A mitochondrion-targeted dual-site fluorescent probe for the discriminative detection of SO₃²⁻ and HSO₃⁻ in living HepG-2 cells

Abstract

Sulfur dioxide, known as an environmental pollutant, produced during industrial productions is also a common food additive that is permitted worldwide. In living organisms, sulfur dioxide forms hydrates of sulfite (SO₂·H₂O), bisulfite (HSO₃⁻) and sulfite (SO₃²⁻) under physiological pH conditions; these three exist in a dynamic balance and play a role in maintaining redox balance, further participating in a wide range of physiological and pathological processes. On the basis of the differences in nucleophilicity between SO₃²⁻ and HSO₃⁻, for the first time, we built a mitochondrion-targeted dual-site fluorescent probe (Mito-CDTH-CHO) based on benzopyran for the highly specific detection of SO₃²⁻ and HSO₃⁻ with two diverse emission channels. Mito-CDTH-CHO can discriminatively respond to the levels of HSO₃⁻ and SO₃²⁻. Besides, its advantages of low cytotoxicity, superior biocompatibility and excellent mitochondrial enrichment ability contribute to the detection and observation of the distribution of sulfur dioxide derivatives in living organisms as well as allowing further studies on the physiological functions of sulfur dioxide.

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1. Introduction

Sulfur dioxide (SO₂), the most common and simple irritating gas, is one of the main pollutants in the atmosphere.¹ In recent years, increasing physiological functions of sulfur dioxide have been discovered in mammals.^2–4 How does SO₂ work in the internal environment of living organisms? It has been reported in numerous studies that SO₂ is not independent in action or directly affects, but dissociated to SO₃²⁻ and HSO₃⁻ (SO₂ derivatives) in neutral fluid or plasma (HSO₃⁻/SO₃²⁻, 1 : 3 M/M),^5,6 which mainly account for its toxicity. There is a dynamic conversion equilibrium between sulfur dioxide and sulfite, which also exists in bisulfite and sulfite.⁷

Among them, HSO₃⁻/SO₃²⁻ at high concentrations are catalyzed to generate a variety of sulfur oxy radicals, which are known to give rise to negligible damage to the body.⁸ Numerous studies have confirmed that abnormally high sulfite levels are closely related to respiratory diseases,⁹ cardiovascular diseases¹⁰ as well as neurological diseases, such as migraine, stroke, brain cancer,¹¹ lung cancer¹² and liver cancer.¹³ Besides, clinical studies suggest that the concentration of sulfur dioxide gas ranges from 1 to 2000 μM in living organisms, and the total concentration of serum sulfite ranges from 0 to 10 μM in healthy donors. HSO₃⁻/SO₃²⁻ also relax aortic rings in a dose-dependent manner at high concentrations ranging from 0.5 to 12 mM.¹⁰ However, whether HSO₃⁻and SO₃²⁻ are independent or synergistic in action remains largely unknown. Therefore, the accurate and independent determination of the levels of SO₃²⁻ and HSO₃⁻ is fairly necessary and valuable for further investigating the physiological functions of sulfur dioxide in living organisms.

Over the past decades, fluorescence imaging technology has drawn considerable attention benefiting from its outstanding performances, such as eminent non-invasiveness, excellent signal-to-noise ratio, high sensitivity, extraordinary reliability, cheap availability and easy operation.¹⁴ Since Qian and Zhang et al. first reported a fluorescent probe based on the Michael addition reaction for HSO₃⁻ in 2013,¹⁵ a large number of fluorescent probes have been developed to detect HSO₃⁻/SO₃²⁻ in recent years.^16–20 Although they work on a simple mechanism and are facile to synthesize, they cannot exactly distinguish between HSO₃⁻ and SO₃²⁻, thus producing the same fluorescence signal. Herein, a dual-site fluorescent probe for SO₂ derivatives (HSO₃⁻ and SO₃²⁻) based on benzopyran, Mito-CDTH-CHO, was designed and synthesized. The probe is capable of discriminatively responding to the levels of HSO₃⁻ and SO₃²⁻ with distinct fluorescence signals. Furthermore, Mito-CDTH-CHO exhibited superior selectivity, lower cytotoxicity, good sensitivity, and readily available for fluorescence imaging in vitro and in vivo. To the best of our knowledge, the probes that possess obvious fluorescence sensing for HSO₃⁻ and SO₃²⁻ are still rare.

