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A near-infrared ratiometric fluorescence probe base on spiropyran derivative for pH and its application in living cells
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117350
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A near-infrared ratiometric fluorescence probe base on spiropyran derivative for pH and its application in living cells Kangming Xiong a, Caixia Yin a,⁎, Yongkang Yue a, Fangjun Huo b,⁎ a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China b Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
a r t i c l e
i n f o
Article history: Received 19 February 2019 Received in revised form 24 June 2019 Accepted 7 July 2019 Available online 08 July 2019 Keywords: Intracellular pH Near-infrared Fluorescence probe Cell imaging
a b s t r a c t The intracellular pH has a significant effect on several essential biological processes such as material transfer, enzymatic action, cell apoptosis et al. Thus, it is necessary to monitor pH fluctuation in living cells. Here, we designed a near-infrared ratiometric fluorescence probe for pH detection. The spectroscopic responses of probe to pH variations were investigated in CH3OH/PBS (v/v, 1:1) mixed solution at different pH values. The experimental results showed that the probe is sensitive to acidity, and the pKa of probe is calculated to be 4.85. When the pH was decreased from 9.0 to 1.0, the color of probe solution change from purple to yellow was found by naked eye. Moreover, the probe can be a practical tool for assessing cellular pH by cell imaging. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Intracellular pH has a remarkable impact on cell cycle-related life activities, such as material transfer [1], enzymatic action [2], cell apoptosis [3] et al. Abnormal pH often leads to cell dysfunction, which easily results in cancer [4] and Alzheimer's diseases [5]. Moreover, changes of biomembrane pH can be observed in cancer disease, and its cellular pH is always lower than normal cells [6]. Therefore, accurate determination of cellular pH can provide important information for biomedical studies. Several methods are currently being used to detect pH change: such as UV–vis absorption spectroscopy [7,8], fluorescence spectroscopy [9,10], nuclear magnetic resonance [11,12] and electrochemistry [13,14]. Among these techniques, fluorescence spectroscopy has received more and more attention due to its convenient operation, realtime detection, and high sensitivity [15]. For this purpose, many pH fluorescence probes have been explored [16–23]. However, in complex biological systems, many of them undergo various disadvantages, such as photobleaching, short wavelength and low selectivity upon pH change. So, it is essential to design a near-infrared ratiometric fluorescence probe for pH with the features of good photostability, large stokes shift and high selectivity. In recent years, the NIR fluorescence probes for pH has attracted increasing attention due to the central role of pH in many cellular events and the effects on abnormal cell growth and division found in diseases such as inflammation and cancer [24,25].
⁎ Corresponding authors. E-mail addresses: [email protected] (C. Yin), [email protected] (F. Huo).
https://doi.org/10.1016/j.saa.2019.117350 1386-1425/© 2019 Elsevier B.V. All rights reserved.
In this work, a near-infrared ratiometric fluorescence probe base on spiropyran derivative for pH was reported. Herein, we employed a cyanine dye as a NIR fluorophore and used a spiropyran as a reaction site for closed-loop to open-loop. From the UV–Vis and fluorescence spectra of probe to pH variation, we found that the probe was sensitive to acidity. And the pKa of probe was calculated to be 4.85. In addition, the probe also can be a practical tool for assessing cellular pH by cell imaging. 2. Experimental 2.1. Materials 2 Hydroxy 1 naphthaldehyde, 2,3,3 Trimethylindolenine and iodoethane were purchased from Aladdin Industrial Corporation. Nigericin was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Anionic salts were purchased from Shanghai Experiment Reagent Co., Ltd. All other chemicals used were of analytical grade. Deionized water was used to prepare all aqueous solutions. 2.2. Instruments A pH meter (Mettler Toledo, Switzerland) was used to determine the pH values of phosphate buffer solution and methanol-phosphate mixed solution. Ultraviolet-visible (UV–Vis) spectra were collected on U-3900 UV–Vis spectrophotometer. Fluorescence spectra were recorded on Hitachi F-7000 spectrophotometer. 1H NMR and 13C NMR spectra were recorded on Bruker AVANCE-600 MHz and 150 MHz NMR spectrometers, respectively. HRMS was carried out on a Thermo Scientific
