Article pubs.acs.org/ac
Mitochondria-Targeted Ratiometric Fluorescent Nanosensor for Simultaneous Biosensing and Imaging of O2•− and pH in Live Cells Hong Huang, Fangyuan Dong, and Yang Tian* Shanghai State Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, East China Normal University, Dongchuan Road 500, Shanghai 200241, China S Supporting Information *
ABSTRACT: Intracellular pH undertakes critical functions in the formation of a proton gradient and electrochemical potential that drives the adenosine triphosphate synthesis. It is also involved in various metabolic processes occurring in mitochondria, such as the generation of reactive oxygen species, calcium regulation, as well as the triggering of cell proliferation and apoptosis. Meanwhile, the aberrant accumulation of O2•− within mitochondria is frequently intertwined with mitochondrial dysfunction and disease development. To disentangle the complicated inter-relationship between pH and O2•− in the signal transduction and homeostasis in mitochondria, herein we developed a mitochondria-targeted single fluorescent probe for simultaneous sensing and imaging of pH and O2•− in mitochondria. CdSe/ZnS quantum dots encapsulated in silica shell was designed as an inner reference element for providing a built-in correction, as well as employed as a carrier to assemble the responsive elements for O2•− and pH, together with mitochondriatargeted molecule. The developed nanosensor demonstrated high accuracy and selectivity for pH and O2•− sensing, against other ROS, metal ions, and amino acids. The remarkable analytical performance of the present nanosensor, as well as good biocompatibility, established an accurate and selective approach for real-time imaging and biosensing of O2•− and pH in mitochondria of live cells. O2•− and pH in cell function and apoptosis, for elucidating mitochondrial physiology and pathology. Over the past decades, several elegant methods have been reported for monitoring of O2•− or pH, including electron paramagnetic resonance (EPR), fluorescent spectroscopy, electrochemical approaches, and so on.11−17 Fluorescence technique is a powerful tool for studying cell biology because of their noninvasive feature, high spatial, and temporal resolution.18−21 In recent years, a number of attractive fluorescent probes specific for either O2•− or pH have been reported.22−25 Our group is very interested in determination of ROS and other related molecules involved in oxidative stress. 26−30 We have designed and developed several fluorescent biosensors for O2•−, •OH, pH, and Cu2+ in live cells and tissues.31−34 However, to the best of knowledge, there is still no report on the simultaneous determination of O2•− and pH in live cells. To achieve the dual-response probe for monitoring of mitochondrial O2•− and pH, fluorescent probe should possess distinct emission profiles to avoid the spectral overlap and capability to discriminate O2•− and pH without cross-talk, as well as mitochondria-targeting ability.29 Thus, it is
M
itochondria play a crucial role in energy metabolism and apoptosis of aerobic organisms.1 On the basis of plentiful chemical composition, mitochondria are involved in a wide variety of essential physiological processes.2 Studies have shown that mitochondria are the main intracellular source of reactive oxygen species (ROS), an inevitable by products of mitochondrial respiration when electrons leak from the electron transfer chain.3 ROS, exist in several different interconvertible forms (e.g., superoxide anion radical (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), etc.), are responsible for cell signaling and numerous diseases, including obesity, diabetes, neurodegenerative disorders, and cancers.4−6 In particular, the aberrant accumulation of O2•−, the primary species of ROS, within mitochondria is frequently intertwined with mitochondrial dysfunction and disease development.6 Thus, exploring mitochondrial O2•− has received considerable attention. Meanwhile, intracellular pH undertakes critical functions in the formation of a proton gradient and electrochemical potential that drives the adenosine triphosphate synthesis.7 It is also involved in various metabolic processes occurring in mitochondria, such as the generation of ROS8 and calcium regulation,9 as well as the triggering of cell proliferation and apoptosis.10 Thus, it is of significant importance to real-time and simultaneous determination of O 2 •− and pH in mitochondria, and to understand the interplaying roles of © 2016 American Chemical Society
