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homocysteine
Analytica Chimica Acta xxx (2015) 1e8
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A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine Xi Dai a, 1, Zhao-Yang Wang b, 1, Zhi-Fang Du a, c, Jie Cui a, Jun-Ying Miao b, **, Bao-Xiang Zhao a, * a b c
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China Taishan College, Shandong University, Jinan 250100, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A colorimetric, ratiometric and water-soluble fluorescent probe was developed. The probe could simultaneous distinguish GSH and Cys/Hcy by visual determination. This probe was successfully used to achieve living cell ratio imaging.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 August 2015 Received in revised form 16 October 2015 Accepted 21 October 2015 Available online xxx
A chlorinated coumarin-aldehyde was developed as a colorimetric and ratiometric fluorescent probe for distinguishing glutathione (GSH), cystenine (Cys) and homocysteine (Hcy). The GSH-induced substitution-cyclization and Cys/Hcy-induced substitution-rearrangement cascades lead to the corresponding thiol-coumarin-iminium cation and amino-coumarin-aldehyde with distinct photophysical properties. The probe can be used to simultaneously detect GSH and Cys/Hcy by visual determination based on distinct different colors e red and pale-yellow in PBS buffer solution by two reaction sites. From the linear relationship of fluorescence intensity and biothiols concentrations, it was determined that the limits of detection for GSH, Hcy and Cys are 0.08, 0.09 and 0.18 mM, respectively. Furthermore, the probe was successfully used in living cell imaging with low cell toxicity. © 2015 Published by Elsevier B.V.
Keywords: Ratiometric Fluorescent probe Glutathione Coumarin Cell imaging
1. Introduction Low molecular weight thiols, like glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), are crucial cellular components that
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (B.-X. Zhao). 1 Equal contribution.
(J.-Y.
Miao),
[email protected]
play numerous roles in metabolism and homeostasis as well as in varying functions of physiological and pathological processes [1e3]. Abnormal levels of biothiols are related to many diseases, such as neurodegenerative diseases, liver damage and renal diseases [4e7]. Fluorescent probes are powerful molecular tools for monitoring trace amounts of analytes in live cells or tissues because of their simplicity and high sensitivity [8e20]. However, the discrimination between three biothiols is still challenging because of their similarity in structure and reactivity. Numerous fluorescent probes have been developed for the
http://dx.doi.org/10.1016/j.aca.2015.10.023 0003-2670/© 2015 Published by Elsevier B.V.
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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detection of biothiols by utilizing different mechanisms, such as cleavage reaction, Michael addition, redox reaction and nucleophilic substitution [21e24]. The addition and cyclization reaction of Cys/Hcy with aldehydes or acrylates and native chemical ligation (NCL) reaction have been developed to discriminate Cys and Hcy over GSH [25e35]. Relatively speaking, the discriminating of GSH over Cys/Hcy remains a tough mission. Our group reported two fluorescent probes to detect GSH over Cys and Hcy using the acrylate derivatives [36,37]. The Michael addition products of the probes with GSH have strong fluorescence; on the contrary, the addition-cyclization products of the probes with Cys/Hcy are nonfluorescence. Yang group demonstrated a GSH fluorescent probe based on the thiol-halogen nucleophilic substitution reaction between GSH and a monochlorinated Bodipy [38]. The attractive strategy is distinct from that induced nucleophilic substitutionrearrangement cascade reaction by Cys/Hcy. In recent years, some fluorescent probes have been designed based on this strategy [39e41]. Furthermore, a chlorinated coumarin-hemicyanine dye with three potential reaction sites was reported for sensing GSH and Cys through different emission channels [41]. However, a simple and effective fluorescent probe for simultaneously sensing GSH, Cys and Hcy is extremely necessary. Herein, we combine the above strategies to study a colorimetric and ratiometric fluorescent probe, 4-chloro-7-(diethylamino)-3carbaldehyde coumarin, with two reaction sites. The probe could simultaneously distinguish GSH and Cys/Hcy through different emission channels in PBS buffer solution. No matter in vivo or in vitro, we could observe the significant different colors by visual determination. Furthermore, the probe was successfully used in living cell ratio imaging. 2. Experimental 2.1. Apparatus and chemicals Thin-layer chromatography (TLC) was conducted on silica gel 60 F254 plates (Merck KGaA) and column chromatography was conducted over silica gel (mesh 200e300). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were carried out on a Bruker Avance 400 spectrometer, using DMSO as solvent and tetramethylsilane (TMS) as internal standard. Melting points were determined on an XD-4 digital micro melting point apparatus. IR spectra were performed with an IR spectrophotometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were obtained on a Q-TOF6510 spectrograph (Agilent). UVevis spectra were measured by using a Hitachi U-4100 spectrophotometer. Fluorescent measurements were recorded on a PerkineElmer LS-55 luminescence spectrophotometer. Quartz cuvettes with a 1 cm path length and 3 mL volume were used for all measurements. The pH measurements were done on a Model PHS3C pH meter. Unless otherwise stated, all reagents were purchased from J&K, Sinopharm Chemical Reagent Co. and Kermel and used without further purification. Twice-distilled water was used throughout all experiments. 2.2. Synthesis of 4-chloro-7-(diethylamino)-3-carbaldehyde coumarin (probe ACCA) 7-(Diethylamino)-4-hydroxy coumarin (4) was synthesized according to literature methods [41]. Under nitrogen, dry DMF (2.5 mL) was added dropwise to POCl3 (1.0 mL) at room temperature. After stirred for 30 min, compound 4 (3 mmol) dissolved in 10 mL DMF was added dropwise to the above solution. The solution was stirred at 60 C for 10 h until TLC indicated the end of the reaction. After cooling to room temperature, the mixture was poured into ice water (50 mL), and was adjusted to neutral with diluted
