A Mitochondria-Targeted Ratiometric Fluorescent pH Probe - ACS

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A Mitochondria-Targeted Ratiometric Fluorescent pH Probe Xiangjun Liu, Linlin Wang, Tao Bing, Nan Zhang, and Dihua Shangguan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00061 • Publication Date (Web): 26 Feb 2019 Downloaded from http://pubs.acs.org on February 28, 2019

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ACS Applied Bio Materials

A Mitochondria-Targeted Ratiometric Fluorescent pH Probe Xiangjun Liu*,†,‡,+, Linlin Wang†,‡,+, Tao Bing†,‡, Nan Zhang†,‡,and Dihua Shangguan*,†,‡ †

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical

Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100190, China ‡

University of Chinese Academy of Sciences, Beijing, 100049, China

+

These authors contributed equally to this work

* Corresponding Author: E-mail: [email protected], [email protected]

ABSTRACT: The pH of mitochondria (pHm) is crucial in physiological processes. Here we report a new mitochondria-targeted ratiometric pH-sensitive fluorescent probe for monitoring the mitochondrial pH change. This probe, IR-ANNA, was constructed by coupling a pH-sensitive naphthalimide derivative ANNA, and a mitochondria-targeted pH-insensitive cyanine dye. IRANNA showed good mitochondria-targeted ability, excellent pH response, and low cytotoxicity, which makes IR-ANNA suitable for monitoring pHm in living cells. Moreover, IR-ANNA has been successfully applied for monitoring the pH change of mitochondria under the stimuli of FCCP, NAC, and H2O2 in living cells.

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KEYWORDS: Mitochondrial targeting; Ratiometric fluorescent probe; pH imaging; Cyanine; Naphthalimide

1. INTRODUCTION Mitochondria, a well-known energy supplying organelle in almost all eukaryotic cells, play vital roles in cellular metabolism, including production of reactive oxygen species (ROS),1,2 regulation of Ca2+ homeostasis,3,4 cell apoptosis and death.5,6 Alkaline pH (pHm ~ 8) is necessary for mitochondria to perform its normal physiological functions. For example, the alkaline matrix can lead to the formation of proton gradient, which is essential to maintain the proton motive potential to generate ATP.7,8 Mitochondria is the main source of ROS, some of which acted as cell signalling molecules. But excess ROS can cause mitochondrial damage and acidification, which lead to mitochondrial autophagy or cell apoptosis.8,9 Because of the critical importance of pH on the function of mitochondria, monitoring the pHm change inside living cells is essential for understanding the physiology and pathology of mitochondria. Fluorescent probes are widely considered as the most powerful technique for pH sensing, owing to their good sensitivity and high spatial resolution.10-15 Essentially, an excellent pHm fluorescent probe should target mitochondria with high selectivity.16-20 Notably, the intensity of turn-on or turn-off fluorescent sensors can be affected by some factors, such as local concentration, intracellular microenvironment, and instrumental parameters, which lead to the concealment of minor deviation of pHm. Ratiometric fluorescent probe, through the self-calibration of two emissions, can effectively remove these interferes and obtain an accurate detection.13,19-22 For example, Wu et al. reported a pyridinium functioned 7-hydroxy coumarin for ratiometric fluorescence detection of mitochondrial pH recently.23 Chen et al. developed a fluorescein/cyanine hybrid sensor for rariometric pHm imaging.24 Some aromatic cations, e.g. triphenylphosphine and


