English

• 0.   Introduction

  Sulfur dioxide (SO₂) is among the air pollutants, which combines with water, forming sulfuric acid- the main component of acid rain^[1-3]. Once SO₂ inhaled into the human body, it produces sulfite and bisulfite^[4], which can further lead to lower respiratory infection (e.g. bronchitis, pneumonia), even lung cancer^[5]. In neutral fluid and plasma, SO₂ is broken down to its derivatives, sulfite and bisulfite (3:1)^[6] with a total sulfite concentration of 0~9.85 μmol·L^-1 measured in serum samples^[7]. However, serum sulfite levels are markedly increased in patients with acute pneumonia and renal failure^[8]. Endogenous bisulfite derivatives are generated from the metabolism of L-cysteine; and aspartate aminotransferase (AAT) (the key enzyme for production of bisulfite derivatives^[9]), is overexpressed in cytosol and mitochondria^[10]. Exposure to high doses of SO₂ levels are linked with many cardiovascular diseases and lung cancer^[11]. SO₂ and bisulfite deriva-tives play an important role in mammalian organisms^[12] and are considered among the so called gasotrans-mitters, such as NO, CO and H₂S^[13-14]. Therefore, it is important and significant to illustrate the formation and biological roles of SO₂ and its derivatives by a powerful molecular sensor. However, there are still many challenges in real-time detection of bisulfite derivatives in biological samples^{[7, 15-16]}.

  Generally, SO₂ and its bisulfite derivatives have been determined using high-performance liquid chromatography, ultraviolet-visible absorption spectro-photometry, mass spectrometry, capillary electro-phoresis, and potentiometry^[17-21]. Compared with the aforementioned techniques, fluorescence imaging has a number of advantages, such as robust response, high sensitivity, real-time and visual detection in living cells and biological tissues^[22-23]. However, current fluorescent imaging probes suffer from shallow tissue penetration, high photodamage, and autofluorescence background interference, which limit their in vivo applications for rapid detection of biological targets. In contrast, both two-photon imaging microscopy (TPIM) and two-photon lifetime imaging microscopy (TPLIM), have excellent spatial and temporal resolu-tion, anti-photobleaching, non-invasive, low phototoxi-city, strong penetration, and higher resolution^[24-29]. Despite these advantages, their applications are still limited. It has to be noted that the lifetime of tradi-tional two-photon organic small molecule fluorescent dye at nanosecond timescale, is similar to cellular background autofluorescence (usually less than 10 ns), which in turn could limit their contrast and sensitivity. These challenges have urged us to develop more effective imaging materials.

  Phosphorescent metal-based materials have the advantages of high luminescence quantum yield, long-lived phosphorescence, and large stokes shift^[30-31]. Phosphorescent iridium complexes, as a typical phos-phorescent reagent, are representative luminescent probes for phosphorescence lifetime imaging micros-copy (PLIM) with lifetimes at microsecond timescale. PLIM has attracted great attention in biosensing and bioimaging, owing to its excellent photophysical properties^[32-33]. Two-photon phosphorescence lifetime imaging microscopy (TPPLIM) that uses the near-infrared (NIR) light as excitation source, which has deep penetration depth can effectively eliminate the background influence and demonstrates micro-environmental variations of the chromophore in a quantitative manner^[34-35]. As reported, the iridium com-plexes are sensitive to diverse cellular microenviron-ment variations, such as pH^[32], oxygen^[36], viscosity^[37], and metal ions^[38]. Although the iridium complexes as phosphorescent probes have been developed for detecting bisulfites derivatives^[39-44], it is worth noting that no TPPLIM probe for visualization of the generated SO₂ derivatives in mitochondria has been reported.

  Herein, we present a long life-time (~110 ns) reactive phosphorescent cyclometalated iridium(Ⅲ) complex probe (named as Ir-EA) for detecting sulfur dioxide (SO₂) derivatives (SO₃^2- and HSO₃^-) in vitro and in vivo (Scheme 1). The reactivity of bisulfite toward Ir-EA probe (through Michael addition reaction) was sensitive over the other biologically relevant active small molecules in the light of significant luminescence enhancement, incre-ases in the lifetime of phosphorescent, and extremely low detection limit in aqueous solution^[45-47]. Interes-tingly, Ir-EA is preferentially accumulated in the mitochondria due to its lipophilic-cationic characteris-tics and thence can detect bisulfites derivatives in real time at subcellular level with superior two-photon effect. More importantly and compared to previous reports, Ir-EA has been greatly improved in terms of cytotoxicity^[40], selectivity^[41], and sensitivity^[42] (Table S1), which are more appropriate for real-time detection of biological bisulfite derivatives at long-time term. Our work provides a novel example for the design and potential application of TPPLIM probe for the detection of bisulfite in the circumstance of in vitro and in vivo phosphorescence imaging.

  Scheme 1

  

  Scheme 1.  Schematic reaction mechanism between bisulfite and Ir-EA

  下载: 全尺寸图片 幻灯片

  1.   Experimental

  1.1   Synthesis and characterization

  All solvents (analytical grade) and reagents were purchased from commercial sources unless otherwise specified. The compounds [Ir(ppy-CHO)₂Cl]₂ and L-EA were synthesized according to literature procedure^[48-49].