NaOH solution. The ensuing precipitate was filtered and washed with water. The crude product was purified by the recrystallization from absolute ethanol to give 4-chloro-7-(diethylamino)-3carbaldehyde coumarin in 73% yield. Yellow solid; mp: 138e140 C. IR (KBr) n: 3090 (ArH), 2972 (eCH3), 2928 (eCH2e), 2879 (HeC]O), 1719 (C]O), 1177(CeOeC). 1H NMR (DMSO, 400 MHz): d ¼ 10.30 (1H, s, eCHO), 7.85 (1H, d, J ¼ 9.3 Hz, ArH), 6.70 (1H, dd, J ¼ 2.4 and 9.3 Hz, ArH), 6.44 (1H, d, J ¼ 2.4 Hz, ArH), 3.49 (4H, q, J ¼ 7.2 Hz, eCH2e), 1.26 (6H, t, J ¼ 7.2 Hz, eCH3). 13C NMR (100 MHz, DMSO-d6): 12.44 (2C), 45.40 (2C), 96.75, 107.82, 110.60, 111.15, 129.31, 153.58, 154.00, 156.45, 159.95, 187.02. HRMS: m/z [MþH]þ calcd for [C14H14ClNO3 þ H]þ: 280.0740, found 280.0731. 2.3. Preparation for UVevis and fluorescence spectral measurements Probe ACCA was dissolved in DMSO to afford the stock solution (2 103 M). Amino acids (GSH, Cys, Hcy, arg, asp, glu, gly, his, lys, pro, ser, thr, trp), cation (Ca2þ, Fe3þ, Kþ, Mg2þ, Naþ, Zn2þ), H2O2, glucose were all dissolved in deionized water at a concentration of 2 102 M for absorption and fluorescence spectral analysis. Test solutions were prepared by placing 100 mL or 50 mL of the stock solution and an appropriate aliquot of each testing species solution into a 10-mL volumetric flask, and the solution was diluted to 10 mL in an aqueous solution with pH 7.4 (PBS buffer). The resulting solution was shaken well and incubated for 60 min at room temperature before recording the spectra. 2.4. HPLC-MS traces HPLC-MS spectra were recorded with a Thermo LCQ Fleet coupled with a thermo Ultimate 3000 HPLC system. HPLC analysis was accomplished with an Atlanti C18 reversed-phase column (2.1 150 mm), with CH3CN (0.1% of HCOOH) and water as the eluent. Injection volume: 5 mM; mobile phase: A-0.1% methanoic acid/acetonitrile, B-water; gradient elution: 0e7.9 min, 20%A; 8e15 min 90%A; flow rate: 0.2 mL min1. 2.5. Cell culture and cell imaging HeLa cells were cultured in RPMI-1640 with 10% CBS at 2 104 cells per well. The probe was dissolved in DMSO at a storage concentration of 10 mM. Cells were adherent-cultured in 24-well culture plates for 12 h. After washing away the culture medium with phosphate-buffered saline solution (PBS), HeLa cells of control group were loaded with 10.0 mM probe solution at 37 C for 40 min. Other test groups were pretreated with GSH (0.5 mM) or Cys (0.1, 0.5, 1.0 and 5.0 mM) at 37 C for 40 min, followed by incubation with 10.0 mM probe solution for 40 min. Then washed 2 times with PBS and underwent imaging measurement by ultraviolet light with a confocal microscope (LSM700). Fluorescence values were quantified by the fluorescence analysis software Image J. The exciting light was 405 nm for the emission range of the blue channel (405e475 nm). The exciting light was 488 nm for the emission range of the green channel (488e520 nm) and the red channel was (560e700 nm). 2.6. Cytotoxicity assay The in vitro cytotoxicity of the probe to HeLa cells was measured by a standard sulforhodamine B (SRB) assay. Briefly, HeLa cells were loaded in 96-well culture plates at 4 104 cells per well. After culture for 24 h, cells were incubated with fresh RPMI 1640 containing 1.0, 5.0 and 10.0 mM probe for 24 h, respectively. Then cells were fixed with 4% TCA for 1 h at 4 C, and then washed 5 times
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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with deionized water; 50 mL SRB was added to each well, and after sufficient reaction with cells, the remaining SRB was removed by washing each well with 1% acetic acid solution, and 100 mL TriseHCl was used to dissolve the SRB. Absorbance at 540 nm was measured in a 96-well multiwell-plate reader (TECAN). 2.7. Photo-bleaching experiments HeLa cells were irradiated with 0, 60, 120, 240, 480 s under different excitation wavelength, and the fluorescent images were captured by a confocal microscope (LSM700). The fluorescence intensity per cell was calculated by the formula: (Total fluorescence value - background fluorescence value)/cell number. 3. Results and discussion 3.1. Design and synthesis of the probe Coumarin derivatives are popular with their good photostability and high fluorescent quantum yield, and have been widely used in fluorescent probes and dyes [42,43]. The substituent groups at 3, 4, 7, 8-position of coumarin moiety have important effect on its fluorescence. Therefore, we use one electron-withdrawing group at the 3-position and one electron-donating group at the 7-position to induce strong fluorescence. Meanwhile, integrating two reaction sites with an aldehyde group and a chlorine leaving group into a coumarin-based fluorescent probe can guarantee the synergetic dual-response to discrimination of GSH and Cys/Hcy. The probe ACCA, 4-chloro-7-(diethylamino)-3-carbaldehyde coumarin, was easily synthesized as shown in Scheme S1 and confirmed by NMR, HRMS, IR and X-ray single crystal diffraction (CCDC NO. 1417230) [41]. 3.2. Mechanism and reaction time of the probe in sensing biothiols Aldehyde and chlorine groups were combined as two neighboring reaction sites, then a series of cascade reaction involving substitution, rearrangement, cyclization and condensation were figured to come. Scheme 1 illustrated the proposed sensing mechanism. Firstly, a chlorine leaving group in 4-position of coumarin would be replaced by the mercapto group of thiols to produce thio-coumarin (2a, 2b and 5), and like Smiles rearrangement, 2a and 2b would be rearranged to form amino-coumarin 3a and 3b [38e40]. On the contrary, 5 cannot be rearranged but intramolecular imine condensation can take place to form a cyclic iminium cation 6 [40,41]. The primary amine of compound 5 reacts
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more easily with aldehyde to form an imine instead of attacking carbon in 4-position to form a ten-membered cyclic intermediate. The imine could be a steadier structure because of the hydrogen bond between NH and the carbonyl of coumarin [44]. Now the products 3a and 3b still had a free aldehyde group. We speculated that the reaction of excess Cys and Hcy with probe ACCA would produce 4a and 4b, respectively [45e47]. The presumptive products 3a and 4a of the reaction of the probe with Cys were verified by electrospray ionization mass spectral analyses which showed the peaks at m/z 365.1212 and 468.1300 (calcd. [MþH]þ 365.1171 and 468.1263, Scheme S1, Fig. S1), respectively. Similar to the reaction of ACCA with Cys, the products of the reaction of the probe with Hcy or GSH also can be verified, the peaks of products 3b and 4b are 380.1618 and 496.1518 (calcd. [Mþ2H]2þ 380.1406, 496.1576, Fig. S2); the peaks of products 5 and 6 are 551.1801 and 533.1656 (calcd. [MþH]þ 551.1812 and [M]þ 533.1701, Fig. S3). Moreover, the reaction process was traced by HPLC-MS. The HPLC peak of probe ACCA was observed at 9.43 min retention time (Fig. S4a). We injected the incubation mixture of 5 mM ACCA with 1 mM Cys at 24 C into a HPLC-MS system for analysis. Obviously, the HPLC peak of ACCA decreased within 1 min and a new peak at 7.67 min retention time appeared, corresponding to 3a (Fig. S4b). 40 min later, the probe or compound 3a reacted with excessive Cys to produce compound 4a, corresponding to another new peak appeared at 5.46 min retention time. According to the abundance of peak, 3a is the main product (Fig. S4c). Furthermore, the products of the probe with GSH were verified by HPLC-MS which showed the peaks at 5.01 and 4.56 min retention time, corresponding to 5 and 6, respectively (Fig. S5). Finally, the intensity of the peaks showed that compound 5 and 6 is the main products. On the other hand, the time-dependent spectra of probe ACCA with GSH, Cys and Hcy were evaluated in PBS buffer solution at room temperature, which also evidenced the sensing mechanism. The absorption of probe ACCA free is 457 nm. Addition of Cys to the solution of probe ACCA led to the absorption at 457 nm quenching, followed by an increase of a blue-shifted new absorption at 379 nm (Fig. S6a). Compared to the model compound A (Absmax ¼ 372 nm) (Scheme S3), the absorption at 379 nm should be assigned to the coumarin substituted by amino at 4-position [48]. It was difficult to monitor the S substitute product (2a) by UVevis and HPLC-MS, but based on the reported mechanism [41], compound 2a should be in existence momently. The addition of Cys to the solution of probe ACCA led to the absorption at 457 nm quenching, followed by an increase of a new absorption at 379 nm (Fig. S6a). Normally, the addition of Cys led to the decrease of the emission at 503 nm with
Scheme 1. Proposed reaction mechanism of the probe with Cys, Hcy, and GSH.
