A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke

Analytica Chimica Acta xxx (xxxx) xxx

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A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke Guo-Jiang Mao a, *, Zhen-Zhen Liang a, Guang-Qi Gao a, Ying-Ying Wang a, Xin-Yu Guo a, Li Su a, Hua Zhang a, **, Qiu-Juan Ma b, Guisheng Zhang a, *** a Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, 453007, PR China b School of Pharmacology, Henan University of Traditional Chinese Medicine, Zhengzhou, 450046, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


A photostable Si-rhodamine-based near-infrared fluorescent probe, Lyso-NIR-pH, was developed for pH in lysosomes.
Lyso-NIR-pH was successfully applied to monitor lysosomal pH increases induced by chloroquine and apoptosis.
Lyso-NIR-pH was successfully applied to monitor lysosomal pH increases during heat stroke.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 September 2019 Accepted 18 September 2019 Available online xxx

Heat stroke is a symptom of hyperthermia with a temperature of more than 40  C, which usually leads to all kinds of physical discomfort and even death. It is necessary to study the mechanism of action of heat stroke on cells or organelles (such as cytotoxicity of heat) and the processes of cells or organelles during heat stroke. Recent studies have shown that there is a certain correlation between heat stroke and lysosome acidity. In order to clarify their relationship, Lyso-NIR-pH, a photostable Si-rhodamine-based near-infrared fluorescent probe, was developed for sensing pH changes in lysosomes during heat stroke in this paper. For Lyso-NIR-pH, a morpholine group is employed as the lysosome-targeting unit and a Hþ-triggered openable deoxylactam is employed as the response unit to pH. Lyso-NIR-pH can detect pH with a high selectivity and a sensitivity, and its pKa is 4.63. Lyso-NIR-pH also has outstanding imaging performances, such as excellent lysosome-targeting ability, low autofluorescence and photostable fluorescence signal, which are in favor of long-term imaging of pH with accurate fluorescence signals. Moreover, we successfully applied Lyso-NIR-pH to monitor lysosomal pH increases induced by chloroquine and apoptosis in live cells. Finally, we successfully applied Lyso-NIR-pH for monitoring changes of lysosomal pH during heat stroke. These results confirmed that Lyso-NIR-pH is a powerful tool to monitor pH change in lysosomes and study its possible effects. © 2019 Elsevier B.V. All rights reserved.

Keywords: Fluorescent probe Lysosomal pH Heat stroke Near-infrared Si-rhodamine

* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (G.-J. Mao), [email protected] (H. Zhang), [email protected] (G. Zhang). https://doi.org/10.1016/j.aca.2019.09.053 0003-2670/© 2019 Elsevier B.V. All rights reserved.

Please cite this article as: G.-J. Mao et al., A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.053