  Synthesis of [Ir(ppy-CHO)₂(bpy)]PF₆: [Ir(ppy-CHO)₂Cl]₂ (1.70 g, 1.43 mmol) and 2, 2′-bipyridine (466 mg, 3.00 mmol) were mixed with 30 mL CH₂Cl₂ and then allowed to cool at r.t. after stirring for 6 h at 65 ℃ under nitrogen. Afterward, NH₄PF₆ (2.40 g, 14.72 mmol) was added and stirred a further 4 h for anion exchange. Next, the filtrate was evaporated, and 10 mL anhydrous ether was added for typical precipitation. After filtration, drying, and purification (using silica gel column chromatography), 1.99 g of yellow solid was obtained. Yield: 81.03%.

  Synthesis of Ir-EA: [Ir(ppy-CHO)₂(bpy)]PF₆ (0.50 g 0.58 mmol) and ethyl cyanoacetate (68 mg, 0.60 mmol) were dissolved in 35 mL ethanol, 20 μL piperidine was added, and the mixture was stirred at 40 ℃ for 5 h. The filtrate was obtained under reduced pressure, and 377 mg orange red solid was collected after purification through silica gel column chromatography with CH₂Cl₂ and CH₃OH as eluant (50:1 to 10:1, V/V). Yield: 61.72%.

  ¹H NMR (400 MHz, DMSO-d₆): δ 8.78 (d, J=8.2 Hz, 2H), 8.34 (d, J=8.2 Hz, 2H), 8.29~8.21 (t, 2H), 8.14 ~8.06 (t, 6H), 8.03~7.96 (t, 2H), 7.92 (d, J=5.4 Hz, 2H), 7.66 (t, J=6.4 Hz, 4H), 7.56 (d, J=8.3, 1.5 Hz, 2H), 7.23 (t, J=12.8, 6.6 Hz, 2H), 7.09 (s, 2H), 4.25 (q, J=7.1 Hz, 4H), 1.25 (t, J=7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO -d₆): δ 165.37 (s), 162.40 (s), 156.01 (s), 154.98 (s), 150.48 (s), 150.06 (s), 149.85 (s), 149.70 (s), 140.59 (s), 139.72 (s), 132.74 (s), 132.54 (s), 129.42 (s), 127.56 (s), 126.26 (s), 125.70 (s), 122.51 (s), 115.79 (s), 102.65 (s), 62.71 (s), 14.42 (s). ESI-MS (MeOH): m/z Calcd. for [M-PF₆]⁺, 903.01; Found: 903.46.

  1.2   Crystallographic structure determination

  Diffusion of diethyl ether into the CH₃OH solution got the crystals of Ir-EA qualified for X-ray analysis at room temperature. X-ray diffraction measurements were carried out on a Bruker Smart 1000 CCD diffractometer with Cu Kα radiation (λ=0.154 178 nm) at 150 K. The crystal structures of Ir-EA were solved through direct methods with program SHELXS and refined by the full-matrix least-squares program SHELXL^[50].

  CCDC: 1914016.

  1.3   Procedures for spectra measurement

  Stock solution of Ir-EA (20 mmol·L^-1) was dissolved in DMSO and diluted to final concentration (20 μmol·L^-1) with test solvent prior to application. After 5 min stabilization in quartz cells (1 cm×1 cm), all electronic absorption and emission spectra were recorded. Stock solution of bisulfite was obtained at a concentration of 50 mmol·L^-1.

  1.4   Confocal imaging

  For live cell confocal laser scanning microscopy experiment, cells were first seeded in a glass dish at a density of 1×10⁴ cells and then incubated for 48 h, cell images were captured with Carl Zeiss LSM 710 confocal microscopy.

  1.5   In vivo imaging of zebrafish larvae

  Zebrafish larvae were bred in zebrafish embryo culture media for two days after hatching. The healthy larvae were selected and incubated with Ir-EA (5 μmol·L^-1) for 2 h and/or additional treatment with bisulfite (50 μmol·L^-1) before visualized using confocal microscopy (λ_ex=810 nm (TPPIM); λ_em=(600±20) nm).

  2.   Results and discussion

  2.1   Chemical synthesis and characterization

  Ir-EA was synthesized according to literature methods (Scheme S1)^[48-49] and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, electrospray ionization mass spectrometry (ESI-MS) (Fig.S1~S5), and X-ray crystallography (Scheme 1 and Table S2). The absorption and phosphorescence spectra of Ir-EA in phosphate buffer saline (PBS), CH₃CN, and CH₂Cl₂ are characterized as shown in Fig.S6 and Table S3. The mechanism of phosphorescence quenching in Ir-EA is mostly attributed to the strong electron-withdrawing groups^[51-52]. Upon excitation at 405 nm, Ir-EA was stimulated to produce weak red phosphorescence at 700 nm in PBS (Fig.S6B) with low phosphorescence quantum yields (0.22%) and short phosphorescent lifetimes (Table S3). The solubility of Ir-EA in a given solvent is largely a function of the polarity of the solvent, which is considered as a source of variation in properties.