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Q Exactive MS instrument. Fluorescence imaging was conducted using Airyscan confocal laser scanning microscope. 2.3. Preparation and characterization of probe Probe was conveniently synthesized via the condensation of 2 Hydroxy 1 naphthaldehyde (1) and 1 ethyl 2,3,3 trimethyl 3H indolium iodide salt (2) in ethanol. The synthesis route of probe was shown in Scheme 1. The compound 2 was synthesized according to the literature [26]. Compound 1 (0.516 g, 3.0 mmol) and compound 2 (0.945 g, 3.0 mmol) were dissolved in 20.0 mL ethanol, then 100.0 μL acetic acid was added, the mixture was refluxed for 10 h. After that, the reaction mixture was evaporated under reduced pressure and then was purified by column chromatography on silica gel (EtOAc/petroleum ether/CH2Cl2, 1:3:1 v/v/v) to give the probe as pink solid (0.674 g, 65.9% yield). 1H NMR (600 MHz, Chloroform d): δ 8.03 (s, 1H), 7.73 (s, 1H), 7.61 (s, 1H), 7.57–7.56 (d, 1H), 7.51–7.50 (d, 1H), 7.33–7.32 (d, 1H), 7.18–7.17 (d, 1H), 7.09 (s, 1H), 6.96 (s, 1H), 6.84–6.83 (d, 1H), 6.56 (s, 1H), 5.79–5.78 (d, 1H), 3.39–3.37 (t, 1H), 3.22–3.20 (t, 1H), 1.33 (s, 3H), 1.20 (s, 3H), 1.17–1.16 (d, 3H); 13C NMR (150 MHz, Chloroform d): δ 152.5, 147.1, 136.5, 129.9, 129.8, 128.7, 128.6, 127.5, 126.7, 124.7, 123.2, 121.7, 120.6, 118.5, 118.2, 117.6, 110.3, 106.2, 104.6, 52.0, 37.8, 26.0, 20.1, 14.4. HRMS m/z: [M + H]+ calculated for C24H24NO+: 342.18524; measured: 342.18568 (Fig. S1). 2.4. General procedure for spectra measurements The stock solution of probe (2.0 mM) was dissolved in methanol. − − − 2− Stock solutions (200.0 mM) of anionic salts (NO− 3 , I , F , Br , S2O5 , 2− 2− S2O8 , S ), amino acids (Lys, Gly, Leu, Met, Phe, GSH, Hcy, Cys) and metal salts (Na+, K+, Zn2+, Mn2+, Mg2+, Cd2+) were prepared in deionized water. UV–Vis and fluorescence spectra were obtained in 2.0 mL different pH values of CH3OH/PBS (v/v, 1:1) mixed solution at room temperature. The fluorescence excitation and emission slit widths were set as 10 nm and 20 nm, respectively. 2.5. Cell culture and imaging A549 cells and were grown in Dulbecco's modified Eagle's medium (DMEM) in an atmosphere of 5% CO2 at 37 °C. Then A549 cells were plated on 6-well plate and allowed to adhere for 24 h. In the pHdependent fluorescence evolution experiments, probe (20.0 μM) was added to the DMEM (2.0 mL) at pH = 4.0, pH = 6.0 and pH = 8.0 in the presence of 1.0 μM of nigericin for 30 min prior to imaging, respectively. A549 cells were imaged by Airyscan confocal laser scanning microscope. λex = 561 nm, λem = 570–620 nm for the red channel; λex = 488 nm, λem = 520–560 nm for the green channel. 3. Results and discussions 3.1. Solvent dependence study The solvent of a system is often considered as an important factor on affecting spectral properties of probe. The effect of different solvent conditions on the spectral properties of probe was investigated in Fig. S2. As
exhibited in Fig. S2, we could find that CH3OH/H2O = 1000/1000 (v/v) mixed solution was more effective for spectral detection of probe. Here, we chose different pH values of CH3OH/PBS (v/v, 1:1) mixed solution as the system for spectral detection of probe. 