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pyridine, have been mostly used to target mitochondria.11,17,19,23 Cyanine dyes, because of their good mitochondrial targeting ability and long emission wavelength, have attracted more attention recently.20,24 Herein, we report a new, small-molecule ratiometric pHm fluorescent probe, IR-ANNA, consisting of a cyanine with a long emission at 720 nm as reference signal and a pH-sensitive naphthalimide derivative ANNA. The targeting ability of IR-ANNA to mitochondria was investigated through co-localization study. And the ratiometric pHm imaging was performed by confocal microscopy. Additionally, the effects of FCCP, NAC, and H2O2 on the pH of mitochondria in living cells were investigated. 2. EXPERIMENT SECTION 2.1. Materials and Instruments. IR-780 and carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) were purchased from Sigma-Aldrich. 4-Bromo-1,8-naphthalic anhydride was purchased from Liaoning Liangang Dye Chemical Co. Ltd. 3-Amino-1,2,4-triazole and ethylenediamine were purchased from Alfa Aesar. N-acetylcysteine (NAC), nigericin sodium salt,

HEPES,

and

6-aminocaproic

acid

were

purchased

from

J&K.

Ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBT) were purchased from GL Biochem. Arginine (Arg), Lysine (Lys), Cysteine (Cys), Glutathione (GSH), and Histidine (His) were purchased from Beijing XinJingKe Biotechnology Ltd. LysTracker Red (LTRed) was purchased from beyotime Ltd. MitoTracker Red (MTRed) was purchased from KeyGEN BioTECH Ltd. All other reagents were purchased from Beijing Chemical Plant. Standard pH buffer solutions were obtained by mixing 200 mM Na2HPO4 and 200 mM NaH2PO4 at different volume ratios. ESI-MS was measured on a LC-MS 2010A system (Shimadzu). MALDI-TOF HR-MS was measured on a solarix mass spectrometer (Bruker). 1H NMR was recorded on a Avance III 500WB nuclear magnetic resonance spectrometer (Bruker). Fluorescence spectra were collected on a F-4600 fluorescent spectrophotometer (Hitachi). Absorption spectra were recorded on a UH5300 spectrophotometer (Hitachi). Fluorescence images



were recorded on an FV1000-IX81 confocal microscope (Olympus). The absorbance for Cell Counting Kit-8 (CCK-8) analysis was recorded on a SpectraMax M5 Reader (Molecular Devices). 2.2. Synthesis of IR-ANNA (Scheme S1). Compound ANNA-NH2. ANNA was synthesized according to our reported method previously.25 ANNA (27 mg, 0.069 mmol) dissolved in 4.5 mL DMF, then EDC (25 mg, 0.13 mmol) and HOBT (18 mg, 0.13 mmol) added to the reaction mixture. After stirred 30 min at room temperature (rt), 8 μL of ethylenediamine added, and stirred for 24 h at rt. Then the product was purified by silica column chromatography and eluted with dichloromethane/methanol/NH3.H2O (5:1:0.1, v/v/v) to obtain red solid ANNA-NH2. MS (ESI): m/z calcd for C22H23N7O3 [M]+ 434.2, found 434.3. Compound IR-ANNA. IR-780 (11 mg, 0.017 mmol) and ANNA-NH2 (15 mg, 0.035 mmol) dissolved in 2.0 mL DMF. After reaction 8 h at 80 oC, the product was purified by silica column chromatography and eluted with dichloromethane/methanol (30:1, v/v) to obtain the final solid. 1H

NMR (500 MHz, DMSO): δ 8.91 (s, 1H), 8.42 (d, J = 8.1 Hz, 1H), 8.33 – 8.18 (m, 2H), 7.57