  2.2   Detection of sulfur dioxide derivatives in aqueous solution

  As shown in Fig. 1A, we first examined the spectral signal response of Ir-EA toward bisulfite. Ir-EA exhibited intense absorption in the region between 230 and 280 nm with the highest absorption band at 250 nm (ε=3.43×10⁴ L·mol^-1·cm^-1) and 350 nm (ε=4.43×10⁴ L·mol^-1·cm^-1), in a mixed solution of PBS/DMSO (9:1, V/V). With the addition of bisulfite into the solution of Ir-EA, the original absorption peak at 350 nm gradually decreased with simultaneous increase in the absorption band centered at 250 nm, with isosbestic point observed at 285 nm. The reaction was gradually saturated when 10 equivalent bisulfites were added. Under visible light irradiation, the color of solution gradually changed from yellow to clear colorless (Inset of Fig. 1A). As shown in Fig. 1B, upon the addition of increasing bisulfite, the intensity of phosphorescence located at 600 nm was enhanced significantly, displaying a ca. 69-fold enhancement at c_HSO3^-=100 μmol·L^-1. The change could be seen with the naked eye. Phosphorescence of the Ir-EA solution gradually changed to bright yellow (from colorless) upon irradiation with 365 nm UV light. More importantly, upon addition of bisulfite in a range of 0~40 μmol·L^-1 (R²=0.990), the phosphorescence intensity of Ir-EA increased linearly (Fig. 1D) and then gradually reached a plateau with increasing bisulfite concentration (Fig.S7). It is worth mentioning that the detection limit of Ir-EA was calculated to be 65 nmol·L^-1 for bisulfite at a signal-to-noise ratio (S/N) of 3. The value is lower than most of iridium probes used to detect bisulfite derivatives (Table S1). Fig.S8 shows that the reaction can be completed within 5 min at 50 μmol·L^-1 bisulfite concentration and the kinetic constants k=0.018 4 s^-1, which indicates that Ir-EA has a short response time to bisulfite. We also explored the changes in the phosphorescence lifetimes (PL) of Ir-EA at a range of 0~10 eq. (0~100 μmol·L^-1) of bisulfite. With the addition of bisulfite, the PL of Ir-EA increased from 110 to 254 ns (Fig. 1C), which is much longer than typical organic probes (usually less than 10 ns). The significant change of lifetime makes it possible to strongly avoid auto-fluorescent background through TPPLIM^[37].

  Figure 1

  

  Figure 1.  Changes in UV-Vis absorption (A) and emission (B) spectra of Ir-EA with the addition of increasing HSO_3^- in PBS/DMSO (9:1, V/V) solution (λ_ex=405 nm); (C) Changes in the luminescence lifetime of Ir-EA with the addition of increasing HSO_3^-; (D) Phosphorescence intensity changes at 600 nm of Ir-EA depend on the concentration of HSO_3^-

  Inset: Ir-EA solution in the absence or presence of HSO_3^- observed by natural light (A) or under 365 nm UV lamp (B)

  下载: 全尺寸图片 幻灯片

  2.3   Specificity of Ir-EA for bisulfite derivatives

  We next examined the specificity of Ir-EA for bisulfite derivatives. In addition to bisulfite derivat-ives, there are several sulfur-containing substances, such as bio-thiol, hydrogen sulfide, and thiocyanate ion in cells, which may have great influence on the specific recognition of Ir-EA toward bisulfite deriva-tives^[53-54]. In order to eliminate the possible influence of these interferences, we investigated the specificity of Ir-EA for detecting bisulfite derivatives in solution environment. In the presence of various reactive sulfur species (S^2-, HS^-, and SCN^-), reactive oxygen species (H₂O₂ and ClO^-), bio-relevant species (GSH and Cys), common anions (CO₃^2-, CH₃COO^-, C₂O₄^2-, H₂PO₄^-, HPO₄^2-, NO₃^-, NO₂^-, NH₄⁺, OH^-, F^-, Cl^-, ClO₄^-, SO₄^2-, S₂O₃^2-, CN^-, EDTA, and citric acid), and several metal cations (Ca²⁺, Al³⁺, Zn²⁺, Fe³⁺, Fe²⁺, Cu²⁺, Mn²⁺, and Co³⁺) at a high concentration (1 mmol·L^-1), the phosphores-cence intensity was recorded after it was stable. As shown in Fig.S9, all these disturbing reactive species could not induce an obvious change in the emission of Ir-EA and similar rapid enhancement of phosphores-cence intensity was observed after addition of bisulfite. To further specify the specificity of Ir-EA towards bisulfite, we also evaluated the phosphorescent response to pH. In the absence of bisulfite, the phosphorescence intensity of Ir-EA was weak and stable at pH value range of 4.5~8.5 (Fig.S10). Upon addition of bisulfite, there was still a significant enhancement of the phosphorescence intensity after reacting with bisulfite. Therefore, at the physiological pH^[55], there was a minimal affection of phosphorescent response of Ir-EA. All these results suggest that Ir-EA is a specific, highly sensitive bioprobe and has the potential to detect bisulfite in biological environments.