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1. Introduction Heat stroke is a symptom of hyperthermia with a temperature of more than 40  C, which usually leads to dizziness, mental disorder, loss of consciousness, organ failure and even death [1]. Current studies have shown that central nervous system dysfunction, dry skin, individuals on medications (or drug users), some genetic factors, and environmental factors can lead to heat stroke [1e3]. However, the mechanism of action of heat stroke on cells or organelles (such as cytotoxicity of heat) and the processes of cells or organelles during heat stroke are not fully clear [4]. Lysosomes are important organelles in cells, where proteins, nucleic acids, polysaccharides and other biological macromolecules are decomposed, and called as the “digestive organs” in cells [5,6]. The pH value of lysosomes is usually acidic, ranging from 3.8 to 5.5. Abnormal variation in the lysosomal pH causes defects in lysosomal function, which leads to including lysosome storage disease, inflammation [7,8]. Some studies have found that the gradient decay of lysosomal proton after addition of alkaline drugs or during apoptosis process seems to cause the acidification of the cytosol and lysosomal pH increase [9,34], which would be used for monitoring apoptosis. Thus, it is important to investigate lysosomal pH in living cells to understand its physiological and pathological processes. Moreover, recent studies have shown that heat stress can activate lysosome-mitochondria apoptotic pathway and increase the membrane permeability of lysosomes to induce the abnormal changes of pH in lysosomes and trigger a series of symptoms [10]. Therefore, heat stroke has a certain mechanism of action on lysosomes, but the mechanism of heat cytotoxicity of heat stroke is not fully clear. It is necessary to accurately measure the acidity changes of lysosomes under heat stress (such as heat shock) and further study on the interaction between lysosome behavior and heat stroke, which would help us to better comprehend the mechanism of heat stroke in organelles. Due to high sensitivity, excellent spatial and temporal resolution, and nondisruptive feature of fluorescent probes, they have been widely applied in various fields [11e15]. Until now, a lot of fluorescent probes for pH have been developed [16e27]. Although some probes have recently been reported for monitoring lysosomal pH change [28e43] and some of them were applied for monitoring lysosomal pH change induced by chloroquine and apoptosis, fluorescent probes for sensing pH changes in lysosomes during heat stroke are rare. Until now, only three fluorescent probes for imaging pH in lysosomes during heat shock have been developed [44e46]. Ma’s group developed a ratiometric probe to monitor pH in lysosomes during heat stroke based on cyanine dye. Subsequently, Huang’s group took hemicyanine dye to prepare a fluorescent probe for ratiometric imaging

pH in lysosomes during heat shock. Yin’s group reported a probe based on naphthalimide to image lysosomal pH during heat stroke. The above probes have their own advantages and have successfully obtained the information between heat stroke and lysosomal pH. However, the two probes from Ma’s group and Huang’s group are based on moderate photostable cyanine dyes, which are not conducive to long-time imaging in vivo with accurate fluorescence signal. Moreover, the excitations and emissions of the probes from Huang’s group and Yin’s group are both located in shortwavelength region, which usually suffered from many problems, such as phototoxicity, autofluorescence in vivo, and photobleaching. Near-infrared probe based on long wavelength excitation and emission can just overcome the above problems [11,47]. Sirhodamine is a near-infrared (NIR) dye with outstanding properties, and has been widely used in imaging [47e57] with many advantages including minimizing photodamage, increasing tissue penetration depth, and minimizing autofluorescence. Especially, Sirhodamine has an excellent photostability, which is far superior to traditional NIR cyanine dyes and in favor of long-time imaging in vivo with stable and accurate fluorescence signals [47e49]. Based on the outstanding photostability and near-infrared emission of Sirhodamine, a near-infrared fluorescent probe (Lyso-NIR-pH) for sensing pH changes in lysosomes during heat stroke was developed in this paper. For Lyso-NIR-pH, a morpholine group is employed as the lysosome-targeting unit and a Hþ-triggered openable deoxylactam is employed as the response unit to pH (Scheme 1). LysoNIR-pH can detect pH with a high sensitivity, a high selectivity, and a pKa of 4.63. The probe also has outstanding imaging performances, such as excellent lysosome-targeting ability, low autofluorescence, and photostable fluorescence, which are in favor of long-time monitoring of pH in vivo. Additionally, we successfully applied Lyso-NIR-pH to monitor increases in pH of lysosomes induced by chloroquine and apoptosis in live cells. Finally, LysoNIR-pH was successfully employed for monitoring changes of lysosomal pH during heat stroke. 2. Experimental 2.1. Reagents and apparatus Reagents and apparatus are listed in supporting information. 2.2. Syntheses The syntheses of Compound 2 and Lyso-NIR-pH were depicted in Scheme 2. Compound 1 were synthesized following the literature procedure [57]. The detailed synthesis procedure of Compound 2 and Lyso-NIR-pH were list in the supporting information.

Scheme 1. The response mechanism of Lyso-NIR-pH to pH in lysosome.

Please cite this article as: G.-J. Mao et al., A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.053

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Scheme 2. Syntheses of Lyso-NIR-pH. (a) (1) phosphorus oxychloride, 1, 2-dichloroethane, 85  C, 4 h; (2) 4-(2-aminoethyl)-morpholine, triethylamine, CH3CN. (b) THF, LiAlH4, 0.5 h.

