A red fluorescent BODIPY probe for iridium (III) ion and its application in living cells

A new red fluorescent probe 1 based on BODIPY skeleton has been successfully synthesized through introduction of 2-(thiophen-2-yl) quinoline moiety at meso- and 3-position, which exhibits excellent optical performance, including high fluorescence quantum yield, large pseudo Stokes' shift as well as high selectivity and sensitivity towards iridium (III) ion in aqueous solution and in living cells.

Iridium is a rare but very useful metal that can be used in many fields including catalytic converters, luminescent materials, anti-cancer drugs, electrodes, fuel cells and jewellery etc. [34 -38]. Especially in organic chemistry, like other heavy-metal catalysts such as palladium and platinum, iridium compounds as the catalysts could also effectively promote the transformation between various functional groups [39,40]. However, the wide use of these heavy-metals has resulted in an increase of residue levels into environment, especially ambient air and soil, which is harmful to public health. As some research findings showed, the iridium exposure could affect an immune imbalance and cause contact urticaria with anaphylactic reactions [41,42]. Thus, the oral permissible daily exposure (PDE) for iridium impurity is less than 100 mg per day by US Pharmacopeia [43]. Therefore, the recognition and detection of these heavy-metal ions have been a considerably significant and active research area. Numerous methods, particularly fluorescent methods, have been developed for the detection of palladium and platinum [44 -53]. Among those, Kaur et al. reported the 'off-on' type fluorescence probe based on the BODIPY core for Pd 2þ detection [49]. To the best of our knowledge, only an example was reported for Ir 3þ detection to date [54]. Accordingly, exploring highly selective and sensitive probes for Ir 3þ detection is very necessary due to the potential hazards to human health. In our previous work, we reported a BODIPY probe with a 2-(thiophen-2-yl)quinoline group as a chelator can selectively and sensitively detect the Fe 3þ ion [55,56]. Herein, we report that functionalization of the pyrrole moieties of BODIPY with 2-(thiophen-2-yl)quinoline (TQ) tunes the dye for Ir 3þ detection. Furthermore, fluorescence microscopy experiments demonstrated that resulting probe 1 could be used to monitor Ir 3þ ion in living cells.

Reagents and instruments
Unless otherwise noted, the reagents or solvents were obtained from commercial suppliers and used without further purification. Metal salts, such as IrCl 3 2 . 4H 2 O, were used for making metal solutions. All air and moisture sensitive reactions were carried out under an argon atmosphere. Dry CH 2 Cl 2 was obtained by refluxing and distilling over CaH 2 under nitrogen. Triethylamine was obtained by simple distillation.
The 1 H NMR and 13 C NMR spectroscopic measurements were made by using a Bruker AVF-400 or a Bruker 700 MHz spectrometer. The measurements for 1 H NMR and 1 H-1 H COSY were performed at 700 MHz (DRX-700) or 400 MHz (DRX-400). The measurements for 13 C NMR were performed at 175 MHz (DRX-700). Mass spectra were measured with a Bruker ultraflex MALDI TOF MS spectrometer and Thermo Scientific LTQ Orbitrap XL spectrometer. Fluorescence spectral measurements were carried out by using a Hitachi F-4600 fluorescence spectrophotometer. Electronic absorption spectra were recorded with Shimadzu UV-2550 spectrophotometers.

Synthetic procedures for probe 1
The synthesis of probe 1 is outlined in scheme 1. The compounds 2 and 3 were produced according to a published procedure [55]. Probe 1 was prepared through Knoevenagel condensation of compound 2 and 5-(Quinolin-2-yl)thiophene-2-carbaldehyde 3 according to the below procedure and characterized by MALDI-TOF MS, HR-MS, 1 H NMR, 13 CNMR.