Received: September 3, 2016 Accepted: November 14, 2016 Published: November 14, 2016 12294
DOI: 10.1021/acs.analchem.6b03470 Anal. Chem. 2016, 88, 12294−12302
Article
Analytical Chemistry
Scheme 1. Schematic Illustration of the Working Principle of the Developed HE + FITC + TPP-SiO2@QD Nanosensor for pH, O2•−, and pH/O2•− Sensing
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an ongoing challenge for simultaneously monitoring of O2•− and pH and elucidating their interplaying roles in mitochondria using a single probe. In this work, we first developed a single ratiometric fluorescent nanosensor for simultaneous detecting and imaging of pH and O2•− in mitochondria of live cells. As shown in Scheme 1, CdSe/ZnS quantum dots (QD) was encapsulated in silica shell to form core−shell SiO2@QD nanoparticles. The SiO2@QD with emission at 710−800 nm not only plays as an inner reference element for providing a built-in correction, but also acts as a carrier to assemble the responsive molecules for O2•− and pH, as well as mitochondria-targeted molecule (4carboxybutyl)triphenylphosphonium bromide (TPP). Then, the specific indicator for O2•−-hydroethidine (HE), pHsensitive molecule-fluorescein isothiocyanate (FITC), and TPP were conjugated onto amine SiO2@QD nanoparticles to form a single fluorescent nanosensor for simultaneous determination of pH and O2•−. The developed nanosensor showed high accuracy and selectivity for pH and O2•− sensing, against other ROS, metal ions, and amino acids. The remarkable analytical performance of the present nanosensor, as well as good biocompatibility, provided a reliable approach for real-time imaging and biosensing of O2•− and pH in mitochondria of live cells. Moreover, this nanosenor enabled us understanding the relationship between O2•− and pH in the mitochondria.
EXPERIMENTAL SECTION
Reagents and Chemicals. Qdot 800 ITK carboxyl CdSe/ ZnS core−shell quantum dots (QD800, 8 μM, emission maxima centered at 800 nm) and MitoTracker Deep Red FM (Mito Tracker) were supplied by Invitrogen Corporation (California, U.S.A.). Cyclohexane, Triton X-100, n-hexanol, anhydrous dimethyl sulfoxide (DMSO), ammonia aqueous solution (25−28%), tetraethyl orthosilicate (TEOS), (3aminopropyl)triethoxysilane (APTES), (4-carboxybutyl)triphenylphosphonium bromide (TPP), nigericin, fluorescein isothiocyanate (FITC), H2O2, NaNO2, and NaClO were received from Aladdin Chemistry Co. Ltd. (Shanghai, China). 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS), 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hydroethidine (HE), p-phenylene diisothiocyanate (DITC), S-nitroso-N-acetyl-DL-penicillamine (SNAP), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), xanthine oxidase (XO), xanthine, cysteine (Cys), methionine (Met), isoleucine (Lle), leucine (Leu), alanine (Ala), histidine (His), glutamine (Glu), tryptophan (Trp), threonine (Thr), glutathione (GSH), 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and KO2 were all obtained from Sigma-Aldrich (Missouri, U.S.A.). Angeli’s salt was purchased from Santa Cruz Biotechnology (California, U.S.A.). Phosphate buffered saline (PBS) was obtained from HyClone (Utah, U.S.A.). Metal chloride salts including KCl, NaCl, CaCl2, MgCl2·6H2O, AlCl3, BaCl2·2H2O, CdCl2·2.5H2O, FeCl3·6H2O, FeCl2·4H2O, CoCl2· 6H2O, CrCl3·6H2O, NiCl2·6H2O, CuCl2·2H2O, ZnCl2, and 12295
DOI: 10.1021/acs.analchem.6b03470 Anal. Chem. 2016, 88, 12294−12302