Compound 2 and Lyso-NIR-pH were characterized by 1H NMR, 13C NMR and MS in the supporting information. 2.3. Spectrum measurement procedure Spectrum measurement procedures are listed in supporting information. 2.4. Cytotoxicity assay Cytotoxicities assay of Lyso-NIR-pH for HeLa cells and A549 cells were list the supporting information. 2.5. Fluorescence imaging in cells For colocalization experiments, A549 cells or HeLa cells were firstly incubated with Lyso-NIR-pH (5.0 mM) and MitoTracker Green FM (500 nM) or LysoSensorTM Green DND-26 (1.0 mM) at 37  C for 30 min, then washed with Dulbecco’s phosphate buffered saline (DPBS) three times and imaged. For LysoSensorTM Green DND-26, the excitation and emission were set at 488 nm and between 505 and 545 nm, respectively. For MitoTracker Green FM, the excitation and emission were set at 488 nm and between 505 and 545 nm, respectively. For Lyso-NIR-pH, the excitation and emission were set at 635 nm and between 650 and 720 nm, respectively. The change of lysosomal pH was induced by chloroquine (100 mM), and the apoptosis was induced by dexamethasone (2 mM). 2.6. Imaging lysosomal pH in buffers with various pH Procedures of imaging lysosomal pH in buffers with various pH were list in the supporting information. 2.7. Imaging lysosomal pH during heat shock HeLa cells or A549 cells were firstly incubated with Lyso-NIRpH (5 mM) at 37  C for 30 min, then washed three times with DPBS (pH 7.4) and imaged at 37  C (control), 41  C and 45  C, respectively. The excitation and emission were set at 635 nm and between 650 and 720 nm, respectively. 3. Result and discussion 3.1. Analytical response of Lyso-NIR-pH to pH Firstly, the response of Lyso-NIR-pH to pH was investigated. The UVeVis spectra and emission spectra of Lyso-NIR-pH at different pH were investigated (Fig. 1a and Fig. 1b). When pH changed from 7.4 to 3, the emission intensity at 675 nm and UVeVis absorption intensity at 655 nm both displayed gradual enhancements due to Hþ-triggered ring-opening reaction process of deoxylactam of the

probe. Lyso-NIR-pH shows a 1400-fold increase in fluorescence between pH 7.4 and pH 3. In Fig. 1c, the analysis of fluorescence intensity changes as a function of pH by using the HendersonHasselbalch equation: log[(Imax-I)/(I-Imin)] ¼ pH-pKa, where I is the observed fluorescence intensity at 675 nm, Imax and Imin are the corresponding maximum and minimum respectively, yielded a pKa of 4.63. The pKa of the probe is located in the range of lysosomal pH fluctuation (3.8e5.5), which indicates that the probe can meet the needs of monitoring lysosomal pH. Also, the probe showed a high selectivity for pH over biologically-related species (Fig. 1d and Fig. S1). The fluorescence intensity of Lyso-NIR-pH did not obviously changed after adding 40 equiv of metal ions and ammonium ion. Also, various common anions all exhibited negligible interference. After adding 1 mM of amino acids and glutathione, the fluorescence intensity of Lyso-NIR-pH remained low. Moreover, the probe Lyso-NIR-pH displayed an excellent reversibility. In Fig. S2, when the pH is switched between 3.5 and 7.0 three times, the fluorescence intensity of Lyso-NIR-pH maintained approximately above 90% of the original signal. These results confirmed that the probe has an outstanding analytical performance to pH and was a powerful tool for monitoring pH. 3.2. Proposed mechanism studies of Lyso-NIR-pH for pH Based on some previous similar studies [32,36] and the optical performance of Lyso-NIR-pH induced by pH changes, proposed mechanism of Lyso-NIR-pH for pH is due to the occurrence of the protonation process depicted in Scheme 1. To verify the proposed mechanism of Lyso-NIR-pH for pH, 1H NMR spectra about LysoNIR-pH and Lyso-NIR-pH (þCF3COOD) were investigated (Fig. S3). As shown in Fig. S3, the addition of trifluoroacetic acid led to downfield shifts of the proton signals of morpholine moiety in Lyso-NIR-pH (a-a0 , b-b0 and g-g0 ), and a new proton signal appeared at d’. These changes could be attributed to the protonation of the nitrogen atom in the morpholine moiety. Meanwhile, the slight downfield shift of the proton signals of aromatic rings occur red, which is indicative of the enhanced delocalization of electrons in xanthenes under acidic conditions. The above results are in accordance with a previous similar study [36]. At the same time, another new proton multiplet signal appeared at ε0 , which can be attributed to the proton signal of the -NH-group after Hþ-triggered deoxylactam ring-opening. Overall, the above results indicate that the proposed mechanism of Lyso-NIR-pH for sensing pH includes the extension of conjugation after ring-opening induced by Hþ and the protonation of the nitrogen atom in the morpholine moiety (Fig. 1). 3.3. Lysosome-targeting ability of Lyso-NIR-pH Subsequently, in order to demonstrate the universal applicability of the probe’s lysosome-targeting ability, Lyso-NIR-pH was assessed in HeLa cells and A549 cells. Firstly, The cytotoxicities of