(d, J = 12.8 Hz, 1H), 7.44 (dd, J = 17.4, 7.4 Hz, 2H), 7.26 (t, J = 7.6 Hz, 1H), 7.12 (d, J = 7.9 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 6.66 (s, 1H), 5.75 (d, J = 14.8 Hz, 1H), 5.39 – 5.28 (m, 1H), 4.08 – 4.00 (m, 1H), 3.90 (s, 2H), 3.76 (d, J = 4.6 Hz, 1H), 3.37 (d, J = 27.0 Hz, 2H), 2.62 (d, J = 24.0 Hz, 1H), 2.54 (d, J = 5.3 Hz, 1H), 2.17 (t, J = 7.2 Hz, 1H), 2.00 (dt, J = 12.2, 6.8 Hz, 2H), 1.71 (td, J = 14.2, 6.7 Hz, 3H), 1.60 (s, 6H), 1.46 (d, J = 4.1 Hz, 1H), 1.37 – 1.15 (m, 12H), 0.92 (t, J = 7.4 Hz, 3H), 0.85 (dd, J = 15.9, 8.8 Hz, 3H). HR-MS (MALDI): m/z calcd for C58H66 N9O3+ [M]+ 936.5283, found 936.5280. 2.3. UV-Vis Absorption and Fluorescence Spectra Measurement. Stock solution of IRANNA (2.0 mM) was prepared in DMSO. IR-ANNA was dissolved in different solvent and the absorption spectra were recorded immediately. For fluorescence spectra measurement, 2.0 μM of IR-ANNA was dissolved in different solvent and the fluorescence spectra were measured under different excitation wavelengths. For the effect of pH, 2.0 μM of IR-ANNA dissolved in different pH buffer solutions in the presence or absence of CTAB and SDS, then the ﬂuorescence spectra


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were recorded. For the interference study, different interference substances (Ca2+, Mg2+, Hg2+, Cd2+, Co2+, Mn2+, Ni2+, Ba2+, Zn2+, Al3+, Fe3+, Arg, Lys, His, Cys, GSH, H2O2, NAC, and FCCP) were mixed with IR-ANNA (2.0 μM) in PBS containing CTAB, then the ﬂuorescence spectra were measured. 2.4. Cell Culture and Imaging. Hela cells and MCF-7 cells were cultured in 1640 and DMEM respectively

supplemented

with

10%

fetal

bovine

serum

(FBS,

Gibco)

and

1%

penicillin/streptomycin (Hyclone) with 5% CO2 at 37 oC. Before imaging, cells were seeded and cultured in confocal dishes for 24 h, then incubated with 2.0 μM IR-ANNA in fresh media containing FBS for 2 h. After washing three times with PBS, and further treatment according to different experiments, the ratiometric fluorescence imaging of IR-ANNA in Hela and MCF-7 cells were carried out on an Olympus FV1000-IX81 confocal microscope. 2.5. Cytotoxicity. Hela cells and MCF-7 cells were seeded in 96-well plate (5000 cells per well) firstly. After cultured overnight, the media were replaced with fresh media in the presence of different concentration of IR-ANNA. After further incubation 48 h, the media were removed and fresh media (FBS free) contained CCK-8 (10%) and incubated for 30 min. The absorbance at 450 nm was recorded on a ELISA reader (SpectraMax M5). 2.6. Co-localization Study. IR-ANNA stained cells were further incubated with MTRed or LTRed in culture media for 30 min, respectively. After washing three times with PBS, the fluorescence images were obtained immediately, in which the fluorescence of ANNA moiety was collected at 500-545 nm with excitation at 488 nm, the fluorescence of cyanine moiety was collected at 655-755 nm with excitation at 635 nm, and the fluorescence of MTRed or LTRed was collected at 570-625 nm under excitation at 559 nm. 2.7. Intracellular pHm Calibration. The living cells stained IR-ANNA were further incubated in different pH high K+ buffer solutions (20 mM HEPES, 1 mM NaH2PO4, 120 mM KCl, 30 mM NaCl, 1 mM CaCl2, 0.5 mM MgSO4, 5 mM glucose, and 20 mM NaOAc) in the presence of nigericin (10 μM). After 30 min, the fluorescence images were recorded, in which the band of 500-