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12 h. Followed by that, the resultant nanoparticles were washed and dissolved into ethanol. The resulted nanoparticles refer to DITC + FITC + TPP-SiO2@QD hereafter. Finally, 1 mL of HE solution (0.7 mM) was added into DITC + FITC + TPPSiO2@QD nanoparticles and kept stirring in the dark for 12 h to generate the nanohybrid probe HE + FITC + TPP-SiO2@ QD. Cell Culture and MTT Assay. HeLa cells and RAW264.7 murine macrophage cells were seeded into 96-well microtiter plates at a density of 1 × 104 cells per well, and grown in high glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 80 μg mL−1 streptomycin, and 80 U mL−1 penicillin. After being incubated at 37 °C under 5% CO2 for 24 h, the culture media was discarded, and the fresh one containing the nanosensor with different concentrations (0, 20, 40, 80, 120, 160, and 200 μg mL−1) was injected into each well and cultured for 48 h. Five replicate experiments were performed for each concentration. Then, 40 μL of MTT solution (0.5 mg mL−1) was added to the cells for 4 h to allow the formation of formazan crystals. Finally, 150 μL of DMSO was added to each well and absorbance (A) of the resulting mixture was measured by a microplate spectrophotometer (Thermo Scientific, USA). The cell viability values were determined according to the following formula: cell viability (%) = the absorbance of experimental group/the absorbance of the blank control group × 100%.32 Cell Apoptosis Assay. Apoptosis assay was implemented by an Annexin V-FITC Apoptosis Detection Kit according to the manufacture’s protocol. Briefly, HeLa cells and RAW264.7 macrophage cells were incubated with the nanosensor at concentrations of 0, 50, 100, 200, 300, and 400 μg mL−1 for 24 h. After the treatment, the culture medium was collected to retain floating cells and adhered cells were detached using the EDTA-free trypsin. Floating and adhered cells were combined and collected by centrifugation. The cell pellets were redispersed in 195 μL of Annexin V-FITC binding buffer, stained with 5 μL of Annexin V-FITC, and 10 μL of a propidium idiode solution (PI) for 15 min in the dark. The cells were then analyzed by flow cytometer. Cell Imaging. Cells were placed onto 35 mm plastic Petri dishes with 20 mm bottom well and cultivated for 24 h. Then, the medium in the wells was replaced with fresh one containing the nanosensor (200 μg mL−1) and further cultivated for 2 h. Thereafter, the adhered cells were washed thrice with PBS to remove the nanosensors that were not taken up into the cells. The multicolor imaging of nanosensor loaded cells was simultaneously collected from blue channel (FITC), green channel (the oxidation product of HE), and red channel (SiO2@QD) in the wavelength ranges of 510−570, 600−670, and 730−800 nm, respectively. The subcellular distribution of the nanosensor was investigated by colocalization imaging experiments, in which the nanosensor-labeled cells were further stained with 100 nM Mito Tracker for 0.5 h. Following the staining experiment, cells were washed thrice with PBS prior to imaging. Fluorescence imaging of Mito Tracker were acquired with a 633 nm excitation laser and emission was collected at 660−710 nm, while for the nanosensor, the excitation wavelength was 488 nm and collected the emission in the range of 510−570 nm. The intracellular pH of the nanosensor loaded cells was homogenized with high K+ buffer (120 mM KCl, 1 mM CaCl2, 30 mM NaCl, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES, and 20 mM NaOAc) of various pH