3.2. UV–vis and fluorescence spectra of probe to pH variation The UV–Vis and fluorescence spectra of probe to pH variations were tested in CH3OH/PBS (v/v, 1:1) mixed solution at different pH values. As shown in Fig. 1a, under alkaline media (pH 9.0), the probe (30 μM) showed an absorption band in the range 540–590 nm with a maximum at 566 nm. When the pH was decreased from 9.0 to 1.0, the absorption band at 540–590 nm of probe gradually reduced and a new absorption band emerged in the range 400–525 nm with a maximum at 478 nm, accompanied by an isobestic point at 528 nm in the absorption spectra. Meanwhile, there was a color change from purple to yellow which could be easily observed by naked eye. The results hinted that the probe could be used to measure pH as a colorimetric detector. As shown in Fig. 1b, when the pH was decreased from 9.0 to 1.0, the fluorescence intensity of probe (30.0 μM) at 598 nm gradually decreased (λex = 566 nm, slit: 10 nm/20 nm), and a new emission band in the range 520–600 nm emerged (emission peak centered at 558 nm) in Fig. 1c (λex = 478 nm, slit: 10 nm/20 nm). As exhibited in Fig. 1d, nonlinear fitting of sigmoidal curve of fluorescence emission at 558 nm versus pH was plotted over the pH range of 1.0 to 9.0 (λex = 478 nm, slit: 10 nm/20 nm). And the pKa of the probe was calculated to be 4.85 ± 0.19 based on the Henderson-Hasselbalch equation (log[(Imax-I) / (IImin)] = pH-pKa), which provided a basis for detection of pH changes under acidic conditions. On the basis of the linear regression equation F = −4019.7973 ∗ pH + 23,521.4189 (R2 = 0.982), a good linear relationship between the fluorescence intensity at 558 nm and pH in the range 4.4–5.4 was observed. These results clearly showed that the probe could be used to detect the pH variation. 3.3. Selectivity, photostability and reversibility studies of probe Next, we studied the anti-interference ability of probe under pH 1.0 or 9.0 by UV–Vis spectra. The absorption response features of probe to pH fluctuation in the presence of various interferences such as represen− − − 2− 2− 2− tative anions (NO− ), biologically re3 , I , F , Br , S2O5 , S2O8 and S lated amino acids (lysine, glycine, leucine, methionine, phenylalanine, glutathione, homocysteine and cysteine) and important metal ions (Na+, K+, Zn2+, Mn2+, Mg2+ and Cd2+) displayed little influence on the absorption response of probe in CH3OH/PBS (v/v, 1:1) mixed solvent with pH values of 1.0 or 9.0, respectively (Fig. 2a). The photostability of probe was carried out through monitoring the absorption of probe in mixed solvent with pH values at 1.0 and 9.0 for 30 min, respectively. As exhibited in Fig. S3, the absorption remained almost stable, revealing that the probe possessed good photostability. Moreover, the reversibility is basically required for real-time monitoring of pH changes in living organs. The reversibility of probe (30.0 μM) was measured using small volumes of HCl and NaOH solutions back and forth to adjust the pH values of mixed solvent between 1.0 and 9.0, respectively. As shown in Fig. 2b, the emission ratios (I558 nm/I598 nm) of probe changed very little after five cycles at pH = 1.0 and 9.0, respectively. These results indicate that conversion
Scheme 1. The synthesis route of probe.
K. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117350
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Fig. 1. (a) Change of absorption spectra of probe (30.0 μM) at various pH (from 9.0 to 1.0). Inset: photograph of probe solution at pH = 1.0 and 9.0; (b) change of fluorescence spectra of probe (30.0 μM) at various pH (from 9.0 to 1.0) (λex = 566 nm, slit: 10 nm/20 nm); (c) change of fluorescence spectra of probe (30.0 μM) at various pH (from 9.0 to 1.0) (λex = 478 nm, slit: 10 nm/20 nm); (d) nonlinear fitting of sigmoidal curve of fluorescence intensity at 558 nm versus various pH values (from 9.0 to 1.0). Inset: linear relationship between fluorescence intensity at 558 nm and pH in the range pH 4.4–5.4.
− − Fig. 2. (a) Absorption spectra of probe (30.0 μM) at pH 1.0 or 9.0 containing representative anions, biologically related amino acids and important metal ions: 1. Blank; 2. NO− 3 ; 3. I ; 4. F ; 2− + + 2+ 2− 5. Br−; 6. S2O2− ; 7. S O ; 8. S ; 9. Lysine; 10. Glycine; 11. Leucine; 12. Methionine; 13. Phenylalanine; 14. Glutathione; 15. Homocysteine; 16. Cysteine. 17. Na ; 18. K ; 19. Zn ; 20. 5 2 8 Mn2+; 21. Mg2+; 22. Cd2+; (b) evolution of I558/I598 in the mixed solvent with alternating cycles of pH values at 1.0 and 9.0.