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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460 nm excitation and the increase of the emission at 470 nm with 360 nm excitation (Fig. S6b,c). After the emission at 503 nm totally quenched in 34 min, the emission at 470 nm still slow increased. This phenomenon proved that the sense mechanism initially involved substitution-rearrangement between probe ACCA and Cys followed by cyclization reaction. Next, addition of GSH to the solution of probe ACCA led to the decrease of the initial absorption peak at 457 nm, followed by a simultaneous increase of two new absorptions at 383 and 489 nm (Fig. 1a). After 5 min, the absorption at 383 nm remains increasing, but along with the absorption at 489 decreased. The higher absorption at 489 nm could be assigned to compound 5, and then the absorption at 489 nm decreased as compound 5 transformed to a cyclic iminium cation 6. The phenomena correspond to model compounds B (Absmax ¼ 468 nm) and C (Absmax ¼ 486 nm) [49]. The S substitute product (5) induces a red-shift, and the cyclic iminium cation (6) from the intracondensation of compound 5 brings a further red-shift. Interestingly, the probe alone showed an emission at 503 nm, however, the addition of GSH led to the decrease of the emission at 503 nm along with the increase of a new emission at 546 nm, which could be assigned to 5 and 6, compared with reference compound B and C, and reached equilibrium within 40 min (Fig. 1b). Therefore, the time-dependent fluorescence spectra showed that the sensing time of the probe toward biothiols all reached equilibrium within 60 min. 3.3. pH effect The pH effects on the detection of biothiols with ACCA were measured in aqueous solution with dual excitation wavelength (Fig. S7). Probe ACCA was stable enough in the range of pH 5.0e10.0. The ratiometric fluorescence response (I546/I503) of ACCA toward GSH could function and stabilize in the range of pH 7.0e8.0, but probe ACCA did not respond toward Cys/Hcy with 460 nm excitation (Fig. S7a). On the other hand, probe ACCA could respond to Cys and Hcy over a wide pH range from 7.5 to 9.5 with 360 nm excitation (Fig. S7b). 3.4. UVevis spectra studies of probe ACCA with biothiols All the samples were investigated in PBS buffer solution without any toxic organic solvents, which showed excellent quality and potential applications in living cell image. Probe ACCA alone was yellow with an absorption maximum at 457 nm (Fig. S8). Upon addition of GSH to the solution of ACCA, absorbance at 457 nm disappeared, while two new peaks at 383 nm and 489 nm appeared. And the solution turned into red. Similarly, addition of
Cys and Hcy induced a new peak at 379 nm, and the solution was pale-yellow. Therefore, probe ACCA could discriminate GSH and Cys/Hcy by visual determination. With gradually addition of 1.0 equiv. GSH to the solution of probe ACCA, the absorption band at 457 nm decreased accompanying red-shift to 489 nm (Fig. 2a). Further, increasing the concentrations of GSH (1.5e10.0 equiv.), the absorption band at 489 nm decreased and that at 383 nm increased (Fig. 2b). The linear ratio responses (A489/A457) were in the range of 2e30 mM to give a detection limit of 0.35 mM (Fig. 2d). Upon addition of Cys, the absorption at 457 nm decreased and a new band at 379 nm gradually increased (Fig. 3a). The absorption ratio (A379/A457) is linearly proportional to the concentrations of Cys from 8 to 80 mM, and the detection limit for Cys was 0.22 mM (Fig. 3b). Similar results were observed for Hcy, and the linearly proportional to the concentration of Hcy (A379/A457) ranged from 8 to 100 mM and the limit of detection was 0.32 mM (Fig. S9). The larger response range of Cys/ Hcy than that of GSH is in accordance with the speculated sensing mechanism, and the whole titration progress also identified with that. 3.5. Fluorescence spectra studies of probe ACCA with biothiols Furthermore, we observed the fluorescence response of the probe ACCA toward biothiols and other analytes in PBS solution at room temperature. When various analytes (GSH, Cys, Hcy, arg, asp, glu, gly, his, lys, pro, ser, thr, trp, Ca2þ, Fe3þ, Kþ, Mg2þ, Naþ, Zn2þ, H2O2, glucose) were added to the solution of probe ACCA, the obvious fluorescence peak remained at 503 nm except for biothiols with 460 nm excitation. The addition of GSH induced a new emission peak at 546 nm, accompanied by a distinct change from green to red fluorescence; but the addition of Cys/Hcy induced fluorescence quenching (Fig. 4a). Further, we recorded the fluorescence spectra at 360 nm excitation. Obviously, probe ACCA itself had almost no emission, while Cys/Hcy caused a new fluorescence peak at 470 nm and showed blue fluorescence (Fig. 4b). The intensity ratio values (I546/I503) of probe ACCA with GSH increased 8fold (from 0.47 to 3.93) at 460 nm excitation wavelength. With dual excitation wavelengths, the ratio values (I470/I503) of probe ACCA with Cys increased 378-fold (from 0.073 to 27.60), and the maximum ratio values of the probe with GSH was 1.86. Therefore, there is scarce interference from GSH when selective detection of Cys/Hcy with excitation at 360 nm. Then, the effects of interference from the above-mentioned analytes on the detection of GSH and Cys/Hcy were evaluated at two excitation wavelengths (Fig. S10). The scarce interference demonstrated that probe ACCA could be used for simultaneous ratiometric detection of GSH and Cys/Hcy
Fig. 1. (a) Time-dependent absorbance changes of probe ACCA (20 mM) with GSH (10 equiv.) in PBS buffer (pH 7.4); (b) Time-dependent fluorescence changes of probe ACCA (10 mM) with GSH (10 equiv.) in PBS buffer (pH 7.4, lex ¼ 460 nm, slit: 10.0 nm/6.0 nm), the insert plot of fluorescence intensity at 503 (-) and 546 ( ) is a function of GSH at different time.