2. Experimental section

2.1. Materials and instruments

All the reagents were supplied by commercial suppliers and were directly used without further purification. Absorption spectra were recorded on a UNICO UV-4802 spectrophotometer. Fluorescence spectra were obtained on a fluorescence spectrophotometer (Lengguang tech CO., Ltd. F97XP, China). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AVANCE III 400 Nanobay at 500 MHz for ¹H NMR and 300 MHz for ¹³C NMR (TMS as an internal standard). High-resolution mass spectra (HRMS) were recorded on a MicrOTOF Bruker. The pH values were measured with an acidity meter (alkalis, pH 400, China).

2.2. Synthesis of probe Mito-CDTH-CHO

The probe was synthesized according to the reported literature via a facile two-step reaction.^21,22 The synthesis routes of the probe are depicted in Scheme S1,† and it was characterized via high-resolution mass spectrometry, ¹H NMR and ¹³C NMR (see ESI†).

Synthesis of Mito-CDTH. Freshly distilled cyclohexanone was added dropwise to a solution of concentrated H₂SO₄ cooled down to 0 °C in advance. To a concentrated H₂SO₄ solution of 2-(4-diethylamino-2-hydroxybenzoyl), benzoic acid was added dropwise in freshly distilled cyclohexanone at 0 °C. Further, heating up to 90 °C, the mixture was vigorously stirred for 2 h, poured into ice, the perchloric acid (70%) was added, the supernatant was filtered off, and the residue was washed with cold water for three times. The residue was dried under vacuum and further purified via silica gel column chromatography (CH₂Cl₂ : MeOH = 20 : 1, v/v) to afford Mito-CDTH as a bright red solid (372 mg, 68%). ESI-MS calcd for C₂₄H₂₆NO₃ [M + H]⁺ 376.1921, found 376.1913.

Synthesis of Mito-CDTH-CHO. To an acetic acid solution (30 ml) of Mito-CDTH (376 mg, 1 mmol), terephthalaldehyde (268 mg, 2 mmol) was added. The reaction solution was stirred at 110 °C for 3 h, and the solvent was evaporated under a reduced pressure. The crude product was extracted with CH₂Cl₂ (100 ml) and water (300 ml), washed with a saturated ammonium chloride solution and dried over anhydrous sodium sulfate. After evaporating using a rotary evaporator, the purple target compound (300 mg, 61%) was obtained via silica gel column chromatography (CH₂Cl₂ : MeOH = 50 : 1, v/v).

¹H NMR (300 MHz, DMSO-d₆) δ 10.03 (s, 1H), 7.95 (d, J = 7.8 Hz, 3H), 7.86–7.63 (m, 4H), 7.47 (s, 1H), 7.34 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 2.3 Hz, 1H), 6.52–6.35 (m, 2H), 3.36 (q, J = 7.0 Hz, 4H), 2.80 (d, J = 15.6 Hz, 1H), 2.67 (s, 1H), 1.91 (d, J = 12.9 Hz, 1H), 1.62 (d, J = 9.5 Hz, 3H), 1.11 (t, J = 6.9 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆) δ 191.99, 149.12, 142.35, 134.78, 134.40, 132.23, 129.65, 129.53, 129.04, 127.80, 124.44, 123.99, 123.39, 109.19, 96.69, 43.43, 26.41, 22.36, 21.54, 12.05. ESI-MS calcd for C₃₂H₃₀NO₄ [M + H]⁺ 492.2133, found 492.2160.

2.3. Preparation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), active sulfur species (RSS) and anions

H₂O₂ was prepared by the direct dilution of a commercial hydrogen peroxide stock solution. NaClO was obtained by the dilution of a commercial hypochlorite solution in purified water and measured using a spectrophotometer (ε_{292 nm} = 350 M⁻¹ cm⁻¹). TBHP was prepared by the dilution of a commercial tert-butyl hydroperoxide stock solution. Peroxynitrite stock (ONOO⁻) was prepared by a previously reported procedure²³ and measured using a spectrophotometer (ε_{302 nm} = 1670 M⁻¹ cm⁻¹) 1 M of stock solutions (Cys, GSH, HS⁻, SO₄²⁻, S₂O₃²⁻, S²⁻, SCN⁻, H₂O₂, ClO⁻, TBHP, Hcy, NO₂⁻, Sx²⁻, Cl⁻, Br⁻, I⁻, CO₃²⁻, HCO₃⁻, PO₄²⁻, HPO₄⁻, AcO⁻, SO₃²⁻, and HSO₃⁻) were prepared by the dissolution of 10 mmol solid in purified water, and diluted to the desired concentrations when needed.