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behavior, the 1H NMR spectra of probe in methanol d4 D2O (1:1, v/ v) with small amount of HCl were determined. Fig. 4 showed the 1 H NMR comparison of probe in methanol d4 D2O (1:1, v/v) before and after the addition of HCl. Upon adding HCl to the solution of probe, the chemical shift value of proton Ha was shifted to downfield distinctly from 6.56 to 8.96 ppm because that the junction of pyran group and indolin group is closed-loop to open-loop to form a large conjugate plane. Meanwhile, probe-2 has a large absorption peak at 478 nm and a strong emission peak at 558 nm. Furthermore, the relative molecular mass of probe-2 was tested by high resolution mass spectrometry, with the m/z of probe-2 being 342.18515 for C24H24NO+ (Fig. S4). Thus, the above results are in good agreement with the proposed recognition mechanism. 3.6. Cellular imaging
Fig. 3. Time-course measurements of the UV–vis absorption at 478 nm of probe upon a pH change from 1.0 to 9.0 and then from 9.0 to 1.0.
between acid and alkaline form of the probe is reversible. Thus, the probe can be a practical tool for assessing pH change. 3.4. Time-dependence study To evaluate the applicability of the probe, the time-dependence of probe was measured. As shown in Fig. 3, the absorption of probe was rapidly switched back and forth between pH = 1.0 and pH = 9.0 within 10 s, respectively. The result demonstrates that the probe can be a practical tool for assessing cellular pH in real time.
Furthermore, we examined the application of probe for assessing cellular pH in A549 cells by cell imaging. To ensure the accuracy of pH measurement in the stain, in vivo calibration of fluorescence signals of probe was required. Here we used nigericin to elicit rapid external and internal pH equilibration. This is a standard approach to calibrate the pH value ex vivo [27,28]. As shown in Fig. 5, the A549 cells incubated with probe (20.0 μM) at pH 4.0 exhibit strong green fluorescence in green channel and non-fluorescence in the red channel. When the pH of the media increased from 4.0 to 6.0, the fluorescence in the green channel gradually attenuated and the fluorescence in the red channel appeared. When the pH of the media increased to 8.0, bright fluorescence in the red channel was observed and the fluorescence in the green channel almost disappeared. Above results show that probe can serve as a ratiometric probe for accurate pH imaging of A549 cells. 4. Conclusion
3.5. Proposed mechanism Spiropyran molecule is formed by the condensation reaction of pyran-aldehyde and indolin. It has two isomers, one is that pyran group and indolin group are vertically linked in space forming a closed-loop spiropyran molecule, and its central C atom is sp3 hybridization. Thus the whole molecule is not conjugated. At this time, the two groups of probe are independent of each other, and the probe has only ultraviolet absorption. The other is that the two groups are conjugated together and share electronic configurations. The Cspiro\\O bond at the junction of pyran group and indolin group breaks and forms a large conjugate plane accompanying with the corresponding Cspiro atom changing from sp 3 hybridization to sp2 hybridization, which will produce a large absorption in the visible region and lead to the appearance of fluorescence. When the probe is dissolved in CH3 OH/PBS (v/v, 1:1) mixed solution, the junction of pyran group and indolin group is closed-loop to openloop to form probe-1 which has a large absorption peak centered at 566 nm and a weak emission peak centered at 598 nm. The above spectra results agree well with our proposed closed-loop to open-loop mechanism (Scheme 2). To study the proton-binding
In this work, a near-infrared ratiometric fluorescent probe based on spiropyran derivative for pH was reported. When the pH was decreased from 9.0 to 1.0, the color of probe solution change from purple to yellow was found by naked eye. From the UV–Vis and fluorescence spectra of probe to pH variation, we found that the probe was sensitive to acidity. Besides, the pKa of the probe was calculated to be 4.85, which provided a basis for detection of pH change under acidic conditions. The conversion between acid and alkaline form of the probe is reversible and rapid, so the probe can be a practical tool for assessing pH change in real time. Furthermore, the probe has been successfully applied to monitor cellular pH in A549 cells. Based on the above experimental results, we hope that our work can promote the fluorescent labeling of tumor cells in the diagnosis of diseases. Acknowledgments We thank the National Natural Science Foundation of China (No. 21775096, 21705102, 21672131), One Hundred People Plan of Shanxi Province, Shanxi Province “1331 Project” Key Innovation
Scheme 2. The proposed recognition mechanism.
K. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117350
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Fig. 4. The 1H NMR comparison of probe in methanol d4 D2O (1:1, v/v) before and after the addition of HCl.
Team Construction Plan Cultivation Team (2018-CT-1), Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), Shanxi
Collaborative Innovation Center of High Value-added Utilization of Coal-related Wastes, China Institute for Radiation Production and Scientific Instrument Center of Shanxi University (201512).