Procedures for metal ion sensing
Stock solutions of various metal ions (0.1 M), such as Ir 3þ , Rh 3þ , Au 3þ , Fe 3þ , Al 3þ , Cd 2þ , Cr 3þ , Cu 2þ , Hg 2þ , Ni 2þ , Pb 2þ , Zn 2þ , Co 2þ , Ag þ , were prepared in deionized water. Palladium (II) solution (0.1 M) and platinum (II) solution (0.01 M) were prepared in dilute hydrochloric acid. For titration, probe 1 (3 ml, 10 mM, dimethyl sulfoxide solution) was added to the quartz optical cell of 1 cm optical path length. The metal solution was gradually added to probe 1 using a micro-pipette. For selectivity royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181090 evaluation, the test samples were prepared by adding appropriate amounts of metal ion stock solution to probe 1 (3 ml, 10 mM, dimethyl sulfoxide solution). Fluorescence was measured for 590 nm excitation within 595 -800 nm.

Cell culture and fluorescence bioimaging
The Hela cell line was provided by the Institute of Biochemistry and Cell Biology, SIBS, CAS (China). Cells were grown in high glucose Dulbecco's Modified Eagle Medium (DMEM, 4.5 g of glucose l 21 ) supplemented with 10% fetal bovine serum (FBS) at 378C and 5% CO 2 . Cells (5 Â 108 l 21 ) were plated on 14 mm glass coverslips and allowed to adhere for 24 h. Experiments to assess Ir 3þ uptake were performed over 1 h in the same medium supplemented with 100 mM IrCl 3 . 3H 2 O.
Immediately before the experiments, cells were washed with PBS buffer and then incubated with 5 mM probe 1 in PBS buffer for 30 min at 378C. Cell imaging was then carried out after washing the cells with PBS buffer. Confocal fluorescence imaging was performed with a Zeiss LSM 710 laser scanning microscope and a 63Â oil-immersion objective lens. Cells incubated with probe 1 were excited at 590 nm using a multi-line argon laser.

Theoretical calculations
Geometry optimization and TD-DFT calculations were carried out for probe 1 and probe 1 with Ir 3þ using the B3LYP functional of the Spartan 16 software packages with 6-31G(d) basis sets.

Results and discussion
3.1. Characterization of the probe 1 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181090 3 635 nm decreased remarkably with a virtually unchanged peak position. Upon addition of more than 100 equiv. of Ir 3þ , the red shift in the fluorescence peak of probe 1 about 30 nm can be observed. As shown in electronic supplementary material, figure S9, the fluorescence response of probe 1 towards Ir 3þ is normalized as R ¼ 1 2 I i /I max , where I max is fluorescence response of probe 1 without Ir 3þ , I i is luminescent intensity of probe 1 with Ir 3þ [47]. The detection limit of probe 1 for Ir 3þ is determined as 2.55 ppm based on the signal-to-noise ratio of three. The good linear relationship between the fluorescence response and concentration of Ir 3þ is obtained with a 0.9910 correlation coefficient.
To gauge the recognition specificity of probe 1 to Ir 3þ ion, the spectral response features of probe 1 to various interference metal ions with much higher concentration were estimated. The selectivity of probe 1 with Ir 3þ is examined according to its bonds with various metal ions as shown in figure 3. When the metal ions ([metal]/[ probe 1] ¼ 200:1), such as Fe 3þ , Al 3þ , Cd 2þ , Ag þ , Cr 3þ , Cu 2þ , Hg 2þ , Ni 2þ , Pb 2þ , Zn 2þ , are added to the probe 1 solution (10 mM in DMSO), the fluorescence spectrum peak and intensity is nearly unchanged. After the addition of the metal ions (Co 2þ , Rh 3þ , Pd 2þ , Au 3þ , Pt 2þ , Ir 3þ ) under the same conditions, the fluorescence intensity decreased by 20%, 20%, 20%, 16%, 33%, 94%, respectively. The above results demonstrate that its Ir 3þ response is not interfered in the background containing appropriate metal ions. That probe 1 could selectively monitor Ir 3þ ion was possible because of the stable binding with sulfur and nitrogen atom in TQ parts. To further demonstrate this mechanism, the binding mode of Ir 3þ ion with probe 1 or compound 2 is investigated by 1 H NMR spectroscopy as shown in figure 4 and electronic supplementary material, figure S7. The 1 H NMR spectra of probe 1 and compound 2 in the presence of different concentrations of Ir 3þ are recorded in DMSO-d 6 and compared to the spectrum of the ion-free probe. During 1 H NMR spectra of compound 2, the protons (Hc and Hd) of the thiophene group at 8.14 ppm and 7.32 ppm are affected significantly by Ir 3þ . Upon the addition of Ir 3þ , significant downfield shifts of protons (D Hc ¼ 0.08 ppm) and (D Hd ¼ 0.07 ppm) corresponding to the protons of the thiophene group are observed, indicating that Ir 3þ coordinates to the sulfur atom of compound 2. Furthermore, the proton (Hb) of the quinoline group at 8.24 ppm exhibits a downfield shift (D Hb ¼ 0.09 ppm), indicating that Ir 3þ coordinates to the nitrogen atom of compound 2. The results indicate that the sulfur atom and nitrogen atom of compound 2 may be involved in coordination to Ir 3þ . Figure 4 shows that the protons (Hc, Hb, Hd and Hc 0 , Hb 0 , Hd 0 ) of probe 1 have significant downfield shifts as well, which indicate that probe 1 and compound 2 have similarities to binding of Ir 3þ ion. Probe 1 based on oxidative PET is in the fluorescence 'on' state when in the ion-free form. Coordination of TQ moiety at meso-position of probe 1 with Ir 3þ ion makes it electron poor and lowers the LUMO of TQ moiety. Our theoretical calculation revealed that the LUMO orbital energy of probe 1 with Ir 3þ is lower, compared to the probe 1(22.68 eV versus 24.01 eV) (electronic supplementary material, figure S8), so that PET from the excited BODIPY moiety to the TQ moiety at meso-position becomes possible, leading to fluorescence quenching. Probe 1 possesses an electrondonating group (TQ moiety at 3-position) conjugated to an electron-withdrawing group (BODIPY moiety); it undergoes ICT from the donor to the acceptor upon photo-excitation.     royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 181090 Ir 3þ ion with the TQ moiety at 3-position enhances the electron-withdrawing character and consequently red shifted the fluorescence spectra.

