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Fluorescent probes based on benzothiazole-spiropyran derivatives for pH monitoring in vitro and in vivo
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 225 (2020) 117506
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Fluorescent probes based on benzothiazole-spiropyran derivatives for pH monitoring in vitro and in vivo Jieji Zhu a, Qi Gao b, Qingxiao Tong a,⁎, Guangfu Wu a,c,⁎⁎ a b c
Department of Chemistry, Shantou University, Guangdong 515063, PR China Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Ave., Hong Kong, China Department of Biomedical, Biological & Chemical Engineering, University of Missouri-Columbia, Agricultural Engineering Building, 65211, MO, United States of America
a r t i c l e
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Article history: Received 28 January 2019 Received in revised form 27 August 2019 Accepted 31 August 2019 Available online 02 September 2019 Keywords: Benzothiazole Spiropyrans Fluorescent probe pH In vivo
a b s t r a c t In this study, by coupling benzothiazole and spiropyrans, three fluorescent probes HBT-pH 1, HBT-pH 2, and HBT-pH 3 were developed for pH variation monitoring. All these probes exhibited remarkable changes of absorption and emission accompanying its protonation under acidic conditions. HBT-pH 1 exhibited OFF-ON response when pH value was changed from 12.00 to 2.02, whereas ratiometric responses (large Stokes shifts) were obtained for HBT-pH 2 and HBT-pH 3. The response was attributed to the open-loop of spiropyran under acidic conditions, which was confirmed by 1H NMR. The pKa values of 6.57, 4.90, and 3.95 were obtained for HBT-pH 1, HBT-pH 2, and HBT-pH 3, respectively, indicating they were suitable for pH variation monitoring. Furthermore, low cytotoxicity and cell imaging of pH changes with HBT-pH 2 in living cells were successfully demonstrated, suggesting potential application in early diagnosis of pH-related diseases. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Maintaining stability of pH value is essential for all living organisms to survive. Intracellular pH (pHi) plays a key role in many physiological and pathological processes such as cell proliferation and apoptosis, endocytosis, ion transport, and multidrug resistance [1,2]. The concentration of H+ in extracellular fluid is about 40 nM (pH = 7.4), and the variation of H+ is between 0.1 and 0.2 pH units [3]. The pH of acidic organelles (endosomes, lysosomes, etc.) is between 4.5 and 5.5. Lysosome is the digestive organ of a cell, which contains N60 kinds of acidic hydrolases. It is beneficial to the degradation of proteins when cell metabolism happens in acidic environment. Abnormal pHi changes can lead to organelle dysfunction, cardiopulmonary and nervous system diseases, cancers, and Alzheimer's diseases [4]. The bio-activities of cancer cells are strongest at pH = 6.85–6.95. Moreover, acidic condition is necessary for cancer cell metastasis. Therefore, acidity is regarded as the main cause of cancer. Accurate determination of pH value is of great significance to public health and human diseases. In tradition, pH value is usually measured with pH test paper or glass electrode. However, pH test paper has poor accuracy and it is based on subjective judgement;
⁎ Corresponding author. ⁎⁎ Correspondence to: G. Wu, Department of Biomedical, Biological & Chemical Engineering, University of Missouri-Columbia, Agricultural Engineering Building, 65211, MO, United States of America. E-mail addresses: [email protected] (Q. Tong), [email protected], [email protected] (G. Wu).
https://doi.org/10.1016/j.saa.2019.117506 1386-1425/© 2019 Elsevier B.V. All rights reserved.
electrochemical interference, possible mechanical damages and defects will do harm to pH monitoring [5]. Fluorescence method is highly sensitive, selective, and nondestructive. This method is very suitable for turbid and inhomogeneous systems as well as fluorescence microscopy research. For fluorescence microscopy research, the information of dynamic distribution and regional changes of intracellular and extracellular pH in real time can be recorded, which paves the way for physiological and pathological research. A large number of fluorescent probes have been reported for metal ions, anions, pH, and bio-molecules detection in the past few decades [6–16]. Strong electron-donating groups such as amino and N,Ndimethylamine, have been introduced into the probe molecules [10]. Electron-donating ability was weakened when amino protonation occurred under acidic conditions. The amino protonation blocked the PET or ICT process [14], leading to changes in fluorescence signals. Another strategy is based on that the ring of spiro or spiropyran opens under acidic conditions, resulting in changes in fluorescence signals [15,16]. Due to poor light resistance and strong short-wavelength fluorescence absorption for cells and living tissues, fluorescence probes with near-infrared (NIR) emission (650–900 nm) attracted wide attentions [17]. It is because the fluorescence in NIR region shows good tissue penetration and weak tissue autofluorescence interference. Generally, NIR probes have long excitation wavelength, which can reduce cell damage and equip the NIR probes with the abilities of bio-detection and bio-imaging. 2-(2-hydroxyphenyl) benzothiazole (HBT) is a classic molecule with intramolecular proton transfer (ESIPT) excited state [18]. It exhibits
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fluorescence emission at 350–400 nm and 500–550 nm, at which the longer-wavelength emission (500–550 nm) was assigned to the ESIPT progress. Spiropyrans are well-known photochromic materials [19,20]. Under photochemical irradiation, ring-closing spiropyran form will be changed into ring-opening merocyanine form. Furthermore, sipropyran derivatives could also undergo the reversible changes from ringopening form to ring-closing form when the environment is changed from acetic to alkaline. Inspired by this mechanism, HBT was used to couple with spiropyrans in this study for developing three fluorescent pH probes HBT-pH 1, HBT-pH 2 and HBT-pH 3. HBT and spiropyran was connected by strong electron-withdrawing and π-conjugated groups (Scheme 1). The π-conjugated systems in these structures are expected to keep ring-closing spiropyran form in a neutral or basic condition which inhibited ESIPT progress, resulting in quenched longerwavelength emission. On the contrary, the ring-opening progress can enhance longer-wavelength emission if pH decreased.