Fig. 5. Fluorescence confocal images of probe (20.0 μM) in A549 cells at pH 4.0 (A1–A4), 6.0 (B1–B4) and 8.0 (C1–C4). The red channel were collected at 570–620 nm (Ex = 561 nm); the green channel were collected at 520–560 nm (Ex = 488 nm). Scale bar = 20.0 μm.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A near-infrared ratiometric fluorescence probe base on spiropyran derivative for pH and its application in living cells Kangming Xiong a, Caixia Yin a,⁎, Yongkang Yue a, Fangjun Huo b,⁎ a Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China b Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China
a r t i c l e
i n f o
Article history: Received 19 February 2019 Received in revised form 24 June 2019 Accepted 7 July 2019 Available online 08 July 2019 Keywords: Intracellular pH Near-infrared Fluorescence probe Cell imaging
a b s t r a c t The intracellular pH has a significant effect on several essential biological processes such as material transfer, enzymatic action, cell apoptosis et al. Thus, it is necessary to monitor pH fluctuation in living cells. Here, we designed a near-infrared ratiometric fluorescence probe for pH detection. The spectroscopic responses of probe to pH variations were investigated in CH3OH/PBS (v/v, 1:1) mixed solution at different pH values. The experimental results showed that the probe is sensitive to acidity, and the pKa of probe is calculated to be 4.85. When the pH was decreased from 9.0 to 1.0, the color of probe solution change from purple to yellow was found by naked eye. Moreover, the probe can be a practical tool for assessing cellular pH by cell imaging. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Intracellular pH has a remarkable impact on cell cycle-related life activities, such as material transfer [1], enzymatic action [2], cell apoptosis [3] et al. Abnormal pH often leads to cell dysfunction, which easily results in cancer [4] and Alzheimer's diseases [5]. Moreover, changes of biomembrane pH can be observed in cancer disease, and its cellular pH is always lower than normal cells [6]. Therefore, accurate determination of cellular pH can provide important information for biomedical studies. Several methods are currently being used to detect pH change: such as UV–vis absorption spectroscopy [7,8], fluorescence spectroscopy [9,10], nuclear magnetic resonance [11,12] and electrochemistry [13,14]. Among these techniques, fluorescence spectroscopy has received more and more attention due to its convenient operation, realtime detection, and high sensitivity [15]. For this purpose, many pH fluorescence probes have been explored [16–23]. However, in complex biological systems, many of them undergo various disadvantages, such as photobleaching, short wavelength and low selectivity upon pH change. So, it is essential to design a near-infrared ratiometric fluorescence probe for pH with the features of good photostability, large stokes shift and high selectivity. In recent years, the NIR fluorescence probes for pH has attracted increasing attention due to the central role of pH in many cellular events and the effects on abnormal cell growth and division found in diseases such as inflammation and cancer [24,25].
⁎ Corresponding authors. E-mail addresses: [email protected] (C. Yin), [email protected] (F. Huo).
https://doi.org/10.1016/j.saa.2019.117350 1386-1425/© 2019 Elsevier B.V. All rights reserved.
In this work, a near-infrared ratiometric fluorescence probe base on spiropyran derivative for pH was reported. Herein, we employed a cyanine dye as a NIR fluorophore and used a spiropyran as a reaction site for closed-loop to open-loop. From the UV–Vis and fluorescence spectra of probe to pH variation, we found that the probe was sensitive to acidity. And the pKa of probe was calculated to be 4.85. In addition, the probe also can be a practical tool for assessing cellular pH by cell imaging. 2. Experimental 2.1. Materials 2 Hydroxy 1 naphthaldehyde, 2,3,3 Trimethylindolenine and iodoethane were purchased from Aladdin Industrial Corporation. Nigericin was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Anionic salts were purchased from Shanghai Experiment Reagent Co., Ltd. All other chemicals used were of analytical grade. Deionized water was used to prepare all aqueous solutions. 2.2. Instruments A pH meter (Mettler Toledo, Switzerland) was used to determine the pH values of phosphate buffer solution and methanol-phosphate mixed solution. Ultraviolet-visible (UV–Vis) spectra were collected on U-3900 UV–Vis spectrophotometer. Fluorescence spectra were recorded on Hitachi F-7000 spectrophotometer. 1H NMR and 13C NMR spectra were recorded on Bruker AVANCE-600 MHz and 150 MHz NMR spectrometers, respectively. HRMS was carried out on a Thermo Scientific
