Synergistic Coordination and Hydrogen Bonding Interaction Modulate

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Synergistic Coordination and Hydrogen Bonding Interaction Modulate the Emission of Iridium Complex for Highly Sensitive Glutamine Imaging in Live Cells Qin Jiang,† Ming Wang,†,‡ Lifen Yang,*,† Hui Chen,*,†,‡ and Lanqun Mao*,†,‡ †

Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Analytical Chemistry for Living Biosystems and Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: Highly selective detection of intracellular glutamine (Gln) is very essential to understand the roles of Gln in some biological processes. Here, we report a new fluorescent method for selective imaging of Gln in live cells with an aldehyde-containing iridium complex, [Ir(pba)2(DMSO)2]PF6 (Hpba = 4-(2-pyridiyl)benzaldehyde) (Ir1), as the probe. Density functional theory (DFT) calculation and experimental results suggest that the coordination and hydrogen bonding interaction between Ir1 and Gln synergistically stabilize the Ir1−Gln complex, modulate charge-transfer characteristics and emission of Ir1, and as a consequence, enable Ir1 as the probe for the fluorescent sensing of Gln. The sensing strategy is well-responsive to Gln without interference from other amino acids or Gln-containing peptides and is demonstrated to be useful for in situ Gln imaging in live cells. The study provides a new method for fluorescent imaging of Gln in live cells, which is envisioned to find interesting applications in understanding the roles of Gln in some physiological processes. lutamine (Gln) is a “non-essential” amino acid that could be produced from tricarboxylic acid (TCA) metabolism inside cells.1 Gln regulates various biological events and controls cell proliferation and survival. For example, a tumor cell consumes Gln at a high rate over other amino acids, providing excessive nutrient to support the rapid growth of tumor cells.2 Also, it has been reported that Gln protects DNA from damage in the brain, and a lowered level of endogenous Gln could be associated with Alzheimer’s disease (AD).3 Therefore, effective detection of Gln is of great importance to understand and uncover the mechanism of a large variety of biological events, including cancers. So far, positron emission tomography (PET)4 and hyperpolarization magnetic resonance (MR)5 have been used for Gln detection. However, the use of specially labeled Gln tracer in these methods has limited their availability for Gln detection. In this regard, fluorescent sensing provides a facile approach for direct Gln detection and imaging in live cells. However, such potentiality has not been explored yet mainly because of the similarity in chemical structure between Gln and other amino acids, which substantially creates great difficulties in designing selective chemical probes for Gln sensing. Recently, luminescent cyclometalated iridium(III) complexes have been emerging as an attractive platform for fluorescent sensing and cellular imaging, by precisely modulating the coordination chemistry and charge-transfer characteristics of iridium(III) complexes.6−11 For example, an azo-featured iridium(III) complex, i.e., [Ir(ppy)2(azobpy)Ir(ppy)2]2+ [ppy

G

© XXXX American Chemical Society

= 2-phenyl-pyridine, azobpy = 4,4′-azobis(2,2′- bipyridine) has been designed for endogenous sulphite and bisulphite detection and imaging in live cells.10 Additionally, Gupta et al. engineered the chemical environment and intramolecular hydrogen bond of cyclometalated iridium(III) complexes for endoplasmicreticulum-targeted cellular imaging.11 With these in mind, we reason that by rationally modulating the chemical environment, such as the coordination and hydrogen bonding interaction of iridium complex in a synergistic manner, one would achieve the selectivity of iridium complex for Gln sensing and imaging in live cells. Herein, we report a proof-of-concept study of designing iridium complex featuring aldehyde group [Ir(pba)2(DMSO)2]PF6 (Hpba = 4-(2-pyridiyl)benzaldehyde), Ir1, for selective Gln sensing and imaging in live cells. As shown in Scheme 1, the coordination of the carboxylate and amine of Gln with a cationic iridium center and the hydrogen bond between the aldehyde of Ir1 and the amide terminal of Gln synergistically stabilize the Ir1−Gln complex. The formation of Ir1−Gln complex effectively tunes the ρ−π conjugation between aldehyde and the phenyl moiety of Ir1, switching the excited states of Ir1 from (π−π*) (pba-) 3IL to [dπ(Ir)-πN∧N*] 3 MLCT and [πC∧N-πN∧N*] 3LLCT, which results in a blue Received: August 29, 2016 Accepted: September 30, 2016

