits high sensitivity and selectivity toward Cys and Hcy in phosphate buffer solution. Moreover, fluorescence imaging of Hela cells and mice tissue was carried out successfully for detecting biothiols in vivo. Then the detection of biothiols in animal serum achieved good results. All of these indicate probe 1 has potential to be a
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biocompatible disease surveillance tool and protein marker. Due to the simple structure and synthetic steps of probe 1, we plan to modify the structure to achieve better water solubility, target ability and selectivity. In conclusion, we will be more indepth research the fluorescent probe which can specifically identify small intracellular molecular and protein.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant no. 21431002).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.04.098.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A colorimetric and fluorescent probe for detecting intracellular biothiols 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 1 March 2016 Received in revised form 28 April 2016 Accepted 29 April 2016 Available online 30 April 2016
A new rapid and highly sensitive coumarin-based probe (probe 1) has been designed and synthesized for detecting intracellular thiols. Probe 1 was prepared by a 4-step procedure as a latent fluorescence probe to achieve high sensitivity and fluorescence turn-on response toward cysteine and homocysteine over GSH and other various natural amino acids under physiological conditions. Owing to specific cyclization between thiols and aldehyde group, probe 1 displayed a highly selectivity toward cysteine and homocysteine. Above all, probe 1 was successfully used for fluorescence imaging of biothiols in Hela cells, and quantitative determination had been achieved within a certain range. Then specific fluorescence imaging of mice organ tissues was obtained for proving the permeability of probe 1. Simultaneously, the viability was measured to be more than 80%, which shows probe 1 can be a rapid and biocompatible probe for biothiols in cells. Furthermore, the measurement of thiols detection in 5 kinds of animal serum showed that probe 1 can be used in determination of biothiols in blood. & 2016 Elsevier B.V. All rights reserved.
Keywords: Coumarin Fluorescent Thiols Colorimetric Probe Bioimaging
1. Introduction Compounds with thiols (–SH) functionality are very important as low molecular weight aliphatic thiols containing amino acids (cysteine and Homocysteine) and peptides (glutathione) play pivotal roles in biological systems. For example, cysteine (Cys) is the only amino acid with a thiols functional group that serves as a unique unit in protein construction, enzyme active sites and cofactors (Albers et al., 2013; Lipton et al., 2002; Marino and Gladyshev, 2010). On the other hand, glutathione (GSH) is critical in maintaining redox homeostasis in the intracellular environment, which is important for maintenance of cellular defense against reactive oxygen species and for a number of biological processes (Kanzok et al., 2000; Wu et al., 2004). Recently, much research interest has been paid to Homocysteine (Hcy) because of its special role as a biomarker in many diseases (Austin et al., 2004; Xiao et al., 2011). Generally, the levels of intracellular biothiols have been associated to toxic agents and diseases. A low level of biothiols would be a dangerous signal for various syndromes, such as slow growth in children, hair depigmentation, lethargy, psoriasis, liver damage, substance abuse, muscle and fat loss, edema and weakness. While an elevated level of biothiols in human plasma is a risk factor for cardiovascular, Alzheimer's disease, neural tube n
Corresponding author. E-mail address: [email protected] (W. Liu).
http://dx.doi.org/10.1016/j.bios.2016.04.098 0956-5663/& 2016 Elsevier B.V. All rights reserved.