Practical application of probe 1
The practical application of probe 1 as Ir 3þ probe in living cells has been evaluated. As shown in figure 5, after incubating the Hela cells with 5 mM probe 1 for 30 min at 378C, a significant green emission from the intracellular region could be observed upon excitation at 590 nm (figure 5a). Supplementing cells with 100 mM Ir 3þ in the growth medium for 1 h at 378C, microscope images showed only weak intracellular fluorescence (figure 5d). Both bright field and overlay measurements with Ir 3þ and probe 1 after treatment confirmed that the cells are viable throughout the imaging experiments (figure 5b,c,e,f ). In addition, large signal ratios (I2/I1 . 20) are observed at the cytoplasm (region 2) and the nucleus (region 1) due to weak nuclear uptake of probe 1 (electronic supplementary material, figure S10). These data lead to exclusive staining in the cytoplasm, which indicates that probe 1 is cellpermeable. These results indicate that probe 1 can be used as an effective intracellular Ir 3þ imaging agent. The cell viability of probe 1 is evaluated through an MTT assay ( figure 6). Cell viability is monitored for 5 and 10 h after treatment with probe 1 over a wide range of concentrations (0-80 mM). This demonstrates that probe 1 does not negatively affect the cell viability over the range of concentrations studied, therefore, can clearly be used for the intracellular detection of the Ir 3þ ion.

Conclusion
In summary, we have designed a red BODIPY probe with 2-(thiophen-2-yl)quinoline substituent at mesoand 3-position, which exhibits excellent optical performance with a pseudo Stokes' shift (300 nm) and strong emission. The high selectivity and sensitivity of probe 1 towards Ir 3þ indicate that probe 1 can be used as a fluorescence turn-off sensor for Ir 3þ in living cells. Probe 1 in fluorescence imaging can avoid photobleaching of emissive dyes and deepen penetration depths. This study displays an effective rationale to design chemosensors for rapid Ir 3þ sensing in biological systems. Meanwhile, there is still considerable scope for further research in this area for the detection of biologically noble metals.