  2.4   Sensing mechanism of Ir-EA for bisulfite derivatives

  To investigate the sensing mechanism, Job′s plot was drawn, and the result indicated the interaction between Ir-EA and bisulfite through a 1:2 binding ration (Fig.S11). Additionally, the ESI-MS of Ir-EA after reaction with bisulfite showed new peaks at 1 111.21 (Fig.S12), suggesting that the addition products reacted with two bisulfite (m/z=1 111.12 ca. for [Ir-EA-PF₆+2NaSO₃]⁺). To investigate the reaction process^[56] and verify the additive position (Scheme 1B), the structural change of ligand L-EA reacted with bisulfite was monitored by ¹H NMR spectrum and 2D COSY spectrum. The proton signal of double bond in L-EA (H_a at δ 8.45) was vanished with the addition of bisulfite to L-EA in DMSO-d₆/D₂O (4:1, V/V) (Fig. 2B). In the meantime, two new double peaks (H_a′ at δ 4.28, H_b at δ 4.70) appeared, which correspond to change in the chemical shift of protons in original double bond and the new protons obtained by the addition reaction. The results in the 2D COSY spectrum also confirm that the two protons are adjacent, and thereby inferring that the bisulfite was added to carbon atoms attached to benzene in double bond (Fig. 2C).

  Figure 2

  

  Figure 2.  Schematic reaction of bisulfite with L-EA (A); Partial ¹H NMR (B) and 2D COSY (C) spectrum of L-EA upon addition of bisulfite (5 eq.) in DMSO-d₆/D₂O (4:1, V/V)

  下载: 全尺寸图片 幻灯片

  2.5   Intracellular bisulfite imaging

  As the basis of bioimaging application, we firstly investigated the cytotoxicity of Ir-EA in living cells using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazo-lium bromide (MTT) assays. MTT data demonstrated that the Ir-EA had low cytotoxicity on A549 and HepG2 cells (IC₅₀ > 100 μmol·L^-1; Fig.S13). Mitoc-hondrial membrane potential (MMP) damage is often a momentous marker of apoptosis and other modes of cell death. JC-1 is a commercial probe, which is able to label the MMP specifically. Mitochondrial depolarization leads to a decrease of JC-1 aggregates formation and an increase of monomer, which is indicated by a reduction of the red fluorescence intensity and increases in the green fluorescence intensity. In further MMP experiments, we used a commercial drug carbonyl cyanide m-chlorophenyl hydrazine (CCCP), which can lead to mitochondrial apoptosis, as a positive control. Ir-EA did not exhibit a decrease in MMP production 6 h post-incubation in CCCP treated and blank control cells (Fig.S14). These results are consistent with the toxicity data, suggesting that Ir-EA has a good biocompatibility.

  Subsequently, the distribution of Ir-EA in subcellular organelles was checked by colocalization experiments with Mito-Tracker Red (MTR) and Lyso-Tracker Red (LTR) in A549 cells. An excellent overlap between the phosphorescence of Ir-EA and that of MTR was viewed (Fig. 3A). Additionally, the intensity scatter plots of the two channels (red and green) are overlapped with the Pearson′s colocalization coefficient of 0.86 for Ir-EA. Compared with mito-chondria, Ir-EA is rarely found in lysosomes as imaged by confocal microscopy. These results suggest that Ir-EA is mainly located in the mitochondria and thus possess the potential application to detect biological bisulfite derivatives in mitochondria specifi-cally. The significant change of phosphorescent property of Ir-EA promoted us to evaluate its further application for bioimaging bisulfite derivatives in living A549 cells. As shown in Fig.S15a, only weak phosphorescence from Ir-EA was observed in control. However, a turn-on response of Ir-EA toward bisulfite was observed in A549 cells with remarkable phos-phorescence enhancement (Fig.S15b~d). Furthermore, the phosphorescent signal can still overlap with MTR (Pearson′s colocalization coefficient: 0.91) (Fig. 3B).

  Figure 3

  

  Figure 3.  Colocalization images of A549 cells cultured with Ir-EA (10 μmol·L^-1, 1 h) and MTR/LTR (100 nmol·L^-1, 30 min) (A), and Ir-EA+HSO₃^- and MTR (B)

  Ir-EA: λ_ex=810 nm, λ_em=(600±20) nm (red); MTR/LTR: λ_ex=543 nm, λ_em =(560±10) nm (green); Overlay: overlay of the 2nd and 3rd columns; Scale bars: 20 μm