Micronass UK, Waters LCT Premier XE. Steady-state emission spectra were recorded at ambient temperature on a Hitachi F-7000 Spectrophotometer and UV/Vis spectra were recorded on a Perkin-Elmer Lambda 950 UV–visible spectrophotometer. pH value was adjusted using Mettler Toledo pH Meter. 2.3. Synthesis of HBT-based fluorescence probes As shown in Scheme 1, the starting material HBT-CHO was synthesized from HBT with hexamethyltetramine in trifluoroacetic acid in 35% yield. In the presence of piperidine, the target product HBT-pH 1 was obtained in 30% yield from the reaction of HBT-CHO with 1, 4dimethylpyridin-1-ium iodide in ethanol. The crude product was purified by column chromatography. The synthesize route of HBT-pH 2 and HBT-pH 3 were the same as HBT-pH 1. All of the new compounds were characterized by 1H NMR and MS. Details can be found in Supporting information.
2. Experimental section 3. Results and discussion 2.1. Materials and instruments 3.1. Solvation effect Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. The salts used in the stock aqueous solutions of ions were Zn(ClO4)2·6H2O, Cd(ClO4) 2·6H2O, Hg(ClO4)2·3H2O, Pb(ClO4)2·3H2O, AgClO4, Fe(ClO4)2·H2O, Co (ClO4)2·6H2O, Ni(ClO4)2·6H2O, Cu(ClO4)2·6H2O, Ba(ClO4)2, NaClO4, Ca(ClO4)2·4H2O, Mg(ClO4)2. 2.2. Instrumentation 1 H NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz) using TMS as internal standard. Mass spectra were obtained with Waters GCT premier, LCMS-2010 and GCT CA127
We firstly studied the photophysical properties of HBT, HBT-pH 1, HBT-pH 2 and HBT-pH 3. As a reference, HBT exhibited emission at around 460 nm when it was dissolved in CH3CN, CH3OH, and DMSO, which is ascribed to the enol form. HBT exhibited emission at around 506 nm when it was dissolved in n-hexane and dichloromethane, which was ascribed to keto form. As HBT was connected with electron withdrawing groups, all three probes showed intramolecular charge transfer (ICT) character (Fig. 1). The maximum emission wavelength of HBT-pH 1, HBT-pH 2 and HBT-pH 3 in DMSO were 660 nm, 675 nm and 715 nm, respectively. Compared with HBT, the emission wavelength of three probes was in NIR reign, which proved our design
Scheme 1. Synthesis of HBT-pH 1, HBT-pH 2 and HBT-pH 3.
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Fig. 1. The normalized fluorescence of HBT (a), HBT-pH 1 (b), HBT-pH 2 (c) and HBT-pH 3 (d) in different solvents.
strategy is effective. The long wavelength emission can effectively reduce the cell damage for cell imaging. 3.2. Evaluation of HBT-based fluorescent probes for pH monitoring The performance of these three fluorescent probes for pH monitoring were then investigated and analyzed. Spectral recordings were performed in PBS buffer. The PBS buffer was 10 mM Na2HPO4-NaH2PO4 containing 10% DMSO. The pH value was adjusted with 0.1 M HCl and 0.1 M NaOH solution, affording PBS buffer solutions (DMSO: PBS = 1: 9, v/v) with different pH value. As shown in Fig. 2(a), the decrease of pH value caused a significant fluorescence enhancement at 640 nm. When pH value was changed from 12.00 to 11.00, the fluorescence exhibited negligible change. Much larger increase was obtained when pH was changed from 11.00 to 3.00. When pH value was changed from 3.00 to 2.02, small decrease of fluorescence was observed, indicating that the response was in the pH range of 12.00–2.02. Overall, HBT-pH 1 displayed an OFF-ON response from 12.00 to 2.02 with a 36-fold enhancement. It is noteworthy that the pKa of HBT-pH 1 was calculated to be 6.57 based on Henderson-Hasselbalch equation [6] as shown in Fig. 2(b), suggesting that HBT-pH 1 could serve as a functional pH probe for the weak acidic environment in vivo like cytoplasm. Besides, the solution of HBT-pH 1 displayed distinct color change from pink to colorless along with changing pH value from 12.00 to 2.02. An isosbestic point at 405 nm was observed in the absorbance of HBT-pH 1 (Fig. S1), indicating a new compound from the interaction between HBT-pH 1 and proton. Compared with HBT-pH 1, HBT-pH 2 and HBT-pH 3 showed different response performance in different pH values as shown in Fig. 2(c)– (f). The fluorescence response of HBT-pH 2 to pH was shown in Fig. 2 (c). Under basic condition (pH = 7.3), HBT-pH 2 showed a maximum fluorescence intensity at 520 nm (I520) due to the fluorescent spiro form of HBT-pH 2. When pH was decreased, I520 decreased with a
concomitant increase of fluorescence intensity at 640 nm, suggesting a ratiometric response. A clear isoemission point at 584 nm was observed. The pKa of HBT-pH 2 was calculated to be 4.90, suggesting that HBT-pH 2 could serve as a functional pH probe for medium acidic environment in vivo like lysosome [21]. The influence of pH on the fluorescence of HBT-pH 3 in the above-mentioned system is shown in Fig. 2(e) and (f). Under alkaline condition, strong fluorescence at 525 nm was obtained, which was ascribed to the screw ring configuration of HBT-pH 3. The fluorescence at 525 nm decreased and the fluorescence at 675 nm was enhanced gradually with the increase of acidity of solution. The appearance of an isoemission point at 600 nm suggested a ratiometric response of pH for HBT-pH 3. The influences of pH change on the absorbance of HBT-pH 2 and HBT-pH 3 were explored as well. HBT-pH 2 displayed a strong absorption peak at 560 nm in PBS buffer containing 10% DMSO at pH 7.30. Gradual decrease of pH from 7.30 to 2.02 (Fig. S2a) or gradual increase of pH from 7.30 to 12.00 resulted in significant absorbance enhancement at 425 nm (Fig. S2b), affording an isosbestic point at 480 nm. The increase of pH value of HBT-pH 2 solution from 7.30 to 12.00 caused distinct color changes from purple to yellow green while the decrease of pH value of HBT-pH 2 solution from 7.30 to 2.02 caused distinct color changes from purple to yellow. These two different response performances indicate HBT-pH 2 is sensitive to pH change. For HBT-pH 3, when the pH was changed from 7.30 to 2.02, the absorption at 583 nm decreases gradually and the absorption at 440 nm increases gradually, with an isosbestic point at 512 nm. The solution was changed from blue to yellow (Fig. S3a). When the pH was changed from 7.30 to 12.00, the absorption decreased gradually at 583 nm and 360 nm while the absorption at 440 nm increased gradually, giving an isosbestic point at 480 nm. The solution was changed from blue to green (Fig. S3b). Fluorescence quantum yield of fluorescent probe is an important consideration to evaluate a fluorescent probe performance. To futher interrogate the response results, the absolute quantum yields of three