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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Fig. 2. UVevis absorption titration spectra of ACCA (20 mM) toward different concentration of (a) GSH (0e1.0 equiv.), (b) GSH (1.5e10.0 equiv.) and (c) GSH (0e10.0 equiv.) in PBS buffer solution (pH 7.4); (d) The plot of ratiometric responses (A489/A457) of ACCA (20 mM) vs equivalents of GSH. Data are mean ± SE (bars) (n ¼ 3).
Fig. 3. (a) UVeVis absorption titration spectra of ACCA (20 mM) toward different concentration of Cys (0e10.0 equiv.) in PBS buffer solution (pH 7.4); (b) The plot of ratiometric responses (A379/A457) of ACCA (20 mM) vs equivalents of Cys. Data are mean ± SE (bars) (n ¼ 3).
with dual emission bands. Upon addition of GSH (0e100 mM), the emission intensity at 503 nm gradually decreased with the appearance of a new emission peak at 546 nm (Fig. 5). Meanwhile a clear iso-emission point was observed at 533 nm. The intensity ratio (I546/I503) lineally increased when the concentration of GSH changed from 0 to 40 mM and the limit of detection was determined to be 0.18 mM (Fig. 5b). We further investigated the fluorescence titration response of probe ACCA toward Hcy/Cys at 360 and 460 nm excitations. Addition of increasing concentrations of Cys to the solution of the probe quenched fluorescence at 503 nm rapidly and elicited a dramatic fluorescence enhancement at 470 nm slowly (Fig. 6). After addition of 4.0 equiv. Cys, the emission at 503 nm totally quenched, but that
at 470 nm still weakly increased. The phenomena were accordance with two reaction steps. There is a good linearity in the range 5e40 mM, and the limit of detection is 0.08 mM (Fig. 6c). With single excitation wavelength 360 nm, the probe is a turn-on fluorescent probe for Cys and the limit of detection is 0.16 mM (Fig. S11). The similar phenomena of probe ACCA with Hcy were shown in Fig. S12 and the limit of detection is 0.09 mM based on the good linearity in the range 5e40 mM (Fig. S13). 3.6. Cell imaging of probe ACCA In view of the outstanding property of probe ACCA in vitro, we investigated the capability of the probe to selectively respond to
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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Fig. 4. Fluorescence spectra of ACCA (10 mM) with various analytes (10 equiv.) in PBS buffer solution (pH 7.4, slit: 10.0 nm/5.0 nm) (a) lex ¼ 460 nm; (b) lex ¼ 360 nm. Inset photograph is ACCA with or without biothiols in UV light.
Fig. 5. (a) Fluorescence spectra of ACCA (10 mM) with GSH (0e10.0 equiv.) in PBS buffer (pH 7.4, lex ¼ 460 nm, slit: 10.0 nm/6.0 nm); (b) The plot of ratiometric responses (I546/I503) of ACCA (10 mM) vs equivalents of GSH. Data are mean ± SE (bars) (n ¼ 3).
Fig. 6. Fluorescence spectra of ACCA (10 mM) with Cys (0e10.0 equiv.) in PBS buffer (pH 7.4, slit: 10.0 nm/6.0 nm) (a) lex ¼ 360 nm; (b) lex ¼ 460 nm; (c) The plot of ratiometric responses (I470/I503) of ACCA (10 mM) vs equivalents of Cys. Data are mean ± SE (bars) (n ¼ 3).
GSH and Cys with dual excitation wavelengths in vivo. Firstly, sulforhodamine B assays were performed in HeLa cells with different concentrations of probe ACCA for 24 h to examine its cytotoxicity (Fig. S14). The unchanging relative viability of HeLa cells demonstrated that the probe was of low toxicity to further imaging experiments. When HeLa cells were incubated with probe ACCA (10 mM) for 40 min, they gave bright green fluorescence and faint red fluorescence (Fig. 7). On the contrary, HeLa cells were pretreated with 0.5 mM GSH for 40 min, and then incubated with probe ACCA for another 40 min. A bright fluorescence in red channel was observed and that in green channel is dark (Fig. 7). The marked enhancement of the ratio of red and green channels exhibited probe ACCA could respond to GSH in dual-color imaging.
Subsequently, the cell imaging of probe ACCA with different concentrations of Cys was performed. As shown in Fig. 8, fluorescence intensity increased with increasing of the concentrations of Cys, and then the quantified data of fluorescence intensity demonstrated that probe ACCA could respond to Cys with turn-on fluorescence. To confirm the fluorescence signals from cells attributed to the reaction of ACCA with biothiols, HeLa cells were preincubated with N- ethylmaleimide (50 mM) for 20 min to remove the endogenous intracellular thiols, and then incubated with probe ACCA for 40 min. The probe alone show green stronger fluorescence and weaker blue fluorescence (Figs. S15, S16). Subsequently, the photostability of ACCA at different channels with two excitation wavelengths was evaluated (Figs. S17, S18). The stable quantified
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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Fig. 7. (a) Fluorescence ratio images of HeLa cells. Left: HeLa cells were treated with 10 mM ACCA for 40 min; right: HeLa cells were pre-incubated with 0.5 mM GSH for 40 min, then with 10 mM ACCA for 40 min. The exciting light was 488 nm, and the emission range of the green channel was 488e520 nm and the red channel was 560e700 nm. (b) The ratio of fluorescence intensity (blue/red). Results are presented as means ± SE. (n > 3, **, p < 0.01 vs. control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. (a) Fluorescence images of HeLa cells. HeLa cells were pre-incubated with different concentrations of Cys (0, 0.1, 0.5, 1 and 5 mM) for 40 min, then with 10 mM ACCA for 40 min. The exciting light of the blue channel was 405 nm, the emission range of that was 405e475 nm. (b) Fluorescence intensity quantitation. Results are presented as means ± SE. (n > 3, **, p < 0.01 vs. control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
data of fluorescence intensity display that probe ACCA has excellent photostability.