  下载: 全尺寸图片 幻灯片

  Owing to the high sensitivity and selectivity to Ir-EA for exogenous detection of bisulfite, its feasibility was then explored to determine endogenous bisulfite in living cells. Thiosulphate sulfurtransferase (TST) is a mitochondrial enzyme involved in the most immediate pathways to generate endogenous bisulfite derivatives from thiosulfate. First, TST react with Na₂S₂O₃ to generate a sulfur-substituted enzyme^[57-58]. Next, the enzyme-bound sulfur is converted to a thiophilic acceptor, such as glutathione (GSH), to generate thiocyanate or disulfide with the concomitant formation of sulfite or bisulfite. However, TST could be restrained by 2, 4, 6-trinitrobenzenesulfonate (TNBS)^[59-60]. HepG2 cells were treated with Na₂S₂O₃/GSH for 2 h to generate the endogenous bisulfite derivatives and then were incubated with Ir-EA for an additional 1 h. As depicted in Fig.S16, HepG2 cells treated with either GSH or Na₂S₂O₃ in the presence of Ir-EA yield a similar phosphorescent intensity compared to the control, which is not a detectable signal. At variance, the phosphorescence intensity was significantly enhanced in HepG2 cells treated with Na₂S₂O₃ /GSH by TPPIM (Fig. 4A). Additionally, when pre-incubated with TNBS, even HepG2 cells incubated with Na₂S₂O₃/GSH could not engender a significant phosphorescent signal (Fig. 4A), resulting from that TST was deactivated by TNBS. These imaging results suggest that Ir-EA is capable of detecting biological bisulfite derivatives endogenously generated by enzyme in living cells.

  Figure 4

  

  Figure 4.  (A) HepG2 cells stained with 10 μmol·L^-1 Ir-EA (2 h) (a, control), 500 μmol·L^-1 GSH+250 μmol·L^-1 Na₂S₂O₃ (2 h)+ Ir-EA (2 h) (b), 1 500 μmol·L^-1 GSH+750 μmol·L^-1 Na₂S₂O₃ (2 h)+Ir-EA (2 h) (c), and 100 mmol·L^-1 TNBS+ 1 500 μmol·L^-1 GSH+750 μmol·L^-1 Na₂S₂O₃ (2 h)+Ir-EA (2 h) (d); (B) TPPIM and TPPLIM images of A549 cells stained with 10 μmol·L^-1 Ir-EA (2 h) (a) and 500 μmol·L^-1 GSH+250 μmol·L^-1 Na₂S₂O₃ (2 h)+Ir-EA (2 h) (b)

  λ_ex=810 nm, λ_em=(600±20) nm; Scale bars: 20 μm

  下载: 全尺寸图片 幻灯片

  TPPLIM effectively eliminates background fluore-scence using deep penetrating near infrared (NIR) light excitation, reflecting the micro-environment changes of chromophores^{[37, 61]}. The phosphorescence lifetimes of Ir-EA toward bisulfite have a significant increase over a timescale of several hundred nano-seconds, which is more versatile than traditional organic fluorescent probes, and are more suitable for TPPLIM. Inspired by this, TPPLIM was also used to investigate the distribution of Ir-EA toward bisulfite derivatives in A549 cells. As shown in Fig. 4B, the mitochondria of A549 cells (control) exhibit only weak phosphorescence and relatively short lifetimes (109 ns) from Ir-EA. However, the mean phosphorescent lifetime (201 ns) was increased when the cells were pre-incubated by GSH (500 μmol·L^-1) and Na₂S₂O₃ (250 μmol·L^-1). The results are in line with those recorded in aqueous (Fig. 1C). The application of this method would provide a new strategy to design TPPIM and TPPLIM probes for detection of exogenous and endogenous bisulfites. Subsequent efforts have been expanded to further elucidate the imaging of Ir-EA as a phosphorescent probe for sensitive imaging of bisulfite derivatives in vivo.

  Zebrafish has emerged as an excellent model for genetics and genomics^[62-64]. Encouraged by the outs-tanding imaging effect of Ir-EA for endogenous and exogenous bisulfite derivatives in living cells, we further elucidated the application of Ir-EA as a sensitive phosphorescent probe for the detection of bisulfite derivatives in vivo. As has been observed in Fig. 5, there is extremely weak phosphorescence in 2-day-old zebrafish embryo pre-incubated with Ir-EA (5 μmol·L^-1) for 2 h under confocal microscopy. However, after adding bisulfite (50 μmol·L^-1), the phosphores-cence was enhanced in zebrafish embryo as shown in the TPPIM images. Therefore, the results of in vivo imaging show that Ir-EA is capable to enter zebrafish and respond effectively to bisulfite derivatives with no observable toxic effect.

  Figure 5

  

  Figure 5.  Two-day-old control zebrafish embryo (A) or pre-incubated with Ir-EA (5 μmol·L^-1) for 2 h (B), and Ir-EA (5 μmol·L^-1) for 2 h and then incubated with HSO₃^- (50 μmol·L^-1) for 30 min (C)

  λ_ex=810 nm, λ_em=(600±20) nm; Scale bars: 200 μm

  下载: 全尺寸图片 幻灯片

  As we discussed in the introduction, although some iridium complexes have been used for the detection of SO₂ in cells or organisms (Table S1), Ir-EA is the first example to take advantage of its long phosphorescence lifetime for the detection of SO₂ derivatives. Ir-EA is a specific and highly sensitive probe with low cytotoxicity and could be utilized for the imaging of exogenous and endogenous bisulfite derivatives in mitochondria of living cells by TPPIM and TPPLIM, which is better than most of iridium probes for bisulfite derivatives.