600 nm ascribed to ANNA, the band of 655-755 ascribed to cyanine moiety. Pseudo-color ratio images were obtained from the fluorescence ratio of red to corresponding green channel. The response curve of the ratio to different pHm were calculated from the ratio images. 2.8. Effect of FCCP, NAC, and H2O2 on the pHm. HeLa cells incubated with 2.0 μM IRANNA in 1640 with 10% FBS at 37 oC for 2 h. Then the IR-ANNA stained cells were washed with PBS twice, and further incubated with FCCP (10 μM), NAC (1.0 mM), and H2O2 (100 μM) in PBS at 37 oC for 20 min, 1.0 h, and 1.0 h, respectively, and then the fluorescence images were recorded immediately. 3. RESULTS and DISCUSSION 3.1. Design and Determination Mechanism of IR-ANNA. The design and determination mechanism of IR-ANNA is shown in Scheme 1. IR-ANNA is composed of a pH-sensitive naphthalimide dye (named as ANNA) and a cyanine dye. ANNA is a pH-sensitive, non-organelle specific molecule reported by our group previously,25 showing weak green emission at acidic pH, and enhanced green emission along with the pH increasing. A cyanine dye, IR-780, was selected as the reference fluorophore for ratiometric fluorescence sensing because of its mitochondriatargeted ability, pH-insensitivity, long wavelength emission and easy modification.26 IR-ANNA was prepared via the condensation of ANNA-NH2 with IR-780 in DMF (Scheme S1), in which ANNA-NH2 was prepared by the reaction of carboxyl group of ANNA with ethylenediamine to give a terminal amino group.

Scheme 1. Mechanism of ratiometric pH response of IR-ANNA.


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3.2. Absorption and Emission Properties of IR-ANNA. The absorption and emission spectra of IR-ANNA in different solvents were studied. The typical absorption peak at about 650 nm ascribed to the cyanine moiety of IR-ANNA was observed in all tested solvents. And the absorption band from 400 to 500 nm was the absorption of ANNA moiety (Figure S1). The fluorescence spectra of IR-ANNA showed a strong emission band at about 510 nm in DMF, DMSO, alcohol, and methanol under excitation at 495 nm (Figure S2). Upon excitation at 635 nm (Figure S3), IR-ANNA showed an emission band from 730 to 750 nm in different organic solvents, owing to the emission of cyanine moiety. However, no emission was observed in water and different pH buffer solutions under excitation at 635 nm (Figure S3, S4). The absorption spectra of IR-ANNA showed a broad absorption around 600-800 nm in different pH buffers (Figure S5). These results suggest that IR-ANNA prefer to aggregate in water and buffers. Considering the different lipid environments in cells, we studied the emission spectra of IRANNA in different surfactants, including SDS (an anionic surfactant) and CTAB (a cationic surfactant). The absorption spectra of IR-ANNA in SDS or CTAB solutions (Figure S6) showed that the broad absorption around 600-800 nm in PBS changed to a sharp absorption peak at about 650 nm, which suggests that aggregated IR-ANNA transform to monomer. Moreover, in CTAB solution, IR-ANNA showed a strong absorption peak at 488 nm (contributed by ANNA moiety) compared with that in SDS or PBS (Figure S6), and this absorption increased with the rising of pH (Figure S7). These results indicate that ANNA moiety is mostly existed in its base form in CTAB micelles even though the bulk pH is 4.71.25 In SDS solution (Figure S7), the ANNA moiety showed an absorption peak around 450 nm when the bulk pH is 4.71 and 7.35, and the absorption peak at 488 nm appeared when the bulk pH increased to 8.26. The emission spectra are shown in Figure 1a, compared with in PBS, IR-ANNA showed a strong emission at about 720 nm upon excitation at 635 nm in both SDS and CTAB buffers. However, the emission spectra of ANNA moiety were much different in SDS and CTAB buffers. Upon excitation at 455 nm, the emission at about 505 nm greatly increased only in CTAB buffer, and the emission in SDS buffer almost did not change compared with that in PBS buffer only (Figure 1b). We further studied the fluorescence spectra of



