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A pH-Switchable Fluorescent Probe for SpatiallyConfined Visualization of Intracellular Hydrogen Peroxide Jun Liu, Jing Ren, Xiaojia Bao, Wei Gao, Chuanliu Wu, and Yibing Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00654 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 6, 2016
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Analytical Chemistry
A pH-Switchable Fluorescent Probe for Spatially-Confined Visualization of Intracellular Hydrogen Peroxide Jun Liu, Jing Ren, Xiaojia Bao, Wei Gao, Chuanliu Wu* and Yibing Zhao* The MOE Key Laboratory of Spectrochemical Analysis and Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, P.R.China Corresponding Author
*
[email protected];
[email protected] Phone: +86 (0)592 2183206 Fax: +86 (0)592 2183206
ABSTRACT: Intracellular H2O2 plays an important role in regulating a variety of cellular functions. Fluorescent probes that can make response to intracellular levels of H2O2 would provide valuable tools for revealing the functions of H 2O2 in living organisms. However, traditional pH-insensitive probes and lysosome-targetable probes can only provide spatially nonspecific visualization of intracellular H2O2 and specific sensing of lysosomal H2O2, respectively. In this work, we developed a H2O2-responsive and pHswitchable fluorescent probe (HP-L1) which can make response sequentially to intracellular H2O2 and lysosomal pH. The fluorescent probe is comprised of a H2O2-responsive boronate moiety and a pH-switchable spirobenzopyran fluorophore. When the probe was applied for intracellular H2O2 sensing, only fluorescent emission from lysosomes is visible, and the fluorescence from other regions is not able to be obviously detected, which is due to the pH-switchable property of the spirobenzopyran fluorophore. Thus, the developed fluorescence probe enables the spatially-confined (i.e., lysosome-specific) visualization of the intracellular H2O2. We envisioned that this kind of fluorescent probe (or the proposed sensing strategy) would allow the visualization of the overall levels of intracellular H2O2 without interferences of possible fluorescent signals from other sources (e.g., dyes for cellular staining and multiplex analysis).
INTRODUCTION Reactive oxygen species (ROS), including superoxide anion, hydroxyl radical, and hydrogen peroxide (H2O2), regulate a wide variety of physiological events in living organisms.1-3 Among them, H2O2 is the most stable one, which has the lowest reactivity and the highest intracellular concentration (~10-8–10-4 M).1 H2O2 is mainly produced by mitochondrial respiration, which is tightly regulated by cellular exposures to various stimuli, such as cytokines and growth factors.2 As a nonpolar molecule, H2O2 is able to diffuse across membranes, and can activate diverse signaling pathways to stimulate cell proliferation, differentiation, migration, or apoptosis.1,4 More importantly, recent studies have revealed that the response of cellular signals to H2O2 is concentration-dependent and cell-type-specific.5-6 For example, lower levels of intracellular H2O2 are more relevant to the regulation of cell proliferation and growth arrest, whereas significantly elevated concentrations of H2O2 can cause oxidative damage of cellular proteins and trigger cellular senescence and apoptosis.1 Currently, the interplay between the cell-type and the concentration of H2O2 on the specific cellular signaling is still unclear. In this context, fluorescent probes that can respond specifically and sensitively to the variation of intracellular H2O2 would provide invaluable tools for exploring the function of H2O2 in living cells or in tissues.7-12 It has been found that functions of lysosomes might be subject to regulations by the fluctuation of intracellular levels of H2O2.1315 However, the mechanism for regulating the functions of lysosomes by H2O2 and the concentration-dependent effects of lysosomal or intracellular H2O2 remains unclear. Though in recent years many fluorescent probes have been developed for