  3.   Conclusions

  In this work, a new iridium(Ⅲ) probe Ir-EA with long phosphorescence lifetime has been designed and employed as a highly sensitive two-photon phosphore-scent probe for real-time detection of bisulfite deriva-tives in vitro and in vivo. The probe Ir-EA displayed a rapid response to sulfur dioxide derivatives with significant phosphorescence enhancement, good selectivity, low detection limit (65 nmol·L^-1), and low cytotoxicity. For the first time, Ir-EA was successfully applied for detection of exogenous and endogenous bisulfite in living cells using TPPLIM. We anticipate that our study could provide a basis to develop a highly sensitive and selective probe for real-time detection of biological bisulfite derivatives in living cells, and we believe that this work would provide new methods and strategies to designing bioimaging reagents and bio-probes.

  Supporting information is available at http://www.wjhxxb.cn

  
  
• 1. [1]

     Chen T M, Kuschner W G, Gokhale J, et al. Am. J. Med. Sci., 2007, 333:249-256 doi: 10.1097/MAJ.0b013e31803b900f

  2. [2]

     Rich D Q, Schwartz J, Mittleman M A, et al. Am. J. Epidemiol., 2005, 161:1123-1132 doi: 10.1093/aje/kwi143

  3. [3]

     Singh A, Agrawal M. J. Environ. Biol., 2007, 29:15-24

  4. [4]

     Sang N, Yun Y, Li H, et al. Toxicol. Sci., 2010, 114:226-236 doi: 10.1093/toxsci/kfq010

  5. [5]

     Lee W J, Teschke K, Kauppinen T, et al. Environ. Health Perspect., 2002, 110:991-995 doi: 10.1289/ehp.02110991

  6. [6]

     Shapiro R. Mutat. Res.:Rev. Genet. Toxicol., 1977, 39:149-175 doi: 10.1016/0165-1110(77)90020-3

  7. [7]

     Ji A J, Savon S R, Jacobsen D W. Clin. Chem., 1995, 41:897-903 doi: 10.1093/clinchem/41.6.897

  8. [8]

     Mitsuhashi H, Ikeuchi H, Yamashita S, et al. Shock, 2004, 21:99-102

  9. [9]

     Tsuzuki T, Obaru K, Setoyama C, et al. J. Mol. Biol., 1987, 198:21-31 doi: 10.1016/0022-2836(87)90454-2

  10. [10]

      Mitsuhashi H, Yamashita S, Ikeuchi H, et al. Shock, 2005, 24:529-534 doi: 10.1097/01.shk.0000183393.83272.de

  11. [11]

      Liang Y, Liu D, Ochs T, et al. Lab. Invest., 2011, 91:12-23 doi: 10.1038/labinvest.2010.156

  12. [12]

      Wang X B, Jin H F, Tang C S, et al. Eur. J. Pharmacol., 2011, 670:1-6 doi: 10.1016/j.ejphar.2011.08.031

  13. [13]

      Wang X B, Jin H F, Tang C S, et al. Clin. Exp. Pharmacol. Physiol., 2010, 37:745-752

  14. [14]

      Li X, Bazer F W, Gao H, et al. Amino Acids, 2009, 37:65-78 doi: 10.1007/s00726-009-0264-5

  15. [15]

      Kajiyama H, Nojima Y, Mitsuhashi H, et al. J. Am. Soc. Nephrol., 2000, 11:923-927

  16. [16]

      Wang K N, Cao Q, Liu L Y, et al. Chem. Sci., 2019, 10:10053-10064 doi: 10.1039/C9SC03594J

  17. [17]

      Theisen S, Hansch R, Kothe L, et al. Biosens. Bioelectron., 2010, 26:175-181 doi: 10.1016/j.bios.2010.06.009

  18. [18]

      Zeng L, Gupta P, Chen Y, et al. Chem. Soc. Rev., 2017, 46:5771-5804 doi: 10.1039/C7CS00195A

  19. [19]

      Sun M, Dai W, Liu D Q. J. Mass Spectrom., 2008, 43:383-393 doi: 10.1002/jms.1335

  20. [20]

      Daunoravicius Z, Padarauskas A. Electrophoresis, 2002, 23:2439-2444 doi: 10.1002/1522-2683(200208)23:15<2439::AID-ELPS2439>3.0.CO;2-Z

  21. [21]

      Liang X, Zhong T, Quan B, et al. Sens. Actuators B, 2008, 134:25-30 doi: 10.1016/j.snb.2008.04.003

  22. [22]

      Yin J, Hu Y, Yoon J. Chem. Soc. Rev., 2015, 44:4619-4644 doi: 10.1039/C4CS00275J

  23. [23]