defect, inflammatory bowel disease, osteoporosis, cancer, and AIDS (Refsum et al., 1998; Seshadri et al., 2002; Shahrokhian, 2001; Townsend et al., 2003). Therefore, the determination and quantification of cellular biothiols is of great importance and has attracted much attention. Of all the traditional detection methods for biothiols, one of the most common analytical techniques is liquid chromatography (LC) (Ivanov et al., 2000; Michaelsen et al., 2009; Refsum et al., 2004; Tcherkas and Denisenko, 2001). Although this method offers high accuracy, it typically requires staff with a certain skill level, cumbersome preconditioning procedures and expensive instruments for the analysis, which makes it unsuitable for on-site trials and household testing. Among the available techniques to detect and quantify thiols, fluorescent methods have attracted increasing interest due to their high sensitivity, inexpensiveness, selectivity, easy operation and nondestructive (Chen et al., 2010; Kaur et al., 2012; Moragues et al., 2011; Wang et al., 2014). Accordingly, during the past decade, considerable efforts have been devoted to developing fluorescent probes for thiols (Jung et al., 2013; Kim et al., 2008; Yin et al., 2013). Among these probes, there are two main mechanisms used to design fluorescent probes for thiols. One is based on cleavage reaction mechanism (Bouffard et al., 2008; Ji et al., 2009; Pires and Chmielewski, 2008; Tang et al., 2007; Yuan et al., 2012), the other one is based on nucleophilic reaction mechanism (Jung et al., 2013; Yin et al., 2013). By comparing the two kinds of mechanisms, nucleophilic reaction mechanism are more actively developed because of the diversity of
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the electrophiles. Especially in recent years, the selective reaction of aldehydes with thiols to form thiazolidines was applied to the detection of Cys and Hcy, as sensors with an aldehyde functionality can form a rapid ring with 1, 3- or 1, 2-aminothiols, while other biothiols, like GSH, cannot (Rusin et al., 2004; Wang et al., 2005; Yang et al., 2011). However, it has been known that the selective detection of Cys, Hcy and GSH is still a challenge due to their reactivity of thiols and similarity in structure. Based on the abovementioned consideration and our earlier work (Chen et al., 2015; Shi et al., 2014; Yang et al., 2014), we report a new type fluorescent probe (probe 1) containing coumarin fluorophore and aldehydes moiety. Coumarin was selected as the fluorophore by reason of 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). Simultaneously, aldehydes was chosen because it serves as not only an electrophile but also a quencher of the coumarin fluorophore. The aldehydes moiety plays an electron acceptor for the photoinduced electron transfer (PET) process, leading to a low fluorescence of probe 1. After treatment with thiols, ring between fluorophore and thiols are formed, and the blocking of PET process can cause recovery of fluorescence (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 Cys, Hcy, GSH, Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln, Trp, Ser, Gly, Tyr, Asn and Asp in ultrapure water were prepared, and stock solution of probe 1 was prepared in 10 mL of DMSO. In a typical experiment, examine solutions were prepared by placing 10 μL of the probe stock solution into the solution of 2 mL phosphate buffer. Fluorescence spectra were measured after addition of analytes for 10 min at 20 °C. An excitation and emission slit widths of 5.0 nm were used for the fluorescent measurements. Phosphate Buffered Saline (PBS) buffer was prepared with the following method. Dissolve 1.775 g of Dibasic Sodium Phosphate in ultrapure water, and dilute to 250 mL, which is the solution A.
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Then dissolve 0.680 g of Potassium Phosphate Monobasic in ultrapure water, and dilute to 100 mL, which is the solution B. Afterwards, solution B was added to solution A until the pH comes to 7.40, then the final PBS buffer was preserver at 4 °C. 2.3. Cell and tissue culture Hela cells were obtained from the school of life science of Lanzhou University (Gansu, China). The cells were propagated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 μg/mL), and streptomycin (100 μg/mL). Cells were maintained under a humidified atmosphere of 5% CO2 and at 37 °C incubator. The cytotoxic effect of compound probe was determined by an MTT assay following the manufacturer instruction (Sigma-Aldrich, MO). Hela cells were initially propagated in a 25 cm2 tissue culture flask in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 μg/mL), and streptomycin (100 μg/mL) in a CO2 incubator. For cytotoxicity assay, cells were seeded into 96-well plates (approximately 104 cells per well), and various concentrations of compound probe (20, 40, 60, 80, 100 and 120 μM) made in DMEM were added to the cells and incubated for 24 h. Solvent control samples (cells treated with DMSO alone) was also included in parallel sets. Following incubation, the growth media was removed, and fresh DMEM containing MTT solution 5 mg/mL was added. The plate was incubated for 2–3 h at 37 °C. Subsequently, the supernatant was removed, the insoluble colored formazan product was solubilized in DMSO, and its absorbance was measured in a microtiter plate reader (Infinite M200, TECAN, Switzerland) at 480 nm. The assay was performed in five sets for each concentration of compound probe complex. Data analysis and calculation of standard deviation was performed with Microsoft Excel 2010 (Microsoft Corporation). For statistical analysis, a one way analysis of variance (ANOVA) was performed using Sigma plot. 2.4. Fluorescence microscope experiment Fluorescence images of dye labeled cells and tissues were obtained by Olympus FV1000 laser confocal microscope IΧ81 with 40 objective (cells) and 10 objective (tissues), numerical aperture (NA) ¼0.65. The images signals at 400–500 nm range were collected by internal PMTs in a 12 bit unsigned 1024*1024 pixels at 40 Hz scan speed. 2.5. Synthesis Initially, compound 3 was synthesized according to the previous report (Scheme S1) (Bochkov et al., 2013; Liu et al., 2014). Then a solution of compound 3 (1.165 g, 5 mmol), phosphorus oxychloride (0.7 mL, 6.75 mmol) in 5 mL N, N-Dimethylformamide (DMF) and stirred at room temperature for 24 h. Then the mixture was poured into aqueous sodium acetate (5 g in 100 mL) and
Scheme 1. Design concept of probe 1 for thiols.