ANNA moiety at different pH in SDS and CTAB solutions. Upon excitation at 488 nm, the emission at about 507 nm appeared only in the basic solution (Figure 1c), and the florescence intensity increased greatly when pH higher than 7.10 in the presence of SDS. Whereas in the presence of CTAB, the emission at about 503 nm appeared at pH 4.33 and increased along with the pH increasing (Figure 1d). The fluorescence intensity of IR-ANNA in CTAB solution at pH 4.71 was similar with that in SDS solution at pH 8.46. These results suggest that the distinct variations of the absorption and emission spectra of ANNA moiety may be attributed to the different local pH of the micelle microenvironments that IR-ANNA existed in. The interface of SDS micelles is highly negatively charged, resulting in the local pH at the interface vicinity greatly lower than that in bulk solution. Whereas, the interface of CTAB micelles is highly positively charged, resulting in the local pH at the interface vicinity greatly higher than that in bulk solution.27,28 Since the emissions of cyanine moiety at 720 nm were almost not changed in the presence of CTAB (Figure S8) or SDS (Figure S9) at different pH, this cyanine moiety could act as a good fluorescence reference unit.

Figure 1. Fluorescence emission spectra of IR-ANNA in the presence of CTAB or SDS upon excitation at 635 (a) and 455 (b) nm. Fluorescence emission spectra of IR-ANNA in different pH buffer containing SDS (c) or


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CTAB (d) upon excitation at 488 nm. Response curve of fluorescence intensity ratio vs pH value in the presence of SDS (e) and CTAB (f).

The ratio of fluorescence intensity at 507 nm to that at 720 nm, F507/F720, increased slowly along with the increasing of the pH from 4.33 to 7.10, and greatly increased from 7.10 to 8.46 in the presence of SDS with a linear relationship in the pH range from 7.10 to 8.46 (Figure 1e, and inset). Similarly, in the presence of CTAB, the ratio of F503/F720 increased upon increasing the pH from 4.33 to 8.46 (Figure 1f). And a broad linear range of the ratio (F503/F720) versus pH from 5.65 to 8.46 was obtained (Figure 1f, inset). These results support the ratiometric pH-sensing mechanism (Scheme 1) of IR-ANNA. 3.3. Interference Tests and Cytotoxicity. The interference of some biological species on the pH detection was further investigated in PBS buffer contained CTAB. The fluorescence intensity ratio of F720/F503 showed a negligible change in the presence of many metal ions (Ca2+, Mg2+, Hg2+, Cd2+, Co2+, Mn2+, Ni2+, Ba2+, Zn2+, Al3+, and Fe3+), some amino acids (Arg, Lys, and His), some redox related species (Cys, GSH, H2O2, and NAC), as well as a mitochondrial uncoupling agent, FCCP (Figure 2a). These results demonstrate the good specificity of IR-ANNA for ratiometric fluorescence determination of pH, and indicate that IR-ANNA can be used to determine intracellular pH without interference.

Figure 2. (a) Fluorescence ratio (F720/F503) of IR-ANNA in the presence of various metal ions, some amino acids and redox species (Ca2+ and Mg2+, 100 μM; other metal ions, 10 μM; Cys, GSH, Arg, Lys, His, and H2O2, 100 μM; NAC, 1.0 mM, FCCP, 10 μM). (b) Cytotoxicity of IR-ANNA to Hela and MCF-7 cells.



