The ratiometric fluorescent probe with high quantum yield for quantitative imaging of intracellular pH

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The ratiometric fluorescent probe with high quantum yield for quantitative imaging of intracellular pH Bo Lina,b,1, Li Fana,1, Zhou Yinga, Jinyin Gea, Xueli Wangb, Tongxin Zhangb, Chuan Donga, ⁎ ⁎⁎ Shaomin Shuanga, , Man Shing Wonga,b, a b

College of Chemistry and Chemical Engineering, Institute of Environmental Science, Shanxi University, Taiyuan, 030006, China Department of Chemistry and Institute of Advanced Materials, Hong Kong Baptist University, Hong Kong, China

ARTICLE INFO

ABSTRACT

Keywords: Fluorescent probe High quantum yield Ratiometric pH imaging

Intracellular pH, especially cytoplasmic pH (~7.2) plays a crucial role in cell functions and metabolism. A ratiometric fluorescent probe namely, 6-(2-(benzothiazol-2-yl)vinyl)naphthalen-2-ol (BTNO) was facilely synthesized by the condensation of 6-hydroxy-2-naphthaldehyde and 2-methylbenzothiazole. BTNO exhibited a remarkable ratiometric emission (F456/F526) enhancement in response to a pH change with a linear range of pH = 9.50–7.00 and a pKa value of 7.91 ± 0.03, which is desirable for measuring and monitoring the cytoplasmic pH fluctuations. In addition, because of the high fluorescence quantum yield of BTNO (Φ = 0.88 in DMSO and 0.61 in water relative to quinine sulfate solution in 0.1 M H2SO4), the interferences of the probe on the physiological functions could be greatly reduced. This could also provide enhanced measurement sensitivity. The successful demonstration of BTNO in detecting and monitoring the intracellular pH changes in live HeLa cells via a ratiometric approach confirmed that BTNO held a practical potential in biomedical research.

1. Introduction Intracellular pH (pHi) plays a key role in various physiological processes of cells including reproduction and apoptosis, ion transport and homeostasis, enzyme activity and redox signaling [1–6]. There are many relatively independent subcellular compartments in eukaryotic cells, and an appropriate pH inside individual district is established and balanced such as lysosomal pH between 4.5 and 5.0 [1,2,7,8], Golgi pH site in 6.0–6.7 [1,2,9,10], mitochondrial pH near 8.0 [1,2,11–16] and pH of cytoplasmic matrix approximately 7.2 [1,2,5]. The pH difference among compartments is one of the vital foundations to maintain and control normal physiological functions of the cells [1,2,5]. Earlier studies revealed that intracellular dysfunctions were associated with destroyed homeostasis or even slight fluctuations of the pH in cells. Moreover, some diseases such as inflammation [17–20], cancer [5,21,22] and Alzheimer's disease [5,23,24] were also closely related to intracellular acidification. Thus, real-time monitoring intracellular pH changes in situ can provide direct information of cellular metabolism and further understanding the processes of physiology and pathology. Fluorescent probes are a powerful tool in real-time bioimaging and bioanalysis due to their non-invasiveness, simple operation, excellent

sensitivity, and selectivity [25,26]. Furthermore, in combination with confocal laser scanning microscopy [27], fluorescent method could provide high temporal and spatial resolution in fluorescent imaging. Hence, a variety of fluorescent probes were used for detection of intracellular pH [28,29]. For instance, green fluorescent proteins (GFP) [30], nanoprobes [31–35], organic small molecules [36,37]. Among them, organic small molecules are one of the most important fluorescent probes because (1) they hold are applicable to any testing objects, including the human body, (2) they are relatively cost-effective and easier to handle, and (3) they enablecan usually to provide high signal-to-noise ratios (up to over 1000) due toas a result of ingenious chemical design [37]. Till now, numerous organic small molecular probes have been developed for intracellular pH measurement and imaging [4,5,38–42]. However, the fluorescence signals of a large number of probes vulnerable to influence from various complex environmental factors, such as photo-bleaching, local probe concentration and inhomogeneous cellular distribution [43–45]. Fortunately, most of the aforementioned interferences can be reduced via high fluorescence quantum yield and self-calibration of probe, and thus allow for a more precise intracellular pH measurement with a quantitative manner. The self-calibration based on ratiometric measurement can be implemented

