A mitochondria-targeted near-infrared probe for colorimetric and ratiometric fluorescence detection of hypochlorite in living cells

Abstract

In this study, we report a near-infrared (NIR) fluorescent probe (CMBI) for the detection of hypochlorite (ClO⁻), which displays colorimetric and ratiometric fluorescence dual responses towards ClO⁻. The probe can detect ClO⁻ with high selectivity, fast response (within 90 s) as well as low detection limit (33 nM). An oxidation reaction was proposed for the sensing mechanism, which was confirmed by ¹H NMR and HR-MS spectra. Fluorescence co-localization studies demonstrated that CMBI was a specific mitochondria-targeted fluorescent probe for ClO⁻ with excellent cell membrane permeability. Furthermore, confocal fluorescence images of HeLa cell indicated that CMBI could be used for monitoring intracellular ClO⁻ in living cells by ratiometric fluorescence imaging.

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Introduction

Mitochondria is the principal energy production compartment of eukaryotic cells, which can utilize oxygen to generate biochemical energy in the form of adenosine triphosphate (ATP).¹ Mitochondria also play important roles in a variety of vital biological processes, such as regulation of the cytosolic calcium homeostasis,² cellular redox state,³ initiation of apoptosis⁴ and generation of reactive oxygen species (ROS).⁵ It has been revealed that mitochondria are the main source of intracellular ROS, which are responsible for cell signaling and numerous human diseases (e.g. cancers, diabetes, obesity and ageing).⁶ Among various ROS, hypochlorous acid/hypochlorite (HClO/ClO⁻) plays a key role in innate immunity, which is typically generated from peroxidation of hydrogen peroxide (H₂O₂) and chloride ions (Cl⁻) catalyzed by the enzyme myeloperoxidase (MPO).⁷ Appropriate level of ClO⁻ is required for the host to defense the invasion of some pathogens in human immune system.⁸ Nevertheless, the over production of ClO⁻ from the mitochondria leads to tissue damage and diseases including arthritis,⁹ atherosclerosis,¹⁰ cancer¹¹ and neurodegeneration.¹² Thus, it is highly in demand to develop an efficient method for monitoring HClO/ClO⁻ in mitochondria.

Fluorescent probes have attracted much attention due to their simplicity, high temporal and spatial resolution, high sensitivity as well as nondestructive imaging properties.¹³ Up to now, several fluorescent probes for HClO/ClO⁻ have been developed on the basis of HClO-mediated oxidation reaction of various functional groups such as C=C bond,¹⁴ C=N bond,¹⁵ ether,¹⁶ hydrazide,¹⁷ oxime,¹⁸ p-methoxyphenol,¹⁹ selenide,²⁰ thioether,²¹ thiospirolactone²² and others.^23–40 However, most of these probes showed emissions in the visible region, which suffered from the interference from background. By contrast, near-infrared (NIR) fluorescent probes with emission in the 650–900 nm region are more favourable for imaging and quantitative measurements in living systems, because their emission can penetrate deeply through tissues with low auto-fluorescence background and reduce photo-damage to biological samples.⁴¹ The ideal fluorescent probe for the detection of ClO⁻ in living systems should have the merits including: (i) NIR emission suitable for imaging in vivo; (ii) remarkable selectivity for ClO⁻ over other ROS/reactive nitrogen species (RNS); (iii) high sensitivity with low limit of detection; (iv) good mitochondria-targeting property because the most intracellular ROS are produced in mitochondria; (v) noticeable ratiometric fluorescence for more accurate and quantitative measurement, since ratiometric fluorescent probe provides an alternative approach that can eliminate most of interferences by measuring the ratio of fluorescence intensities at two different emission wavelengths.⁴²

In this work, we proposed a NIR fluorescent probe (CMBI) for specific detection of ClO⁻. As shown in Scheme 1, the probe was constructed by the condensation of 7-diethylaminocoumarin with benzo[e]indolium. Owing to the extended π-conjugation and strong intramolecular charge transfer (ICT), CMBI will display NIR emission. In CMBI, the C=C bond between 7-diethylaminocoumarin and benzo[e]indolium group is fragile to the oxidation of ClO⁻. When the C=C bond is destroyed by ClO⁻, the probe will display a colorimetric and ratiometric fluorescence dual responses towards ClO⁻, which is suitable for ratiometric fluorescence detection of HClO/ClO⁻.

Scheme 1 (a) Design strategy and (b) synthetic route for probe CMBI.

Experimental section

Reagents and instrumentation

Unless otherwise stated, all reagents were purchased from commercial suppliers (Aladdin-Reagent, Sigma-Aldrich, TCI). All chemicals and solvents were of analytical grade and used without further purifications. The ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AV-400 spectrometer with tetramethylsilane (TMS) as the internal standard. The chemical shift was recorded in ppm and the following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets. Mass spectra were measured on a HP-1100 LC-MS spectrometer. UV-vis spectra were recorded on Hitachi UV-3310 spectrometer. Fluorescence spectra were recorded on a Hitachi FL-4500 fluorometer. Confocal microscopy fluorescence images were acquired on a Nikon A1 confocal laser-scanning microscope with a 100 objective lens. The pH values were determined by a PHS-3C pH meter, which was purchased from the Shanghai Yoke Instrument Co., Ltd. The solvents used for UV-vis and fluorescence measurements are of HPLC grade. MitoTracker Green FM was purchased from Invitrogen Corporation.