PbCl2 were bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All the chemicals were of analytical grade and used as received. Superoxide anion (O2•−) was derived from KO2 dissolved in anhydrous DMSO solution. The concentration of O2•− was determined by measuring the reduction of ferricytochrome c spectrophotometrically using an UV−vis spectrophotometer and the extinction coefficient of ferrocytochrome c at 550 nm (21.1 mM−1 cm−1). For the selectivity and competition experiments, singlet oxygen (1O2) was generated by the reaction of NaClO (25 μM) with H2O2 (25 μM). Nitric oxide (NO) and nitroxyl (HNO) were, respectively, produced from the solution of SNAP and Angeli’s salt. Peroxynitrite (ONOO−) was obtained by the chemical reaction between NaNO2 (25 μM) and H2O2 (25 μM). Alkyl peroxyl radical (ROO•) was derived from thermolysis of AAPH (25 μM) in air-saturated aqueous solution at 37 °C. Hydroxyl radical (•OH) was supplied via Fenton reaction between Fe2+ and H2O2. Instruments and Apparatus. Fluorescence spectra were measured at room temperature on an F-4600 fluorescent spectrophotometer (Hitachi, Japan). The morphology of QD800 and SiO2@QD were observed by a JEM-2100F transmission electron microscopy (TEM) working at an acceleration voltage of 200 kV (Hitachi, Japan). Fourier transform infrared spectroscopy (FTIR) spectra of the samples were collected on a Nicolet iS10 FTIR spectrometer (Thermo Scientific, U.S.A.). Cell apoptosis assay experiments were conducted using a BD FACSCalibur flow cytometer (BD Biosciences, U.S.A.). Confocal fluorescence images were obtained by a TCS SP8 laser scanning microscope equipped with a 63× oil immersion objective (Leica, Germany). Preparation of the Fluorescent Nanosensor. In order to obtain the nanosensor, CdSe/ZnS QD800 were initially incorporated into silica matrix through a water-in-oil microemulsion process according to our previously reported method.34 Briefly, 20 μL of QD800 was mixed with 1.87 mL of cyclohexane, 0.45 mL of Triton X-100 and n-hexanol for 10 min. Then, 50 μL of aqueous ammonia was added dropwise. After 30 min of stirring to ensure homogeneity of the microemulsion, 50 μL of TEOS was introduced. The reaction system was left under stirring for 24 h to obtain the core−shell structured SiO2@QD nanoparticles. To endow the silica surface with amino groups, 25 μL of APTES was put into the above solution and the polymerization process was allowed to proceed for 12 h. The reaction was determined by acetone, and the resultant particles were harvested by centrifugation. Then, the as-prepared amine SiO2@QD nanoparticles were conjugated with TPP using EDC and NHS as the coupling agents. In detail, 2 mL of TPP solution (10 mM) was activated with EDC/NHS (50 mg/50 mg) for 2 h at room temperature, followed by the introduction of amine SiO2@QD nanoparticles and reacted for another 12 h. Afterward, the resulted nanoparticles were centrifuged, rinsed several times with ethanol and redispersed into ethanol to get a homogeneous solution. The as-prepared nanoparticles were denoted as TPPSiO2@QD. Subsequently, FITC and DITC were modified onto TPPSiO2@QD nanoparticles through the reaction between the isothiocyanate group of FITC and DITC with the amino group on SiO2@QD surface. Typically, 0.2 mL of FITC solution (14.7 μM) was introduced into the TPP-SiO2@QD solution. After 2 h, 4 mL of DITC solution (26.6 μM) was added and stirred for 12296
DOI: 10.1021/acs.analchem.6b03470 Anal. Chem. 2016, 88, 12294−12302
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Figure 1. (A, B) TEM images of (A) QD800 and (B) SiO2@QD nanoparticles; (C) Fluorescence spectra of (a) FITC, (b) HE, (c) QD800 nanoparticles, (d) the as-prepared HE + FITC + TPP-SiO2@QD nanosensor, and (e) the nanosensor after being reacted with O2•−, upon excitation at 488 nm; (D) FTIR spectra of (a) SiO2@QD nanoparticles, (b) TPP-SiO2@QD, (c) DITC + FITC + TPP-SiO2@QD, and (d) HE + FITC + TPP-SiO2@QD.
values (7.9, 7.4, 6.9, 6.4, and 5.9) in the presence of 10 μM nigericin at 37 °C for 30 min.35 To globally elevate intracellular O2•− stores, exogenous O2•− generated from the enzymatic reaction of Xanthine/XO system was introduced directly into the Petri dish and further incubated for 30 min.36 For the O2•− blocking experiments, cells were precultured with 2 mM GSH or 300 μM DIDS for 30 min, before homogenizing the intracellular pH or stimulating by exogenous O2•−.31,37