Corresponding author. Corresponding author. College of Chemistry and Chemical Engineering, Institute of Environmental Science, Shanxi University, Taiyuan, 030006, China. E-mail addresses: [email protected] (S. Shuang), [email protected] (M.S. Wong). 1 These authors contributed equally to this work. ⁎

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https://doi.org/10.1016/j.talanta.2019.120279 Received 26 April 2019; Received in revised form 13 August 2019; Accepted 17 August 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Bo Lin, et al., Talanta, https://doi.org/10.1016/j.talanta.2019.120279

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by concurrently recording fluorescence intensities at two wavelengths and calculating their ratios [15,45]. Numerous ratiometric fluorescent probes for measuring intracellular pH have been reported in recent years. However, most of these probes were used for measuring pH of acidic compartments such as lysosomes [46–48] and E. coli cytoplasm [49–51]. Small amount of ratiometric fluorescent probes could be used to real-time quantitatively monitor intracellular pH in the near neutral region. The current some probes use pyrene [52], curcumin [53] and hemicyanine [53] as fluorophore. However, the fluorescence quantum yields of above probes are relatively low. To further improve fluorescence quantum yield of probe, Kim [54] et al. constructed a ratiometric fluorescent probe via the combination of rhodamine and fluorescein to monitor a wide range of pH values in cells. The corresponding quantum yield of the probe was greatly enhanced. The strategy of selecting high fluorescence quantum yield fluorophore provides the opportunity for fabrication of favoriable ratiometric pH probe. Benzothiazole-based dyes have been extensively used as versatile and practical fluorescent probes due to theirs high quantum yield, excellent photostability and ratiometric availability. Recently, we have developed a benzothiazole-based pH probe namely, BTDB with ratiometric emission characteristics and large Stokes shift for extremely acidic sensing [55]. Herein, we reported a novel ratiometric pH probe, 6-(2-(benzothiazol-2-yl)vinyl)naphthalen-2-ol (BTNO, Scheme 1), in which the ethylene bridge was conjugated with a naphthol electron donor (D) and a benzothiazole electron acceptor (A). Importantly, BTNO shows a high fluorescence quantum yield (Φ = 0.88 in DMSO and 0.61 in water relative to quinine sulfate solution in 0.1 M H2SO4), which can effectively reduce the excitation light intensity used preventing it from damaging biological samples as well as drastically improve the sensitivity of fluorescence measurement. Furthermore, BTNO was successfully used to detect and monitor the pH changes in live cells via a ratiometric method.

Fig. 1. Changes of the absorption spectra of BTNO (75 μM) with decreasing the pH from 11.50 to 5.00. Inset: the color of solution changes from yellow to yellow-green with the pH decreasing. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2. Results and discussion 2.1. pH-dependent optical properties of BTNO The absorption and fluorescence emission spectra were first carried out at various pH values from 11.50 to 4.00. As shown in Fig. 1, when pH value is 11.50, BTNO exhibited strong absorption at 385 nm (ε385 4 −1 cm−1). Whereas the pH decreasing from nm = 1.64 × 10 L mol 11.50 to 4.00, the absorption intensity at 385 nm reduced gradually, simultaneously, a new peak appeared at 321 nm and increased dramatically (ε321 nm = 1.36 × 104 L mol−1 cm−1 at pH 4.00). Meanwhile, a well-explicit isosbestic point at 349 nm was observed and the solution color changed from yellow to colorless via naked eye. The excitation spectra of BTNO in Fig. S1 shows that the maximum excitation wavelengths are 385 and 331 nm at pH 11.50 and 4.00,

Fig. 2. Changes of the fluorescence spectra of BTNO (10 μM) with decreasing pH from 11.50 to 4.00 Inset: the color of fluorescence changes from green to aquamarine with the pH decreasing. The excitation wavelength was set at 349 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Scheme 1. Synthetic scheme of BTNO and its structures in neutral and alkaline conditions. 2