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stirred for another 1 h. The precipitate formed was filtered off and washed with water. Then 20% HCl was added and the mixture further heated for 30 min at 60 °C, then cooled and extracted with dichloromethane. The organic solvent was removed by vacuum, and chromatography on silica gel using EA/PE (v/v, 1:2) as the eluent afford red solid (yield 60%). m.p. 135 °C. m/z 260.1135 [M – H þ ]. 1H NMR (400 MHz, Chloroform-d) δ 10.31 (s, 1H), 7.85 (d, J ¼9.3 Hz, 1H), 6.69 (d, J ¼9.4 Hz, 1H), 6.44 (s, 1H), 3.49 (q, J ¼7.4 Hz, 4H), 1.26 (t, J ¼7.4 Hz, 6H). 13C NMR (101 MHz, Chloroform-d) δ 187.07, 160.00, 156.50, 154.16, 153.74, 129.34, 110.98, 110.66, 107.72, 96.65, 45.43, 12.53.
3. Results and discussion 3.1. UV–vis absorption and fluorescence spectra The absorption and fluorescence emission spectra of probe 1 (10 μM) in PBS buffer (20 mM, pH 7.4) are shown in Fig. 1(a). Owing to the influence of PET effect caused by aldehydes moiety, probe 1 has a very weak fluorescence that can be hardly observed with the naked eye. After treated with thiols (20 eq.) for 10 min at room temperature (20 °C), probe 1 exhibited an absorption peak shift from 457 nm to 384 nm, and meanwhile, the fluorescence
intensity at 480 nm caught a 5-fold enhancement. To acquire a better understanding of the reaction of probe 1, time dependent modulations in the fluorescence spectra of probe 1 with thiols were monitored. Probe 1 (10 μM) was treated with 20 eq. of Cys, Hcy and GSH, respectively. Then, the fluorescence signal at 480 nm was plotted as a function of time (Fig. 1(c)). As a result, we cannot observe an obvious enhancement of the fluorescence signal of GSH within 10 min. Therefore, probe 1 has the capacity to separate Cys and Hcy with GSH. The fluorescence spectra clearly show an increase in the emission intensity of probe 1 in PBS buffer as the concentration of Cys increases (Fig. 1(d)). The fluorescence intensity at 480 nm were plotted as a function of Cys concentration, and the plots show a proportional relationship between emission intensities at 480 nm and the corresponding concentrations in Fig. 1(d). Meanwhile, The detection limit of the probe for Cys was determined to be 0.874 μM. All these data attest to that probe 1 can be a sensitive quantitative detection for Cys and Hcy. 3.2. Selectivity studies To examine the selectivity toward thiols, probe 1 was treated with Cys, Hcy, GSH and 19 kinds of other common amino acids including Glu, Phe, Ala, Pro, Thr, His, Ile, Arg, Lys, Val, Leu, Met, Gln,
Fig. 1. (a) UV/vis absorption spectral changes of probe 1 (10 μM) upon treatment with Cys, Hcy and GSH (20 eq.) 10 min later at 20 °C in PBS buffer. (b) Fluorescence spectra of probe 1 (10 μM) upon treated with Cys, Hcy and GSH for 10 min at 20 °C in PBS buffer. (c) Time-dependent fluorescence intensity of probe 1 (10 μM) at 480 nm in the presence of 20 eq. Cys, Hcy or GSH in PBS buffer. (d) Fluorescence emission spectra of probe 1 (10 μM) in the presence of gradually varied concentration of Cys (0–70 eq.) in PBS buffer. Inset: The change in the fluorescence intensity of probe 1 (10 μM) at 480 nm against varied concentrations of Cys from 0 to 20 eq. in PBS buffer. Ex¼ 384 nm. (1 eq.¼ 10 μM).