monitoring H2O2 in living cells or in some specific subcellular organelles (e.g., in the mitochondria),8,10,16-19 it is still a challenge to specifically monitor the lysosomal (or intracellular) H2O2 correlated to the regulation of lysosome function. In addition, lysosomes are substantially more acidic (pH 4–6) than other places within cells,20-23 a feature that has been widely explored for sensing applications or exploited as an endogenous stimulus for the activation of fluorescent signals.21 We hypothesized that the interplay between lysosomal protons and H2O2 within cells might on one hand influence the performance of the sensing of intracellular H2O2 by fluorescent probes, but on the other hand provide an opportunity to monitor specifically the lysosomal H2O2 (without influence from H2O2 within the cytoplasm other subcellular organelles) or for lysosome-specific visualization of fluorescent signals generated from reactions of probes with intracellular H2O2. Fluorescent probes that are able to make response sequentially to intracellular H2O2 and lysosomal pH are highly desired. In this study, we design and synthesize a H2O2-responsive and pH-switchable fluorescent probe for the spatially-confined (i.e., lysosome-specific) visualization of intracellular H2O2. The fluorescent probe exploits boronate as a H2O2-sensing unit and photochromic spirobenzopyran as a pH-switchable fluorophore. Unlike other traditional strategies for monitoring overall levels of H2O2 in cells using pH-insensitive probes or visualizing specifically the lysosomal H2O2 through lysosome-targetable fluorescent probes (Scheme 1a),11,14-15,19,24-25 our strategy enables not only the fluorescent sensing of intracellular H2O2 without spatial confinement (i.e., whole cell level), but also allows specific visualization of the fluorescent signals restricted to lysosomes. We proposed that while the sensing probes diffusing
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into the cells can spontaneously diffuse within the cells and react with analytes, the intensity of the fluorescent signals detected from lysosomes could reflects the overall levels of the intracellular H2O2 (Scheme 1b). Lysosomal H2O2 levels might also be more specifically and sensitively detected while the responsiveness of the probes to H2O2 is insensitive to pH or is more sensitive under acidic pH compared to neutral condition. As the demand for cell-based multiplex analysis and extensive staining of cells is growing, we envisioned that the spatiallyconfined (or rather, lysosome-specific) visualization strategy would reserve plenty of space (e.g., the cytoplasm) for multiplexed fluorescent imaging analysis.
Scheme 1. a) Traditional strategies for the sensing of H2O2: 1) pH-insensitive probes for intracellular H2O2; 2) lysosome-targetable probes for lysosomal H2O2. b) Our H2O2-responsive and pH-switchable fluorescent probe for the spatially-confined (i.e., lysosome-specific) imaging of intracellular H2O2.
EXPERIMENTAL SECTION Materials and apparatus. All chemicals were purchased from major suppliers such as Energy Chemical (Shanghai), Alfa Aesar (Shanghai), TCI (Shanghai), J&K (Beijing), and used as received. 4-(Diethylamino)salicylaldehyde, 2,4-dihydroxybenzaldehyde were obtained from Energy Chemical. 2-Hydroxy-3formylnaphthalene was purchased from TCI. Iodoethane was purchased from J&K. 4-(Bromomethyl)benzeneboronic acid pinacol ester was obtained from Alfa Aesar. Hela cells were purchased from CoBioer Biosciences CO., LTD (Nanjing, China). Dulbecco's Modified Eagle Medium (DMEM) with high glucose and phosphate buffered saline (PBS) were obtained from Thermo Scientific (Beijing, China). Eppendorf tubes (1.5 mL), glass bottom cell culture dishes and cell culture dishes were purchased from JET BIOFIL (Guangzhou, China). Phorbol 12-myristate13acetate (PMA) and LysoTracker Green DND-26 were purchased from Beyotime. UV-Vis absorption and fluorescence spectra were recorded using a U-3900H spectrophotometer (Hitachi) and a F7000 fluorescence spectrophotometer (Hitachi), respectively. The 1 H NMR spectra were recorded respectively at 500 and 125 MHz on a Bruker Advance-500 spectrometer, using tetramethylsilane (TMS) as the internal standard. Electrospray ionization mass spectroscopy (ESI-MS) was carried out on a Bruker Esquire 3000 plus mass spectrometer. All the measurements were operated at room temperature (25 °C). Confocal cell images were taken by Leica TCS SP5X Confocal Microscope. Synthesis of L1. 