The cytotoxicity of IR-ANNA towards Hela and MCF-7 cells were investigated by CCK-8 assay. After incubation with different concentrations of IR-ANNA for 48 hours, no significant cytotoxicity was found even the concentration of IR-ANNA high to 100 μM (Figure 2b). The low cytotoxicity suggests that IR-ANNA is a potential and safe fluorescent probe for monitoring pHm change of living cells. 3.4. Localization of IR-ANNA in Living Cells. IR-780 was reported to selectively accumulate in mitochondria of tumour cells with strong fluorescence.26 To confirm the targeting ability of IRANNA to mitochondria, co-staining experiment of IR-ANNA and Mito Tracker Red (MTRed, a commercially available mitochondrial probe) was performed in Hela cells. Confocal images showed that the fluorescence of ANNA and cyanine moieties were obtained in two channels upon excitation at 488 and 635 nm respectively (Figure 3a, b). And the fluorescent images in both channels were overlapped very well with the fluorescent image of MTRed (Figure 3e, f), the Pearson's correlation coefficients (PCC) were 0.83 and 0.80, respectively (Figure 3g, h). Good overlap between fluorescent images of IR-ANNA and MTRed with PCC of 0.81 and 0.83 were also observed in MCF-7 cells (Figure S10). In contrast, poor overlap between the fluorescent images of IR-ANNA and LTRed (a lysosome specific dye, Lyso Tracker Red) was obtained with a low PCC of 0.30 and 0.56 in Hela (Figure 3, i-p) and MCF-7 cells (Figure S11). These results confirm that IR-ANNA can be specifically accumulated in mitochondria of living cells.


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Figure 3. Confocal images costained by IR-ANNA and MTRed (a-g) or LTRed (i-p) of Hela cells. (a, i) Pseudoblue fluorescence of IR-ANNA (λex 488 nm); (b, j) deep red fluorescence of IR-ANNA (λex 635 nm); (c) pseudogreen fluorescence of MTRed (λex 559 nm); (d, l) bright field; (e) overlay of (a) and (c); (f) overlay of (b) and (c); (g) correlation of IR-ANNA (a, 488 nm) and MTRed (c); (h) correlation of IR-ANNA (b, 635 nm) and MTRed (c); (k) pseudo-green fluorescence of LTRed (λex 559 nm); (m) overlay of (i) and (k); (n) overlay of (j) and (k); (o) correlation of IR-ANNA (i, 488 nm) and LTRed (k); (p) correlation of IR-ANNA (j, 635 nm) and LTRed (k).

3.5. Ratiometric pHm Imaging of IR-ANNA in Living Cells. The ratiometric pHm imaging of IR-ANNA was studied using confocal microscopy in two channels ascribed to ANNA and cyanine



moieties respectively (Figure 3a, b, i and j). For quantitative determination of pHm in cells, the valid intracellular pH calibration was carried out firstly using high K+ buffer at different pH values containing nigericin in Hela cells. As shown in Figure 4, the fluorescence intensity in green channel (4a, Green) from ANNA moiety gradually increased along with the increasing of pH from 5.99 to 7.50, and with a very slight decrease with the pH further increasing to 8.46. While the fluorescence intensity in red channel from cyanine moiety (4a, Red) remained almost constant from pH 5.99 to 7.50, and gradually decreased upon the pH further increasing to 8.46. The merged images (4a, Merged) showed obvious changes from red to green colour. Moreover, the pseudocoloured ratio images (4a, Ratio (R/G)), which obtained from the fluorescence intensity ratio of red to green channels, showed more obvious colour changes. Similar results were obtained in MCF-7 cells (Figure S12).


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Figure 4. (a) Confocal fluorescence images of Hela cells incubated by IR-ANNA upon further incubation in high K+ HEPES buffers at different pH containing 10.0 μM nigericin. (b) Response curve of the ratio (Fgreen/Fred) to different pH based on the images. (c) Confocal images of Hela cells incubated by IR-ANNA. Green channel



image (λex 488 nm); Red channel image (λex 635 nm); Merged image was overlay of green and red channel image; Ratio (R/G) image was obtained from the fluorescence ratio of red to corresponding green channel.