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Q Exactive MS instrument. Fluorescence imaging was conducted using Airyscan confocal laser scanning microscope. 2.3. Preparation and characterization of probe Probe was conveniently synthesized via the condensation of 2 Hydroxy 1 naphthaldehyde (1) and 1 ethyl 2,3,3 trimethyl 3H indolium iodide salt (2) in ethanol. The synthesis route of probe was shown in Scheme 1. The compound 2 was synthesized according to the literature [26]. Compound 1 (0.516 g, 3.0 mmol) and compound 2 (0.945 g, 3.0 mmol) were dissolved in 20.0 mL ethanol, then 100.0 μL acetic acid was added, the mixture was refluxed for 10 h. After that, the reaction mixture was evaporated under reduced pressure and then was purified by column chromatography on silica gel (EtOAc/petroleum ether/CH2Cl2, 1:3:1 v/v/v) to give the probe as pink solid (0.674 g, 65.9% yield). 1H NMR (600 MHz, Chloroform d): δ 8.03 (s, 1H), 7.73 (s, 1H), 7.61 (s, 1H), 7.57–7.56 (d, 1H), 7.51–7.50 (d, 1H), 7.33–7.32 (d, 1H), 7.18–7.17 (d, 1H), 7.09 (s, 1H), 6.96 (s, 1H), 6.84–6.83 (d, 1H), 6.56 (s, 1H), 5.79–5.78 (d, 1H), 3.39–3.37 (t, 1H), 3.22–3.20 (t, 1H), 1.33 (s, 3H), 1.20 (s, 3H), 1.17–1.16 (d, 3H); 13C NMR (150 MHz, Chloroform d): δ 152.5, 147.1, 136.5, 129.9, 129.8, 128.7, 128.6, 127.5, 126.7, 124.7, 123.2, 121.7, 120.6, 118.5, 118.2, 117.6, 110.3, 106.2, 104.6, 52.0, 37.8, 26.0, 20.1, 14.4. HRMS m/z: [M + H]+ calculated for C24H24NO+: 342.18524; measured: 342.18568 (Fig. S1). 2.4. General procedure for spectra measurements The stock solution of probe (2.0 mM) was dissolved in methanol. − − − 2− Stock solutions (200.0 mM) of anionic salts (NO− 3 , I , F , Br , S2O5 , 2− 2− S2O8 , S ), amino acids (Lys, Gly, Leu, Met, Phe, GSH, Hcy, Cys) and metal salts (Na+, K+, Zn2+, Mn2+, Mg2+, Cd2+) were prepared in deionized water. UV–Vis and fluorescence spectra were obtained in 2.0 mL different pH values of CH3OH/PBS (v/v, 1:1) mixed solution at room temperature. The fluorescence excitation and emission slit widths were set as 10 nm and 20 nm, respectively. 2.5. Cell culture and imaging A549 cells and were grown in Dulbecco's modified Eagle's medium (DMEM) in an atmosphere of 5% CO2 at 37 °C. Then A549 cells were plated on 6-well plate and allowed to adhere for 24 h. In the pHdependent fluorescence evolution experiments, probe (20.0 μM) was added to the DMEM (2.0 mL) at pH = 4.0, pH = 6.0 and pH = 8.0 in the presence of 1.0 μM of nigericin for 30 min prior to imaging, respectively. A549 cells were imaged by Airyscan confocal laser scanning microscope. λex = 561 nm, λem = 570–620 nm for the red channel; λex = 488 nm, λem = 520–560 nm for the green channel. 3. Results and discussions 3.1. Solvent dependence study The solvent of a system is often considered as an important factor on affecting spectral properties of probe. The effect of different solvent conditions on the spectral properties of probe was investigated in Fig. S2. As