Synthesis of 3-ethyl-1,1,2-trimethyl-1H-benz[e]indolium iodide (2)

1,1,2-Trimethyl-1H-benzo[e]indole (314 mg, 1.5 mmol) and ethyl iodide (468 mg, 3.0 mmol) were placed in a 50 mL round bottom flask. Then, 20 mL of anhydrous CH₃CN was added into the flask, and the mixture was refluxed for 12 h. After the reaction was completed, the reaction mixture was cooled to room temperature and produce some precipitates. The solid was collected by filtration, and washed with cold EtOH for twice. The crude product was re-crystallized from EtOH to afford product 2 as a blue solid (493 mg, yield: 90%). ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.38 (d, J = 8.2 Hz, 1H, ArH), 8.30 (d, J = 8.9 Hz, 1H, ArH), 8.22 (d, J = 8.2 Hz, 1H, ArH), 8.16 (d, J = 8.9 Hz, 1H, ArH), 7.79 (t, J = 7.5 Hz, 1H, ArH), 7.73 (t, J = 7.5 Hz, 1H, ArH), 4.62 (q, J = 7.3 Hz, 2H, NCH₂), 2.94 (s, 3H, CH₃), 1.76 (s, 6H, CH₃), 1.50 (t, J = 7.3 Hz, 3H, NCH₂CH₃).

Synthesis of probe CMBI

7-Diethylaminocoumarin-3-aldehyde (1)⁴³ (294 mg, 1.2 mmol) and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indolium iodide (2) (365 mg, 1.0 mmol) were placed in a 50 mL round bottom flask with 20 mL anhydrous EtOH. Then, piperidine (0.1 mL) and acetic acid (0.1 mL) were added. The reaction mixture was heated at 65 °C for 6 h in nitrogen atmosphere. After the reaction was completed (monitored by TLC plate), the reaction mixture was cooled to room temperature and concentrated under vacuum to give a solid. The crude product was purified by column chromatography (silica gel, EtOH : DCM = 1 : 50–1 : 30) to afford probe CMBI as a purple solid (397 mg, yield: 67%). ¹H NMR (400 MHz, DMSO-d₆) δ/ppm 8.87 (s, 1H, ArH), 8.44 (d, J = 8.4 Hz, 1H, ArH), 8.39 (d, J = 15.9 Hz, 1H, =CH), 8.28 (d, J = 9.0 Hz, 1H, ArH), 8.21 (d, J = 8.4 Hz, 1H, ArH), 8.11 (d, J = 9.0 Hz, 1H, ArH), 7.91 (d, J = 15.9 Hz, 1H, =CH), 7.79 (t, J = 7.5 Hz, 1H, ArH), 7.71 (t, J = 7.5 Hz, 1H, ArH), 7.62 (d, J = 9.2 Hz, 1H, ArH), 6.94 (dd, J = 9.2, 2.0 Hz, 1H, ArH), 6.74 (d, J = 2.0 Hz, 1H, ArH), 4.63 (q, J = 7.2 Hz, 2H, CH₂), 3.58 (q, J = 7.0 Hz, 4H, NCH₂), 2.01 (s, 6H, CH₃), 1.52 (t, J = 7.2 Hz, 3H, CH₂CH₃), 1.19 (t, J = 7.0 Hz, 6H, NCH₂CH₃). ¹³C NMR (100 MHz, DMSO-d₆) δ/ppm 181.51, 160.01, 158.07, 154.40, 150.22, 149.17, 138.72, 138.22, 133.39, 132.79, 131.50, 130.49, 128.81, 127.37, 123.50, 113.32, 112.77, 111.79, 109.92, 109.49, 97.08, 56.49, 53.60, 45.32, 42.42, 26.44, 13.74, 12.97. HR-MS (ESI): m/z calculated for [C₃₁H₃₃N₂O₂]⁺ 465.2542, found 465.2596.

Conversion of CMBI by NaClO

CMBI (59 mg, 0.1 mmol) was dissolved in 10 mL of EtOH/PBS mixture solution (v/v = 1/9, pH 7.4, 10 mM), and then was treated with 10% NaClO aqueous solution (3.0 mL) for 5 min. The reaction mixture was extracted with DCM (10 mL × 3). The combined organic extracts were dried over anhydrous MgSO₄, filtered, and concentrated under vacuum to give a solid. The residue was purified by column chromatography (silica gel, DCM) to afford a yellow solid (15 mg, yield: 61%).

General procedure for the spectral measurement

Stock solutions of probe CMBI (1 mM) was prepared in HPLC grade EtOH. Stock solutions of analytes (2.5–5 mM) were prepared in distilled water. For optical measurements, CMBI was diluted to 10 μM in EtOH/PBS solution (v/v = 1/9, pH 7.4, 10 mM), and 3.0 mL of the resulting solution was placed in a quartz cell of 1.0 cm optical path length each time. The UV-vis or fluorescent spectra titrations were recorded upon addition of ClO⁻. All spectroscopic experiments were carried out at room temperature. The excitation wavelength was 390 nm and the excitation slit/emission width was set as 2.5 nm/5.0 nm (for blue fluorescence) or the excitation wavelength was 585 nm and the excitation slit/emission width was set as 5.0 nm/5.0 nm (for red fluorescence).