[3]
4. Conclusions In summary, we have developed a colorimetric and ratiometric fluorescent probe for detecting simultaneously GSH and Cys/Hcy in PBS buffer solution. The chlorinated coumarin-aldehyde fluorescent probe with two reaction sites, could distinguish GSH and Cys/ Hcy from dual excitation wavelengths and emission channels. The sense mechanism was confirmed by HRMS, HPLC-MS, timedependent spectra and fluorescence titration spectra. The significant different colors were observed by visual determination no matter in vivo or in vitro. The probe had a low detection limit about 108 mM for biothiols, furthermore, probe ACCA was successfully used in living cell ratio imaging. Acknowledgments This study was supported by the Natural Science Foundation of Shandong Province (ZR2014BM004) and the National Natural Science Foundation of China (91313303). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2015.10.023. References [1] K. Kusmierek, G. Chwatko, R. Glowacki, E. Bald, Determination of endogenous thiols and thiol drugs in urine by HPLC with ultraviolet detection, J. Chromatogr. B 877 (2009) 3300e3308. [2] A. Pastore, A. Alisi, G. di Giovamberardino, A. Crudele, S. Ceccarelli, N. Panera, C. Dionisi-Vici, V. Nobili, Plasma levels of homocysteine and cysteine increased
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A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine Xi Dai a, 1, Zhao-Yang Wang b, 1, Zhi-Fang Du a, c, Jie Cui a, Jun-Ying Miao b, **, Bao-Xiang Zhao a, * a b c
Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China Taishan College, Shandong University, Jinan 250100, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A colorimetric, ratiometric and water-soluble fluorescent probe was developed. The probe could simultaneous distinguish GSH and Cys/Hcy by visual determination. This probe was successfully used to achieve living cell ratio imaging.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 August 2015 Received in revised form 16 October 2015 Accepted 21 October 2015 Available online xxx
A chlorinated coumarin-aldehyde was developed as a colorimetric and ratiometric fluorescent probe for distinguishing glutathione (GSH), cystenine (Cys) and homocysteine (Hcy). The GSH-induced substitution-cyclization and Cys/Hcy-induced substitution-rearrangement cascades lead to the corresponding thiol-coumarin-iminium cation and amino-coumarin-aldehyde with distinct photophysical properties. The probe can be used to simultaneously detect GSH and Cys/Hcy by visual determination based on distinct different colors e red and pale-yellow in PBS buffer solution by two reaction sites. From the linear relationship of fluorescence intensity and biothiols concentrations, it was determined that the limits of detection for GSH, Hcy and Cys are 0.08, 0.09 and 0.18 mM, respectively. Furthermore, the probe was successfully used in living cell imaging with low cell toxicity. © 2015 Published by Elsevier B.V.
Keywords: Ratiometric Fluorescent probe Glutathione Coumarin Cell imaging
1. Introduction Low molecular weight thiols, like glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), are crucial cellular components that
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (B.-X. Zhao). 1 Equal contribution.
(J.-Y.
Miao),
[email protected]
play numerous roles in metabolism and homeostasis as well as in varying functions of physiological and pathological processes [1e3]. Abnormal levels of biothiols are related to many diseases, such as neurodegenerative diseases, liver damage and renal diseases [4e7]. Fluorescent probes are powerful molecular tools for monitoring trace amounts of analytes in live cells or tissues because of their simplicity and high sensitivity [8e20]. However, the discrimination between three biothiols is still challenging because of their similarity in structure and reactivity. Numerous fluorescent probes have been developed for the
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Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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detection of biothiols by utilizing different mechanisms, such as cleavage reaction, Michael addition, redox reaction and nucleophilic substitution [21e24]. The addition and cyclization reaction of Cys/Hcy with aldehydes or acrylates and native chemical ligation (NCL) reaction have been developed to discriminate Cys and Hcy over GSH [25e35]. Relatively speaking, the discriminating of GSH over Cys/Hcy remains a tough mission. Our group reported two fluorescent probes to detect GSH over Cys and Hcy using the acrylate derivatives [36,37]. The Michael addition products of the probes with GSH have strong fluorescence; on the contrary, the addition-cyclization products of the probes with Cys/Hcy are nonfluorescence. Yang group demonstrated a GSH fluorescent probe based on the thiol-halogen nucleophilic substitution reaction between GSH and a monochlorinated Bodipy [38]. The attractive strategy is distinct from that induced nucleophilic substitutionrearrangement cascade reaction by Cys/Hcy. In recent years, some fluorescent probes have been designed based on this strategy [39e41]. Furthermore, a chlorinated coumarin-hemicyanine dye with three potential reaction sites was reported for sensing GSH and Cys through different emission channels [41]. However, a simple and effective fluorescent probe for simultaneously sensing GSH, Cys and Hcy is extremely necessary. Herein, we combine the above strategies to study a colorimetric and ratiometric fluorescent probe, 4-chloro-7-(diethylamino)-3carbaldehyde coumarin, with two reaction sites. The probe could simultaneously distinguish GSH and Cys/Hcy through different emission channels in PBS buffer solution. No matter in vivo or in vitro, we could observe the significant different colors by visual determination. Furthermore, the probe was successfully used in living cell ratio imaging. 2. Experimental 2.1. Apparatus and chemicals Thin-layer chromatography (TLC) was conducted on silica gel 60 F254 plates (Merck KGaA) and column chromatography was conducted over silica gel (mesh 200e300). 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were carried out on a Bruker Avance 400 spectrometer, using DMSO as solvent and tetramethylsilane (TMS) as internal standard. Melting points were determined on an XD-4 digital micro melting point apparatus. IR spectra were performed with an IR spectrophotometer VERTEX 70 FT-IR (Bruker Optics). HRMS spectra were obtained on a Q-TOF6510 spectrograph (Agilent). UVevis spectra were measured by using a Hitachi U-4100 spectrophotometer. Fluorescent measurements were recorded on a PerkineElmer LS-55 luminescence spectrophotometer. Quartz cuvettes with a 1 cm path length and 3 mL volume were used for all measurements. The pH measurements were done on a Model PHS3C pH meter. Unless otherwise stated, all reagents were purchased from J&K, Sinopharm Chemical Reagent Co. and Kermel and used without further purification. Twice-distilled water was used throughout all experiments. 2.2. Synthesis of 4-chloro-7-(diethylamino)-3-carbaldehyde coumarin (probe ACCA) 7-(Diethylamino)-4-hydroxy coumarin (4) was synthesized according to literature methods [41]. Under nitrogen, dry DMF (2.5 mL) was added dropwise to POCl3 (1.0 mL) at room temperature. After stirred for 30 min, compound 4 (3 mmol) dissolved in 10 mL DMF was added dropwise to the above solution. The solution was stirred at 60 C for 10 h until TLC indicated the end of the reaction. After cooling to room temperature, the mixture was poured into ice water (50 mL), and was adjusted to neutral with diluted
