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A novel phosphorescent probe based on ion-paired iridium(III) complex has been designed and synthesized by incorporating α,β-unsaturated ketone moiety in the cationic component. The phosphorescent intensity of cationic component is sensitive to bithiols, such as cysteine and homocysteine, based on the addition reaction of bithiols with α,β-unsaturated ketone moiety, while that of the anionic component remains unchanged. Thus, this ion-paired iridium(III) complex can be used for ratiometric luminescence sensing and imaging of intracellular biothiols with excellent sensing performance. Moreover, the long phosphorescence lifetime of the cationic component is also sensitive to bithiols. Hence, this ion-paired iridium(III) complex has been further used for time-resolved luminescence imaging of intracellular biothiols. As far as we know, this is the first report about molecular probe for both ratiometric and time-resolved luminescence imaging of intracellular biothiols.
© 2016 Optical Society of America
1. Introduction
Over the past decade, phosphorescent transition-metal complexes, especially cyclometalated iridium(III) complexes, have attracted considerable research interest due to their intriguing photophysical properties, such as highly efficient room-temperature phosphorescence, excellent color tunability, and high photo- and chemical stability [1–5]. To date, iridium(III) complexes have been successfully applied in various optical and electronic fields, especially as emitters in organic light-emitting diodes (OLED) for neutral complexes and light-emitting electrochemical cells (LEC) for cationic ones (Fig. 1(a)) [6–10]. To achieve their applications, much effort has been devoted to the development of excellent iridium(III) complexes by designing and modifying their ligand structures [11–13]. Recently, several groups have reported an interesting class of ion-paired iridium(III) complexes, which are composed of a pair of complex components with opposite charges (Fig. 1(a)) [14–18]. By varying the ligand structures for each ionic component, it is convenient to tune the photophysical properties of two ionic components, respectively. Thus, the phosphorescent properties of ion-paired iridium(III) complexes were changed. The application of ion-paired iridium(III) complexes in OLED has been realized [14,19]. Now, it becomes an important topic to further exploit the applications of this interesting class of complexes.
Fig. 1 (a) Classification of phosphorescent iridium(III) complexes. (b) Design strategy of phosphorescent ion-paired iridium(III) complexes for ratiometric biodetection.
Most recently, the application of phosphorescent iridium(III) complexes in biodetection has been attracting increasing attentions due to their excellent phosphorescent properties, especially their long phosphorescence lifetime in the range of from several hundred nanoseconds to several microseconds, which is beneficial for the elimination of the interference from the short-lived background fluorescence and scattered light [20–23]. To date, most reported phosphorescent iridium(III) complexes exhibit single emission band and the application in biodetection is based on the variation in its intensity. The accuracy of such intensity-based detection, however, would be influenced by external and environmental factors, such as probe concentration, excitation power, etc [24,25]. Ratiometric detection, by measuring the ratio changes of emission intensities at two different wavelengths, is a feasible method to minimize the above influences [25–31]. For iridium(III) complexes, it is usually difficult to design ratiometric probes for lack of effective and versatile strategy to rationally control their excited-state properties and design complexes with two separated emission bands [31,32]. Ion-paired iridium(III) complexes exhibit two emission bands from cationic and anionic components respectively, and the wavelengths of two emission bands can be easily tuned through the modification of ligand structures of two components [14,15]. Thus, ion-paired iridium(III) complexes with two separated emission bands and sensing ability are anticipated to be an excellent platform to realize ratiometric measurement (Fig. 1(b)). In addition, the long phosphorescent lifetime of iridium(III) complexes makes them a good candidate for time-resolved luminescence imaging utilizing photoluminescence lifetime imaging (PLIM) and time-gated luminescence imaging microscopy (TGLI), which can effectively eliminate the undesirable short-lived background fluorescence [33–35].
Herein, we designed and synthesized an interesting ion-paired iridium(III) complex with α,β-unsaturated ketone moiety in cationic component for ratiometric and time-resolved luminescence sensing and imaging of intracellular biothiols. Cysteine (Cys) and homocysteine (Hcy) were chosen as the target analytes because they play very crucial roles in physiological processes and participate in the processes of reversible redox reactions and cellular growth [36–38].