2.4. pH value adjustment

The pH values of the solutions were directly obtained by preparing a series of specific pH buffers, including acetate buffer, phosphate buffer, and sodium hydroxide/potassium chloride/boric acid buffer.

2.5. Spectral analysis

1 mM of the probe stock solution was prepared by dissolving 1 mg Mito-CDTH-CHO in 2 ml anhydrous ethanol, and diluted with PBS solution (10 mM, pH = 7.4, 6.0 or 8.0, containing 2% EtOH) for final test solutions.

2.6. Cell cytotoxicity assay

The cytotoxicity was measured using a CCK-8 kit. Hela cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics at 37 °C under 5% CO₂ for 24 h. Hela cells were cultured with a fresh medium containing various concentrations of Mito-CDTH-CHO (0–40 μM) for another 12 h. Next, Hela cells were washed three times with PBS and incubated with diluted CCK-8 reagent for 1 h, and then the cell viability was determined by a microplate reader. The procedure was repeated three times for each concentration.

2.7. Cell culture and imaging

HepG-2 cells were cultured in a DMEM medium (containing 1% penicillin/streptomycin and 10% FBS) under an air condition at 37 °C under 5% CO₂. HepG-2 cells at the logarithmic growth phase were implanted into 25 mm glass-bottomed dishes and incubated overnight. After the attachment of cells, the cells were treated with different pH values (pH = 6.0, 7.4 and 8.0) of the DMEM medium for 3 h. The pH of the DMEM medium was adjusted by adding a specific concentration of hydrochloric acid or sodium hydroxide.²³ HepG-2 cells were washed with PBS three times and incubated with Mito-CDTH-CHO (20 μM) in an untreated DMEM medium. Confocal fluorescence images were recorded using a Zeiss LSM 800 confocal laser scanning microscope. The green channel was collected at 460–520 nm at an excitation of 390, and the blue fluorescence channel was covered over the range of 420–470 nm at an excitation of 370 nm.

3. Results and discussions

3.1. Design and synthesis of Mito-CDTH-CHO

According to the previous reports on the response mechanism of detecting sulfur dioxide type fluorescent probes,^24–30 we proposed that (Scheme 1), on the one hand, the oxygen positive ion on the benzopyran ring acts as a strong electron-withdrawing group, which reduces the electron cloud density of the C=C double bond and enable the C=C double bond strong electrophilicity. On the other hand, the C=O double bond conjugated to the benzene ring also possesses weaker electrophilicity, which makes the strong nucleophilic HSO₃⁻ attack the C=O and C=C double bonds. When the probe Mito-CDTH-CHO conjugate structure is broken, strong blue fluorescence is emitted. The weaker nucleophilic SO₃²⁻ could only attack the C=C double bond, hence exhibited red-shifted green fluorescence.

Scheme 1 Rational design and sensing mechanism of the probe for HSO₃⁻ and SO₃²⁻.

3.2. Vitro sensing of Mito-CDTH-CHO for SO₃²⁻/HSO₃⁻

First of all, we tested the UV-Vis absorption of Mito-CDTH-CHO in the absence and presence of SO₂ derivatives (SO₃²⁻ and HSO₃⁻) in PBS buffer. There were mainly two absorption peaks at 300–700 nm, centered at 330 nm and 553 nm, respectively. Mito-CDTH-CHO exhibited similar UV spectra changes after it reacted with SO₃²⁻ (Fig. 1a) and HSO₃⁻ (Fig. 1b). The absorption intensity at 553 nm sharply decreased, which means for the breaking of the conjugate system, and a new absorption at 365 nm was elevated.

Fig. 1 Fluorescence and UV responses of Mito-CDTH-CHO toward SO₂ derivatives. (a) The UV-Vis absorption spectra change of Mito-CDTH-CHO (20 μM) with Na₂SO₃ (20 μM) in PBS buffer (pH 7.4, containing 2% EtOH). (b) UV-Vis absorption spectra change of Mito-CDTH-CHO (20 μM) with NaHSO₃ (20 μM) in PBS buffer (pH 6.0, containing 2% EtOH). (c) Fluorescence spectra changes of Mito-CDTH-CHO (20 μM) with 50 μM SO₂ derivatives in PBS buffer (containing 2% EtOH). Black: Mito-CDTH-CHO; blue: NaHSO₃, λ_ex = 370 nm; green: Na₂SO₃, λ_ex = 390 nm. (d) Fluorescence spectra changes of Mito-CDTH-CHO (20 μM) in the presence of various concentrations Na₂SO₃ (0–600 μM), λ_ex = 390 nm.