NaOH solution. The ensuing precipitate was filtered and washed with water. The crude product was purified by the recrystallization from absolute ethanol to give 4-chloro-7-(diethylamino)-3carbaldehyde coumarin in 73% yield. Yellow solid; mp: 138e140 C. IR (KBr) n: 3090 (ArH), 2972 (eCH3), 2928 (eCH2e), 2879 (HeC]O), 1719 (C]O), 1177(CeOeC). 1H NMR (DMSO, 400 MHz): d ¼ 10.30 (1H, s, eCHO), 7.85 (1H, d, J ¼ 9.3 Hz, ArH), 6.70 (1H, dd, J ¼ 2.4 and 9.3 Hz, ArH), 6.44 (1H, d, J ¼ 2.4 Hz, ArH), 3.49 (4H, q, J ¼ 7.2 Hz, eCH2e), 1.26 (6H, t, J ¼ 7.2 Hz, eCH3). 13C NMR (100 MHz, DMSO-d6): 12.44 (2C), 45.40 (2C), 96.75, 107.82, 110.60, 111.15, 129.31, 153.58, 154.00, 156.45, 159.95, 187.02. HRMS: m/z [MþH]þ calcd for [C14H14ClNO3 þ H]þ: 280.0740, found 280.0731. 2.3. Preparation for UVevis and fluorescence spectral measurements Probe ACCA was dissolved in DMSO to afford the stock solution (2 103 M). Amino acids (GSH, Cys, Hcy, arg, asp, glu, gly, his, lys, pro, ser, thr, trp), cation (Ca2þ, Fe3þ, Kþ, Mg2þ, Naþ, Zn2þ), H2O2, glucose were all dissolved in deionized water at a concentration of 2 102 M for absorption and fluorescence spectral analysis. Test solutions were prepared by placing 100 mL or 50 mL of the stock solution and an appropriate aliquot of each testing species solution into a 10-mL volumetric flask, and the solution was diluted to 10 mL in an aqueous solution with pH 7.4 (PBS buffer). The resulting solution was shaken well and incubated for 60 min at room temperature before recording the spectra. 2.4. HPLC-MS traces HPLC-MS spectra were recorded with a Thermo LCQ Fleet coupled with a thermo Ultimate 3000 HPLC system. HPLC analysis was accomplished with an Atlanti C18 reversed-phase column (2.1 150 mm), with CH3CN (0.1% of HCOOH) and water as the eluent. Injection volume: 5 mM; mobile phase: A-0.1% methanoic acid/acetonitrile, B-water; gradient elution: 0e7.9 min, 20%A; 8e15 min 90%A; flow rate: 0.2 mL min1. 2.5. Cell culture and cell imaging HeLa cells were cultured in RPMI-1640 with 10% CBS at 2 104 cells per well. The probe was dissolved in DMSO at a storage concentration of 10 mM. Cells were adherent-cultured in 24-well culture plates for 12 h. After washing away the culture medium with phosphate-buffered saline solution (PBS), HeLa cells of control group were loaded with 10.0 mM probe solution at 37 C for 40 min. Other test groups were pretreated with GSH (0.5 mM) or Cys (0.1, 0.5, 1.0 and 5.0 mM) at 37 C for 40 min, followed by incubation with 10.0 mM probe solution for 40 min. Then washed 2 times with PBS and underwent imaging measurement by ultraviolet light with a confocal microscope (LSM700). Fluorescence values were quantified by the fluorescence analysis software Image J. The exciting light was 405 nm for the emission range of the blue channel (405e475 nm). The exciting light was 488 nm for the emission range of the green channel (488e520 nm) and the red channel was (560e700 nm). 2.6. Cytotoxicity assay The in vitro cytotoxicity of the probe to HeLa cells was measured by a standard sulforhodamine B (SRB) assay. Briefly, HeLa cells were loaded in 96-well culture plates at 4 104 cells per well. After culture for 24 h, cells were incubated with fresh RPMI 1640 containing 1.0, 5.0 and 10.0 mM probe for 24 h, respectively. Then cells were fixed with 4% TCA for 1 h at 4 C, and then washed 5 times
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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with deionized water; 50 mL SRB was added to each well, and after sufficient reaction with cells, the remaining SRB was removed by washing each well with 1% acetic acid solution, and 100 mL TriseHCl was used to dissolve the SRB. Absorbance at 540 nm was measured in a 96-well multiwell-plate reader (TECAN). 2.7. Photo-bleaching experiments HeLa cells were irradiated with 0, 60, 120, 240, 480 s under different excitation wavelength, and the fluorescent images were captured by a confocal microscope (LSM700). The fluorescence intensity per cell was calculated by the formula: (Total fluorescence value - background fluorescence value)/cell number. 3. Results and discussion 3.1. Design and synthesis of the probe Coumarin derivatives are popular with their good photostability and high fluorescent quantum yield, and have been widely used in fluorescent probes and dyes [42,43]. The substituent groups at 3, 4, 7, 8-position of coumarin moiety have important effect on its fluorescence. Therefore, we use one electron-withdrawing group at the 3-position and one electron-donating group at the 7-position to induce strong fluorescence. Meanwhile, integrating two reaction sites with an aldehyde group and a chlorine leaving group into a coumarin-based fluorescent probe can guarantee the synergetic dual-response to discrimination of GSH and Cys/Hcy. The probe ACCA, 4-chloro-7-(diethylamino)-3-carbaldehyde coumarin, was easily synthesized as shown in Scheme S1 and confirmed by NMR, HRMS, IR and X-ray single crystal diffraction (CCDC NO. 1417230) [41]. 3.2. Mechanism and reaction time of the probe in sensing biothiols Aldehyde and chlorine groups were combined as two neighboring reaction sites, then a series of cascade reaction involving substitution, rearrangement, cyclization and condensation were figured to come. Scheme 1 illustrated the proposed sensing mechanism. Firstly, a chlorine leaving group in 4-position of coumarin would be replaced by the mercapto group of thiols to produce thio-coumarin (2a, 2b and 5), and like Smiles rearrangement, 2a and 2b would be rearranged to form amino-coumarin 3a and 3b [38e40]. On the contrary, 5 cannot be rearranged but intramolecular imine condensation can take place to form a cyclic iminium cation 6 [40,41]. The primary amine of compound 5 reacts
3
more easily with aldehyde to form an imine instead of attacking carbon in 4-position to form a ten-membered cyclic intermediate. The imine could be a steadier structure because of the hydrogen bond between NH and the carbonyl of coumarin [44]. Now the products 3a and 3b still had a free aldehyde group. We speculated that the reaction of excess Cys and Hcy with probe ACCA would produce 4a and 4b, respectively [45e47]. The presumptive products 3a and 4a of the reaction of the probe with Cys were verified by electrospray ionization mass spectral analyses which showed the peaks at m/z 365.1212 and 468.1300 (calcd. [MþH]þ 365.1171 and 468.1263, Scheme S1, Fig. S1), respectively. Similar to the reaction of ACCA with Cys, the products of the reaction of the probe with Hcy or GSH also can be verified, the peaks of products 3b and 4b are 380.1618 and 496.1518 (calcd. [Mþ2H]2þ 380.1406, 496.1576, Fig. S2); the peaks of products 5 and 6 are 551.1801 and 533.1656 (calcd. [MþH]þ 551.1812 and [M]þ 533.1701, Fig. S3). Moreover, the reaction process was traced by HPLC-MS. The HPLC peak of probe ACCA was observed at 9.43 min retention time (Fig. S4a). We injected the incubation mixture of 5 mM ACCA with 1 mM Cys at 24 C into a HPLC-MS system for analysis. Obviously, the HPLC peak of ACCA decreased within 1 min and a new peak at 7.67 min retention time appeared, corresponding to 3a (Fig. S4b). 40 min later, the probe or compound 3a reacted with excessive Cys to produce compound 4a, corresponding to another new peak appeared at 5.46 min retention time. According to the abundance of peak, 3a is the main product (Fig. S4c). Furthermore, the products of the probe with GSH were verified by HPLC-MS which showed the peaks at 5.01 and 4.56 min retention time, corresponding to 5 and 6, respectively (Fig. S5). Finally, the intensity of the peaks showed that compound 5 and 6 is the main products. On the other hand, the time-dependent spectra of probe ACCA with GSH, Cys and Hcy were evaluated in PBS buffer solution at room temperature, which also evidenced the sensing mechanism. The absorption of probe ACCA free is 457 nm. Addition of Cys to the solution of probe ACCA led to the absorption at 457 nm quenching, followed by an increase of a blue-shifted new absorption at 379 nm (Fig. S6a). Compared to the model compound A (Absmax ¼ 372 nm) (Scheme S3), the absorption at 379 nm should be assigned to the coumarin substituted by amino at 4-position [48]. It was difficult to monitor the S substitute product (2a) by UVevis and HPLC-MS, but based on the reported mechanism [41], compound 2a should be in existence momently. The addition of Cys to the solution of probe ACCA led to the absorption at 457 nm quenching, followed by an increase of a new absorption at 379 nm (Fig. S6a). Normally, the addition of Cys led to the decrease of the emission at 503 nm with
Scheme 1. Proposed reaction mechanism of the probe with Cys, Hcy, and GSH.