2. General experimental information
The 1H and 13C NMR spectra were recorded from Bruker Ultra Shield Plus 400 MHz NMR instrument at 298 K using d-substituted solvents. Chemical shifts are referenced against external Me4Si (1H, 13C) in units of ppm. Mass spectra were obtained on Bruker autoflex matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. Shimadzu UV-3600 UV-VIS-NIR spectrophotometer and Edinburgh FL 920 spectrophotometer were used to measure the UV-visible absorption and photoluminescence data, respectively. Confocal luminescence imaging was obtained on Olympus FV1000 laser scanning confocal microscope. The time-resolved luminescence imaging equipment is integrated with the Olympus FV1000 laser scanning confocal microscope. The lifetime values were calculated with professional software provided by PicoQuant Company. The theoretical calculation was carried out by the Gaussian 09 package. The complex structures were optimized by B3LYP density functional theory. The LANL2DZ basis set was utilized to describe the core electrons of the iridium atom and the 6-31G* basis set was used to analyze all other atoms.
Characterization data of C1. 1H NMR (400 MHz, MeOD) δ: 8.75(s, 2H), 8.42 (q, J = 9.0 Hz, 4H), 8.28 (d, J = 5.9 Hz, 2H), 8.21 (d, J = 7.8 Hz, 2H), 8.17 (s, 2H), 8.13 (s, 2H), 8.04 (d, J = 9.1 Hz, 4H), 7.85 (dd, J = 16.3, 7.3 Hz, 4H), 7.62 (d, J = 15.5 Hz, 2H), 7.44 (d, J = 8.9 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.20 (t, J = 7.4 Hz, 2H), 7.12-7.05 (m, 2H), 6.84 (t, J = 7.8 Hz, 2H), 6.78 (d, J = 9.2 Hz, 4H), 6.53 (d, J = 7.2 Hz, 2H), 3.11 (d, J = 11.8 Hz, 12H); 13C NMR (100 MHz, CDCl3) δ: 186.36, 169.84, 155.82, 153.94, 150.89, 147.90, 147.38, 146.06, 145.50, 140.10, 135.77, 134.57, 131.58, 131.30, 130.92, 130.58, 129.18, 127.61, 127.24, 126.90, 125.97, 124.75, 124.68, 123.22, 122.17, 117.75, 111.04, 39.99. MS (MALDI-TOF-MS) [m/z]: 1103.270 (C1-PF6)+.
Characterization data of A1C1. 1H NMR (400 MHz, DMSO-d6) δ: 9.41 (d, J = 5.7 Hz, 2H), 8.82 (s, 2H), 8.48 (s, 4H), 8.24 (d, J = 8.0 Hz, 2H), 8.17 (d, J = 15.7 Hz, 2H), 8.07 (d, J = 5.6 Hz, 2H), 8.03-7.93 (m, 8H), 7.89-7.85 (m, 4H), 7.59 (d, J = 7.9 Hz, 2H), 7.50 (d, J = 15.6 Hz, 2H), 7.37 (t, J = 7.4 Hz, 2H), 7.23 (d, J = 8.9 Hz, 4H), 7.16(t, J = 8.1 Hz 2H), 7.10-7.05 (m, 2H), 6.80 (m, 2H), 6.70 (d, J = 8.7 Hz, 6H), 6.58 (t, J = 7.2 Hz, 2H), 6.38 (d, J = 7.5 Hz, 2H), 6.02 (d, J = 7.2 Hz, 2H), 2.99 (s, 12H); 13C NMR (100 MHz, DMSO-d6) δ: 185.82, 170.01, 168.12, 155.88, 154.28, 153.56, 151.13, 147.90, 147.18, 146.13, 145.98, 144.72, 141.01, 137.03, 134.17, 131.71, 131.53, 131.09, 131.02, 130.51, 129.89, 128.95, 128.04, 127.89, 127.32, 124.55, 124.47, 124.13, 123.49, 123.16, 123.06, 120.93, 119.53, 118.52, 111.31, 79.44, 79.29, 79.07, 78.74, 39.96. MS (MALDI-TOF-MS) [m/z]: 1103.27 (cationic component), 553.25 (anionic component).