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DOI: 10.1021/acs.analchem.6b03383 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Scheme 1. Chemical Structure of Ir1 and Synergistic Coordination and Hydrogen Bonding Interaction between Ir1 and Gln

Scheme 2. Synthesis of Cyclometalated Iridium(III) Complex Ir1

shift and enhancement of the emission of Ir1.12−14 Such a property essentially forms a straightforward basis for the selective and sensitive Gln sensing with Ir1 as the probe. Cell studies show that the Ir1 probe exhibits low cytotoxicity and could be used for in situ fluorescent imaging of exogenous and endogenous Gln in live cells. To the best of our knowledge, this study represents the first study on designing fluorescent Gln sensing and imaging in live cells by modulating the chemical environment of iridium complex, which holds a great promise to illuminate the roles of Gln in some physiological and pathological processes.

(μ-Cl)2Ir(pba)2] (dimer 1). The crude dimer 1 (93.48 mg, 0.08 mmol) and DMSO (30 mL) was heated to reflux for an additional 2 h before adding a 4-fold excess of KPF6 at room temperature. The suspension was stirred for 1 h, and the insoluble inorganic salts were removed via filtration. The solvent was evaporated under reduced pressure, and the crude product was further purified using flash chromatography on silica gel eluted with CH2Cl2/acetone (30/1, v/v). Ir1 was obtained as a yellow solid with 65% yield, the chemical structure of Ir1 was characterized and confirmed using 1H NMR, FT-IR spectrum, ESI-MS, and elemental analysis. 1H NMR (300 MHz, DMSO-d6, TMS): δ ppm: 9.86 (s, 1H; CHO), 9.62 (s, 3H), 8.45 (d, 2H), 8.21 (t, 2H), 8.04 (m, 2H), 7.76 (t, 1H), 7.64 (t, 1H), 7.43 (m, 2H), 6.75 (s, 1H), 6.14 (s, 1H). FT-IR (KBr, cm−1): 1684 (s, νCO). ESI-MS (m/z): 712.92, 635.00, 557.08 (corresponding to [Ir(pba)2(DMSO)2]+, [Ir(pba)2(DMSO)]+, and [Ir(pba)2]+, respectively) (Figure S1). Anal. Calcd for C28H28IrN2S2O4PF6·2.5C3H6O: C, 42.50; H, 4.33; N, 2.81. Found: C, 42.89; H, 3.85; N, 3.29. Photophysical Characterization of Ir1. The absorption and emission spectra of Ir1 were measured in a DMSO/PBS solution (1/49, v/v, pH 7.0, 10 mM) at room temperature. To determine the quantum yield of Ir1, quinoline sulfate in 0.05 M sulfuric acid solution was used as a reference (QE = 0.55). For the fluorescent detection of Gln, different concentrations of amino acids and peptides were added to a DMSO/PBS solution of Ir1 (10 μM), respectively. Cell Culture and Cytotoxicity Assay. Human cervical cancer cells (HeLa) cells were provided by Peking Union Medical College Hospital (China). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in the presence of 5% CO2. For cytotoxicity assay of the Ir1 probe, HeLa cells were seeded at a density of 1 × 104 /well in a 96-well cell culture plate at 37 °C under a 5% CO2 atmosphere for 24 h. Ir1 at different concentrations of 100, 80, 60, 40, 20 μM were added to cells, followed by 24 h of incubation before the cytotoxicity assay using the CCK-8 kit. CLSM Imaging. HeLa cells were first seeded in 35 mm glass-bottomed dishes at a density of 106 cells/well and then treated with Ir1 (20 μM) for 60 min at 37 °C under 5% CO2 atmosphere before CLSM imaging. For mitochondria staining, HeLa cells were incubated with 2 μM rhodamine123 for 10 min. The excitation wavelength of Ir1 probe was 405 nm, and the rhodamine123 was 488 nm. The emissions were collected