Determination of the detection limit

The detection limit of probe CMBI for NaClO was calculated based on fluorescence titration. To determine the S/N ratio, the fluorescence intensity of CMBI without ClO⁻ at 658 nm was measured by five times and the standard deviation of blank measurements was determined. Under the present conditions, the ratio values of fluorescence intensity (F₄₇₅/F₆₅₈) was plotted as a concentration of ClO⁻. The detection limit was then calculated with the equation:

Detection limit = 3σ/k

where σ is the standard deviation of blank measurements, k is the slope between the ratio values of fluorescence intensity (F₄₇₅/F₆₅₈) of CMBI versus ClO⁻ concentration.

Cell culture and fluorescence imaging

HeLa cells (Perking Union Medical College, China) were cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone), supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin (100 units per mL)–streptomycin (100 μg mL⁻¹) liquid (Invitrogen) at 37 °C in a humidified incubator containing 5% CO₂ in air. The cells were incubated for 24 h before dye loading on an uncoated 35 mm diameter glass-bottomed dish (NEST). Then, the cells were incubated with serum-free DMEM and 10 μM CMBI at 37 °C for 30 min, washed twice with PBS to remove excess probe, and mounted on the microscope stage. Fluorescence images were captured by a Nikon A1 confocal laser-scanning microscope equipped with a live cell workstation. To explore the ability of the probe for sensing ClO⁻ in living cells, the live HeLa cells were in situ treated with 100 μM ClO⁻ and then the same set of cells was used for confocal laser-scanning microscopy measurement at different time. For colocalization experiments, the cells were incubated with serum-free DMEM containing 10 μM CMBI and 200 nM MitoTracker Green FM (a commercially available mitochondria specific staining dye) at 37 °C for 30 min, washed twice with PBS to remove excess probe, then incubated with serum-free DMEM and mounted on the microscope stage.

Results and discussion

Design and synthesis

In this work, 7-diethylaminocoumarin was employed as the building block to prepare fluorescent probe CMBI in view of its excellent photophysical property, such as large Stokes shift and high fluorescence quantum yield.⁴⁴ Whereas, the UV-vis absorption and emission spectra of 7-diethylaminocoumarin mainly locate at the region of 400–500 nm, which cannot penetrate deeply through tissues and has strong interference from auto-fluorescence background. NIR fluorescent probes with emission in the 650–900 nm region are more favourable for imaging and quantitative measurements in living systems, because they can overcome these defects encountered by common fluorescent probe. To push the emission into NIR region, 7-diethylaminocoumarin was conjugated with benzo[e]indolium by an ethylene group (Scheme 1). Benzo[e]indolium could extend the π-conjugation, increase the water-solubility and help probe to accumulate selectively in mitochondria of live cells.⁴⁵ Such a “push–pull” characteristic CMBI with large π-conjugation will display NIR fluorescence. It was envisioned that the large π-conjugation of CMBI would be destroyed while the bridge C=C bond was oxidized by ClO⁻.¹⁴ Consequently, the original red fluorescence from CMBI will fade away, meanwhile the blue fluorescence from coumarin moiety will come out. Therefore, CMBI will exhibit a colorimetric and ratiometric fluorescence dual responses towards ClO⁻.

The fluorescent probe CMBI was easily synthesized by condensation of 7-diethylaminocoumarin-3-aldehyde (1) with 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indolium iodide (2) in the presence of piperidine/AcOH (Scheme 1b).^45a,46 The structure of probe CMBI was fully characterized by ¹H NMR, ¹³C NMR and HR-MS (Fig. S1–S3†). Detailed synthetic procedures and structure analysis are described in the Experimental section and ESI.†

Colorimetric and ratiometric response of CMBI towards ClO⁻

The preliminary assay of CMBI for the detection of ClO⁻ was explored, which was performed in EtOH/PBS solution (v/v = 1/9, pH 7.4, 10 mM) at room temperature. The probe CMBI solution exhibited violet color and red fluorescence. Upon addition 10 equiv. of ClO⁻, the violet color of CMBI solution immediately changed to colourless and the fluorescence changed from red to blue (Fig. 1 inset).

Fig. 1 (a) UV-vis absorption and (b) fluorescence spectra changes of 10 μM CMBI in EtOH/PBS solution (v/v = 1/9, pH 7.4, 10 mM) upon addition of increasing amount of ClO⁻ (0–100 μM). Each spectrum was recorded after 3 min. Inset: (a) the color and (b) fluorescence images of CMBI in the absence/presence of ClO⁻.

Next, we explored the spectra titrations of CMBI with ClO⁻. As shown in Fig. 1 and Table S1,† CMBI displayed a strong absorption band centered at 592 nm and an anticipated NIR emission band centered at 658 nm. Upon addition of increasing amounts of ClO⁻, the fluorescence intensity of CMBI at 658 nm gradually decreased with the simultaneous appearance of a new blue-shifted emission band centered at 475 nm. Meanwhile, the maximum absorption wavelength blue-shifted from 592 nm to 400 nm, which suggested that the large π-conjugation of CMBI was interrupted by ClO⁻. When the concentration of ClO⁻ increased to 10 equiv. with respect to CMBI, the newly formed fluorescence band reached to a plateau with 370-fold enhancement in the ratiometric value (F₄₇₅/F₆₅₈), suggesting that CMBI could serve as a ratiometric fluorescent probe for ClO⁻. It is worthy to note that such a significant ratio signal change at two wavelengths is highly desirable for ratiometric fluorescent probes, as the sensitivity and the dynamic range of ratiometric probes are controlled by the ratios.