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Fig. 2. Up: natural amino acids selectivity and competitiveness of probe 1 (2 μM) to 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. (Gray lines mean the fluorescence intensity of probe 1 and various amino acids; red lines mean the competitiveness which react with probe 1 between Cys and various amino acids; green bold line means the fluorescence intensity of probe 1 in PBS; other bold lines mean the fluorescence intensity of probe 1 treated with Cys, Hcy or GSH) Down: color changes and fluorescence changes excited by a UV lamp (365 nm) in probe 1 upon addition of Cys, Hcy, GSH and various amino acids. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Trp, Ser, Gly, Tyr, Asn and Asp (20 eq.) in PBS buffer (pH 7.4) solution under the same conditions. Then, the fluorescence emission of samples were measured. As might be expected, a significant increase in fluorescence intensity was observed only the addition of Cys or Hcy. The other 19 kinds of amino acids and GSH exhibited no noticeable fluorescence change (Fig. 2). These results clearly demonstrated probe 1 to be a highly selective probe towards sulfydryl than other nucleophiles such as amino and hydroxyl. Furthermore, digital photograph of probe 1 with different amino acids under ambient light and 365 nm UV lamp was caught out (Fig. 2). As a consequence, a dramatic color change from yellow to colorless can be observed by naked eyes, and an obvious transform form weak green to strong blue fluorescent was obtained. These results indicated thiols can be detected through both colorimetric and fluorescence methods by probe 1. 3.3. Reaction kinetics and mechanism To confirm the formation of 1-Cys, probe 1 was treated with Cys, and the reaction product was isolated. The partial 1H NMR spectra of 1 and the isolated 1-Cys are shown in Fig. S11. The resonance signal corresponding to the aldehyde proton at 10.09 ppm disappeared; however, concomitantly, a new peak at 4.99 ppm assigned to the methine proton of the thiazolidine diastereomer emerged, consistent with the previous reports (Hu et al., 2011; Son et al., 2014; Wang et al., 2005). Besides, the formation of 1-Cys can
be verified by the mass spectra as shown in Fig. S15. 3.4. Suitable pH To test the capability of probe 1 to detect thiols, fluorescence spectra of probe 1 with 20 eq. Cys was obtained at different pH values (from 2 to 13, prepared with NaOH and HCl). After that, the relationship between fluorescence intensity at 480 nm and the pH value of examine solution was rendered in Fig. S4-left. The results shown that probe 1 exhibit an obvious response in slightly alkaline solution, because the deprotonated thiolate amino is more nucleophilic than its neutral form (Song et al., 2013). Since a preferable effect of the detection, a further measurement was taken in PBS buffer (Fig. S4-right, 5.8–8.0 20 mM). For detecting thiols in vivo, the PBS buffer of pH 7.4 was selected for the general measurement conditions, which is physiological condition. Thus, within the biologically relevant pH range (5.8–8.0), probe 1 could be used to detect intracellular thiols without interference. 3.5. Cytotoxicity In addition, the cell viability of 1 to Hela (Fig. S7) was evaluated using MTT assay. After treating with 120 μM of 1 at 37 °C, the viability was more than 80%, which indicates the low cytotoxicity and good biocompatibility of probe 1. Consequently, the highly selective probe 1 can rapidly detect Cys in living cells under the
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Fig. 3. Fluorescence images and bright field images of Hela cells. Up: Hela cells treated with different concentration of probe 1 for 10 min. Down: Hela cells treated with probe 1 (30 μM) for different times.