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Fig. 2. (a) Fluorescence spectra of HBT-pH 1 (10 μM), (c) HBT-pH 2 (10 μM), and (e) HBT-pH 3 (10 μM) in PBS buffer with different pH values. The PBS buffer was 10 mM Na2HPO4NaH2PO4 containing 10% DMSO. The pH value was adjusted with 0.1 M HCl and 0.1 M NaOH solution, affording PBS buffer solutions (DMSO: PBS = 1: 9, v/v, pH 2.02–12.0). (b) The fluorescent intensity of HBT-pH 1 at 640 nm, (d) HBT-pH 2 at 640 nm, and (f) HBT-pH 3 at 675 nm upon exposed to various pH (2.02–12.00). The red lines in (b), (d), and (f) are the fitting lines based on Henderson-Hasselbalch equation [6]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
porbes at different pH values were tested (Fig. S4). Results were consistent with fluorescence spectra. Probe HBT-pH 2 and HBT-pH 3 exhibited higher quantum yield in acid or base buffer than in neutral buffer, implying their capabilities of ratiometric response. 3.3. Response Mechanism The pH response mechanisms for all the above three probes were then carefully studied. Based on the spectral experiments, the pH response mechanisms of HBT-pH 1, HBT-pH 2, and HBT-pH 3 were deduced as shown in Fig. 3. HBT-pH 1 maintains spiropyran configuration under strong alkalinity (pH 11–12). The molecule is highly distorted, and fluorescence is quenched. Under weak alkalinity to acidity (pH 10–2), the spiral ring is destroyed. The carbon atoms in the center of the spiral ring are changed from sp3 hybridization to sp2 hybridization, affording two aromatic parts. The aromatic system changed from orthogonal to coplanar, the charge rearranged, the molecular conjugation increased, and the fluorescence enhanced by ESIPT. This explanation also can be applied to HBT-pH 2, and HBT-pH 3. The pH can cause the transformation between distortion structure
(spiropyran) and coplanar structure (two aromatic parts). The conjugated degree (CD) of these three probes is CDHBT-pH 1 N CDHBT-pH 2 N CDHBT-pH 3, therefore, the emission wavelength of HBT-pH 3 is larger than those of HBT-pH 1 and HBT-pH 2. In order to verify the hypothesized mechanism, probes were titrated by 1H NMR using DMSO d6 as solvent, concentrated hydrochloric acid and triethylamine as acid-base regulator. The results were well consistent with our hypothesis (Figs. S6–S10). 3.4. Selectivity and reversibility Selectivity and reversibility were two important considerations to evaluate a fluorescent probe. In Fig. 4a, c, and e, black arrow, red arrow, and blue arrow indicate that probe exposed to interference species, probe exposed to H+ ion, and the mixture of probe and interference species exposed to H+ ion, respectively. The blank referred to that the fluorescence was obtained under neutral condition (pH = 7.30). Other ions and anions (Zn2+, Cd2+, Hg2+, Pb2+, Ag+, Fe3+, Co2 2− + , Ni2+, Cu2+, F−, Cl−, Br−, I−, CO2− ) rarely exerted obvious effects 3 ,O when probes were employed for pH changes monitoring except for that
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Fig. 3. (a) Response mechanism of HBT-pH 1, (b) HBT-pH 2, and (c) HBT-pH 3 to pH changes.
Ag+ and Cu2+ decreased the fluorescence of HBT-pH 1. However, neither Ag+ nor Cu2+ affected the application for the complexes were unstable in acidic environment. The selectivity of these three probes were further interrogated when they were exposed to different reaction species such as oxygen, nitrogen and sulfur reactive species under both neutral (pH = 7.3) and acid (pH = 2.1) conditions (Fig. S5). Negligible influence on the fluorescence of these three pH probes was obtained when 5 equiv. reactive species were added. Detailed information can be found in the supporting information. As shown in Fig. 4(a), (c), and (e), HBT-pH 1, HBT-pH 2, and HBT-pH 3 solutions were sensitive to the pH changes rather than the added ions and anions. Moreover, negligible changes were observed for these three probes by adding other ions to probe solutions under acidic conditions. These two experiments indicated excellent selectivity and anti-interference for three probes. Considering the pH value inside the living cells is always in the state of oscillation, it requires that pH probe should have good ability of reversible response. The oscillation experimental results of three probes showed that the pH value oscillates between 2.02 and 7.30. The reversibility of the response of the probe was examined by successive addition of concentrated NaOH and HCl solutions. After four cycles, the fluorescence exhibited negligible changes for three probes, indicating good ability of reversible response as shown in Fig. 4(b), (d), and (f). These three probes also displayed excellent photostability within 30 min (Figs. S11–13). The excitation wavelength was set at 365 nm and the pH was 2.02, 4.91 and 7.30, respectively. These equip these three probes with high possibilities of cell imaging.