To explain the fluorescence response distinction, the dual-site fluorescence response of probe toward SO₃²⁻ and HSO₃⁻ was investigated. As shown in Fig. 1c, the probe Mito-CDTH-CHO (20 μM) had almost no fluorescence in the absence of SO₃²⁻/HSO₃⁻ at 400–600 nm. However, after the reaction with 50 of μM Na₂SO₃, the strong green fluorescence was emitted (λ_ex = 390 nm, λ_em = 492 nm). When at the same concentration in the case of NaHSO₃, the solution exhibited luminous blue emission at a shorter wavelength (λ_ex = 370 nm, λ_em = 456 nm). These results provide a preliminary proof that the probe Mito-CDTH-CHO conjugate structure is destroyed by SO₃²⁻ and HSO₃⁻.

As shown in Fig. 1d and 2a, for SO₃²⁻, with the addition of the Na₂SO₃ (0–600 μM), the emission intensity at 492 nm increased significantly (pH = 7.4, 37 °C, λ_ex = 390 nm), and an excellent linear relationship (R² = 0.992) was obtained in the range of 10–100 μM. Moreover, the detection limit was calculated to be 100 nM. For HSO₃⁻, Fig. 2b and c revealed that the fluorescence intensity at 456 nm constantly increased after the addition of 0–1000 μM NaHSO₃ in the phosphate buffer solution (pH = 6.0, 37 °C, λ_ex = 370 nm), in a wide linear range (40–200 μM). The detection limit was 80 nM (Fig. 2c).

Fig. 2 Fluorescence responses of Mito-CDTH-CHO toward SO₂ derivatives. (a) The linear curve of Mito-CDTH-CHO (20 μM) fluorescence intensity at 492 nm with Na₂SO₃ concentrations range from 10–100 μM. (b) Fluorescence spectra changes of Mito-CDTH-CHO (20 μM) after the addition of various concentrations NaHSO₃ (0–1000 μM). PBS buffer, pH 6.0, containing 2% EtOH, λ_ex = 370 nm. (c) The linear curve of Mito-CDTH-CHO (20 μM) fluorescence intensity at 456 nm with NaHSO₃ concentrations range from 40–200 μM in PBS buffer. (d) The fluorescence intensity values of Mito-CDTH-CHO (20 μM) after interacting with 500 μM SO₂ derivatives, reactive nitrogen species, reactive oxygen species, active sulfur species and anions. (1) Mito-CDTH-CHO, (2) Cys, (3) GSH, (4) HS⁻, (5) SO₄²⁻, (6) S₂O₃²⁻, (7) S²⁻, (8) SCN⁻, (9) H₂O₂, (10) NaClO, (11) TBHP, (12) Hcy, (13) NO₂⁻, (14) Sx²⁻, (15) Cl⁻, (16) Br⁻, (17) I⁻, (18) CO₃²⁻, (19) HCO₃⁻, (20) PO₄²⁻, (21) HPO₄⁻, (22) AcO⁻, (23) SO₃²⁻, (24) HSO₃⁻.

Afterward, we evaluated the selectivity and pH stability of the probe Mito-CDTH-CHO towards sulfur dioxide derivatives. As described in Fig. 2d, SO₃²⁻/HSO₃⁻ led to a significant fluorescence enhancement at 492 nm and 456 nm, respectively. While other interfering substances, including reactive nitrogen species (NO₂⁻ and NO₃⁻), reactive oxygen species (ClO⁻, H₂O₂, TBHP), active sulfur species (SO₄²⁻, HS⁻, S₂O₃²⁻, S²⁻, SCN⁻, Sx²⁻, Cys, Hcy, GSH) and anions (I⁻, Br⁻, Cl⁻, CO₃²⁻, HCO₃⁻, PO₄³⁻, HPO₄²⁻, ACO⁻), did not produce remarkable fluorescence response. From what had been discussed above, Mito-CDTH-CHO presented excellent selectivity toward SO₃²⁻ and HSO₃⁻ in separated emission regions (492 and 456 nm).