The ratio of Fgreen/Fred of IR-ANNA in Hela cells coming from the pseudo-color ratio images increased from pH 5.99 to 8.46 (Figure 4b), with a linear range from pH 6.49 to 8.46, which indicates that IR-ANNA can be used for the ratiometric pHm imaging and detection. The confocal images obtained from Hela cells were shown in Figure 4c. Based on the fluorescence intensity ratio of green to red channel ascribed to ANNA and cyanine moiety, the average pH of the mitochondria in Hela cells was determined to be 7.99 ± 0.03, which is in agreement with the earlier studies.17,24,29 3.6. Effects of Chemicals on pHm. Stimulation from exotic chemicals can influence the pH of mitochondria of living cells greatly. In order to test the feasibility of monitoring pHm variation with IR-ANNA, Hela cells stained with IR-ANNA were treated with FCCP, a typical protonophoric uncoupler of mitochondrial oxidative phosphorylation; and two redox chemicals, NAC (a GSH precursor) and H2O2 respectively. As shown in Figure 5, after cultured IR-ANNA stained Hela cells in PBS for 20 min, the average pHm was almost kept unchanged, and calculated to be about 8.00 (Figure 5, PBS); whereas after incubated in PBS containing FCCP for 20 min, the average pHm decreased greatly to about 6.62 (Figure 5, FCCP). These results confirm that FCCP caused the mitochondrial membrane potential out of balance, and resulted in obvious acidification of mitochondria.27 The treatment of NAC decreased the pHm in Hela cells to about 6.92. which indicate that NAC, as a proton donor, also caused the acidification of mitochondria (Figure 5, NAC). H2O2 is well-known that can cause redistribution of H+ from acidified organelles to other organelles through impairing the vacuolar proton pump (V-ATPase), which the hydrolysis of ATP could cause acidification of mitochondria.23,31 Our results showed that H2O2 acidified the mitochondria of Hela cells to about 6.98 (Figure 5, H2O2).


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Figure 5. Confocal fluorescence images of IR-ANNA pre-stained Hela cells after further incubated with PBS only (20 min) and PBS containing FCCP (20 min), NAC (1.0 h), or H2O2 (1.0 h), respectively. Green channel image (λex 488 nm); Red channel image (λex 635 nm); Merged image was overlay of green and red channel image; Ratio (R/G) image was obtained from the fluorescence ratio of red to corresponding green channel.

4. CONCLUSION In summary, by coupling a pH-sensitive ANNA moiety and a mitochondria-targeted pHinsensitive cyanine moiety, a novel ratiometric fluorescent pH probe (IR-ANNA) was developed in this study. This probe showed excellent response to pH variations. The common species in cells, such as metal ions, amino acids and redox related species almost did not affect the pH detection. IR-ANNA showed good mitochondrial targeting ability, and was used to quantitative determination of mitochondrial pH in living cells successfully. Upon the stimulus by FCCP, NAC,



or H2O2, obvious acidification of mitochondria was observed, which demonstrates that IR-ANNA could be used for pHm monitoring via a ratiometric fluorescence strategy. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthetic route of IR-ANNA, Absorption spectra of IR-ANNA in different solvents, Fluorescence spectra of IR-ANNA in different solvents under excitation at 495 and 635 nm, Fluorescence spectra of IR-ANNA in different pH buffers under excitation 635 nm, Absorption spectra of IR-ANNA in different pH buffers, Absorption spectra of IR-ANNA in PBS containing CTAB or SDS in acidic and basic pH buffers, Fluorescence spectra of IR-ANNA in the presence of CTAB and SDS at different pH buffers under excitation at 635 nm, Confocal images costained by IR-ANNA and MTRed of MCF-7 cells, Confocal images costained by IRANNA and LTRed of MCF-7 cells, Confocal fluorescence images of MCF-7 cells incubated by IR-ANNA upon further incubation in high K+ HEPES buffers at different pH containing 10.0 μM nigericin, 1H NMR and MALDI mass spectrum of IR-ANNA AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. ORCID Xiangjun Liu: 0000-0002-0119-2134 Tao Bing: 0000-0002-5121-053X Dihua Shangguan: 0000-0002-5746-803X


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A Mitochondria-Targeted Ratiometric Fluorescent pH Probe - ACS

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