Please cite this article as: G.-J. Mao et al., A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.053

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Fig. 1. (a, b) Fluorescence emission spectra (a) and UVevis spectra (b) of Lyso-NIR-pH (5 mM) in the presence of different pH, respectively. (c) The pH titration curve was plotted by NIR fluorescence as a function of pH. (d) Fluorescence intensity of Lyso-NIR-pH (5 mM) in the presence of other biologically relevant species in Britton-Robinson buffer solution at  pH 7.4. From left to right: (1) control (pH ¼ 5); (2) Naþ; (3) Kþ; (4) Ca2þ; (5) Mg2þ; (6) Fe3þ; (7) Cu2þ; (8) Zn2þ; (9) Mn2þ; (10) Ni2þ; (11) Cd2þ; (12) Co2þ; (13) NHþ 4 ; (14) CH3COO ;    2 2 2 2   2 (15) CO2 3 ; (16) SO4 ; (17) F ; (18) Br ; (19) I ; (20) S ; (21) SO3 (22) S2O3 ; (23) NO2 ; (24) H2PO4 ; (25) HPO4 ; (26) Glutathione; (27) Arginine; (28) Valine; (29) Tryptophan; (30) Cysteine; (31) Glycine; (32) Homocysteine. Concentration: 200 mM for (2)e(25); 1 mM for others.

Lyso-NIR-pH in HeLa cells and A549 cells were investigated (Fig. S4). High cell viabilities (more than 88%) were observed in the presence of a series of concentration of Lyso-NIR-pH (0e30 mM), which indicated that Lyso-NIR-pH possesses low cytotoxicity to cells. Next, we applied Lyso-NIR-pH into colocalization experiments in HeLa cells and A549 cells to assess the lysosome-targeting ability of Lyso-NIR-pH (Fig. 2 and Fig. S5). The cells stained with Lyso-NIR-pH show obvious red fluorescence (Fig. 2b and Fig. S5b). And the cells stained with a commercially lysosomal tracker (LysoSensorTM Green DND-26) exhibit noticeable green fluorescence (Fig. 2a and Fig. S5a). The fluorescence overlaps between the above two channels are high(Fig. 2c and Fig. S5c), with high Pearson’s colocalization coefficients (0.91 for HeLa cells and 0.90 for A549 cells) and the changes in the fluorescence intensity profile of regions of interest (red straight line in Fig. 2a and Fig. S5a) tend towards synchronization (Fig. 2e and Fig. S5e). At the same time, we also examined the mitochondria-targetable ability of the probe Lyso-NIR-pH with a commercially mitochondrial tracker (MitoTracker Green FM). As shown in Fig. 2f-j and Fig. S5f-S5j, there is poor overlap between the probe and LysoTracker Green DND-26, with low Pearson’s correlation coefficients(0.22 for HeLa cells and 0.43 for A549 cells) and bad linear overlap (Fig. 2h-j and Fig. S5hS5j). The results strongly confirm that Lyso-NIR-pH can specifically localize in lysosome. Moreover, photostability of Lyso-NIRpH was investigated (Fig. 3). As shown in the figures, the long-time irradiation didn’t resulted in a remarkable fluorescence decrease, and its photostability is superior than Cy5-N3 in cells. The result indicated that Lyso-NIR-pH has an outstanding photostability and can meet the need for long-term imaging in vivo.