1,2,3,3-tetramethyl-3H-indolium iodide (1.88 g, 10 mmol) and 4-(dimethylamino) benzaldehyde (1.93 g, 10
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mmol) were dissolved in 20 mL CH3CN, and the reaction mixture was refluxed with stirring for 12 h and then evaporated in vacuo. The residue was purified by column chromatography on silica gel (CH2Cl2/MeOH, 20:1 vol/vol) to give L1 (1.2 g) as a dark purple solid. Yield: 59%. 1H NMR (500 MHz, d-DMSO, ppm): δ 11.05 (s, 1H), 8.43 (d, J = 15.0 Hz, 1H), 7.99 (s, 1H), 7.72 (d, J = 7.4 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.20 (s, 1H), 6.52 (dd, J = 9.3, 2.1 Hz, 1H), 6.22 (d, J = 2.2 Hz, 1H), 4.39 (d, J = 7.0 Hz, 2H), 3.49 (m, J = 17.3, 8.6 Hz, 4H), 1.70 (s, 6H), 1.36 (t, J = 7.2 Hz, 3H), 1.18 (t, J = 7.0 Hz, 6H). ESI-MS: m/z calcd for [C24H31N2O]+, 363.24; found, 363.2471 (M) +. Synthesis of HP-L1. A mixture of L1 (363 mg, 1 mmol), 4(bromomethyl)benzeneboronic acid pinacol ester (356 mg, 1.2 mmol), and Cs2CO3 (490 mg, 1.5 mmol) in N,Ndimethylformamide (DMF; 15 mL) was heated at 80 °C for 8 h. After cooling, 30 mL water was added into the mixture and extracted with CH2Cl2 (10 mL × 3). The organic solutions were combined, washed with water and brine, and dried with Na2SO4. The solvents were evaporated to give the crude product, which was purified by flash chromatography (silica gel, dichloromethane/0-2% methanol) to give the desired products as a pale solid (0.24 g). Yield: 53%. 1H NMR (500 MHz, d-DMSO, ppm): δ 8.44 (d, J = 13.9 Hz, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.697.57 (m, 4H), 7.48-7.40 (m, 4H), 7.18 (d, J = 15.5 Hz, 1H), 6.60 (d, J = 9.2 Hz, 1H), 6.40 (s, 1H), 5.39 (s, 2H), 4.71 (s, 2H), 3.59 (dd, J = 13.7, 6.7 Hz, 4H), 1.68 (s, 6H), 1.32 (s, 12H), 1.18 (dd, J = 9.2, 4.7 Hz, 6H), 1.07 (s, 3H). ESI-TOF: m/z calcd for [C37H48BN2O3]+, 579.38; found, 579.3759 (M) +. Cell culture. Hela cells were maintained in DMEM medium (high glucose) supplemented with 10% FBS and 1% penicillin/ streptomycin (penicillin: 10,000 U∙mL–1, streptomycin: 10,000 U∙mL–1) at 37 °C in a humidified atmosphere containing 5% CO2. Cells were passaged at about 80 % cell confluency using a 0.25% trypsin solution. Confocal imaging of cells. Cells were plated into glass bottom cell culture dishes at a total of 105 cells/well. After 24 h at 37 °C, 5% CO2, the cells were grown to about 80% confluence. The medium was then removed and the cells were washed once with PBS. Cells were then incubated with 1 mL complete medium containing 5 μM L1 (or 5 μM HP-L1) and 1 μM LysoTracker Green DND-26 at 37 °C, 5 % CO2 for 30 min. After that, medium was removed and cells were washed twice with PBS. Cells were then imaged using a Leica TCS SP5X Confocal Microscope System at different detection channels (LysoTracker Green DND26 channel: λex = 488 nm, λem = 520–560 nm; L1/HP-L1 channel: λex = 543 nm, λem = 600–670 nm). Confocal imaging of cells treated with PMA or H2O2. Hela cells were seeded as described above. Cells were preincubated with 1 mL complete medium containing 2 μg/mL PMA (or 50/100 μM H2O2) at 37 °C, 5 % CO2 for 30 min. After that, medium was removed and cells were washed once with PBS and then were treated with 1 mL complete medium containing 5 μM HP-L1 and 1 μM LysoTracker Green DND-26 at 37 °C, 5 % CO2 for 30 min. After that, medium was removed and cells were washed twice with PBS. Cells were then imaged using a Leica TCS SP5X Confocal Microscope System.