condition of low cytotoxicity. 3.6. Cell imaging and possible detection of intracellular thiols The suitable amphipathicity and insignificant cytotoxicity of probe 1 offered the possibility of using this reagent for in vivo fluorescence imaging in living cells and tissue. To test the capability of probe 1 to image Cys in living cells, we further applied probe 1 to cells. Hela was used and a gradual increase of blue fluorescence over time was observed after incubated with 30 μM probe 1 for 5–60 min at 37 °C, the strongest intracellular fluorescence can be cached in 30 min. However, when cells were not incubated with probe 1, there was no intracellular background fluorescence under the same bioimaging conditions. The result demonstrated that probe 1 can easily penetrate cell membranes
and make fast fluorescent labeling. As another control experiment, when Hela cell were pre-treated with N-ethylmaleimide (NEM, a thiol-blocking reagent) for 30 min to remove the endogenous cellular thiols and then incubated with 30 μM of probe 1 for 10 min under the same condition, intracellular fluorescence cannot be observed, which further confirmed that the fluorescence turn-on in Fig. 3-down was caused by reacting probe 1 with the cellular thiols (Fig. 4). We simultaneously studied the quantitative determination property of probe 1 to thiols in Hela. A distinct blue fluorescence could be observed when the concentration of 1 increased to 30 μM, and an enhancement of brightness occurred with the addition of more probe after treated with 1 for 10 min (Fig. 3-up). Predictably, the brightness of cells demonstrated a linear increase with a growing concentration of 1 in the range of 0–50 μM (Fig.
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Fig. 4. Fluorescence images and bright field images of mice liver tissue treated with difference methods.
showed fluorescence due to the reaction between thiols and probe 1. Next, the fluorescence intensity did not become strong when the tissue slice was pre-treated with 20 mM of GSH, which proved that even high concentration of GSH will not interfere with the determination of Cys/Hcy. Furthermore, when tissue slice were pre-treated with 20 mM of Cys and further incubated with the probe, much stronger fluorescence was observed. These results indicate that the blue fluorescence emission in tissue is due to the formation of the 1-Cys adduct. 3.8. Thiols detection in animal serum
Fig. 5. The fluorescence intensity at 480 nm of different kind of animal (Chicken, Horse, Sheep, Cattle and Goat) serums, the number in the figure is the calculated concentration of thiols.
S6), which proved that probe 1 could also detect thiols quantitatively in vivo.
To ascertain the practical applicability of probe 1, thiols content in the different kinds of animal serum samples were studied. Estimation the concentration of thiols in blood serum is crucial for understanding its role in the pathogenesis of cardiovascular diseases. The serum samples were diluted 10 times with PBS, then make titration curve in these solution. Upon addition of probe 1 (10 μM) to the animal serum solution, we observed obviously enhancement of fluorescence signal at 480 nm, and the concentration calculated with titrating equation is much close to the normal concentration of thiol compounds in animal serum (Fig. 5) (Yang et al., 2014). This result indicates that probe 1 can be potentially employed to detect thiols quantitatively in serum samples.
3.7. Tissue imaging 4. Conclusion We also investigated the utility of this probe to image the process in mice organ tissue. After injected with 100 μL of the probe solution (1 mM/L; DMSO: normal saline ¼1:1), the mice was dissected and frozen, then the photo of different mice organ slices was obtained (Fig. S8). These photos show similar fluorescence intensity, which revealed that our probe has a good permeability in vivo. Meanwhile, the organ slices of liver was incubated with 50 μM probe 1 for 10 min at 37 °C, the dye-stained tissue slice
A colorimetric and turn-on fluorescent probe (probe 1) for rapid detecting biothiols was synthesized. Probe 1 exhibits high sensitivity and selectivity toward Cys and Hcy in phosphate buffer solution. Moreover, fluorescence imaging of Hela cells and mice tissue was carried out successfully for detecting biothiols in vivo. Then the detection of biothiols in animal serum achieved good results. All of these indicate probe 1 has potential to be a
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biocompatible disease surveillance tool and protein marker. Due to the simple structure and synthetic steps of probe 1, we plan to modify the structure to achieve better water solubility, target ability and selectivity. In conclusion, we will be more indepth research the fluorescent probe which can specifically identify small intracellular molecular and protein.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant no. 21431002).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.04.098.
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