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the energy gap (ΔE) between the LUMO and HOMO energy levels increased from 2.22 eV of BTNO-H+ to 3.50 eV of BTNO in the process of the vertical electron excitation of the probe, which is consistent with the blue-shift of the absorption and fluorescence emission spectra of BTNO. 2.3. Selectivity, photostability, and reversibility of BTNO The accuracy of pH detection may be interfered from intracellular various biomolecules and ions. The selectivity response of BTNO (10 μM) to pH in the presence of various physiologically ubiquitous substance were performed at pH 11.50 and 4.00, respectively. As shown in Fig. S4, physiologically ubiquitous metal ions, (for instance K+, Li+, Mg2+ and Ca2+), heavy and transition metal ions (for instance Zn2+, Fe2+, Cu2+, Co2+, Hg2+, Ni2+, Ba2+, Cd2+ and Mn2+), common anions (for instance F−, Br−, I−, SO42 -, S2O32−, SO32−, HS−, NO3−, NO2−, Ac−, HCO3−, ClO4− and ClO−) as well as some other important substances (such as H2O2, O2−, O2−, NO, ONOO−, L-glutathione, homocysteine and cysteine) caused neglect effect on the fluorescence intensity ratios (F456/F526) of BTNO at pH 11.50 and 4.00, respectively. These results indicated that BTNO showed excellent selectivity response to pH over biomolecules and ions under physiological concentrations and the relative errors were less than ± 5%. The photostability of BTNO was measured by monitoring the fluorescence emissions at 526 nm and 456 nm at pH 11.50 and pH 4.00 over a period of 2 h, respectively (Fig. S3). As shown in Fig. 4, the fluorescent emission ratios of BTNO remained almost stable, implying that the BTNO is highly photostable. Good pH-dependent reversibility is a mandatory property for BTNO to monitor pH changes in living organelles in real-time. To evaluate its reversibility, the pH value of the solution was modulated repeatedly between 11.50 and 4.00 via using small volume HCl (1 M) and NaOH (1 M), and the emission ratios (F456/F526) of BTNO were recorded. As shown in Fig. S5, the reaction of BTNO with OH− (or H+) is almost fully reversible. Furthermore, the response and recovery times are rapid in different pH solutions indicating the excellent pH-dependent reversibility of the probe.

Fig. 3. Boltzmann function fitting of the pH-dependent ratio of BTNO at F456/ F526. Inset: the best linearity shown in the pH range of 7.00–9.50.

respectively. Meanwhile, a well-definite equal-excitation point and equal-emission point at 349 and 481 nm were observed, respectively. As depicted in Fig. 2, under the alkaline condition at pH 11.50, BTNO displayed strong emission at 526 nm upon an excitation at 349 nm. However, decreasing the pH from 11.50 to 4.00, the fluorescence intensity at 526 nm reduced gradually, concomitantly, a new peak at 456 nm emerged and enhanced progressively with an equal-emission point at 481 nm. The fluorescence color changed from green to aquamarine. Moreover, BTNO showed significantly large Stokes shift of 141 and 125 nm at pH 11.50 and 4.00, respectively. Such a large shift could effectively reduce the spectral interference from the excitation light. More importantly, BTNO exhibited high fluorescence quantum yield (Φ = 0.88 in DMSO and 0.61 in water) relative to quinine sulfate solution (Φ = 0.54 in 0.1 M H2SO4). The high fluorescence quantum yield could effectively reduce the excitation light intensity used and thus preventing it from damaging biological samples as well as drastically improve the sensitivity of fluorescence measurement. The pKa value of BTNO could be acquired from Boltzmann function fitting the pH dependence ratiometric fluorescence intensity (F456/F526) versus pH. As shown in Fig. 3, the pKa value of BTNO was estimated to be 7.91 ± 0.03, with the linear regression equation of 2.13641–0.21119 × pH (R2 = 0.9995) in the range of 9.50–7.00. The emission ratio (F456/F526) varied from 0.10 at pH 11.50 to 0.76 at pH 4.00 with a change of 7.6-fold between two plateaus of the pH titration curve. Thus, our probe BTNO could be potentially useful for quantitatively monitoring the cytoplasmic matrix pH (pH ~ 7.2) fluctuation by means of the ratiometric fluorescence measurement.