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460 nm excitation and the increase of the emission at 470 nm with 360 nm excitation (Fig. S6b,c). After the emission at 503 nm totally quenched in 34 min, the emission at 470 nm still slow increased. This phenomenon proved that the sense mechanism initially involved substitution-rearrangement between probe ACCA and Cys followed by cyclization reaction. Next, addition of GSH to the solution of probe ACCA led to the decrease of the initial absorption peak at 457 nm, followed by a simultaneous increase of two new absorptions at 383 and 489 nm (Fig. 1a). After 5 min, the absorption at 383 nm remains increasing, but along with the absorption at 489 decreased. The higher absorption at 489 nm could be assigned to compound 5, and then the absorption at 489 nm decreased as compound 5 transformed to a cyclic iminium cation 6. The phenomena correspond to model compounds B (Absmax ¼ 468 nm) and C (Absmax ¼ 486 nm) [49]. The S substitute product (5) induces a red-shift, and the cyclic iminium cation (6) from the intracondensation of compound 5 brings a further red-shift. Interestingly, the probe alone showed an emission at 503 nm, however, the addition of GSH led to the decrease of the emission at 503 nm along with the increase of a new emission at 546 nm, which could be assigned to 5 and 6, compared with reference compound B and C, and reached equilibrium within 40 min (Fig. 1b). Therefore, the time-dependent fluorescence spectra showed that the sensing time of the probe toward biothiols all reached equilibrium within 60 min. 3.3. pH effect The pH effects on the detection of biothiols with ACCA were measured in aqueous solution with dual excitation wavelength (Fig. S7). Probe ACCA was stable enough in the range of pH 5.0e10.0. The ratiometric fluorescence response (I546/I503) of ACCA toward GSH could function and stabilize in the range of pH 7.0e8.0, but probe ACCA did not respond toward Cys/Hcy with 460 nm excitation (Fig. S7a). On the other hand, probe ACCA could respond to Cys and Hcy over a wide pH range from 7.5 to 9.5 with 360 nm excitation (Fig. S7b). 3.4. UVevis spectra studies of probe ACCA with biothiols All the samples were investigated in PBS buffer solution without any toxic organic solvents, which showed excellent quality and potential applications in living cell image. Probe ACCA alone was yellow with an absorption maximum at 457 nm (Fig. S8). Upon addition of GSH to the solution of ACCA, absorbance at 457 nm disappeared, while two new peaks at 383 nm and 489 nm appeared. And the solution turned into red. Similarly, addition of
Cys and Hcy induced a new peak at 379 nm, and the solution was pale-yellow. Therefore, probe ACCA could discriminate GSH and Cys/Hcy by visual determination. With gradually addition of 1.0 equiv. GSH to the solution of probe ACCA, the absorption band at 457 nm decreased accompanying red-shift to 489 nm (Fig. 2a). Further, increasing the concentrations of GSH (1.5e10.0 equiv.), the absorption band at 489 nm decreased and that at 383 nm increased (Fig. 2b). The linear ratio responses (A489/A457) were in the range of 2e30 mM to give a detection limit of 0.35 mM (Fig. 2d). Upon addition of Cys, the absorption at 457 nm decreased and a new band at 379 nm gradually increased (Fig. 3a). The absorption ratio (A379/A457) is linearly proportional to the concentrations of Cys from 8 to 80 mM, and the detection limit for Cys was 0.22 mM (Fig. 3b). Similar results were observed for Hcy, and the linearly proportional to the concentration of Hcy (A379/A457) ranged from 8 to 100 mM and the limit of detection was 0.32 mM (Fig. S9). The larger response range of Cys/ Hcy than that of GSH is in accordance with the speculated sensing mechanism, and the whole titration progress also identified with that. 3.5. Fluorescence spectra studies of probe ACCA with biothiols Furthermore, we observed the fluorescence response of the probe ACCA toward biothiols and other analytes in PBS solution at room temperature. When various analytes (GSH, Cys, Hcy, arg, asp, glu, gly, his, lys, pro, ser, thr, trp, Ca2þ, Fe3þ, Kþ, Mg2þ, Naþ, Zn2þ, H2O2, glucose) were added to the solution of probe ACCA, the obvious fluorescence peak remained at 503 nm except for biothiols with 460 nm excitation. The addition of GSH induced a new emission peak at 546 nm, accompanied by a distinct change from green to red fluorescence; but the addition of Cys/Hcy induced fluorescence quenching (Fig. 4a). Further, we recorded the fluorescence spectra at 360 nm excitation. Obviously, probe ACCA itself had almost no emission, while Cys/Hcy caused a new fluorescence peak at 470 nm and showed blue fluorescence (Fig. 4b). The intensity ratio values (I546/I503) of probe ACCA with GSH increased 8fold (from 0.47 to 3.93) at 460 nm excitation wavelength. With dual excitation wavelengths, the ratio values (I470/I503) of probe ACCA with Cys increased 378-fold (from 0.073 to 27.60), and the maximum ratio values of the probe with GSH was 1.86. Therefore, there is scarce interference from GSH when selective detection of Cys/Hcy with excitation at 360 nm. Then, the effects of interference from the above-mentioned analytes on the detection of GSH and Cys/Hcy were evaluated at two excitation wavelengths (Fig. S10). The scarce interference demonstrated that probe ACCA could be used for simultaneous ratiometric detection of GSH and Cys/Hcy
Fig. 1. (a) Time-dependent absorbance changes of probe ACCA (20 mM) with GSH (10 equiv.) in PBS buffer (pH 7.4); (b) Time-dependent fluorescence changes of probe ACCA (10 mM) with GSH (10 equiv.) in PBS buffer (pH 7.4, lex ¼ 460 nm, slit: 10.0 nm/6.0 nm), the insert plot of fluorescence intensity at 503 (-) and 546 ( ) is a function of GSH at different time.
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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Fig. 2. UVevis absorption titration spectra of ACCA (20 mM) toward different concentration of (a) GSH (0e1.0 equiv.), (b) GSH (1.5e10.0 equiv.) and (c) GSH (0e10.0 equiv.) in PBS buffer solution (pH 7.4); (d) The plot of ratiometric responses (A489/A457) of ACCA (20 mM) vs equivalents of GSH. Data are mean ± SE (bars) (n ¼ 3).