4. Applications in cell imaging 4.1. Cell viability As obtained in the spectral experiments, the pKa (4.90) of HBT-pH 2 matched with that of lysosome (pH = 4.5–5.5), and large excitation (510 nm) and emission wavelength (640 nm) can effectively reduce cell damage and self-absorption, affording HBT-pH 2 high potential for intracellular lysosome pH monitoring and cell imaging. Cytotoxicity is an important factor for cell imaging. HBT-pH 2 was first dissolved in DMSO, then DMSO solution was added to bovine serum culture medium, affording culture medium containing 1.56–50 μM HBT-pH 2. Hela cells were cultured in the above culture medium for three generations, followed by wash with PBS buffer for three times. As shown in Fig. 5, the cell viability of HBT-pH 2 was N90% in the range of 1.56–12.5 μM, indicating that the important physiological activities of cells were not affected by the toxicity of the probe itself under this experimental condition, which satisfied the imaging conditions and had the potential of intracellular pH monitoring. Similar phenomenon can be observed with HBT-pH 1 and HBT-pH 3 as shown in Figs. S14 and S15. We assumed that the unhealthy status of living cells were partially due to the use organic solvent (DMSO) and the probe's toxicity when the probe with higher concentration was used. It is always demanding for fluorescent probes with better water solubility and biocompatibility. The employment of oligo (ethylene glycol) is preferable and efficient to improve the bio-compatibility and water solubility of fluorescent probe, which has been common used in other reports
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Fig. 4. (a) Fluorescent spectra of HBT-pH 1, (c) HBT-pH 2, and (e) HBT-pH 3, and probe mixed with various analytes. (b) Oscillation experiments of HBT-pH 1, (d) HBT-pH 2, and (f) HBTpH 3 with the pH range 2.02–7.30.
[22,23]. The hydrophilic oligo (ethylene glycol) components impose solubility in aqueous environment and enhance bio-compatibility on the overall assembly. Meanwhile, hydrophobic group is essential as well since it can effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. In the future, more efforts will be denoted to the enhancement of bio-compatibility and water solubility of these pH probes by introducing the oligo group. 4.2. Cell imaging
Fig. 5. Cell viability in the presence of HBT-pH 2 at different concentrations (1.56–50 μM). The data were obtained through MTT assay.
Abnormal pH values are often associated with cell dysfunction and some common diseases such as cancer and Alzheimer's disease. In order to investigate the ability of HBT-pH 2 for pH monitoring in living cell, confocal fluorescence microscopy was employed to obtain the fluorescence image of HBT-pH 2 in HeLa cells under different pH values. Hela cells were cultured in DMEM supplemented with 10% fetal bovine serum and 5% CO2 at 37 °C on 96-well plates for 24 h. Probe HBT-pH 2 (10 μM) was firstly added to above cells and the cells were incubated for 30 min. For cell imaging, HBT-pH 2 loaded cells were rinsed three times and incubated with PBS buffer at various pH values (pH = 2.02, 3.02, 3.83, 4.91, 6.00, 7.30, and 12.0) for 10 min, respectively. Confocal
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fluorescence imaging was performed on Leica TCS SP8 confocal microscope system. Fluorescence at two emission channels of 475–550 nm and 600–700 nm were measured at room temperature with an excitation of 405 nm. The images were stacked together. As shown in Fig. 6, when HeLa cells were incubated with HBT-pH 2 (10 μM) at 37 °C for 30 min, green fluorescence (b column) increases with the increase of pH value (2.02–7.30), while red fluorescence in HeLa cells (c column) gradually vanishes. It is more obvious in the mode of merged images (fourth column) in which the fluorescence change from bright orange to pale yellowish-green with the increase of pH value is apparent, indicating the ability of HBT-pH 2 to measure a wide range of intracellular pH values. When pH reached to 12.0, fluorescence in channel 1 becomes much more brilliant, while fluorescence in channel 2 exhibits significant decrease. It is noticeable that cells in round shape under pH N 8.0 was
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obtained, implying unhealthy status of Hela cells under basic conditions. Similar phenomenon can be found in Fig. 6 (pH = 12.0). HBT-pH 2 exhibits excellent cell membrane permeability and staining ability in living cells and the fluorescence intensities within cells changed in concentration dependent manner as shown in Figs. 5 and 6. However, the different fluorescence intensities within single cells demonstrate that intracellular pH is not uniformly distributed. We assume that HBT-pH 2 stained lysosomes in living cells according to a pKa of 4.90 for HBT-pH 2, which is at the fringe of lysosomal pH of 4.8. [24–27] 5. Conclusions In this study, three pH probes HBT-pH 1, HBT-pH 2 and HBT-pH 3 were designed and synthesized based on the open-loop of spiropyran
Fig. 6. Confocal fluorescence images of HeLa cells with HBT-pH 2 in PBS buffers for 30 min at pH 2.02, 3.02, 3.83, 4.91, 6.00, 7.30, and 12.0. (a) column: Bright images. (b) column: fluorescence images collected at the channel of 475–550 nm. (c) Column: fluorescence images collected at the channel of 600–700 nm. (d) Merged images of (a), (b) and (c) columns. All of these images were obtained with an excitation wavelength of 405 nm. The scale bar was 20 μm.