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RESULTS AND DISCUSSION pH-Switchable Fluorophores. Photochromic spirobenzopyrans can undergo a reversible isomerization from the ring-closed (spiro) form to the ring-opened (merocyanine) form under light irradiation.26-28 The deprotonated merocyanine form is thermodynamically unstable and weakly fluorescent, which can transform back into the spiro form in the dark and results in decoloration of the chromophore.27-28 Instead, the protonated merocyanine form is thermodynamically stable and highly fluorescent.27 Therefore, we utilize the spirobenzopyran as a pHswitchable fluorophore for the construction of the fluorescent probe for H2O2. Three different spirobenzopyrans were designed and synthesized (L1, L2, and L3; Scheme 2a). Fluorescence emission spectra of these three compounds in different buffers (pH 4–8) were then recorded using a fluorimeter. Figure 1 showed the fluorescence emission of L1 under different pH and a plot of the fluorescence intensity (L1, L2, and L3) as a function of the pH value. We observed a gradual increase in the fluorescence intensity while the pH of the buffers decreased from 8.0 to 4.0. By analyzing the data by logistic regression, pKa of L1, L2, and L3 can be calculated (5.4, 6.2, and 5.7 respectively) with the Henderson-Hasselbalch equation. Of note, the pKa of L1 is lowest, which should display a higher pH-responsiveness under the lysosomal pH range (pH 4–6) compared to L2 or L3. Considering that the hydroxyl group in L1 might interact with common metal ions in complex biological environments, it is thus important to examine if the presence of metal ions affects the fluorescence emission of L1 (either protonated or deprotonated). Owing to the complexity of the intracellular environment, the effects of other bioactive small molecules on the fluorescence of L1 were also examined, including glutathione (GSH), cysteine (Cys), H2O2, and hypochloric acid (HClO). L1 exhibits excellent response selectivity to protons, that is, the presence of metal ions and other small molecules does not significantly influence the fluorescence intensity (Figure 1c). In addition, we found that L1 displays a good reversibility between pH 5.0 and pH 8.0 due to the rapid kinetics of the protonation and deprotonation of L1. Thus, these results indicate that L1 was an ideal pH-switchable fluorophore for the construction of fluorescent probes for intracellular or lysosomal H2O2 sensing.
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Figure 1. a) Fluorescent emission spectra of L1 (1 μM) in aqueous solutions of different pH; λex=520 nm. b) Plots of the changes of fluorescent intensity as a function of pH for the fluorophores L1, L2, and L3 (λex=520 nm, λem=584 nm). c) Fluorescent intensities of L1 (1 μM) in the presence of foreign species (100 μM) under different pH (1: blank; 2: K+; 3: Na+; 4: Ca2+; 5: Mg2+; 6: Zn2+; 7: Cu2+; 8: Fe2+ 9: Fe3+, 10: GSH; 11: Cys; 12: H2O2; 13: HClO).
pH-Switchable Fluorescent Probe for H2O2. An aryl boronate was then conjugated with the hydroxyl group of L1 to yield the probe HP-L1 (Scheme 2b). Aryl boronate has been widely utilized as a H2O2-sensing unit due to its rapid response rate and excellent response selectivity to H2O2 under physiological conditions.8,15,29 HP-L1 only exhibits a weak fluorescence signal with an emission maximum at 584 nm (λex = 520 nm) due to the quenching effect of the attached boronate. To examine if HP-L1 can make response to H2O2, the fluorescence spectra of HP-L1 incubated with H2O2 of various concentrations (0–100 μM) were recorded. It was found that the addition of H2O2 can trigger a significant enhancement of the fluorescence of HP-L1 under acidic condition (pH 5.0, acetate buffer) due to the H2O2-mediated oxidation and elimination of the aryl boronate (Figure 2a). The rate of the fluorescence response was also examined (Figure S9), and the result indicates a very fast reaction between HP-L1 and H2O2. In addition, a linear relationship between fluorescence intensity and the concentration of H2O2 was observed in the range of 0–80 μM (Figure S10), and the limit of detection calculated following the 3σ IUPAC criteria was 0.23 μM. The fluorescence responses of the probe to H2O2 under pH 6.0 and 7.4 (phosphate buffer) were also examined for comparison. The fluorescence response under acidic condition (pH 5.0 or 6.0) is significantly more sensitive than that under the neutral condition (Figure 2b). Considering that the H2O2-triggered oxidation of the boronate is rapid and efficient under both the acidic and neutral conditions, the relatively higher sensitivity of the fluorescence response under a lower pH should arise from the relatively stronger fluorescence emission of the protonated HP-L1 relative to its deprotonated form. Scheme 3 presents the mechanism of the pH-dependent fluorescence response to H2O2. To further evaluate the selectivity of the probe HP-L1, the fluorescence of HP-L1 in solutions containing other potentially competing ROS (including tertbutylhydroperoxide (TBHP), HClO, NO, and KO2) was recorded. No obvious changes in fluorescence were observed, indicating
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imaged by confocal microscopy. As shown from Figure 3, we observed strong intracellular red fluorescence from L1, indicating that L1 can efficiently diffuse into the cells. More importantly, there is an obvious overlap between the red and green (i.e., the lysosome-tracker) fluorescent regions, implying that L1 diffused into the cytoplasm can further relocate to the lysosomal compartments or L1 can be endocytosed into lysosomes directly. This preliminary result thus indicates the feasibility of visualizing the intracellular sensing signals from the lysosome windows by taking advantage of the pH-switchable feature of the sensing fluorophores.