nanoparticles to generate the nanosensor with a mean diameter of ∼56 ± 5 nm, which is similar to that of the SiO2@QD nanoparticles (Figure S2, Supporting Information). FITC, a fluorophore that is sensitive to pH with a pKa of 6.5 and exhibits an emission peak at 515 nm (Figure 1C, curve a; Figure S3A, Supporting Information).39 HE, a nonfluorescent molecule (Figure 1C, curve b), shows an emission peak at 615 nm after specifically reacted with O2•− to generate the fluorescent oxidation products (Figure S3B, Supporting Information).31 Accordingly, as demonstrated in Figure 1C, the developed nanosensor shows two emission peaks at 518 and 800 nm excited at 488 nm (curve d), corresponding to the emission of FITC and QD nanoparticles surrounded by SiO2 shell (curve c), respectively. However, when the nanosensor reacted with O2•−, as expected, three well-resolved emission peaks were observed at 518, 615, and 800 nm (curve e). The surface functionalization processes were monitored by FTIR spectroscopy. As shown (Figure 1D, curve a), the FTIR spectrum of amine SiO2@QD exhibited two bands at 3276 and 1090 cm−1, which are assigned to the stretching vibrations of N−H and Si−O−Si, respectively. TPP shows the specific absorption band of carboxyl groups (Figure S4, curve a, Supporting Information) at 3362 cm−1 (VO−H), 1705 cm−1 (VCO), and 1106 cm−1 (VC−O).34,40 The new emerging band at 1658 cm−1 in FTIR spectrum of TPP-SiO2@QD nanoparticles (Figure 1D, curve b) indicates the formation of an amide group between TPP and amine
[email protected] FTIR spectrum of FITC (Figure S4, curve b, Supporting Information) gives four characteristic peaks located at 3081, 2024, 1735, and 1596 cm−1, which are ascribed to the vibrations of O−H, NCS, CO, and C−O−C, respectively.32 Meanwhile, DITC also shows the characteristic peak of N
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RESULTS AND DISCUSSION Preparation and Characterization of the Nanosensor. As shown in Figure 1A, QD800 nanoparticles were observed in uniform morphology with about ∼7 ± 3 nm in size. Through the microemulsion process, QD800 nanoparticles were encapsulated into silicon nanoparticles to generate SiO2@QD nanoparticles with an average diameter ∼55 nm (Figure 1B and Figure S1 in the Supporting Information). The encapsulation of QD800 with silica would reduce their cytotoxicity of semiconductor QDs like CdSe/ZnS.38 The thickness of silica shell (∼24 nm, Figure 1B) may prevent fluorescence energy transfer between QDs and other fluorophores, as well as avoid the quenching from metal ions and other biological molecules, thus proving a reliable reference signal. Moreover, the silica shell with amine group may act as a functional substrate for conjugating other recognition molecules onto SiO2@QD surface. Then, for fabricating a mitochondrial-targeted probe, TPP, a mitochondria targeting moiety, was modified onto the surface of amine SiO2@QD nanoparticles using EDC and NHS as the coupling agents. Followed by that, FITC and HE were sequentially conjugated onto the surface of amine SiO2@QD 12297
DOI: 10.1021/acs.analchem.6b03470 Anal. Chem. 2016, 88, 12294−12302
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Figure 2. (A) Fluorescence spectra of the nanosensor upon the exposure to O2•− of different concentrations: 0, 0.5, 2.5, 5, 10, 15, 20, 30, 40, 50, 60, 75, and 90 μM; (B) Plot of Fgreen/Fred as a function of the different concentrations of O2•−; (C) Fluorescence spectra of the nanosensor at different pH values: 7.86, 7.76, 7.65, 7.56, 7.45, 7.34, 7.26, 7.13, 7.03, 6.92, 6.80, 6.70, 6.61, 6.46, 6.33, 6.24, 6.14, 6.06, 5.95, 5.86, 5.76, 5.65, and 5.56; (D) Plot of Fblue/Fred as a function of different pH.
Figure 3. (A) Fluorescence spectra of the nanosensor upon the simultaneous exposure to different concentrations of O2•− (0, 20, 40, 60, and 80 μM) and different pH (7.9, 7.4, 6.9, 6.4, and 5.9); (B) plot of Fgreen/Fred as a function of the different concentrations of O2•−; and (C) plot of Fblue/ Fred as a function of different pH.