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under acidic conditions. As a fluorophore, benzothiazole was coupled into spiropyran structures. Because of the quaternary ammonium salting of N atom, the three probes have good water solubility. All of them displayed good anti-interference abilities and good selectivity, the long emission wavelength (N600 nm), good response to acidic and alkaline environments. The pKa values of HBT-pH 1, HBT-pH 2 and HBT-pH 3 were 6.57, 4.90 and 3.95, respectively, indicating they were suitable for the detection of pH in weak acidic, acidic and strong acidic environments. More importantly, it was confirmed that HBT-pH 2 exhibited low cytotoxicity and the cell imaging of pH changes in living cells were successfully demonstrated. We believe that it has the potential application in early diagnosis of pH-related diseases.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 51673113) and the key project of DEGP (No. 2018KZDXM032). Appendix A. Supplementary data Supplementary data includes synthesis of HBT-pH 1, HBT-pH 2 and HBT-pH 3, absorbance of HBT-pH 1, HBT-pH 2, HBT-pH 3 for pH monitoring, 1H NMR spectra of HBT-pH 1, HBT-pH 2, and HBT-pH 3 in solution with different pH values, and photostability. Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019. 117506.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Fluorescent probes based on benzothiazole-spiropyran derivatives for pH monitoring in vitro and in vivo Jieji Zhu a, Qi Gao b, Qingxiao Tong a,⁎, Guangfu Wu a,c,⁎⁎ a b c
Department of Chemistry, Shantou University, Guangdong 515063, PR China Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Ave., Hong Kong, China Department of Biomedical, Biological & Chemical Engineering, University of Missouri-Columbia, Agricultural Engineering Building, 65211, MO, United States of America
a r t i c l e
i n f o
Article history: Received 28 January 2019 Received in revised form 27 August 2019 Accepted 31 August 2019 Available online 02 September 2019 Keywords: Benzothiazole Spiropyrans Fluorescent probe pH In vivo
a b s t r a c t In this study, by coupling benzothiazole and spiropyrans, three fluorescent probes HBT-pH 1, HBT-pH 2, and HBT-pH 3 were developed for pH variation monitoring. All these probes exhibited remarkable changes of absorption and emission accompanying its protonation under acidic conditions. HBT-pH 1 exhibited OFF-ON response when pH value was changed from 12.00 to 2.02, whereas ratiometric responses (large Stokes shifts) were obtained for HBT-pH 2 and HBT-pH 3. The response was attributed to the open-loop of spiropyran under acidic conditions, which was confirmed by 1H NMR. The pKa values of 6.57, 4.90, and 3.95 were obtained for HBT-pH 1, HBT-pH 2, and HBT-pH 3, respectively, indicating they were suitable for pH variation monitoring. Furthermore, low cytotoxicity and cell imaging of pH changes with HBT-pH 2 in living cells were successfully demonstrated, suggesting potential application in early diagnosis of pH-related diseases. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Maintaining stability of pH value is essential for all living organisms to survive. Intracellular pH (pHi) plays a key role in many physiological and pathological processes such as cell proliferation and apoptosis, endocytosis, ion transport, and multidrug resistance [1,2]. The concentration of H+ in extracellular fluid is about 40 nM (pH = 7.4), and the variation of H+ is between 0.1 and 0.2 pH units [3]. The pH of acidic organelles (endosomes, lysosomes, etc.) is between 4.5 and 5.5. Lysosome is the digestive organ of a cell, which contains N60 kinds of acidic hydrolases. It is beneficial to the degradation of proteins when cell metabolism happens in acidic environment. Abnormal pHi changes can lead to organelle dysfunction, cardiopulmonary and nervous system diseases, cancers, and Alzheimer's diseases [4]. The bio-activities of cancer cells are strongest at pH = 6.85–6.95. Moreover, acidic condition is necessary for cancer cell metastasis. Therefore, acidity is regarded as the main cause of cancer. Accurate determination of pH value is of great significance to public health and human diseases. In tradition, pH value is usually measured with pH test paper or glass electrode. However, pH test paper has poor accuracy and it is based on subjective judgement;
⁎ Corresponding author. ⁎⁎ Correspondence to: G. Wu, Department of Biomedical, Biological & Chemical Engineering, University of Missouri-Columbia, Agricultural Engineering Building, 65211, MO, United States of America. E-mail addresses: [email protected] (Q. Tong), [email protected], [email protected] (G. Wu).
https://doi.org/10.1016/j.saa.2019.117506 1386-1425/© 2019 Elsevier B.V. All rights reserved.
electrochemical interference, possible mechanical damages and defects will do harm to pH monitoring [5]. Fluorescence method is highly sensitive, selective, and nondestructive. This method is very suitable for turbid and inhomogeneous systems as well as fluorescence microscopy research. For fluorescence microscopy research, the information of dynamic distribution and regional changes of intracellular and extracellular pH in real time can be recorded, which paves the way for physiological and pathological research. A large number of fluorescent probes have been reported for metal ions, anions, pH, and bio-molecules detection in the past few decades [6–16]. Strong electron-donating groups such as amino and N,Ndimethylamine, have been introduced into the probe molecules [10]. Electron-donating ability was weakened when amino protonation occurred under acidic conditions. The amino protonation blocked the PET or ICT process [14], leading to changes in fluorescence signals. Another strategy is based on that the ring of spiro or spiropyran opens under acidic conditions, resulting in changes in fluorescence signals [15,16]. Due to poor light resistance and strong short-wavelength fluorescence absorption for cells and living tissues, fluorescence probes with near-infrared (NIR) emission (650–900 nm) attracted wide attentions [17]. It is because the fluorescence in NIR region shows good tissue penetration and weak tissue autofluorescence interference. Generally, NIR probes have long excitation wavelength, which can reduce cell damage and equip the NIR probes with the abilities of bio-detection and bio-imaging. 2-(2-hydroxyphenyl) benzothiazole (HBT) is a classic molecule with intramolecular proton transfer (ESIPT) excited state [18]. It exhibits
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fluorescence emission at 350–400 nm and 500–550 nm, at which the longer-wavelength emission (500–550 nm) was assigned to the ESIPT progress. Spiropyrans are well-known photochromic materials [19,20]. Under photochemical irradiation, ring-closing spiropyran form will be changed into ring-opening merocyanine form. Furthermore, sipropyran derivatives could also undergo the reversible changes from ringopening form to ring-closing form when the environment is changed from acetic to alkaline. Inspired by this mechanism, HBT was used to couple with spiropyrans in this study for developing three fluorescent pH probes HBT-pH 1, HBT-pH 2 and HBT-pH 3. HBT and spiropyran was connected by strong electron-withdrawing and π-conjugated groups (Scheme 1). The π-conjugated systems in these structures are expected to keep ring-closing spiropyran form in a neutral or basic condition which inhibited ESIPT progress, resulting in quenched longerwavelength emission. On the contrary, the ring-opening progress can enhance longer-wavelength emission if pH decreased.