A linear calibration between the corresponding absorbance ratiometric value (A₄₀₀/A₅₉₂) and ClO⁻ concentrations (0–50 μM) was obtained with high coefficient (R² = 0.9919) (Fig. 2a), which indicated that CMBI might be used to quantitatively detect ClO⁻ concentrations. Moreover, by plotting the corresponding fluorescence intensity ratiometric value (F₄₇₅/F₆₅₈) versus the concentration of ClO⁻, a good linear relationship (R² = 0.9924) was obtained with ClO⁻ concentrations ranging from 0 to 50 μM (Fig. 2b). Moreover, CMBI could respond to low concentration of ClO⁻ with detection limit of 33 nM (Fig. S4†), which was comparable to the previously reported ratiometric fluorescent probes for ClO⁻ (Table S2†). These results indicated that CMBI could quantitatively detect ClO⁻ with high sensitivity.

Fig. 2 Linear relationships between (a) UV-vis absorbance ratios (A₄₀₀/A₅₉₂) and (b) fluorescence intensity ratios (F₄₇₅/F₆₅₈) of 10 μM CMBI versus concentrations of ClO⁻ in EtOH/PBS solution (v/v = 1/9, pH 7.4, 10 mM). Each spectrum was recorded after 3 min.

Time and pH dependent fluorescence response of CMBI toward ClO⁻

The time-dependent fluorescence response of CMBI towards ClO⁻ was examined. As shown in Fig. 3a, upon addition of 10 equiv. ClO⁻, the fluorescence intensity (at 475 nm) increased promptly and reached to maximum within 90 s, which indicated that CMBI could serve as a “fast response” fluorescent probe for ClO⁻ and might be suitable for real-time detection of ClO⁻ in living systems.

Fig. 3 (a) Time-resolved fluorescence responses of 10 μM CMBI towards 100 μM ClO⁻ in EtOH/PBS solution (v/v = 1/9, pH 7.4, 10 mM). (b) Ratiometric fluorescence changes of 10 μM CMBI in the absence (■) and presence (●) of 100 μM ClO⁻ in EtOH/buffer solution (v/v = 1/9, 10 mM) with different pH values. Each spectrum was recorded after 3 min.

To examine the feasibility of CMBI as a versatile probe for ClO⁻, pH-dependent fluorescence responses towards ClO⁻ was tested in EtOH/buffer solution (v/v = 1/9) with different pH values. As shown in Fig. 3b, the fluorescence intensity ratios (F₄₇₅/F₆₅₈) of CMBI was almost unchanged from pH 4.5 to 9.5. Upon addition of 10 equiv. ClO⁻, the fluorescence intensity of CMBI increased remarkably at the pH values ranging from 6.5 to 9.5, which was compatible with the pK_a of HClO (pK_a = 7.53).⁴⁷ Therefore, CMBI can used to monitor HClO/ClO⁻ at a wide range of pH values.

Selectivity of CMBI toward ClO⁻

High selectivity is one of the most important requirements for the applicability of fluorescent probes. To evaluate the selectivity for ClO⁻, 10 μM CMBI was treated with various biologically relevant species, including various small molecular species (500 μM) and ROS/RNS (100 μM). As shown in Fig. 4, addition of 100 μM ClO⁻ led to a significant color change and a great fluorescence enhancement of CMBI, which was so obvious that it could be detected by naked eyes (Fig. 4 inset). By contrast, other ROS (H₂O₂, ˙OH, TBHP, TBO˙, ˙O₂⁻ and ¹O₂), RNS (NO), biothiols (Cys, Hcy and GSH) and common anions (F⁻, Cl⁻, Br⁻, N₃⁻, NO₂⁻, NO₃⁻, AcO⁻, HCO₃⁻, HPO₄²⁻, H₂PO₄⁻, SO₄²⁻ and S₂O₃²⁻) only induced a negligible ratiometric response of the probe. To gain some insights of the high selectivity for ClO⁻ over HS⁻ and HSO₃⁻, we compared the kinetic differences of probe CMBI for these analytes. As shown in Fig. S6,† the probe showed much stronger fluorescence response towards ClO⁻ in comparison with HS⁻ and HSO₃⁻. The probe displayed a noticeable colorimetric and ratiometric fluorescence response to ClO⁻ on the basis of oxidation cleavage of alkene group in CMBI, which generated a high emissive diethylaminocoumarin aldehyde. While, HS⁻ and HSO₃⁻ attacked the indolium group according to nucleophilic addition reaction or 1,4-nucleophilic addition reaction, which generated benzo[e]indole and diethylaminocoumarin group.^45,48 Förster resonance energy transfer (FRET) or photo-induced electron transfer (PET) might occur between these two fluorophores, which would largely decreased the fluorescence quantum yield of diethylaminocoumarin. Therefore, this probe is selective for ClO⁻ over HS⁻ and HSO₃⁻ according to fluorescence response. Although CMBI also displayed color change in the presence of ONOO⁻ (Fig. 4 inset and S5†), the fluorescence ratios (F₄₇₅/F₆₅₈ < 0.8) were much weaker compared with that of ClO⁻ (F₄₇₅/F₆₅₈ > 360) (Fig. 4). These results suggest that CMBI has good selectivity for ClO⁻ over other biological species, which enables it to detect ClO⁻ in complex biological environment.

Fig. 4 Fluorescence intensity ratios (F₄₇₅/F₆₅₈) of 10 μM CMBI in EtOH/PBS solution (v/v = 1/9, pH 7.4, 10 mM) in the presence of various small molecular species (500 μM) and ROS/RNS (100 μM). Each spectrum was recorded after 3 min. Inset: (a) the color and (b) fluorescence images of CMBI in the presence of various small molecular species (50 equiv.) and ROS/RNS (10 equiv.) from left to right in the similar sequence as exhibited above.