3.4. Fluorescence imaging of pH in lysosome by Lyso-NIR-pH Next, Lyso-NIR-pH was employed to image pH of lysosomes in HeLa cells and A549 cells (Fig. 4 and Fig. S6). The cells were firstly incubated with Lyso-NIR-pH and then incubated in different pH buffers (4.0, 5.0, and 6.0) in the presence of nigericin, which was used for homogenizing the intracellular pH and extracellular medium [58]. As shown in Fig. 4 and Fig. S6, the red fluorescence gradually decreases with the increase of pH. For HeLa cells, the normalized fluorescence intensities were list as follows: 1 at pH 4.0, 0.79 at pH 5.0, and 0.28 at pH 6.0. For A549 cells, a similar phenomenon was observed. The normalized fluorescence intensities were respectively list as follows: 1 at pH 4.0, 0.71 at pH 5.0, and 0.22 at pH 6.0. These results indicated that Lyso-NIR-pH is a sensitive tool to monitor lysosomal pH.

3.5. Fluorescence monitoring changes of lysosomal pH induced by drug In order to further prove the practical application performance of Lyso-NIR-pH, the probe was used to monitor drug-induced changes in lysosomal pH in vivo. HeLa cells were firstly incubated with Lyso-NIR-pH with a remarkable fluorescence. Subsequently, the same cells were further treated with chloroquine, which is an alkaline drug that can neutralize the lysosomal acidity and result in an increase of lysosomal pH [59]. After adding chloroquine (100 mM), the fluorescence obviously decreased in 150 s (Fig. 5). This result indicates that Lyso-NIR-pH can monitor lysosomal pH.

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Fig. 2. Lysosome colocalization test and Mitochondria colocalization test for Lyso-NIR-pH (5 mM) in HeLa cells. (a)e(e) for lysosome colocalization test with LysoSensorTM Green DND-26 (1.0 mM); (f)e(j) for mitochondria colocalization test with MitoTracker Green FM (500 nM). (a) Fluorescence image of LysoSensorTM Green DND-26; (f) Fluorescence image of MitoTracker Green FM; (b, g) Fluorescence image of Lyso-NIR-pH; (c, h) Overlay of (a) and (b), overlay (f) and (g), respectively; (d, i) Intensity correlation plot of region inside the red ellipse; (e, j) Cross-sectional analysis along the red line. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3. Confocal images of HeLa cells with Cy5-N3 (10 mM) and Lyso-NIR-pH (5 mM), respectively. (a) Images by continuously irradiated by laser (635 nm) for different times. Figures in the figure represent the number of imaging times. (b) Quantification of the relative mean fluorescence levels of cells from the images of Cy5-N3 and Lyso-NIR-pH. Femtosecond pulses at 20% laser power.

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Fig. 4. (a) Confocal images of HeLa cells preloaded on Lyso-NIR-pH (5 mM) and further incubated in buffers with various pH values (4.0, 5.0, and 6.0) in the presence of 10 mM of nigericin for 30 min at 37  C. (b) Quantified relative fluorescence intensity of images.

3.6. Fluorescence monitoring changes of lysosomal pH during apoptosis Lysosomal pH would increase with apoptosis processing, and this is related to lysosomal proton release [9]. We further applied Lyso-NIR-pH to track lysosomal pH during apoptosis (Fig. 6). HeLa cells were firstly cultivated with Lyso-NIR-pH and showed a strong

fluorescence. After adding the dexamethasone (2 mM) that can induce apoptosis [60], the fluorescence intensity showed a timedependent decrease due to the apoptosis-induced increase in lysosomal pH. The dramatically morphological changes of HeLa cells confirmed that apoptosis process occurred. The result further proved that Lyso-NIR-pH can real-time imaging lysosomal pH changes.