exhibited in Fig. S2, we could find that CH3OH/H2O = 1000/1000 (v/v) mixed solution was more effective for spectral detection of probe. Here, we chose different pH values of CH3OH/PBS (v/v, 1:1) mixed solution as the system for spectral detection of probe. 3.2. UV–vis and fluorescence spectra of probe to pH variation The UV–Vis and fluorescence spectra of probe to pH variations were tested in CH3OH/PBS (v/v, 1:1) mixed solution at different pH values. As shown in Fig. 1a, under alkaline media (pH 9.0), the probe (30 μM) showed an absorption band in the range 540–590 nm with a maximum at 566 nm. When the pH was decreased from 9.0 to 1.0, the absorption band at 540–590 nm of probe gradually reduced and a new absorption band emerged in the range 400–525 nm with a maximum at 478 nm, accompanied by an isobestic point at 528 nm in the absorption spectra. Meanwhile, there was a color change from purple to yellow which could be easily observed by naked eye. The results hinted that the probe could be used to measure pH as a colorimetric detector. As shown in Fig. 1b, when the pH was decreased from 9.0 to 1.0, the fluorescence intensity of probe (30.0 μM) at 598 nm gradually decreased (λex = 566 nm, slit: 10 nm/20 nm), and a new emission band in the range 520–600 nm emerged (emission peak centered at 558 nm) in Fig. 1c (λex = 478 nm, slit: 10 nm/20 nm). As exhibited in Fig. 1d, nonlinear fitting of sigmoidal curve of fluorescence emission at 558 nm versus pH was plotted over the pH range of 1.0 to 9.0 (λex = 478 nm, slit: 10 nm/20 nm). And the pKa of the probe was calculated to be 4.85 ± 0.19 based on the Henderson-Hasselbalch equation (log[(Imax-I) / (IImin)] = pH-pKa), which provided a basis for detection of pH changes under acidic conditions. On the basis of the linear regression equation F = −4019.7973 ∗ pH + 23,521.4189 (R2 = 0.982), a good linear relationship between the fluorescence intensity at 558 nm and pH in the range 4.4–5.4 was observed. These results clearly showed that the probe could be used to detect the pH variation. 3.3. Selectivity, photostability and reversibility studies of probe Next, we studied the anti-interference ability of probe under pH 1.0 or 9.0 by UV–Vis spectra. The absorption response features of probe to pH fluctuation in the presence of various interferences such as represen− − − 2− 2− 2− tative anions (NO− ), biologically re3 , I , F , Br , S2O5 , S2O8 and S lated amino acids (lysine, glycine, leucine, methionine, phenylalanine, glutathione, homocysteine and cysteine) and important metal ions (Na+, K+, Zn2+, Mn2+, Mg2+ and Cd2+) displayed little influence on the absorption response of probe in CH3OH/PBS (v/v, 1:1) mixed solvent with pH values of 1.0 or 9.0, respectively (Fig. 2a). The photostability of probe was carried out through monitoring the absorption of probe in mixed solvent with pH values at 1.0 and 9.0 for 30 min, respectively. As exhibited in Fig. S3, the absorption remained almost stable, revealing that the probe possessed good photostability. Moreover, the reversibility is basically required for real-time monitoring of pH changes in living organs. The reversibility of probe (30.0 μM) was measured using small volumes of HCl and NaOH solutions back and forth to adjust the pH values of mixed solvent between 1.0 and 9.0, respectively. As shown in Fig. 2b, the emission ratios (I558 nm/I598 nm) of probe changed very little after five cycles at pH = 1.0 and 9.0, respectively. These results indicate that conversion
Scheme 1. The synthesis route of probe.
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Fig. 1. (a) Change of absorption spectra of probe (30.0 μM) at various pH (from 9.0 to 1.0). Inset: photograph of probe solution at pH = 1.0 and 9.0; (b) change of fluorescence spectra of probe (30.0 μM) at various pH (from 9.0 to 1.0) (λex = 566 nm, slit: 10 nm/20 nm); (c) change of fluorescence spectra of probe (30.0 μM) at various pH (from 9.0 to 1.0) (λex = 478 nm, slit: 10 nm/20 nm); (d) nonlinear fitting of sigmoidal curve of fluorescence intensity at 558 nm versus various pH values (from 9.0 to 1.0). Inset: linear relationship between fluorescence intensity at 558 nm and pH in the range pH 4.4–5.4.
− − Fig. 2. (a) Absorption spectra of probe (30.0 μM) at pH 1.0 or 9.0 containing representative anions, biologically related amino acids and important metal ions: 1. Blank; 2. NO− 3 ; 3. I ; 4. F ; 2− + + 2+ 2− 5. Br−; 6. S2O2− ; 7. S O ; 8. S ; 9. Lysine; 10. Glycine; 11. Leucine; 12. Methionine; 13. Phenylalanine; 14. Glutathione; 15. Homocysteine; 16. Cysteine. 17. Na ; 18. K ; 19. Zn ; 20. 5 2 8 Mn2+; 21. Mg2+; 22. Cd2+; (b) evolution of I558/I598 in the mixed solvent with alternating cycles of pH values at 1.0 and 9.0.