Proposed sensing mechanism

To gain some insights of the sensing mechanism, we studied the oxidation products by ¹H NMR and HR-MS spectrum. The product of CMBI from the oxidation of ClO⁻ was isolated by using a silica gel column and then was subjected to ¹H NMR analysis. The major product was identified as compound 1 (Fig. 5), indicating that compound 1 might be the final product of CMBI after treatment with ClO⁻ (Scheme 2). This speculation was also confirmed by HR-MS, where a noticeable peak at m/z 246.1131 was assigned to [compound 1 + H]⁺ (calculated for [C₁₄H₁₅NO₃ + H]⁺, 246.1130) (Fig. S7†).

Fig. 5 ¹H NMR spectra of (a) the isolated product from CMBI after treatment with NaClO and (b) 7-diethylaminocoumarin-3-aldehyde (compound 1) in CDCl₃ (400 MHz).

Scheme 2 Proposed mechanism for CMBI sensing of ClO⁻.

Cell imaging

Encouraged by these merits described above, we investigated the potential application of CMBI for monitoring ClO⁻ in living cells. Cell cytotoxicity of CMBI was examined by MTT assay, and the results indicated that the probe CMBI have very low cytotoxicity (Fig. S8†). As shown in Fig. 6a and b, HeLa cells exhibited a clear cell profile with red fluorescence and nearly non-fluorescence in blue channel after incubation with 10 μM CMBI for 30 min. When the HeLa cells were further incubated with 100 μM ClO⁻ for 3 min, red fluorescence was reduced and blue fluorescence appeared (Fig. 6d and e). As the incubation time prolonged, the blue fluorescence from HeLa cells became much stronger and the original red fluorescence gradually faded away. The ratiometric images (F_blue/F_red) (Fig. 6c, f and i) were obtained by mediating the blue channel images (Fig. 6a, d and g) with the related red channel images (Fig. 6b, e and h), which was significantly increased after the addition of ClO⁻. Cell staining results indicated that CMBI was cell membrane permeable and could be employed for ratiometric fluorescence imaging of ClO⁻ in living cells.

Fig. 6 Relative confocal fluorescence images of HeLa cells under different conditions with CMBI. HeLa cells treated with 10 μM CMBI (a–c), then further incubated with 100 μM ClO⁻ for 3 min (d–f) and 10 min (g–i). Fluorescence images from left to right: blue channel (λ_ex = 402 nm, λ_em = 425–475 nm), red channel (λ_ex = 638 nm, λ_em = 662–737 nm) and ratiometric images (F_blue/F_red). Scale bar: 20 μm.

To investigate the subcellular localization of CMBI inside cells, commercially available mitochondria specific staining dye (MitoTracker Green FM) was used for co-localization study. HeLa cells were co-incubated with 10 μM CMBI and 200 nM MitoTracker Green FM at 37 °C for 30 min. As shown in Fig. 7a, a clear mitochondria profile with strong green fluorescence was observed, which was ascribed to MitoTracker Green FM. A similar mitochondria profile with red fluorescence was also observed after incubation with CMBI (Fig. 7b), which overlaid very well with the fluorescence of MitoTracker Green FM (Fig. 7c and f). In addition, Pearson's co-localization coefficient, that describes the correlation of the intensity distribution between two channels, was calculated to be 0.94 (Fig. 7e), confirming that CMBI was site-specifically stained in mitochondria of living cells.

Fig. 7 Confocal fluorescence images of HeLa cells stained with (a) 200 nM MitoTracker Green FM (green channel: λ_ex = 488 nm, λ_em = 500–530 nm) and (b) 10 μM CMBI (red channel: λ_ex = 638 nm, λ_em = 662–737 nm). (c) Merged image of (a) and (b). (d) Bright field image. (e) Correlation plot of MitoTracker Green FM and CMBI intensities. (f) Intensity profile of regions of interest (ROI) across HeLa cells. Scale bar: 20 μm.

Conclusions

In summary, we have rationally designed a NIR fluorescent probe for ClO⁻, which displays colorimetric and ratiometric fluorescence dual responses towards ClO⁻ based on oxidation cleavage reaction of C=C bond. This probe have some remarkable advantages including high sensitivity, good selectivity, rapid response (within 90 s) and low detection of limit (33 nM). Fluorescence co-localization studies demonstrated that CMBI was a specific mitochondria-targeted fluorescent probe for ClO⁻ with excellent cell membrane permeability. Confocal fluorescence images of HeLa cell indicated that CMBI could be used for monitoring intracellular ClO⁻ in living cells by ratiometric fluorescence imaging.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 21203138, 31371750, 31560712) and the Natural Science Foundation of Hubei Province (2013CFC007).