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Figure 2. a) Fluorescence emission spectra of HP-L1 (1 μM) in the presence of different concentrations of H2O2 at pH 5.0 (acetate buffer); λex=520 nm. b) Plots of fluorescent intensity of HP-L1 (λex=520 nm, λem=584 nm; of note, the shape of the emission spectra was slight changed compared to that of L1 shown in Figure 1a, which is likely due to the variations of instrumental parameters) under three different pH (5.0, 6.0 and 7.4) as a function of H2O2 concentrations. c) Relative changes in fluorescent intensity of HP-L1 (1 μM) upon the addition of H2O2 or some other competing ROS (50 and 100 μM for the patterned and filled columns, respectively).
Figure 3. Confocal fluorescence images of Hela cells exposed to 5.0 μM L1. Left: red channel for L1; middle: green channel for lysosome-tracker; right: overlay of bright field and fluorescent images. The bottom panels show the enlarged views of the white frames.
We further examined the feasibility of detecting the endogenous H2O2 by the probe HP-L1 in living cells. HP-L1 was found to be not toxic to the cells (determined by MTT assays; Figure S11). By incubating the cells with HP-L1 and LysoTracker Green DND for 30 min at 37 °C, the cells were then imaged using confocal microscopy. PMA, a compound that can be used to stimulate the endogenous generation of H2O2 through triggering assembly of NADPH oxidase on the cell membrane,2930 was also exploited to increase the intracellular levels of H2O2. The pre-stimulated cells (by ~30 min incubation with 2 μg/mL PMA) were imaged for comparison after the incubation with LysoTracker Green DND-26 and HP-L1. As shown from Figure 4, strong intracellular red fluorescence was observed from cells, regardless of whether the cells were stimulated by PMA. Moreover, the red fluorescence obviously overlaps with the green fluorescence from the lysosomal tracker, which indicates that the fluorescent signals mainly came from the lysosomal compartments. The red fluorescence from the PMA-stimulated cells was not obviously enhanced compared to the untreated cells, suggesting very likely that PMA is unable to stimulate the generation of H2O2 in the Hela cells or that the cells might produce a sufficient quantity of endogenous H2O2 (without PMA stimulation) for reacting with the probe HP-L1 present within Scheme 3. A proposed mechanism for describing fluorescence response of the cells. probe HP-L1 to H2O2 and pH. We further treated the cells with exogenous H2O2 (two different concentrations; 50 and 100 μM) and imaged the intracellular H2O2 Fluorescent Imaging of H2O2 in Living Cells. The promising with HP-L1. Once again, we observed strong red fluorescence results in the above sections indicate that the fluorescent response from the lysosomes (Figure 4). No obvious fluorescence from of the probe to H2O2 is highly selective and pH-dependent. This other regions in cells was detected, further suggesting that the intrigues us to examine if the H2O2-responsive and pH-switchable probe HP-L1 only allows the lysosome-specific imaging of fluorescent probe can be exploited to achieve lysosome-specific intracellular H2O2. However, similar to that observed from PMAvisualization of intracellular or lysosomal H2O2. As L1 is the stimulated cells, no significant enhancement in fluorescence was product formed by the reaction of HP-L1 with H2O2, we first observed even when the cells were treated with an extremely high examined if L1 can diffuse into the lysosomes and specifically concentration of H2O2 (e.g., 100 μM), further suggesting that the light up the lysosomal compartments. Hela cells were incubated endogenous H2O2 might be sufficient enough for the complete with lysosome-specific dye (i.e., LysoTracker Green DND-26) consumption of the intracellular HP-L1. In addition, these results and L1 simultaneously for 30 min at 37 °C, and then cells were indicated that the intracellular concentration of the probe should 4 ACS Paragon Plus Environment