The pH interference of the probe for SO₂ derivatives was discussed. In the absence of SO₃²⁻ and HSO₃⁻, the probe had little fluorescence and was unaffected by the variation of pH values (Fig. S5†). When Na₂SO₃ or NaHSO₃ was added, the fluorescence intensity changed with the mutual conversion balance between SO₃²⁻ and HSO₃⁻ in the range of pH 4 to 10 (Fig. S6†). In the range of acidic pH (4–6), HSO₃⁻ ion dominates, thus Mito-CDTH-CHO exhibited stronger fluorescence at 456 nm than in neutral and weak basic pH ranges (Fig. S7†). In basic pH ranges (7–10), SO₃²⁻ accounts for the main part, so the fluorescence intensity increased with the pH value increase (Fig. S6†).

In general, compared to other fluorescence probes sensing SO₂, the most evident superiority of Mito-CDTH-CHO is selectivity for SO₃²⁻ and HSO₃⁻. Most fluorescent probes for detecting SO₂ are not selective toward SO₃²⁻ and HSO₃⁻ due to their very similar chemical properties, showing that the same response toward SO₃²⁻ (HSO₃⁻) when detecting HSO₃⁻ (SO₃²⁻). In addition, superior water solubility, suitable detection limit, and accurate mitochondrial targeting performance also indicate that Mito-CDTH-CHO is a fairly qualified fluorescent probe for the accurate detection of sulfur dioxide derivatives (see Table S1†).

3.3. The proposed mechanism of Mito-CDTH-CHO for SO₂ derivatives detection

In order to illuminate the reaction mechanism between Mito-CDTH-CHO and SO₂ derivatives, the NMR titration experiments in DMSO-d₆/D₂O (8 : 2) were performed. As shown in Fig. 3, a proton at 6.34 ppm represents the double bond conjugated to benzopyrone of the probe Mito-CDTH-CHO. As the concentration of Na₂SO₃ increased from 1 to 10 eq., the proton peak disappeared, while the singlet at 4.89 ppm appeared. The response mechanism of the probe Mito-CDTH-CHO to NaHSO₃ was also confirmed (Fig. 4), except the singlet at 4.89 ppm, an additional hydroxyl proton peak at 5.12 ppm emerged, which is attributed to the difference in nucleophilicity between SO₃²⁻ and HSO₃⁻. These results further verified that the dual-site sensing of Mito-CDTH-CHO toward Na₂SO₃ and NaHSO₃ via different double bond nucleophilic addition reactions.

Fig. 3 The ¹H NMR titration of Mito-CDTH-CHO with Na₂SO₃.

Fig. 4 The ¹H NMR titration of Mito-CDTH-CHO with NaHSO₃.

3.4. Cellular imaging of Mito-CDTH-CHO

In view of the excellent performances of the probe Mito-CDTH-CHO in vitro, the capability of the discriminative detection of SO₃²⁻ and HSO₃⁻ was investigated. Prior to bioimaging experiments, the cytotoxic assay was carried out by a CCK-8 method in HepG-2 cells, the results indicated that Mito-CDTH-CHO had low cytotoxicity (Fig. 5).

Fig. 5 Cell viability of Mito-CDTH-CHO in a standard CCK-8 kit in living HepG-2 cells for 24 h. The experiment was repeated three times (±S.D.).

According to the literature,^31–33 cationic small molecules could enter into mitochondria and interact with anionic species via the electrostatic interaction. The design of Mito-CDTH-CHO is based on our considerations that the benzopyran cation (containing oxygen positive ions) can act as a mitochondrion-targeting moiety. The positive charge and hydrophobic properties of the benzopyran cation are supposed to mediate the localization of Mito-CDTH-CHO inside the mitochondrial membrane. Thus, we speculate that Mito-CDTH-CHO will mainly distribute in the mitochondria. In order to verify our hypothesis, the mitochondrial colocalization experiment was carried out. The commercial mitochondrion tracker (Mito-tracker) and Mito-CDTH-CHO were co-incubated in HepG-2 cells. The fluorescence imaging from Mito-CDTH-CHO in the blue channel (Fig. 6c and g) overlapped well with the Mito-tracker in the green channel (Fig. 6b and f), resulting in the Pearson's correlation coefficient of 0.98. Furthermore, the region of interest (ROI) is illustrated in Fig. 6j, and the normalized fluorescence intensity of Mito-CDTH-CHO changed in coordination with the normalized fluorescence intensity of Mito-Tracker Green. These results suggested that Mito-CDTH-CHO possesses the excellent ability to target mitochondrial of subcellular organelle in HepG-2 cells.