2.4. Cytotoxicity measurements of BTNO It is also crucial to evaluate the cytotoxicity of the probe to living cells and IC50 was widely used as the evaluation criterion [56]. The IC50 value of BTNO was estimated to be 92.33 μM on Graphpad-prism 5.0

2.2. Theoretical calculations In order to better comprehend the optical characteristics of BTNO before and after reaction with proton, the time-dependent density functional theory (TD-DFT) calculations were performed on Gaussian 09 program under B3LYP/6-31 G(d) level. Fig. S2 shows the optimized structure and the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) plots of BTNO and its deprotonated form (BTNO-H+). The HOMO and LUMO electron clouds of BTNO distribute homogeneously in the entire π-conjugated framework of the molecule. Whereas the HOMO electron cloud of BTNO-H+ mainly concentrated in the naphthol moiety (D) and double bond, and the LUMO electron cloud of BTNO-H+ primarily distributed in the benzothiazole moiety (A) and double bond. Such electron cloud distribution is favorable to the intramolecular charge transfer (ICT) from naphthol moiety to benzothiazole unit in the excited state. In addition,

Fig. 4. The changes of BTNO's F456/F526 of with times under pH 11.50 and 4.00, respectively. λex = 349 nm. Excitation and emission bandwidths were both set at 1.2 nm. 3

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Fig. 5. (1-35) Fluorescence images in HeLa cells after treated with 10 μM BTNO at pH 5.50 (the first line), 6.50 (the second line), 7.00 (the third line), 7.40 (the fourth line), 8.00 (the fifth line), 8.50 (the sixth line), 9.50 (the seventh line), respectively. The blue channel and green channel images were collected at 430–480 nm and 500–550 nm, respectively, with excitation at 405 nm. The first, second, third, fourth and fifth row show the corresponding bright-field cells, blue channel, green channel, merged imaging and pseudocolor ratio imaging, respectively. (36) The linear relationship plot of average FBlue/FGreen with pH. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. (1-25), Fluorescence images of 10 μM BTNO stained HeLa cells after addition of NH4Cl (5 mM) to the imaging solution for 10, 20, 30 and 40 min; (26) the pH changes of the cell after being treated with NH4Cl over time in which the pH values were calculated with the equation of FBlue/FGreen = 3.3308–0.313 × pH.

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software according to the results of MTT assay (Fig. S6) [57]. This value clearly indicated that BTNO showed low toxicity to living cells and would be suitable for cell imaging experiments.

pH reversibility, and low cytotoxicity, which is highly desirable for cytoplasmic pH imaging. BTNO also showed excellent cell membrane permeability and has been successfully used for real-time detection and monitoring intracellular pH fluctuations in live cells using ratiometric technique. Our results demonstrated that probe BTNO had a great potential for real-time monitoring the physiological pH fluctuations, and investigating diseases related to cytoplasmic pH change.