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EXPERIMENTAL SECTION Materials and Instruments. All chemicals used for Ir1 synthesis and amino acids were purchased from Aladdin (Shanghai, China). Dipeptide (Gly−Gln and Ala−Gln) and Rhodamine123 were purchased from Sigma-Aldrich and used as received. IrCl3·3H2O was purchased from Shanghai Jiuling Chemical Co. Ltd. Cell Counting Kit-8 (CCK-8) was obtained from Beyotime Institute of Biotechnology. All aqueous solutions used in the experiments were prepared with Milli-Q water (18.2 MΩ, Millipore). Proton NMR (1H NMR) spectra were recorded with a Varian spectrometer at 300 MHz. Mass spectrometry experiments were performed either on a linear ion trap mass spectrometer (Thermo Scientific LTQ XL) equipped with a nano ESI source (ESI-MS) or on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). UV−visible spectra were measured on a TU-1900 spectrophotometer (Beijing Purkinje General Instrument Co. Ltd., China). Fourier Transfer Infrared (FT-IR) spectra (KBr pellet) were collected on a Tensor-27 FTIR spectrometer (Bruker). Steady-state emission experiments were measured on a Hitachi F-4600 spectrometer (Hitachi Co. Ltd., Japan) with Xe lamp as the excitation source at room temperature. Luminescent lifetime measurements were performed with a DIY pico-second time-resolved fluorescence spectrometer. Confocal laser scanning microscopy (CLSM) images were performed on an Olympus FV1000-IX81 CLSM and a Leica TCS SP confocal system (Leica, Germany). Synthesis of Ir1. Cyclometalated iridium(III) complex Ir1 was prepared via a two-step reaction shown below (Scheme 2). Chloro-bridged dinuclear iridium(III) precursor [(pba)2Ir(μCl)2Ir(pba)2] (dimer 1) was first synthesized according to a previous report,15 and the subsequent chloride replacement in [(pba)2Ir(μ-Cl)2Ir(pba)2] by requisite solvent DMSO afforded Ir1. Briefly, IrCl3·3H2O (353 mg, 1 mmol) and Hpba (4-(2pyridiyl)benzaldehyde, 458 mg, 2.5 mmol) were mixed in 2methoxyethanol and water (3/1, v/v) solution and refluxed for 24 h under stirring.21 After the mixture was cooled to room temperature, the yellow precipitate was collected as [(pba)2IrB

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Analytical Chemistry at 420−490 nm and 500−600 nm for the loaded cells, respectively. Lifetime Measurement. Ir1 solution for transient luminescent decay measurements was prepared by dissolving Ir1−Gln complex into DMSO/PBS (1/49, v/v, pH 7.0, 10 mM). Briefly, the 800 nm laser pulses generated from a Ti:sapphire regenerative amplifier (Spitfire, Spectra Physics) were frequency doubled and used as the excitation pulses. The pulse energy was about 100 nJ/pulse at the sample. Fluorescence gathered with the 90-degree-geometry was dispersed by a polychromator (250is, Chromex) and collected with a photon-counting type streak camera (C5680, Hamamatsu Photonics). The spectral resolution was 2 nm, and the temporal resolution was 2−100 ps depending on the delaytime-range setting. All the spectroscopic measurements were carried out at room temperature. Theoretical Calculation. The geometries of Ir1 and its corresponding adducts (Ir1−AA) with various amino acids (Gln, Arg, Ala, Asp, Asn, His, Ile, Cys, Tyr, Lys, Gly, Glu) and GSH were optimized with DFT method in water. Meta-GGA functional M06L,22 which had been previously reported to perform well in the calculation of 4d and 5d transition metal− ligand binding energetics,23 was used here with a polarized valence double-ζ basis set def-SVP24 denoted as B1 for geometry optimizations. For more accurate description of the crucial H-bonding between amino acids with Ir1, the diffuse basis functions on O atoms were used.25 The scalar relativistic effect of iridium atom was considered by the small-core relativistic pseudopotential (ECP-60-MWB).26 Solvent effect was modeled with continuum solvation model SMD throughout.27 The optimized minimum was verified to be true minimum by vibrational analysis to have no imaginary frequency. On the basis of these optimized geometries, single point M06L calculations utilizing multiply polarized valence triple-ζ basis set def-TZVPP28 (denoted as B2, and also augmented with corresponding diffuse functions for O atom) were performed to refine the final reported Gibbs free energies. The calculation of the excited states of the emission to explore the luminescence characters of Ir1 and Ir1−Gln were performed with DFT and time-dependent DFT (TD-DFT) approaches for the triplet (phosphorescence) and singlet (fluorescence) excited states, respectively. For this purpose, the long-range corrected functional CAM-B3LYP,29 which was designed to calculate charge-transfer (CT) excitations encountered in approximate density functional, was used in TD-DFT calculations including excited state geometry optimization in water.30 The excited state geometry optimization were performed with B1 basis set, followed by single point calculations utilizing B2 basis set to determine the emission energies. The solvent effect on the excited states is accounted for SMD model as for the ground state calculations described above.27 All the DFT and TD-DFT calculations were performed with the Gaussian 09 program.31