Fig. 6 Confocal microscopy colocalization images of Mito-CDTH-CHO and Mito-Tracker Green in HepG-2 cells. (a and e) the bright field of HepG-2 cells. (b and f) fluorescence image of Mito-Tracker Green (λ_ex = 488 nm, λ_em = 500–540 nm). (c and g) fluorescence image of Mito-CDTH-CHO (λ_ex = 370 nm, λ_em = 400–460 nm). (d and h) merged image of (b and c), (f and g), respectively. (i) Intensity scatter plot of blue and green channels and (j) normalized intensity profile of the linear region of part a across the HepG-2 cells.

For the sake of better experimental results, we conducted the control experiments with a lyso-tracker. As illustrated in Fig. 7, the green fluorescence of Mito-CDTH-CHO is not overlapped at all with the red fluorescence of LysoTracker RED with the Pearson's correlation coefficients (R_r) of 0.3308 and an overlap coefficient (R) of 0.6277 (Fig. 7e). The green fluorescence of Mito-CDTH-CHO and red fluorescence of LysoTracker RED changes in the intensity profiles of ROIs are not synchronized at all (Fig. 7f). The result further indicates that Mito-CDTH-CHO mainly localizes in the mitochondria of living cells.

Fig. 7 Confocal microscopy colocalization images of Mito-CDTH-CHO and Mito-Tracker RED in HepG-2 cells. (a) Bright field of HepG-2 cells. (b) Fluorescence image of Mito-Tracker RED (λ_ex = 548 nm, λ_em = 560–620 nm). (c) Fluorescence image of Mito-CDTH-CHO (λ_ex = 390 nm, λ_em = 460–520 nm). (d) The merges images of (a and b). (e) Intensity scatter plot of red and green channels. (f) Normalized intensity profile of the linear region of part a across the HepG-2 cells. Scale bar = 20 μm.

HepG-2 cells were pre-treated with a probe (20 μM) in the DMEM medium and then incubated with Na₂SO₃ (200 μM) for 30 min. As shown in Fig. 8, due to the equilibrium conversion between SO₃²⁻ and HSO₃⁻ in a neutral fluid, HepG-2 cells exhibited distinct fluorescence (a₁) in the green channel and weak fluorescence (a₂) in the blue channel. In contrast, after incubating the HepG-2 cells with NaHSO₃ (200 μM) for 30 min, clear fluorescence in the blue channel (b₁) and weak fluorescence (b₂) in the green channel were observed. Inspired by the above experimental results, HepG-2 cells were cultured with NaHSO₃ (200 μM) for 30 min, it is worth noting that the fluorescence in the green channel disappeared (d₁), whereas the fluorescence in the blue channel enhanced (d₂). Similarly, in the HepG-2 cells incubated with Na₂SO₃ in the DMEM medium for 30 min, there was fluorescence enhancement in the green channel (c₁) and almost no fluorescence in the blue channel (c₂). Therefore, probe Mito-CDTH-CHO can detect the intracellular SO₃²⁻ and HSO₃⁻ levels with different fluorescence signals.

Fig. 8 The confocal fluorescence images of probe and SO₂ derivatives in HepG-2 cells. (a₁–a₄) HepG-2 cells were pre-treated with Mito-CDTH-CHO (20 μM), then incubated with Na₂SO₃ (200 μM) for 30 min. (b₁–b₄) HepG-2 cells were incubated with NaHSO₃ (200 μM) for 30 min. (c₁–c₄) After incubated with Na₂SO₃ (200 μM) for 30 min. (d₁–d₄) After incubated with NaHSO₃ (200 μM) for 30 min. All cells were pre-treated with different pH values (pH = 6.0, 7.4 and 8.0) DMEM medium for 3 h.

4. Conclusions

In short, to the best of our knowledge, for the first time, a dual-site fluorescence probe for HSO₃⁻ and SO₃²⁻ with two different emission signals was designed and synthesized. Mito-CDTH-CHO can distinguishingly sense the levels of HSO₃⁻ and SO₃²⁻ with different fluorescence signals under separate pH conditions in living biological systems and possesses low cytotoxicity, excellent biocompatibility and outstanding mitochondrial targeting. In the meantime, the sensing mechanism of the double bond nucleophilic addition was successfully validated using the NMR titration experiments. We envision that Mito-CDTH-CHO could provide a deeper insight into and a better understanding of the physiological and pathological processes of SO₂ derivatives.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Key Laboratory of Materials Chemistry Independent Project Fund ZK201904 (2019). Jiangsu Province Agricultural Science and Technology Independent Innovation Fund Project CX (19) 3101. Special funds for the transformation of scientific and technological achievements SBA 2019030143.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ra01233e

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