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behavior, the 1H NMR spectra of probe in methanol d4 D2O (1:1, v/ v) with small amount of HCl were determined. Fig. 4 showed the 1 H NMR comparison of probe in methanol d4 D2O (1:1, v/v) before and after the addition of HCl. Upon adding HCl to the solution of probe, the chemical shift value of proton Ha was shifted to downfield distinctly from 6.56 to 8.96 ppm because that the junction of pyran group and indolin group is closed-loop to open-loop to form a large conjugate plane. Meanwhile, probe-2 has a large absorption peak at 478 nm and a strong emission peak at 558 nm. Furthermore, the relative molecular mass of probe-2 was tested by high resolution mass spectrometry, with the m/z of probe-2 being 342.18515 for C24H24NO+ (Fig. S4). Thus, the above results are in good agreement with the proposed recognition mechanism. 3.6. Cellular imaging
Fig. 3. Time-course measurements of the UV–vis absorption at 478 nm of probe upon a pH change from 1.0 to 9.0 and then from 9.0 to 1.0.
between acid and alkaline form of the probe is reversible. Thus, the probe can be a practical tool for assessing pH change. 3.4. Time-dependence study To evaluate the applicability of the probe, the time-dependence of probe was measured. As shown in Fig. 3, the absorption of probe was rapidly switched back and forth between pH = 1.0 and pH = 9.0 within 10 s, respectively. The result demonstrates that the probe can be a practical tool for assessing cellular pH in real time.
Furthermore, we examined the application of probe for assessing cellular pH in A549 cells by cell imaging. To ensure the accuracy of pH measurement in the stain, in vivo calibration of fluorescence signals of probe was required. Here we used nigericin to elicit rapid external and internal pH equilibration. This is a standard approach to calibrate the pH value ex vivo [27,28]. As shown in Fig. 5, the A549 cells incubated with probe (20.0 μM) at pH 4.0 exhibit strong green fluorescence in green channel and non-fluorescence in the red channel. When the pH of the media increased from 4.0 to 6.0, the fluorescence in the green channel gradually attenuated and the fluorescence in the red channel appeared. When the pH of the media increased to 8.0, bright fluorescence in the red channel was observed and the fluorescence in the green channel almost disappeared. Above results show that probe can serve as a ratiometric probe for accurate pH imaging of A549 cells. 4. Conclusion
3.5. Proposed mechanism Spiropyran molecule is formed by the condensation reaction of pyran-aldehyde and indolin. It has two isomers, one is that pyran group and indolin group are vertically linked in space forming a closed-loop spiropyran molecule, and its central C atom is sp3 hybridization. Thus the whole molecule is not conjugated. At this time, the two groups of probe are independent of each other, and the probe has only ultraviolet absorption. The other is that the two groups are conjugated together and share electronic configurations. The Cspiro\\O bond at the junction of pyran group and indolin group breaks and forms a large conjugate plane accompanying with the corresponding Cspiro atom changing from sp 3 hybridization to sp2 hybridization, which will produce a large absorption in the visible region and lead to the appearance of fluorescence. When the probe is dissolved in CH3 OH/PBS (v/v, 1:1) mixed solution, the junction of pyran group and indolin group is closed-loop to openloop to form probe-1 which has a large absorption peak centered at 566 nm and a weak emission peak centered at 598 nm. The above spectra results agree well with our proposed closed-loop to open-loop mechanism (Scheme 2). To study the proton-binding
In this work, a near-infrared ratiometric fluorescent probe based on spiropyran derivative for pH was reported. When the pH was decreased from 9.0 to 1.0, the color of probe solution change from purple to yellow was found by naked eye. From the UV–Vis and fluorescence spectra of probe to pH variation, we found that the probe was sensitive to acidity. Besides, the pKa of the probe was calculated to be 4.85, which provided a basis for detection of pH change under acidic conditions. The conversion between acid and alkaline form of the probe is reversible and rapid, so the probe can be a practical tool for assessing pH change in real time. Furthermore, the probe has been successfully applied to monitor cellular pH in A549 cells. Based on the above experimental results, we hope that our work can promote the fluorescent labeling of tumor cells in the diagnosis of diseases. Acknowledgments We thank the National Natural Science Foundation of China (No. 21775096, 21705102, 21672131), One Hundred People Plan of Shanxi Province, Shanxi Province “1331 Project” Key Innovation
Scheme 2. The proposed recognition mechanism.
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Fig. 4. The 1H NMR comparison of probe in methanol d4 D2O (1:1, v/v) before and after the addition of HCl.
Team Construction Plan Cultivation Team (2018-CT-1), Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061), Shanxi
Collaborative Innovation Center of High Value-added Utilization of Coal-related Wastes, China Institute for Radiation Production and Scientific Instrument Center of Shanxi University (201512).
Fig. 5. Fluorescence confocal images of probe (20.0 μM) in A549 cells at pH 4.0 (A1–A4), 6.0 (B1–B4) and 8.0 (C1–C4). The red channel were collected at 570–620 nm (Ex = 561 nm); the green channel were collected at 520–560 nm (Ex = 488 nm). Scale bar = 20.0 μm.
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