Notes and references

1. (a) K. Henze and W. Martin, Nature, 2003, 426, 127; (b) Roopa, N. Kumar, V. Bhalla and M. Kumar, Chem. Commun., 2015, 51, 15614; (c) Z. Xu and L. Xu, Chem. Commun., 2016, 52, 1094.
2. (a) J. M. Baughman, F. Perocchi, H. S. Girgis, M. Plovanich, C. A. Belcher-Timme, Y. Sancak, X. R. Bao, L. Strittmatter, O. Goldberger, R. L. Bogorad, V. Koteliansky and V. K. Mootha, Nature, 2011, 476, 341; (b) K. Mallilankaraman, C. Cárdenas, P. J. Doonan, H. C. Chandramoorthy, K. M. Irrinki, T. Golenár, G. Csordás, P. Madireddi, J. Yang, M. Müller, R. Miller, J. E. Kolesar, J. Molgó, B. Kaufman, G. Hajnóczky, J. K. Foskett and M. Madesh, Nat. Cell Biol., 2012, 14, 1336.
3. A. Chacinska, C. M. Koehler, D. Milenkovic, T. Lithgow and N. Pfanner, Cell, 2009, 138, 628.
4. (a) R. J. Youle and M. Karbowski, Nat. Rev. Mol. Cell Biol., 2005, 6, 657; (b) J. Nunnari and A. Suomalainen, Cell, 2012, 148, 1145; (c) R. H. Malty, M. Jessulat, K. Jin, G. Musso, J. Vlasblom, S. Phanse, Z. Zhang and M. Babu, J. Proteome Res., 2015, 14, 5.
5. H. M. McBride, M. Neuspiel and S. Wasiak, Curr. Biol., 2006, 16, R551.
6. (a) D. C. Wallace, Annu. Rev. Genet., 2005, 39, 359; (b) M. S. Remedi, C. G. Nichols and J. C. Koster, Cell Metab., 2006, 3, 5; (c) M. Valko, D. Leibfritz, J. Moncol, M. T. D. Cronin, M. Mazur and J. Telser, Int. J. Biochem. Cell Biol., 2007, 39, 44.
7. (a) A. J. Kettle and C. C. Winterbourn, Redox Rep., 1997, 3, 3; (b) Z. M. Prokopowicz, F. Arce, R. Biedron, C. L.-L. Chiang, M. Ciszek, D. R. Katz, M. Nowakowska, S. Zapotoczny, J. Marcinkiewicz and B. M. Chain, J. Immunol., 2010, 184, 824; (c) S. J. Klebanoff, J. Leukocyte Biol., 2005, 77, 598.
8. (a) D. Roos and C. C. Winterbourn, Science, 2002, 296, 669; (b) F. C. Fang, Nat. Rev. Microbiol., 2004, 2, 820.
9. M. J. Steinbeck, L. J. Nesti, P. F. Sharkey and J. Parvizi, J. Orthop. Res., 2007, 25, 1128.
10. (a) J. A. Berliner and J. W. Heinecke, Free Radical Biol. Med., 1996, 20, 707; (b) S. Sugiyama, K. Kugiyama, M. Aikawa, S. Nakamura, H. Ogawa and P. Libby, Arterioscler., Thromb., Vasc. Biol., 2004, 24, 1309.
11. D. I. Pattison and M. J. Davies, Biochemistry, 2006, 45, 8152.
12. (a) Y. W. Yap, M. Whiteman and N. S. Cheung, Cell. Signalling, 2007, 19, 219; (b) D. I. Pattison and M. J. Davies, Chem. Res. Toxicol., 2001, 14, 1453.
13. (a) H. S. Jung, J. H. Han, Z. H. Kim, C. Kang and J. S. Kim, Org. Lett., 2011, 13, 5056; (b) L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He and S. Zhu, J. Am. Chem. Soc., 2012, 134, 13510; (c) L.-J. Zhang, Z.-Y. Wang, X.-J. Cao, J.-T. Liu and B.-X. Zhao, Sens. Actuators, B, 2016, 236, 741.
14. (a) F. Liu, T. Wu, J. Cao, H. Zhang, M. Hu, S. Sun, F. Song, J. Fan, J. Wang and X. Peng, Analyst, 2013, 138, 775; (b) J. Park, H. Kim, Y. Choi and Y. Kim, Analyst, 2013, 138, 3368; (c) M. Sun, H. Yu, H. Zhu, F. Ma, S. Zhang, D. Huang and S. Wang, Anal. Chem., 2014, 86, 671; (d) X. Wang, F. Song and X. Peng, Dyes Pigm., 2016, 125, 89; (e) J. Li, P. Li, F. Huo, C. Yin, T. Liu, J. Chao and Y. Zhang, Dyes Pigm., 2016, 130, 209; (f) Y. Wu, J. Wang, F. Zeng, S. Huang, J. Huang, H. Xie, C. Yu and S. Wu, ACS Appl. Mater. Interfaces, 2016, 8, 1511.
15. (a) L. Yuan, W. Lin, J. Song and Y. Yang, Chem. Commun., 2011, 47, 12691; (b) Z. Yang, M. Wang, M. She, B. Yin, Y. Huang, P. Liu, J. Li and S. Zhang, Sens. Actuators, B, 2014, 202, 656; (c) S. Goswami, K. Aich, S. Das, B. Pakhira, K. Ghoshal, C. K. Quah, M. Bhattacharyya, H.-K. Fun and S. Sarkar, Chem.–Asian J., 2015, 10, 694; (d) W.-C. Chen, P. Venkatesan and S.-P. Wu, New J. Chem., 2015, 39, 6892; (e) B. Wang, D. Chen, S. Kambam, F. Wang, Y. Wang, W. Zhang, J. Yin, H. Chen and X. Chen, Dyes Pigm., 2015, 120, 22; (f) W.-L. Wu, Z.-M. Zhao, X. Dai, L. Su and B.-X. Zhao, Sens. Actuators, B, 2016, 232, 390.