2.5. Fluorescence imaging of BTNO in HeLa cells In order to explore the ability of BTNO to measure and image intracellular pH fluctuations, the pH-dependent ratiometric emission responses of BTNO in living HeLa cells were studied. The intracellular pH between 9.50 and 5.50 was homogenized to the surrounding medium using the H+/K+ ionophore, nigericin [58]. As shown in Fig. 5, BTNO dispersed well in the whole cytoplasm between the nucleus and cell membrane, indicating that BTNO had excellent cell membrane permeability. When pH is 9.50, the BTNO-stained HeLa cells exhibited very weak fluorescence in the blue channel (Fig. 5–33), and relatively bright fluorescence in the green channel (Fig. 5–32). With the pH decreasing from 9.50 to 5.50, the blue fluorescence enhanced significantly (the third row in Fig. 5), in contrast, the green fluorescence progressively quenched (the second row in Fig. 5). Bright-field images (the first row in Fig. 5) confirmed that the viability of the cells after BTNO incubation. Moreover, the remarkable pseudocolor ratio (the fifth row in Fig. 5) based on the ratiometric responses of the blue channel to the green channel afforded a characteristic pH-dependent fluorescence signal. Furthermore, the fluorescence intensity ratio exhibited a good linear response in the pH range of 9.50–5.50 (Fig. 5–36, FBlue/ FGreen = 3.3308–0.313 × pH, R2 = 0.99642), which demonstrates that BTNO could be used as a ratiometric pH sensing probe for intracellular pH detection and imaging. To demonstrate the capability of BTNO for monitoring cytoplasmic pH fluctuations in real-time, NH4Cl (5 mM) was used to treat BTNOstained HeLa cells as high concentrations of NH4Cl could dramatically increase cytoplasmic pH [59]. As shown in Fig. 6, upon addition of NH4Cl, the fluorescence intensity in the blue channel progressively faded out over a period of 40 min whereas the green fluorescence intensity steadily increased. Fig. 6–26 indicated that the intracellular pH increased from 6.97 ± 0.10 to 8.14 ± 0.04 over time. It was reported that hydroxyl radicals generated by H2O2 could cause acidification of cells due to the production of some acidic substances such as phosphoric acid [5]. After an addition 0.1 mM of H2O2 into BTNO-stained HeLa cells for 40 min, the fluorescence intensity in the blue channel significantly increased and the fluorescence intensity in the green channel diminished slightly (Fig. S7). Meanwhile, the pH values decreased from 6.92 ± 0.09 to 5.32 ± 0.04 according to the pH calibration curve in Fig. 5–36. These results clearly demonstrated that BTNO can be employed to monitoring cytoplasmic pH fluctuations in living cells via ratiometric imaging.