Micronass UK, Waters LCT Premier XE. Steady-state emission spectra were recorded at ambient temperature on a Hitachi F-7000 Spectrophotometer and UV/Vis spectra were recorded on a Perkin-Elmer Lambda 950 UV–visible spectrophotometer. pH value was adjusted using Mettler Toledo pH Meter. 2.3. Synthesis of HBT-based fluorescence probes As shown in Scheme 1, the starting material HBT-CHO was synthesized from HBT with hexamethyltetramine in trifluoroacetic acid in 35% yield. In the presence of piperidine, the target product HBT-pH 1 was obtained in 30% yield from the reaction of HBT-CHO with 1, 4dimethylpyridin-1-ium iodide in ethanol. The crude product was purified by column chromatography. The synthesize route of HBT-pH 2 and HBT-pH 3 were the same as HBT-pH 1. All of the new compounds were characterized by 1H NMR and MS. Details can be found in Supporting information.
2. Experimental section 3. Results and discussion 2.1. Materials and instruments 3.1. Solvation effect Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. The salts used in the stock aqueous solutions of ions were Zn(ClO4)2·6H2O, Cd(ClO4) 2·6H2O, Hg(ClO4)2·3H2O, Pb(ClO4)2·3H2O, AgClO4, Fe(ClO4)2·H2O, Co (ClO4)2·6H2O, Ni(ClO4)2·6H2O, Cu(ClO4)2·6H2O, Ba(ClO4)2, NaClO4, Ca(ClO4)2·4H2O, Mg(ClO4)2. 2.2. Instrumentation 1 H NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz) using TMS as internal standard. Mass spectra were obtained with Waters GCT premier, LCMS-2010 and GCT CA127
We firstly studied the photophysical properties of HBT, HBT-pH 1, HBT-pH 2 and HBT-pH 3. As a reference, HBT exhibited emission at around 460 nm when it was dissolved in CH3CN, CH3OH, and DMSO, which is ascribed to the enol form. HBT exhibited emission at around 506 nm when it was dissolved in n-hexane and dichloromethane, which was ascribed to keto form. As HBT was connected with electron withdrawing groups, all three probes showed intramolecular charge transfer (ICT) character (Fig. 1). The maximum emission wavelength of HBT-pH 1, HBT-pH 2 and HBT-pH 3 in DMSO were 660 nm, 675 nm and 715 nm, respectively. Compared with HBT, the emission wavelength of three probes was in NIR reign, which proved our design
Scheme 1. Synthesis of HBT-pH 1, HBT-pH 2 and HBT-pH 3.
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Fig. 1. The normalized fluorescence of HBT (a), HBT-pH 1 (b), HBT-pH 2 (c) and HBT-pH 3 (d) in different solvents.
strategy is effective. The long wavelength emission can effectively reduce the cell damage for cell imaging. 3.2. Evaluation of HBT-based fluorescent probes for pH monitoring The performance of these three fluorescent probes for pH monitoring were then investigated and analyzed. Spectral recordings were performed in PBS buffer. The PBS buffer was 10 mM Na2HPO4-NaH2PO4 containing 10% DMSO. The pH value was adjusted with 0.1 M HCl and 0.1 M NaOH solution, affording PBS buffer solutions (DMSO: PBS = 1: 9, v/v) with different pH value. As shown in Fig. 2(a), the decrease of pH value caused a significant fluorescence enhancement at 640 nm. When pH value was changed from 12.00 to 11.00, the fluorescence exhibited negligible change. Much larger increase was obtained when pH was changed from 11.00 to 3.00. When pH value was changed from 3.00 to 2.02, small decrease of fluorescence was observed, indicating that the response was in the pH range of 12.00–2.02. Overall, HBT-pH 1 displayed an OFF-ON response from 12.00 to 2.02 with a 36-fold enhancement. It is noteworthy that the pKa of HBT-pH 1 was calculated to be 6.57 based on Henderson-Hasselbalch equation [6] as shown in Fig. 2(b), suggesting that HBT-pH 1 could serve as a functional pH probe for the weak acidic environment in vivo like cytoplasm. Besides, the solution of HBT-pH 1 displayed distinct color change from pink to colorless along with changing pH value from 12.00 to 2.02. An isosbestic point at 405 nm was observed in the absorbance of HBT-pH 1 (Fig. S1), indicating a new compound from the interaction between HBT-pH 1 and proton. Compared with HBT-pH 1, HBT-pH 2 and HBT-pH 3 showed different response performance in different pH values as shown in Fig. 2(c)– (f). The fluorescence response of HBT-pH 2 to pH was shown in Fig. 2 (c). Under basic condition (pH = 7.3), HBT-pH 2 showed a maximum fluorescence intensity at 520 nm (I520) due to the fluorescent spiro form of HBT-pH 2. When pH was decreased, I520 decreased with a
concomitant increase of fluorescence intensity at 640 nm, suggesting a ratiometric response. A clear isoemission point at 584 nm was observed. The pKa of HBT-pH 2 was calculated to be 4.90, suggesting that HBT-pH 2 could serve as a functional pH probe for medium acidic environment in vivo like lysosome [21]. The influence of pH on the fluorescence of HBT-pH 3 in the above-mentioned system is shown in Fig. 2(e) and (f). Under alkaline condition, strong fluorescence at 525 nm was obtained, which was ascribed to the screw ring configuration of HBT-pH 3. The fluorescence at 525 nm decreased and the fluorescence at 675 nm was enhanced gradually with the increase of acidity of solution. The appearance of an isoemission point at 600 nm suggested a ratiometric response of pH for HBT-pH 3. The influences of pH change on the absorbance of HBT-pH 2 and HBT-pH 3 were explored as well. HBT-pH 2 displayed a strong absorption peak at 560 nm in PBS buffer containing 10% DMSO at pH 7.30. Gradual decrease of pH from 7.30 to 2.02 (Fig. S2a) or gradual increase of pH from 7.30 to 12.00 resulted in significant absorbance enhancement at 425 nm (Fig. S2b), affording an isosbestic point at 480 nm. The increase of pH value of HBT-pH 2 solution from 7.30 to 12.00 caused distinct color changes from purple to yellow green while the decrease of pH value of HBT-pH 2 solution from 7.30 to 2.02 caused distinct color changes from purple to yellow. These two different response performances indicate HBT-pH 2 is sensitive to pH change. For HBT-pH 3, when the pH was changed from 7.30 to 2.02, the absorption at 583 nm decreases gradually and the absorption at 440 nm increases gradually, with an isosbestic point at 512 nm. The solution was changed from blue to yellow (Fig. S3a). When the pH was changed from 7.30 to 12.00, the absorption decreased gradually at 583 nm and 360 nm while the absorption at 440 nm increased gradually, giving an isosbestic point at 480 nm. The solution was changed from blue to green (Fig. S3b). Fluorescence quantum yield of fluorescent probe is an important consideration to evaluate a fluorescent probe performance. To futher interrogate the response results, the absolute quantum yields of three