16. (a) J. Shepherd, S. A. Hilderbrand, P. Waterman, J. W. Heinecke, R. Weissleder and P. Libby, Chem. Biol., 2007, 14, 1221; (b) Y. Zhou, J.-Y. Li, K.-H. Chu, K. Liu, C. Yao and J.-Y. Li, Chem. Commun., 2012, 48, 4677; (c) Y.-X. Liao, M.-D. Wang, K. Li, Z.-X. Yang, J.-T. Hou, M.-Y. Wu, Y.-H. Liu and X.-Q. Yu, RSC Adv., 2015, 5, 18275; (d) X.-H. Zhou, Y.-R. Jiang, X.-J. Zhao and D. Guo, Talanta, 2016, 160, 470.
17. (a) Z. Zhang, Y. Zheng, W. Hang, X. Yan and Y. Zhao, Talanta, 2011, 85, 779; (b) L. Long, D. Zhang, X. Li, J. Zhang, C. Zhang and L. Zhou, Anal. Chim. Acta, 2013, 775, 100; (c) Y.-R. Zhang, X.-P. Chen, J. Shao, J.-Y. Zhang, Q. Yuan, J.-Y. Miao and B.-X. Zhao, Chem. Commun., 2014, 50, 14241; (d) M. Ren, B. Deng, K. Zhou, X. Kong, J.-Y. Wang, G. Xu and W. Lin, J. Mater. Chem. B, 2016, 4, 4739; (e) R. Zhang, J. Zhao, G. Han, Z. Liu, C. Liu, C. Zhang, B. Liu, C. Jiang, R. Liu, T. Zhao, M.-Y. Han and Z. Zhang, J. Am. Chem. Soc., 2016, 138, 3769.
18. (a) W. Lin, L. Long, B. Chen and W. Tan, Chem.–Eur. J., 2009, 15, 2305; (b) J. Shi, Q. Li, X. Zhang, M. Peng, J. Qin and Z. Li, Sens. Actuators, B, 2010, 145, 583; (c) X. Cheng, H. Jia, T. Long, J. Feng, J. Qin and Z. Li, Chem. Commun., 2011, 47, 11978; (d) G. Cheng, J. Fan, W. Sun, K. Sui, X. Jin, J. Wang and X. Peng, Analyst, 2013, 138, 6091; (e) M. Emrullahoğlu, M. Üçüncü and E. Karakuş, Chem. Commun., 2013, 49, 7836.
19. (a) Z.-N. Sun, F.-Q. Liu, Y. Chen, P. K. H. Tam and D. Yang, Org. Lett., 2008, 10, 2171; (b) J. J. Hu, N.-K. Wong, Q. Gu, X. Bai, S. Ye and D. Yang, Org. Lett., 2014, 16, 3544.
20. (a) S.-R. Liu and S.-P. Wu, Org. Lett., 2013, 15, 878; (b) Z. Lou, P. Li, Q. Pan and K. Han, Chem. Commun., 2013, 49, 2445; (c) G. Cheng, J. Fan, W. Sun, J. Cao, C. Hu and X. Peng, Chem. Commun., 2014, 50, 1018; (d) W. Zhang, W. Liu, P. Li, J. kang, J. Wang, H. Wang and B. Tang, Chem. Commun., 2015, 51, 10150.
21. (a) S. Kenmoku, Y. Urano, H. Kojima and T. Nagano, J. Am. Chem. Soc., 2007, 129, 7313; (b) Y. Koide, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, J. Am. Chem. Soc., 2011, 133, 5680; (c) S. R. Liu, M. Vedamalai and S. P. Wu, Anal. Chim. Acta, 2013, 800, 71; (d) J. Jing and J.-L. Zhang, Chem. Sci., 2013, 4, 2947; (e) Q. A. Best, N. Sattenapally, D. J. Dyer, C. N. Scott and M. E. McCarroll, J. Am. Chem. Soc., 2013, 135, 13365; (f) H. Xiao, K. Xin, H. Dou, G. Yin, Y. Quan and R. Wang, Chem. Commun., 2015, 51, 1442; (g) L. Yuan, L. Wang, B. K. Agrawalla, S.-J. Park, H. Zhu, B. Sivaraman, J. Peng, Q.-H. Xu and Y.-T. Chang, J. Am. Chem. Soc., 2015, 137, 5930; (h) X. Wang, L. Zhou, F. Qiang, F. Wang, R. Wang and C. Zhao, Anal. Chim. Acta, 2016, 911, 114; (i) F. Tian, Y. Jia, Y. Zhang, W. Song, G. Zhao, Z. Qu, C. Li, Y. Chen and P. Li, Biosens. Bioelectron., 2016, 86, 68.
22. (a) X.-Q. Zhan, J.-H. Yan, J.-H. Su, Y.-C. Wang, J. He, S.-Y. Wang, H. Zheng and J.-G. Xu, Sens. Actuators, B, 2010, 150, 774; (b) X. Chen, K.-A. Lee, E.-M. Ha, K. M. Lee, Y. Y. Seo, H. K. Choi, H. N. Kim, M. J. Kim, C.-S. Cho, S. Y. Lee, W.-J. Lee and J. Yoon, Chem. Commun., 2011, 47, 4373; (c) Q. Xu, K.-A. Lee, S. Lee, K. M. Lee, W.-J. Lee and J. Yoon, J. Am. Chem. Soc., 2013, 135, 9944; (d) S. Ding, Q. Zhang, S. Xue and G. Feng, Analyst, 2015, 140, 4687; (e) J. Zhou, L. Li, W. Shi, X. Gao, X. Li and H. Ma, Chem. Sci., 2015, 6, 4884.