Acknowledgments We are gratefully for the financial supports of the National Natural Science Foundation of China (No. 21675135, 21874087 and 21575084), the Shanxi Scholarship Council of China (2017 Key-1). Talents Project and General Research Fund (GRF) (HKBU 12301317) by the Research Grant Council of Hong Kong. We also appreciate Lanqi Huang, Di Xu and Wu Chun from Hong Kong Baptist University, Hong Kong for assisting us on performing experiments. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120279. References [1] C. Joseph R, G. Sergio, O. John, Sensors and regulators of intracellular pH, Nat. Rev. Mol. Cell Biol. 11 (1) (2010) 50–61. [2] P. Paroutis, N. Touret, S. Grinstein, The pH of the secretory pathway: measurement, determinants, and regulation, Physiology 19 (19) (2004) 207–215. [3] V.A. G, M.P. B, Spatially and functionally distinct Ca2+ stores in sarcoplasmic and endoplasmic reticulum, Science 275 (5306) (1997) 1643–1648. [4] W. Niu, Z. Wei, J. Jia, S. Shuang, C. Dong, K. Yun, A ratiometric emission NIRfluorescent probe for sensing and imaging pH changes in live cells, Dyes Pigments 152 (2018). [5] L. Yinhui, W. Yijun, Y. Sheng, Z. Yirong, Y. Lin, Z. Jing, Y. Ronghua, Hemicyaninebased high resolution ratiometric near-infrared fluorescent probe for monitoring pH changes in vivo, Anal. Chem. 87 (4) (2015) 2495. [6] L.-Q. Niu, J. Huang, Z.-J. Yan, Y.-H. Men, Y. Luo, X.-M. Zhou, J.-M. Wang, J.H. Wang, Fluorescence detection of intracellular pH changes in the mitochondriaassociated process of mitophagy using a hemicyanine-based fluorescent probe, Spectrochim. Acta A Mol. Biomol. Spectrosc. 207 (2019) 123–131. [7] J. Ge, L. Fan, K. Zhang, T. Ou, Y. Li, C. Zhang, C. Dong, S. Shuang, S.W. Man, A twophoton ratiometric fluorescent probe for effective monitoring of lysosomal pH in live cells and cancer tissues, Sens. Actuators B Chem. 262 (2018). [8] X. Liu, Y. Su, H. Tian, Y. Lei, H. Zhang, X. Song, J.W. Foley, A ratiometric fluorescent probe for lysosomal pH measurement and imaging in living cells using single-wavelength excitation, Anal. Chem. 89 (13) (2017). [9] N. Demaurex, W. Furuya, S. D'Souza, J.S. Bonifacino, S. Grinstein, Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements of pH using retrieval of TGN38 and furin from the cell surface, J. Biol. Chem. 273 (4) (1998) 2044–2051. [10] J.H. Kim, C.A. Lingwood, D.B. Williams, W. Furuya, M.F. Manolson, S. Grinstein, Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor, JCB (J. Cell Biol.) 134 (6) (1996) 1387. [11] M.F.C. Abad, G.D. Benedetto, P.J. Magalhães, L. Filippin, T. Pozzan, Mitochondrial pH monitored by a new engineered GFP mutant, J. Biol. Chem. 279 (12) (2003) 11521–11529. [12] M.H. Lee, N. Park, C. Yi, J.H. Han, J.H. Hong, K.P. Kim, D.H. Kang, J.L. Sessler, C. Kang, J.S. Kim, Mitochondria-immobilized pH-sensitive off-on fluorescent probe, J. Am. Chem. Soc. 136 (40) (2014) 14136–14142. [13] Q. Sujie, L. Qi, L. Weimin, R. Haohui, Z. Hongyan, W. Jiasheng, G. Jiechao, W. Pengfei, Coumarin/fluorescein-fused Fluorescent Dyes for Rapidly Monitoring Mitochondrial pH Changes in Living Cells, Spectrochim. Acta. A: Mol. Biomol. Spectros. (2018). [14] M.Y. Wu, K. Li, Y.H. Liu, K.K. Yu, Y.M. Xie, X.D. Zhou, X.Q. Yu, Mitochondriatargeted ratiometric fluorescent probe for real time monitoring of pH in living cells, Biomaterials 53 (65) (2015) 669–678. [15] F. Wang, D. Liu, Y. Shen, J. Liu, D. Li, X. Tian, Q. Zhang, J. Wu, S. Li, Y. Tian, A twophoton mitochondria-targeted fluorescent probe for the detection of pH fluctuation in tumor and living cells, Dyes and Pigments (2019). [16] B. Lin, L. Fan, J. Ge, W. Zhang, C. Zhang, C. Dong, S. Shuang, A naphthalene-based fluorescent probe with a large Stokes shift for mitochondrial pH imaging, Analyst 143 (20) (2018) 5054–5060. [17] M. Lee, N. Park, C. Yi, J. Han, J. Hong, K. Kim, D. Kang, J. Sessler, C. Kang, J. Kim, Mitochondria-ImmobilizedpH-sensitive off–onfluorescent probe, J. Am. Chem. Soc. 136 (40) (2016) 14136–14142. [18] E. Carafoli, I. Roman, Mitochondria and disease, Mol. Asp. Med. 3 (5) (1980) 295–429.

2.6. Experiments Experimental details can be found on the electronic support information. 3. Conclusions In summary, a novel ratiometric pH fluorescence probe namely, BTNO was facilely synthesized by the condensation of 6-hydroxy-2naphthaldehyde and 2-methylbenzothiazole, which was successfully used for quantitative monitoring and imaging of pH fluctuations in live cells. BTNO exhibited a remarkable ratiometric emission (F456/F526) enhancement in response to a pH changes within a linear range of pH = 9.50–7.00 with a pKa value of 7.91 ± 0.03. Interestingly, BTNO showed a high fluorescence quantum yield of 0.88 in DMSO and 0.61 in water, which could effectively decrease the excitation intensity employed and thus preventing it from damaging biological samples, as well as drastically improve the sensitivity of fluorescence measurement. Moreover, it displayed high selectivity, good photostability, excellent 6

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