      Vendrell M, Zhai D, Er J C, et al. Chem. Rev., 2012, 112:4391-4420 doi: 10.1021/cr200355j

  24. [24]

      赵振盛, 郭旭东, 李沙瑜, 等.化学学报, 2016, 74:593-596ZHAO Zhen-Sheng, GUO Xu-Dong, LI Sha-Yu, et al. Acta Chim. Sinica, 2016, 74:593-596

  25. [25]

      Yang Y, Zhao Q, Feng W, et al. Chem. Rev., 2013, 113:192-270 doi: 10.1021/cr2004103

  26. [26]

      Tang Y, Kong X, Xu A, et al. Angew. Chem. Int. Ed., 2016, 55:3356-3359 doi: 10.1002/anie.201510373

  27. [27]

      Wang J W, Wong A M, Flores J, et al. Cell, 2003, 112:271-282 doi: 10.1016/S0092-8674(03)00004-7

  28. [28]

      Gissibl T, Thiele S, Herkommer A, et al. Nat. Photonics, 2016, 10:554-560 doi: 10.1038/nphoton.2016.121

  29. [29]

      Cao D, Liu Z, Verwilst P, et al. Chem. Rev., 2019, 119:10403-10519 doi: 10.1021/acs.chemrev.9b00145

  30. [30]

      Zhao Q, Huang C, Li F. Chem. Soc. Rev., 2011, 40:2508-2524 doi: 10.1039/c0cs00114g

  31. [31]

      Ma D L, He H Z, Leung K H, et al. Angew. Chem. Int. Ed., 2013, 52:7666-7682 doi: 10.1002/anie.201208414

  32. [32]

      He L, Tan C P, Ye R R, et al. Angew. Chem. Int. Ed., 2014, 53:12137-12141 doi: 10.1002/anie.201407468

  33. [33]

      Li Y, Tan C P, Zhang W, et al. Biomaterials, 2015, 39:95-104 doi: 10.1016/j.biomaterials.2014.10.070

  34. [34]

      Li J, Pu K. Chem. Soc. Rev., 2019, 48:38-71 doi: 10.1039/C8CS00001H

  35. [35]

      Guo Z, Park S, Yoon J, et al. Chem. Soc. Rev., 2014, 43:16-29 doi: 10.1039/C3CS60271K

  36. [36]

      Zhang K Y, Gao P, Sun G, et al. J. Am. Chem. Soc., 2018, 140:7827-7834 doi: 10.1021/jacs.8b02492

  37. [37]

      Hao L, Li Z W, Zhang D Y, et al. Chem. Sci., 2019, 10:1285-1293 doi: 10.1039/C8SC04242J

  38. [38]

      Zhao Q, Cao T, Li F, et al. Organometallics, 2007, 26:2077-2081 doi: 10.1021/om061031r

  39. [39]

      Li G, Chen Y, Wang J, et al. Chem. Sci., 2013, 4:4426-4433 doi: 10.1039/c3sc52301b

  40. [40]

      Li G, Chen Y, Wang J, et al. Biomaterials, 2015, 63:128-136 doi: 10.1016/j.biomaterials.2015.06.014

  41. [41]

      Wang K N, Zhu Y, Xing M, et al. Sens. Actuators B, 2019, 295:215-222 doi: 10.1016/j.snb.2019.05.077

  42. [42]

      Liu J B, Yang C, Ko C N, et al. Sens. Actuators B, 2017, 243:971-976 doi: 10.1016/j.snb.2016.12.083

  43. [43]

      Li X, Zeng R, Xie C, et al. Dyes Pigm., 2019, 165:128-136 doi: 10.1016/j.dyepig.2019.02.018

  44. [44]

      Gao H, Qi H, Peng Y, et al. Analyst, 2018, 143:3670-3676 doi: 10.1039/C8AN00640G

  45. [45]

      Liu Z, Guo S, Piao J, et al. RSC Adv., 2014, 4:54554-54557 doi: 10.1039/C4RA10137E

  46. [46]

      Zhang Y, Guan L, Yu H, et al. Anal. Chem., 2016, 88:4426-4431 doi: 10.1021/acs.analchem.6b00061

  47. [47]

      Zhang W, Liu T, Huo F, et al. Anal. Chem., 2017, 89:8079-8083 doi: 10.1021/acs.analchem.7b01580

  48. [48]

      Li C, Yu M, Sun Y, et al. J. Am. Chem. Soc., 2011, 133:11231-11239 doi: 10.1021/ja202344c

  49. [49]

      Liu Z, Zhou X, Miao Y, et al. Angew. Chem. Int. Ed., 2017, 56:5812-5816 doi: 10.1002/anie.201702114

  50. [50]

      Sheldrick G M. SHELX-97, Program for the Solution and the Refinement of Crystal Structures, University of Göttingen, Germany, 1997.