Fig. 3. (a) UVeVis absorption titration spectra of ACCA (20 mM) toward different concentration of Cys (0e10.0 equiv.) in PBS buffer solution (pH 7.4); (b) The plot of ratiometric responses (A379/A457) of ACCA (20 mM) vs equivalents of Cys. Data are mean ± SE (bars) (n ¼ 3).
with dual emission bands. Upon addition of GSH (0e100 mM), the emission intensity at 503 nm gradually decreased with the appearance of a new emission peak at 546 nm (Fig. 5). Meanwhile a clear iso-emission point was observed at 533 nm. The intensity ratio (I546/I503) lineally increased when the concentration of GSH changed from 0 to 40 mM and the limit of detection was determined to be 0.18 mM (Fig. 5b). We further investigated the fluorescence titration response of probe ACCA toward Hcy/Cys at 360 and 460 nm excitations. Addition of increasing concentrations of Cys to the solution of the probe quenched fluorescence at 503 nm rapidly and elicited a dramatic fluorescence enhancement at 470 nm slowly (Fig. 6). After addition of 4.0 equiv. Cys, the emission at 503 nm totally quenched, but that
at 470 nm still weakly increased. The phenomena were accordance with two reaction steps. There is a good linearity in the range 5e40 mM, and the limit of detection is 0.08 mM (Fig. 6c). With single excitation wavelength 360 nm, the probe is a turn-on fluorescent probe for Cys and the limit of detection is 0.16 mM (Fig. S11). The similar phenomena of probe ACCA with Hcy were shown in Fig. S12 and the limit of detection is 0.09 mM based on the good linearity in the range 5e40 mM (Fig. S13). 3.6. Cell imaging of probe ACCA In view of the outstanding property of probe ACCA in vitro, we investigated the capability of the probe to selectively respond to
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Fig. 4. Fluorescence spectra of ACCA (10 mM) with various analytes (10 equiv.) in PBS buffer solution (pH 7.4, slit: 10.0 nm/5.0 nm) (a) lex ¼ 460 nm; (b) lex ¼ 360 nm. Inset photograph is ACCA with or without biothiols in UV light.
Fig. 5. (a) Fluorescence spectra of ACCA (10 mM) with GSH (0e10.0 equiv.) in PBS buffer (pH 7.4, lex ¼ 460 nm, slit: 10.0 nm/6.0 nm); (b) The plot of ratiometric responses (I546/I503) of ACCA (10 mM) vs equivalents of GSH. Data are mean ± SE (bars) (n ¼ 3).
Fig. 6. Fluorescence spectra of ACCA (10 mM) with Cys (0e10.0 equiv.) in PBS buffer (pH 7.4, slit: 10.0 nm/6.0 nm) (a) lex ¼ 360 nm; (b) lex ¼ 460 nm; (c) The plot of ratiometric responses (I470/I503) of ACCA (10 mM) vs equivalents of Cys. Data are mean ± SE (bars) (n ¼ 3).
GSH and Cys with dual excitation wavelengths in vivo. Firstly, sulforhodamine B assays were performed in HeLa cells with different concentrations of probe ACCA for 24 h to examine its cytotoxicity (Fig. S14). The unchanging relative viability of HeLa cells demonstrated that the probe was of low toxicity to further imaging experiments. When HeLa cells were incubated with probe ACCA (10 mM) for 40 min, they gave bright green fluorescence and faint red fluorescence (Fig. 7). On the contrary, HeLa cells were pretreated with 0.5 mM GSH for 40 min, and then incubated with probe ACCA for another 40 min. A bright fluorescence in red channel was observed and that in green channel is dark (Fig. 7). The marked enhancement of the ratio of red and green channels exhibited probe ACCA could respond to GSH in dual-color imaging.
Subsequently, the cell imaging of probe ACCA with different concentrations of Cys was performed. As shown in Fig. 8, fluorescence intensity increased with increasing of the concentrations of Cys, and then the quantified data of fluorescence intensity demonstrated that probe ACCA could respond to Cys with turn-on fluorescence. To confirm the fluorescence signals from cells attributed to the reaction of ACCA with biothiols, HeLa cells were preincubated with N- ethylmaleimide (50 mM) for 20 min to remove the endogenous intracellular thiols, and then incubated with probe ACCA for 40 min. The probe alone show green stronger fluorescence and weaker blue fluorescence (Figs. S15, S16). Subsequently, the photostability of ACCA at different channels with two excitation wavelengths was evaluated (Figs. S17, S18). The stable quantified
Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023
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Fig. 7. (a) Fluorescence ratio images of HeLa cells. Left: HeLa cells were treated with 10 mM ACCA for 40 min; right: HeLa cells were pre-incubated with 0.5 mM GSH for 40 min, then with 10 mM ACCA for 40 min. The exciting light was 488 nm, and the emission range of the green channel was 488e520 nm and the red channel was 560e700 nm. (b) The ratio of fluorescence intensity (blue/red). Results are presented as means ± SE. (n > 3, **, p < 0.01 vs. control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. (a) Fluorescence images of HeLa cells. HeLa cells were pre-incubated with different concentrations of Cys (0, 0.1, 0.5, 1 and 5 mM) for 40 min, then with 10 mM ACCA for 40 min. The exciting light of the blue channel was 405 nm, the emission range of that was 405e475 nm. (b) Fluorescence intensity quantitation. Results are presented as means ± SE. (n > 3, **, p < 0.01 vs. control). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
data of fluorescence intensity display that probe ACCA has excellent photostability.
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4. Conclusions In summary, we have developed a colorimetric and ratiometric fluorescent probe for detecting simultaneously GSH and Cys/Hcy in PBS buffer solution. The chlorinated coumarin-aldehyde fluorescent probe with two reaction sites, could distinguish GSH and Cys/ Hcy from dual excitation wavelengths and emission channels. The sense mechanism was confirmed by HRMS, HPLC-MS, timedependent spectra and fluorescence titration spectra. The significant different colors were observed by visual determination no matter in vivo or in vitro. The probe had a low detection limit about 108 mM for biothiols, furthermore, probe ACCA was successfully used in living cell ratio imaging. Acknowledgments This study was supported by the Natural Science Foundation of Shandong Province (ZR2014BM004) and the National Natural Science Foundation of China (91313303). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2015.10.023. References [1] K. Kusmierek, G. Chwatko, R. Glowacki, E. Bald, Determination of endogenous thiols and thiol drugs in urine by HPLC with ultraviolet detection, J. Chromatogr. B 877 (2009) 3300e3308. [2] A. Pastore, A. Alisi, G. di Giovamberardino, A. Crudele, S. Ceccarelli, N. Panera, C. Dionisi-Vici, V. Nobili, Plasma levels of homocysteine and cysteine increased
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Please cite this article in press as: X. Dai, et al., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine, Analytica Chimica Acta (2015), http://dx.doi.org/10.1016/j.aca.2015.10.023