Figure 1. UV−vis absorption and photoluminescence spectra of Ir1 solution (10 mM in DMSO) at room temperature.

were observed in the lower energy region of 650−700 nm. Ir1 was emissive with a maximum peak at 557 nm (red curve), the fluorescence lifetime of Ir1 was single-exponential with 100 ns, which was at the same magnitude with that of cyclometalated iridium complexes sharing similar chemical structures.17 The quantum yield of Ir1 was determined to be 0.04 with quinoline sulfate as a reference, showing a decreased fluorescence emission compared to that of complex [Ir(ppy)2 (DMSO)2]+ (ppy = phenylpyridine).18 The weak emission of Ir1 may be partially due to the introduction of electron-withdrawing ligand (pba- moiety), which results in a mixing excited state of (π−π*) (pba-) 3IL and [dπ(Ir)- π*(pba-)] 3MLCT. The theoretical emission of the lowest triplet of Ir1, calculated by density functional theory (DFT) approach, was located at 593 nm at the CAM-B3LYP/B2 level. The calculated emission of Ir1 was in good agreement with the experimental results, indicating that the aldehyde moiety could switch the excited state from (π−π*) (pba-) 3IL to [dπ(Ir)-π*(pba-)] 3MLCT, providing the rationality for the design of fluorescent sensing by modulating the chemical binding of the aldehyde group of Ir1. Selective Detection of Glutamine with Ir1. We demonstrated the use of Ir1 for Gln sensing by titrating the emission of Ir1 with Gln solution. As shown in Figure 2A, upon the addition of increased concentrations of Gln into the Ir1 solution (10 μM in DMSO/PBS, v/v, 1/49, 10 mM), Ir1 emission at 557 nm was decreased gradually whereas a new

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RESULTS AND DISCUSSION Photophysical Property of Ir1. As shown in Figure 1, the UV−vis absorption spectrum of Ir1 shows strong absorption bands in 274−324 nm region and weak bands in 375−420 nm region (blue curve), which were ascribed to the intraligand 1IL (π → π*) (pba−) transition and the spin-allowed metal-toligand charge transfer 1MLCT (dπ(Ir) → π*(pba−) transitions of cyclometalated iridium complex, respectively.16 In addition, spin-forbidden 3MLCT (dπ(Ir) → π*(pba−) absorption peaks

Figure 2. (A) Emission spectra of Ir1 solution (10 mM in DMSO/ PBS, v/v = 1/49) in the presence of increased concentrations of Gln (from bottom to top: 0, 10, 20, 40, 60, 80, 100, 200, 400, 600, and 800 μM); (B) Selective Gln detection using Ir1. Fluorescence responses (F/F0) of Ir1 (10 mM in DMSO/PBS, v/v = 1/49) to various amino acids (800 μM) and Gln-containing peptides (800 μM) (from left to right were 1, Ir1; 2, Gln; 3, Arg; 4, Ala; 5, Asp; 6, Asn; 7, His; 8, Ile; 9, Cys; 10, Tyr; 11, Lys; 12, Gly; 13, Glu; 14, GSH; 15, Ala−Gln; and 16, Gly−Gln. C