  51. [51]

      Zhang F, Liang X, Zhang W, et al. Biosens. Bioelectron., 2017, 87:1005-1011 doi: 10.1016/j.bios.2016.09.067

  52. [52]

      He L, Qiao J, Duan L, et al. Adv. Funct. Mater., 2009, 19:2950-2960 doi: 10.1002/adfm.200900723

  53. [53]

      Jiao X, Li Y, Niu J, et al. Anal. Chem., 2018, 90:533-555 doi: 10.1021/acs.analchem.7b04234

  54. [54]

      Lin V S, Chen W, Xian M, et al. Chem. Soc. Rev., 2015, 44:4596-4618 doi: 10.1039/C4CS00298A

  55. [55]

      Wu J, Pan J, Ye Z, et al. Sens. Actuators B, 2018, 274:274-284 doi: 10.1016/j.snb.2018.07.161

  56. [56]

      Hou J T, Ren W X, Li K, et al. Chem. Soc. Rev., 2017, 46:2076-2090 doi: 10.1039/C6CS00719H

  57. [57]

      Cipollone R, Ascenzi P, Tomao P, et al. J. Mol. Microbiol. Biotechnol., 2008, 15:199-211 doi: 10.1159/000121331

  58. [58]

      Ploegman J H, Drent G, Kalk K H, et al. Nature, 1978, 273:120-124 doi: 10.1038/273120a0

  59. [59]

      Ramasamy S, Singh S, Taniere P, et al. Am. J. Physiol.:Gastrointest. Liver Physiol., 2006, 291:288-296 doi: 10.1152/ajpgi.00324.2005

  60. [60]

      Malliopoulou V, Rakitzis E, Malliopoulou T. Anticancer Res., 1989, 9:1133-1136

  61. [61]

      Zhang K Y, Yu Q, Wei H, et al. Chem. Rev., 2018, 118:1770-1839 doi: 10.1021/acs.chemrev.7b00425

  62. [62]

      Nasevicius A, Ekker S C. Nat. Genet., 2000, 26:216-220 doi: 10.1038/79951

  63. [63]

      Zon L I, Peterson R T. Nat. Rev. Drug Discovery, 2005, 4:35-44 doi: 10.1038/nrd1606

  64. [64]

      Lieschke G J, Currie P D. Nat. Rev. Genet., 2007, 8:353-367 doi: 10.1038/nrg2091

• 

  Scheme 1  Schematic reaction mechanism between bisulfite and Ir-EA

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Changes in UV-Vis absorption (A) and emission (B) spectra of Ir-EA with the addition of increasing HSO_3^- in PBS/DMSO (9:1, V/V) solution (λ_ex=405 nm); (C) Changes in the luminescence lifetime of Ir-EA with the addition of increasing HSO_3^-; (D) Phosphorescence intensity changes at 600 nm of Ir-EA depend on the concentration of HSO_3^-

  Inset: Ir-EA solution in the absence or presence of HSO_3^- observed by natural light (A) or under 365 nm UV lamp (B)

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Schematic reaction of bisulfite with L-EA (A); Partial ¹H NMR (B) and 2D COSY (C) spectrum of L-EA upon addition of bisulfite (5 eq.) in DMSO-d₆/D₂O (4:1, V/V)

  下载: 全尺寸图片 幻灯片
  

  Figure 3  Colocalization images of A549 cells cultured with Ir-EA (10 μmol·L^-1, 1 h) and MTR/LTR (100 nmol·L^-1, 30 min) (A), and Ir-EA+HSO₃^- and MTR (B)

  Ir-EA: λ_ex=810 nm, λ_em=(600±20) nm (red); MTR/LTR: λ_ex=543 nm, λ_em =(560±10) nm (green); Overlay: overlay of the 2nd and 3rd columns; Scale bars: 20 μm

  下载: 全尺寸图片 幻灯片
  

  Figure 4  (A) HepG2 cells stained with 10 μmol·L^-1 Ir-EA (2 h) (a, control), 500 μmol·L^-1 GSH+250 μmol·L^-1 Na₂S₂O₃ (2 h)+ Ir-EA (2 h) (b), 1 500 μmol·L^-1 GSH+750 μmol·L^-1 Na₂S₂O₃ (2 h)+Ir-EA (2 h) (c), and 100 mmol·L^-1 TNBS+ 1 500 μmol·L^-1 GSH+750 μmol·L^-1 Na₂S₂O₃ (2 h)+Ir-EA (2 h) (d); (B) TPPIM and TPPLIM images of A549 cells stained with 10 μmol·L^-1 Ir-EA (2 h) (a) and 500 μmol·L^-1 GSH+250 μmol·L^-1 Na₂S₂O₃ (2 h)+Ir-EA (2 h) (b)

  λ_ex=810 nm, λ_em=(600±20) nm; Scale bars: 20 μm

  下载: 全尺寸图片 幻灯片
  

  Figure 5  Two-day-old control zebrafish embryo (A) or pre-incubated with Ir-EA (5 μmol·L^-1) for 2 h (B), and Ir-EA (5 μmol·L^-1) for 2 h and then incubated with HSO₃^- (50 μmol·L^-1) for 30 min (C)

  λ_ex=810 nm, λ_em=(600±20) nm; Scale bars: 200 μm

  下载: 全尺寸图片 幻灯片
•