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Fig. 2. (a) Fluorescence spectra of HBT-pH 1 (10 μM), (c) HBT-pH 2 (10 μM), and (e) HBT-pH 3 (10 μM) in PBS buffer with different pH values. The PBS buffer was 10 mM Na2HPO4NaH2PO4 containing 10% DMSO. The pH value was adjusted with 0.1 M HCl and 0.1 M NaOH solution, affording PBS buffer solutions (DMSO: PBS = 1: 9, v/v, pH 2.02–12.0). (b) The fluorescent intensity of HBT-pH 1 at 640 nm, (d) HBT-pH 2 at 640 nm, and (f) HBT-pH 3 at 675 nm upon exposed to various pH (2.02–12.00). The red lines in (b), (d), and (f) are the fitting lines based on Henderson-Hasselbalch equation [6]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
porbes at different pH values were tested (Fig. S4). Results were consistent with fluorescence spectra. Probe HBT-pH 2 and HBT-pH 3 exhibited higher quantum yield in acid or base buffer than in neutral buffer, implying their capabilities of ratiometric response. 3.3. Response Mechanism The pH response mechanisms for all the above three probes were then carefully studied. Based on the spectral experiments, the pH response mechanisms of HBT-pH 1, HBT-pH 2, and HBT-pH 3 were deduced as shown in Fig. 3. HBT-pH 1 maintains spiropyran configuration under strong alkalinity (pH 11–12). The molecule is highly distorted, and fluorescence is quenched. Under weak alkalinity to acidity (pH 10–2), the spiral ring is destroyed. The carbon atoms in the center of the spiral ring are changed from sp3 hybridization to sp2 hybridization, affording two aromatic parts. The aromatic system changed from orthogonal to coplanar, the charge rearranged, the molecular conjugation increased, and the fluorescence enhanced by ESIPT. This explanation also can be applied to HBT-pH 2, and HBT-pH 3. The pH can cause the transformation between distortion structure
(spiropyran) and coplanar structure (two aromatic parts). The conjugated degree (CD) of these three probes is CDHBT-pH 1 N CDHBT-pH 2 N CDHBT-pH 3, therefore, the emission wavelength of HBT-pH 3 is larger than those of HBT-pH 1 and HBT-pH 2. In order to verify the hypothesized mechanism, probes were titrated by 1H NMR using DMSO d6 as solvent, concentrated hydrochloric acid and triethylamine as acid-base regulator. The results were well consistent with our hypothesis (Figs. S6–S10). 3.4. Selectivity and reversibility Selectivity and reversibility were two important considerations to evaluate a fluorescent probe. In Fig. 4a, c, and e, black arrow, red arrow, and blue arrow indicate that probe exposed to interference species, probe exposed to H+ ion, and the mixture of probe and interference species exposed to H+ ion, respectively. The blank referred to that the fluorescence was obtained under neutral condition (pH = 7.30). Other ions and anions (Zn2+, Cd2+, Hg2+, Pb2+, Ag+, Fe3+, Co2 2− + , Ni2+, Cu2+, F−, Cl−, Br−, I−, CO2− ) rarely exerted obvious effects 3 ,O when probes were employed for pH changes monitoring except for that
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Fig. 3. (a) Response mechanism of HBT-pH 1, (b) HBT-pH 2, and (c) HBT-pH 3 to pH changes.
Ag+ and Cu2+ decreased the fluorescence of HBT-pH 1. However, neither Ag+ nor Cu2+ affected the application for the complexes were unstable in acidic environment. The selectivity of these three probes were further interrogated when they were exposed to different reaction species such as oxygen, nitrogen and sulfur reactive species under both neutral (pH = 7.3) and acid (pH = 2.1) conditions (Fig. S5). Negligible influence on the fluorescence of these three pH probes was obtained when 5 equiv. reactive species were added. Detailed information can be found in the supporting information. As shown in Fig. 4(a), (c), and (e), HBT-pH 1, HBT-pH 2, and HBT-pH 3 solutions were sensitive to the pH changes rather than the added ions and anions. Moreover, negligible changes were observed for these three probes by adding other ions to probe solutions under acidic conditions. These two experiments indicated excellent selectivity and anti-interference for three probes. Considering the pH value inside the living cells is always in the state of oscillation, it requires that pH probe should have good ability of reversible response. The oscillation experimental results of three probes showed that the pH value oscillates between 2.02 and 7.30. The reversibility of the response of the probe was examined by successive addition of concentrated NaOH and HCl solutions. After four cycles, the fluorescence exhibited negligible changes for three probes, indicating good ability of reversible response as shown in Fig. 4(b), (d), and (f). These three probes also displayed excellent photostability within 30 min (Figs. S11–13). The excitation wavelength was set at 365 nm and the pH was 2.02, 4.91 and 7.30, respectively. These equip these three probes with high possibilities of cell imaging.