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Table 1. Free Energy Changes ΔG (kcal/mol) of Gln Substitution in Ir1−Gln by Other Amino Acids (AA) (Ir1− Gln + AA → Ir1−AA + Gln) Calculated at the M06L/B2 Level

emission at 475 nm was increased, corresponding to a blue shift of about 82 nm. The blue shift of Ir1 emission after Gln addition was due to the coordination of carboxylate and amine of Gln with iridium center, stabilizing the HOMO orbital and resulting in an increased energy gap of Ir1.19 The fluorescence intensity of Ir1 at 475 nm was enhanced up to 200 times when the concentration of Gln was increased from 0 to 800 μM. Moreover, with the addition of 12 kinds of natural amino acids, including arginine (Arg), alanine (Ala), aspartate (Asp), asparagine (Asn), histidine (His), isoleucine (Ile), cysteine (Cys), tyrosine (Tyr), lysine (Lys), glycine (Gly), glutamate (Glu) and glutamine (Gln), and three Gln-containing peptides (GSH, Gly−Gln, Ala−Gln), we did not observed obvious enhancement of Ir1 emission (Figure 2B), though the addition of arginine (Arg) slightly enhanced the emission of Ir1 (10 μM). In addition, we found negligible Ir1 and Ir1−Gln fluorescence changes after 7 days of storage (Figure S2), indicating the high stability of both Ir1 and Ir1−Gln complex for Gln sensing. Taken together, the fluorescence titration results demonstrate that Ir1 exhibits a high selectivity for Gln detection over other amino acids and Gln-containing peptides. We hypothesized that the selectivity was mostly due to the specific interaction between Ir1 and Gln, which facilitates the formation of stable Ir1−Gln complex to modulate the emission of Ir1, as discussed below. Mechanism Study of Ir1 and Gln Binding. To further understand the mechanism that enables selective Gln sensing, we first analyzed the interaction between Ir1 and Gln and characterized the Ir1−Gln complex using mass spectrometry (MS). To this end, Ir1 (10 μM) was incubated with Gln (800 μM) at 37 °C for 1 h, followed by MALDI-TOF mass spectrometry analysis. As shown in Figure 3, a new peak at m/z

amino acid

Arg

Ala

Asp

Asn

His

Ile

ΔG amino acid

2.4 Cys

3.4 Tyr

3.9 Lys

2.3 Gly

2.7 Glu

4.2 GSH

ΔG

1.9

1.2

3.1

3.4

5.8

2.7

suggesting the higher stability of Ir1−Gln than other Ir1−AA complexes. Moreover, the optimized structure of Ir1−Gln (Figure 4A) suggests that the carbonyl group of terminal

Figure 4. (A) Optimized geometry of Ir1−Gln at M06L/B1 level. (B) Chemical structures of Gln and GSH. (C) Optimized chemical structure of Ir1−Gln.

aldehyde in Ir1 forms a hydrogen bond with the amide of Gln, further stabilizing Ir1−Gln complex. In comparison, when the amide hydrogen in Gln is substituted by cysteine in GSH or other Gln-containing peptide (Figure 4B), the hydrogen bond between Gln and Ir1 was disrupted, resulting in weak capability to form complex with Ir1. The DFT calculation and MS spectrometry studies indicate that the coordination and hydrogen bond between Ir1 and Gln synergistically stabilize Ir1−Gln complex, which modulate Ir1 emission for selective Gln detection. Fluorescent Gln Imaging in Live Cells. Having demonstrated the selective Gln detection using Ir1, we next studied Ir1 as a fluorescent indicator to monitor exogenous and endogenous glutamine in live cells. To this end, we first examined the cytotoxicity of Ir1 and evaluated the biocompatibility of Ir1 for live cell studies. Human cervical cancer cells (HeLa) were incubated with varied concentrations of Ir1, followed by cell viability measurement using the CCK-8 assay. As shown in Figure S5, with the concentration of Ir1 exposed to cells increased from 20 μM to 100 μM, the viable cells remained as high as 90% after 24 h treatment, compared to nontreated group, indicating the low cytotoxicity and high biocompatibility of Ir1 for live cell studies. To demonstrate Ir1 for intracellular Gln monitoring, HeLa cells were pretreated with different concentrations of Gln, followed by Ir1 incubation and confocal laser scanning microscopy (CLSM) imaging. As shown in Figure 5, a strong intracellular fluorescence was observed from cells treated with 20 μM Ir1. Moreover, the incubation of the cells with Gln (800 μM) further enhanced fluorescence of Ir1. The CLSM studies indicate the capability of Ir1 as a cell-permeable probe for exogenously supplied Gln imaging in live cells. In addition, we found that Ir1 emission was mostly localized in the cytoplasm by quantifying the fluorescent intensity in nucleus and cytoplasm. As shown in Figure 6A, the treatment of HeLa cells with 20 μM Ir1 resulted in 600-times stronger fluorescence in cytoplasm than that in nucleus, indicating the

Figure 3. MALDI-TOF spectrum of Ir1/Gln complex.