Fig. 5. (a) Confocal images of Lyso-NIR-pH in HeLa cells stimulated with chloroquine (100 mM). (b) Quantified relative fluorescence intensity of images.

Please cite this article as: G.-J. Mao et al., A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.053

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Fig. 6. (a) Time dependent fluorescence changes of Lyso-NIR-pH in HeLa cells during apoptosis induced by dexamethasone (2 mM). (b) Quantified relative fluorescence intensity of images.

Fig. 7. (a) The fluorescence images of Lyso-NIR-pH -loaded HeLa cells under heat shock at 37  C, 41  C, and 45  C for 20 min. (b) Quantified relative fluorescence intensity of images.

3.7. Fluorescence monitoring changes of lysosomal pH during heat stroke At last, Lyso-NIR-pH was applied to investigate changes of lysosomal pH during heat stroke (Fig. 7 and Fig. S7). The heat stroke temperatures were set at 41  C and 45  C. As shown in the figures, the fluorescence intensities obviously decreased with increases of the temperature. For HeLa cells, the normalized fluorescence

intensities were list as follows: 1 at 37  C, 0.71 at 41  C, and 0.56 at 45  C. For A549 cells, the normalized fluorescence intensities were list as follows: 1 at 37  C, 0.75 at 41  C, and 0.60 at 45  C. Moreover, in order to confirm that the fluorescence change of the probe is caused by the change of pH instead of the change of the temperature, the influence of temperature on the fluorescence intensity of Lyso-NIR-pH was determined between 37  C and 45  C in buffers (Fig. 8). As shown in Fig. 8, the fluorescence intensity of the probe

Please cite this article as: G.-J. Mao et al., A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.053

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(17IRTSTHN001), Key Project of Science and Technology of Henan Province (192102210041). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.09.053. References

Fig. 8. Effects of temperature on the fluorescence intensity of Lyso-NIR-pH (5 mM) in Britton-Robinson buffer solution at pH 4.0, 5.0, and 6.0.

almost remained stable with the increase of temperature. Thus, the above results proved that heat stroke can induce an increase of pH in lysosomes, which is consistent with previous reports [44,46]. 4. Conclusion In a word, we have reported a NIR fluorescent probe (Lyso-NIRpH) for lysosomal pH with outstanding photostability. The probe can detect pH with a high selectivity, an excellent sensitivity to pH, and a pKa of 4.63 in vitro, and has an excellent lysosome-targeting ability in cells. Based on outstanding photostability and NIR emission of Si-rhodamine, Lyso-NIR-pH also displayed outstanding imaging performances in vivo including low autofluorescence and photostable fluorescence. Moreover, Lyso-NIR-pH was successfully employed to image lysosomal pH increases induced by chloroquine and apoptosis, respectively. Finally, we successfully applied LysoNIR-pH for monitoring changes of lysosomal pH during heat stroke. All of the results demonstrated that Lyso-NIR-pH is a powerful tool to monitor pH in lysosomes and study on effects of pH on lysosomes. Notes The authors declare no competing financial interest. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grants 21505032, 21722501, 21605039, 21405034 and U1704170), Project funded by China Postdoctoral Science Foundation (Grants 2017M612405), the key scientific research project of higher education of the Henan province (15A150016), Dr. start-up project funding of Henan Normal University (qd14126), the Youth Science Foundation of Henan Normal University (2014QK13), Program for Science Technology Innovation Talents in Universities of Henan Province (18HASTIT001), the Program for Innovative Research Team in University of Henan Province

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Please cite this article as: G.-J. Mao et al., A photostable Si-rhodamine-based near-infrared fluorescent probe for monitoring lysosomal pH during heat stroke, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.09.053