4. Applications in cell imaging 4.1. Cell viability As obtained in the spectral experiments, the pKa (4.90) of HBT-pH 2 matched with that of lysosome (pH = 4.5–5.5), and large excitation (510 nm) and emission wavelength (640 nm) can effectively reduce cell damage and self-absorption, affording HBT-pH 2 high potential for intracellular lysosome pH monitoring and cell imaging. Cytotoxicity is an important factor for cell imaging. HBT-pH 2 was first dissolved in DMSO, then DMSO solution was added to bovine serum culture medium, affording culture medium containing 1.56–50 μM HBT-pH 2. Hela cells were cultured in the above culture medium for three generations, followed by wash with PBS buffer for three times. As shown in Fig. 5, the cell viability of HBT-pH 2 was N90% in the range of 1.56–12.5 μM, indicating that the important physiological activities of cells were not affected by the toxicity of the probe itself under this experimental condition, which satisfied the imaging conditions and had the potential of intracellular pH monitoring. Similar phenomenon can be observed with HBT-pH 1 and HBT-pH 3 as shown in Figs. S14 and S15. We assumed that the unhealthy status of living cells were partially due to the use organic solvent (DMSO) and the probe's toxicity when the probe with higher concentration was used. It is always demanding for fluorescent probes with better water solubility and biocompatibility. The employment of oligo (ethylene glycol) is preferable and efficient to improve the bio-compatibility and water solubility of fluorescent probe, which has been common used in other reports
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Fig. 4. (a) Fluorescent spectra of HBT-pH 1, (c) HBT-pH 2, and (e) HBT-pH 3, and probe mixed with various analytes. (b) Oscillation experiments of HBT-pH 1, (d) HBT-pH 2, and (f) HBTpH 3 with the pH range 2.02–7.30.
[22,23]. The hydrophilic oligo (ethylene glycol) components impose solubility in aqueous environment and enhance bio-compatibility on the overall assembly. Meanwhile, hydrophobic group is essential as well since it can effectively insulate the fluorophores from each other to prevent detrimental interchromophoric interactions and preserve their photophysical properties. In the future, more efforts will be denoted to the enhancement of bio-compatibility and water solubility of these pH probes by introducing the oligo group. 4.2. Cell imaging
Fig. 5. Cell viability in the presence of HBT-pH 2 at different concentrations (1.56–50 μM). The data were obtained through MTT assay.
Abnormal pH values are often associated with cell dysfunction and some common diseases such as cancer and Alzheimer's disease. In order to investigate the ability of HBT-pH 2 for pH monitoring in living cell, confocal fluorescence microscopy was employed to obtain the fluorescence image of HBT-pH 2 in HeLa cells under different pH values. Hela cells were cultured in DMEM supplemented with 10% fetal bovine serum and 5% CO2 at 37 °C on 96-well plates for 24 h. Probe HBT-pH 2 (10 μM) was firstly added to above cells and the cells were incubated for 30 min. For cell imaging, HBT-pH 2 loaded cells were rinsed three times and incubated with PBS buffer at various pH values (pH = 2.02, 3.02, 3.83, 4.91, 6.00, 7.30, and 12.0) for 10 min, respectively. Confocal
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fluorescence imaging was performed on Leica TCS SP8 confocal microscope system. Fluorescence at two emission channels of 475–550 nm and 600–700 nm were measured at room temperature with an excitation of 405 nm. The images were stacked together. As shown in Fig. 6, when HeLa cells were incubated with HBT-pH 2 (10 μM) at 37 °C for 30 min, green fluorescence (b column) increases with the increase of pH value (2.02–7.30), while red fluorescence in HeLa cells (c column) gradually vanishes. It is more obvious in the mode of merged images (fourth column) in which the fluorescence change from bright orange to pale yellowish-green with the increase of pH value is apparent, indicating the ability of HBT-pH 2 to measure a wide range of intracellular pH values. When pH reached to 12.0, fluorescence in channel 1 becomes much more brilliant, while fluorescence in channel 2 exhibits significant decrease. It is noticeable that cells in round shape under pH N 8.0 was
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obtained, implying unhealthy status of Hela cells under basic conditions. Similar phenomenon can be found in Fig. 6 (pH = 12.0). HBT-pH 2 exhibits excellent cell membrane permeability and staining ability in living cells and the fluorescence intensities within cells changed in concentration dependent manner as shown in Figs. 5 and 6. However, the different fluorescence intensities within single cells demonstrate that intracellular pH is not uniformly distributed. We assume that HBT-pH 2 stained lysosomes in living cells according to a pKa of 4.90 for HBT-pH 2, which is at the fringe of lysosomal pH of 4.8. [24–27] 5. Conclusions In this study, three pH probes HBT-pH 1, HBT-pH 2 and HBT-pH 3 were designed and synthesized based on the open-loop of spiropyran
Fig. 6. Confocal fluorescence images of HeLa cells with HBT-pH 2 in PBS buffers for 30 min at pH 2.02, 3.02, 3.83, 4.91, 6.00, 7.30, and 12.0. (a) column: Bright images. (b) column: fluorescence images collected at the channel of 475–550 nm. (c) Column: fluorescence images collected at the channel of 600–700 nm. (d) Merged images of (a), (b) and (c) columns. All of these images were obtained with an excitation wavelength of 405 nm. The scale bar was 20 μm.
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under acidic conditions. As a fluorophore, benzothiazole was coupled into spiropyran structures. Because of the quaternary ammonium salting of N atom, the three probes have good water solubility. All of them displayed good anti-interference abilities and good selectivity, the long emission wavelength (N600 nm), good response to acidic and alkaline environments. The pKa values of HBT-pH 1, HBT-pH 2 and HBT-pH 3 were 6.57, 4.90 and 3.95, respectively, indicating they were suitable for the detection of pH in weak acidic, acidic and strong acidic environments. More importantly, it was confirmed that HBT-pH 2 exhibited low cytotoxicity and the cell imaging of pH changes in living cells were successfully demonstrated. We believe that it has the potential application in early diagnosis of pH-related diseases.
Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 51673113) and the key project of DEGP (No. 2018KZDXM032). Appendix A. Supplementary data Supplementary data includes synthesis of HBT-pH 1, HBT-pH 2 and HBT-pH 3, absorbance of HBT-pH 1, HBT-pH 2, HBT-pH 3 for pH monitoring, 1H NMR spectra of HBT-pH 1, HBT-pH 2, and HBT-pH 3 in solution with different pH values, and photostability. Supplementary data to this article can be found online at https://doi.org/10.1016/j.saa.2019. 117506.
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