CS at 2062 cm−1 (Figure S4, curve c, Supporting Information). After FITC and DITC were conjugated on TPP-SiO2@QD nanoparticles, five peaks were clearly obtained at 3237, 2032, 1744, 1596, and 1090 cm−1 in the FTIR spectrum of DITC + FITC + TPP-SiO2@QD nanoparticles (Figure 1D, curve c), which are attributed to −NH2, NCS, CO, C−O−C, and −Si−O, respectively.39,41 The existence of CO and NCS groups suggests the attachment of FITC and DITC on the surface of TPP-SiO2@QD nanoparticles. After reacted with HE, the disappearance of the absorption band at 2032 cm−1 for NCS and the appeared band at 3364 cm−1 for the N−H stretching of HE (Figure S4, curve d, Supporting Information) implies the successful attachment of HE and formation of the HE + FITC + TPPSiO2@QD nanosensor (Figure 1D, curve d). Ratiometric Responses to O2•−, pH, and pH/O2•−. From Figure 2A, it was found that the green fluorescence ascribed to the oxidation products of HE (Fgreen, 600−670 nm) enhanced gradually with the addition of O2•−. By contrast, the
blue fluorescence (Fblue, 510−570 nm) from FITC and the red fluorescence (Fred: 730−800 nm) from SiO2@QD stayed constant. Thus, the red fluorescence from SiO2@QD was selected as an inner reference to provide a built-in correction for avoid the environmental effect for O2•− determination. As a result, the integrated fluorescence intensity ratio between green and red channels, Fgreen/Fred, gradually enhanced with the increasing concentrations of O2•−. As plotted in Figure 2B, the signal ratio Fgreen/Fred shows a good linearity with O2•− concentration in the range of 0.5−90 μM. The detection limit, at a signal-to-noise ratio of 3, was calculated to be ∼85 nM. Compared with those reported fluorescent probes for O2•−, the present probe demonstrates promising application in monitoring of O2•− with high sensitivity and wide concentration range.42,43 On the other hand, the blue emission, Fblue, from FITC decreased gradually with the decreasing pH values, while negligible changes were observed in the red fluorescence and green fluorescence (Figure 2C). The ratio of the integrated 12298
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Figure 4. (A) Colocalization studies in RAW264.7 macrophage cells that were costained with the nanosensor (200 μg mL−1) and Mito Tracker (100 nM): (a−c) Fluorescence images (a) from the nanosensor (λex = 488 nm, λem = 510−570 nm, blue channel), (b) from the Mito Tracker (λex = 633 nm, λem = 660−710 nm, red channel), and (c) the merged image of (a) and (b); (d) the bright field image; (e) the merged image of (c) and (d). (B) Fluorescence images of RAW264.7 cells cultured in growth media supplemented with different concentrations of O2•−: (a, f, k, p) 0, (b, g, l, q) 20, (c, h, m, r) 40, (d, i, n, s) 60, and (e, j, o, t) 80 μM. (a−e) Fluorescence images from the blue channel (λem = 510−570 nm). (f−j) Fluorescence images from the green channel (λem = 600−670 nm). (k−o) Fluorescence images from the red channel (λem = 730−800 nm). (u) Mean fluorescence intensities from blue channel (blue curve), green channel (green curve), and red channel (red curve) vs different concentrations of O2•−. Plots of (v) Fgreen/Fred and (w) Fblue/Fred vs different concentrations of O2•−.
intensities, Fblue/Fred, gradually decreased with the decreasing of pH. A good linearity between the fluorescence intensity ratio and pH values was clearly observed in the range from 5.56 to 7.86 (Figure 2D), which meets the requirements for the determination of physiological pH values. Then, in order to understand the relationship between pH and O2•‑ in mitochondria, we investigated the fluorescence responses of the nanosensor with simultaneous change of pH and O2•− concentrations. As shown in Figure S5 in the Supporting Information, the reaction time between HE and O2•− reached the plateau after ∼30 min, while FITC responded immediately with the change of pH value within ∼28 s. Thus, the fluorescence spectra of the nanosensor after simultaneous changes of pH and O2•− were acquired after equilibrated for 30 min. As shown in Figure 3A, the blue fluorescence of FITC dramatically decreased as the decreasing pH from 7.9 to 5.9. Meanwhile, the green fluorescence ascribed to the oxidation products of HE gradually enhanced with the rising concentration of O2•− from 0 to 80 μM. The red fluorescence from
SiO2@QD remained constant, resulting in the ratiometric determination of pH and O2•−. Importantly, the nanosensor could separately respond to pH and O2•−, because the two slope coefficients shown in Figure 3B,C agree well with those shown in Figure 2. With the applications to complicated living cells, not only sensitivity but selectivity is also a very important factor for the analytical method. Therefore, selectivity of the proposed sensing system was examined. In the experiments, we measured the changes of fluorescence intensity ratios (ΔFblue/Fred and ΔFgreen/Fred) with the addition of potential interferences, and compared with that of pH or O2•−. Other typical ROS such as 1 O2, H2O2, NO, HNO, ONOO−, ROO•, and •OH, as well as metal ions including K+, Na+, Mg2+, Fe3+, Cu2+, Ca2+, and physiological relevant amino acids were examined under identical conditions. As shown in Figure S6 in Supporting Information, negligible interferences (