701.77, which was ascribed to [Ir(pba)2(Gln)], appeared in the MS spectrum of Ir1/Gln complex, indicating two DMSO molecules in Ir1 were substituted by one Gln molecule (Scheme 1) in the Ir1−Gln complex. In comparison, no change was observed when Ir1 was incubated with other amino acids under the same conditions, indicating the coordination between Ir1 and these amino acids was not stable or strong enough to form a complex and modulate Ir1 emission (Figure S3 and S4). We further calculated the free energy change, ΔG, for the transformation from Ir1−Gln to Ir1−AA, to compare the stability of the complex formed between Ir1 and different amino acids (AA). DFT calculations indicate that all ΔG values of Ir1−Gln to Ir1−AA transformation are positive (Table 1), D

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(20 μM) and costaining mitochondria using rodamine123 demonstrate a high level of colocalization of Ir1 and mitochondria. It has been previously reported that glutamine is an important energy source in mitochondria.20 Therefore, Ir1 could be potentially used to study mitochondrial glutamine metabolism.

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CONCLUSIONS In summary, we have developed a novel and selective fluorescent glutamine probe using an iridium complex, [Ir(pba)2(DMSO)2]PF6. We have found that the coordination and hydrogen bonding interaction between Ir1 and Gln synergistically stabilized the Ir1−Gln complex and modulated Ir1 emission, enabling the selective detection of Gln over other amino acids and in situ glutamine imaging in live cells. We envision that the strategy reported here not only diversifies the chemistry that could be harnessed for the development of iridium complex for molecular imaging but also paves a new way to the study of the role of glutamine in physiological and pathological processes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03383. ESI-MS spectrum of Ir1 complex, photophysical characterization of Ir1 complex, fluorescent spectra of Ir1 and Ir1−Gln complex, MALDI-TOF spectrum of Ir1/Arg complex, MALDI-TOF spectrum of Ir1/His complex, cell viability (%) of HeLa cells, CLSM images of HeLa cells (PDF)

Figure 5. CLSM and bright-field images of HeLa cells (A) without any treatment; (B) incubated with 20 μM Ir1 in DMSO/PBS (pH 7.0, 1/ 49, v/v, 10 mM) for 1 h at 37 °C. (C) Preincubated with 800 μM Gln for 1 h, then incubated with 20 μM Ir1 in DMSO/PBS (pH 7.0, 1/49, v/v, 10 mM) for 1 h at 37 °C (λex: 405 nm, λem: 425−470 nm). Scale bar: 20 μm.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: (86)-10-62559373. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Grant Nos. 21435007, 21321003, 21210007, 91413117 for L.M, 21290194, and 21521062 for H.C.), the National Basic Research Program of China (973 program, 2013CB933704), and the Chinese Academy of Sciences for the financial support.

Figure 6. CLSM imaging (A1) of HeLa cells incubated with 20 μM Ir1 in DMSO/PBS (pH 7.0, 1/49, v/v, 10 mM) for 1 h at 37 °C. Luminescence intensity profiles (A2) across the line shown in panel (A1) corresponding to cytoplasm region (1 and 3), nuclear region (2), and extracellular region (4). Bright-field (B1) and CLSM (B2−B3) images of HeLa cells incubated with 20 μM Ir1 in DMSO/PBS (pH 7.0, 1/49, v/v, 10 mM) for 1 h at 37 °C and then further incubated with 2 μM rodamine123 for 10 min under the same condition: (B2) blue channel at 420−490 nm (λex: 405 nm); (B3) green channel at 500−600 nm (λex: 488 nm); merged image (B4) of blue channel and green channel. Scale bar: 20 μm.

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REFERENCES

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main localization of Ir1 in cytoplasm. Moreover, a detailed cellular compartment staining suggested that Ir1 was selectively and spontaneously localized in mitochondria. As shown in Figure 6B and Figure S6, the treatment of HeLa cells with Ir1 E

DOI: 10.1021/acs.analchem.6b03383 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.6b03383 Anal. Chem. XXXX, XXX, XXX−XXX