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be substantially increased to achieve the imaging analysis when the concentration of the intracellular H2O2 is relatively high. Scheme 4 shows the sensing principle of the H2O2-responsive and pH-switchable fluorescent probe. As that obtained experimentally, only fluorescent signals from the lysosomal compartments can be specifically detected due to the pH-switchable property of the probe, which reflect the overall level of intracellular H2O2, though the probe can react non-selectively with both the H2O2 in the cytoplasm and the lysosomal H2O2, as well as that located in some other intracellular organelles. Finally, we demonstrated that the fluorescence of the activated probes (L1) in other regions of cells (e.g., the cytoplasm) can also be selectively switched on by the acidification of cells (Figure S12), which provides an alternative way to more globally monitor the intracellular levels of H2O2 as the traditional pH-insensitive H2O2 probes did.
the photochromic spirobenzopyran, which is highly fluorescent under acidic conditions, but only fluoresces weakly under neutral conditions. A H2O2-responsive boronate was used as the sensing unit, which can make response rapidly to H2O2 at both acidic and neutral conditions. We further demonstrated that the fluorescent probe can be exploited for the intracellular sensing of endogenous H2O2. Only fluorescent signals from lysosomal compartments can be obviously detected, and the fluorescent signals from other regions is invisible, which is due to the pH-switchable property of the probe. Compared to traditional sensing strategies for monitoring the overall levels of intracellular H2O2 using pHinsensitive probes or sensing more specifically the lysosomal H2O2 using lysosome-targetable fluorescent probes, our sensing strategy enables the visualization of the intracellular H2O2 through the lysosome windows more specifically and very likely without potential interference from fluorescent signals originating from dyes used for other purpose (e.g., the dyes used for intracellular staining and other probes for multiplexed fluorescent imaging analysis).
AUTHOR INFORMATION Corresponding Author *
[email protected];
[email protected] NOTE The authors declare no competing financial interest.
ACKNOWLEDGMENT Figure 4. Confocal fluorescence images of Hela cells exposed to 5.0 μM HPL1 (a–c) or cells pretreated with PMA (d–f) and H2O2 (g–l) exposed to 5.0 μM HP-L1. Top: red channel for HP-L1; middle: green channel for lysosometracker; bottom: overlay of bright field and fluorescent images. The fluorescent emission of the probe HP-L1 within cells only slightly increased even when the concentration of H2O2 increased up to 100 μM.
We would like to acknowledge the financial support of the National Basic Research Program of China (Grant 2014CH932004), the National Natural Science Foundation of China (Grants 21305114, 21375110, and 21475109), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant 21521004), and PCSIRT (Grant IRT13036).
SUPPORTING INFORMATION Synthetic methods of L2 and L3; NMR and MS characterizations of compounds synthesized in this work; Figure S1–S12.
REFERENCES
Scheme 4. A schematic drawing illustrating the sensing principle of the H2O2responsive and pH-switchable fluorescent probe in cells and how the spatiallyconfined (i.e., lysosome-specific) imaging of intracellular H2O2 was achieved. Only fluorescence within lysosomes is visible; that is, the probes reacted with H2O2 became visible until they have diffused into lysosomal compartments.
CONCLUSIONS In summary, we developed a H2O2-responsive and pH-switchable fluorescent probe for lysosome-specific imaging of intracellular H2O2. The fluorophore that we exploited for the probe design is
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Analytical Chemistry
TOC:
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