23. (a) Q. Wang, C. Liu, J. Chang, Y. Lu, S. He, L. Zhao and X. Zeng, Dyes Pigm., 2013, 99, 733; (b) W. Shu, L. Yan, Z. Wang, J. Liu, S. Zhang, C. Liu and B. Zhu, Sens. Actuators, B, 2015, 221, 1130.
24. T. Guo, L. Cui, J. Shen, R. Wang, W. Zhu, Y. Xu and X. Qian, Chem. Commun., 2013, 49, 1862.
25. (a) S. Goswami, A. Manna, S. Paul, C. K. Quah and H.-K. Fun, Chem. Commun., 2013, 49, 11656; (b) J. Fan, H. Mu, H. Zhu, J. Wang and X. Peng, Analyst, 2015, 140, 4594.
26. J. Zhao, H. Li, K. Yang, S. Sun, A. Lu and Y. Xu, New J. Chem., 2014, 38, 3371.
27. L. Wang, L. Long, L. Zhou, Y. Wu, C. Zhang, Z. Han, J. Wang and Z. Da, RSC Adv., 2014, 4, 59535.
28. Y. Yang, C. Yin, F. Huo, J. Chao, Y. Zhang and S. Jin, Sens. Actuators, B, 2014, 199, 226.
29. H. Zhu, J. Fan, J. Wang, H. Mu and X. Peng, J. Am. Chem. Soc., 2014, 136, 12820.
30. P. Venkatesan and S.-P. Wu, Analyst, 2015, 140, 1349.
31. K. Xiong, F. Huo, C. Yin, J. Chao, Y. Zhang and M. Xu, Sens. Actuators, B, 2015, 221, 1508.
32. W.-C. Chen, P. Venkatesan and S.-P. Wu, Anal. Chim. Acta, 2015, 882, 68.
33. Y.-R. Zhang, N. Meng, J.-Y. Miao and B.-X. Zhao, Chem.–Eur. J., 2015, 21, 19058.
34. L. Long, Y. Wu, L. Wang, A. Gong, F. Hu and C. Zhang, Chem. Commun., 2015, 51, 10435.
35. (a) Q. Xu, C. H. Heo, G. Kim, H. W. Lee, H. M. Kim and J. Yoon, Angew. Chem., Int. Ed., 2015, 54, 4890; (b) Q. Xu, C. H. Heo, J. A. Kim, H. S. Lee, Y. Hu, D. Kim, K. M. K. Swamy, G. Kim, S.-J. Nam, H. M. Kim and J. Yoon, Anal. Chem., 2016, 88, 6615.
36. J. Wang, Y. Ni and S. Shao, Talanta, 2016, 147, 468.
37. T. Cheng, J. Zhao, Z. Wang, J. An, Y. Xu, X. Qian and G. Liu, Dyes Pigm., 2016, 126, 218.
38. K. Xiong, F. Huo, C. Yin, Y. Chu, Y. Yang, J. Chao and A. Zheng, Sens. Actuators, B, 2016, 224, 307.
39. J. Li, C. Yin, F. Huo, K. Xiong, J. Chao and Y. Zhang, Sens. Actuators, B, 2016, 231, 547.
40. Y. Yang, F. Huo, C. Yin, M. Xu, Y. Hu, J. Chao, Y. Zhang, T. E. Glass and J. Yoon, J. Mater. Chem. B, 2016, 4, 5101.
41. (a) Z. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014, 43, 16; (b) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622.
42. M. H. Lee, J. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015, 44, 4185.
43. J.-S. Wu, W.-M. Liu, X.-Q. Zhuang, F. Wang, P.-F. Wang, S.-L. Tao, X.-H. Zhang, S.-K. Wu and S.-T. Lee, Org. Lett., 2007, 9, 33.
44. (a) A. Helal, M. H. O. Rashid, C.-H. Choi and H.-S. Kim, Tetrahedron, 2011, 67, 2794; (b) J.-T. Hou, K. Li, K.-K. Yu, M.-Z. Ao, X. Wang and X.-Q. Yu, Analyst, 2013, 138, 6632.
45. (a) J. Xu, J. Pan, X. Jiang, C. Qin, L. Zeng, H. Zhang and J. F. Zhang, Biosens. Bioelectron., 2016, 77, 725; (b) Y. Liu, K. Li, K.-X. Xie, L.-L. Li, K.-K. Yu, X. Wang and X.-Q. Yu, Chem. Commun., 2016, 52, 3430.
46. (a) L. Zeng, N. Fan, J. Zha, X. Hu, B. Fu, C. Qin and L. Wang, Analyst, 2013, 138, 7083; (b) J. Zha, B. Fu, C. Qin, L. Zeng and X. Hu, RSC Adv., 2014, 4, 43110; (c) L. Zhu, J. Xu, Z. Sun, B. Fu, C. Qin, L. Zeng and X. Hu, Chem. Commun., 2015, 51, 1154.
47. Z. Lou, P. Li and K. Han, Acc. Chem. Res., 2015, 48, 1358.
48. K. Xiang, S. Chang, J. Feng, C. Li, W. Ming, Z. Liu, Y. Liu, B. Tian and J. Zhang, Dyes Pigm., 2016, 134, 190.

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Footnote

† Electronic supplementary information (ESI) available: Spectroscopic properties and characterization data. See DOI: 10.1039/c6ra22868b

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