3. Results and discussion
Firstly, cationic complex C1 (Fig. 2(a)) with two α,β-unsaturated ketone moieties in N^N ligand was designed and synthesized. The chemical structure of C1 was characterized via 1H NMR spectroscopy, 13C NMR spectroscopy, and MALDI-TOF-MS spectroscopy. The UV–vis absorption spectrum of C1 in CH3CN/H2O (3:2, v/v) solution (6.2 × 10−6 mol·L−1) is shown in Fig. 2(b). Interestingly, it exhibits an intense and broad absorption band at the visible region from 400 to 550 nm, which is assigned to the intraligand charge transfer (ILCT) transition from 4-(dimethylamino)phenyl (NMe2phenyl) and carbonyl (CO) moieties to 2,2'-bipyridine (bpy) and vinyl (C = C) moieties in N^N ligand according to the theoretical calculation results (Fig. 3 and Table 1) [36,38]. In addition, C1 exhibits weak and broad emission at 560 nm in CH3CN/H2O (3:2, v/v) solution (5.0 × 10−5 mol·L−1) at room temperature, and its luminescence quantum yield was measured to be 0.001. According to the time-dependent density functional theory (TDDFT) calculation results (Fig. 3 and Table 1), the lowest triplet state (T1) of C1 is attributed to the ILCT transition from NMe2phenyl and carbonyl moieties to bpy and vinyl (C = C) moieties in N^N ligand and ligand-to-ligand charge transfer (LLCT) transition from C^N ligand to bpy and vinyl (C = C) moieties in N^N ligand [36,38]. The weakly emissive property of C1 indicates the effective non-radiative deactivation process in ILCT and LLCT excited states due to the strong participation of α,β-unsaturated ketone moieties.
Fig. 2 (a) Chemical structure and sensing mechanism of C1. Changes in UV-vis absorption (b) and phosphorescence spectra (c) of C1 in CH3CN/H2O (3:2, v/v) with various amounts of Cys. Inset in (c): Emission image of C1 in the absence (left) and presence (right) of Cys.
Table 1. Calculated singlet and triplet states of C1 and C1-Cys.
The sensing performance of C1 after adding Cys was then investigated through the variation in absorption and emission spectra. Upon addition of Cys to the solution of C1, the absorption band at 430 nm disappeared. Moreover, the phosphorescent emission intensity of C1 at 560 nm was enhanced significantly with the addition of Cys. Thus, C1 could be used as an OFF–ON type phosphorescent probe for Cys. The luminescence quantum yield and lifetime of the adduct C1-Cys in N2-saturated solution were measured to be 0.10 and 278.1 ns, respectively, which are independent of concentration. These spectral responses indicated that Cys may react with α,β-unsaturated ketone moiety of C1 through the addition reaction (Fig. 2). This mechanism has been confirmed by MALDI-TOF-MS spectroscopy. The results showed that there are two molecular ion peaks at 1224.278 and 1345.203, which were assigned to the molecular ion fragment of the adducts with a 1:1 and 1:2 binding ratio between C1 and Cys. According to the theoretical calculation results (Fig. 3 and Table 1), the excited state of the adduct C1-Cys is attributed to ILCT from NMe2phenyl to bpy and LLCT from NMe2phenyl moiety to cyclometalated C^N ligand, leading to the significant emission enhancement. The similar spectral behaviour was also observed for Hcy. In addition, the selectivity of C1 to Cys and Hcy was studied. For other amino acids and thiol-containing peptides, such as alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), glycine (Gly), glutathione (GSH), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu), methionine (Met), phenylalanine (Phe), serine (Ser), proline (Pro), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val), no evident spectral response was observed, indicating the good selectivity of C1 to thiol-containing Cys and Hcy.
Next, practical application of C1 in luminescence imaging of live Hela cells was investigated using a confocal luminescence microscopy. As demonstrated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, C1 shows low cytotoxicity to living cells. As shown in Fig. 4(a), all Hela cells exhibited bright intracellular luminescence after incubation with C1 for 45 min at 37 °C. The images showed that the luminescence was obvious in the cytoplasm region, not in the nucleus. Furthermore, the selectivity of C1 for the detection of biothiols within living cells was studied through confocal luminescence imaging. When Hela cells were initially pretreated with 200 μM N-ethylmaleimide (NEM, as a thiol-reactive compound) at 37 °C for 30 min and then incubated with C1 (5 × 10−5 mol·L−1) for 45 min at 37 °C, Hela cells displayed very weak emission (Fig. 4(b)). This result suggested that the intense emission is originated from the reaction between C1 and biomolecules containing thiol within living Hela cells, and these facts are in accordance with the sensing results obtained in the solution.
Fig. 4 Luminescence images of C1 in Hela cells before (a) and after (b) treatment of NEM. (c) Photoluminescence lifetime image of C1 in Hela cells. (d-f) Time-gated luminescence images of C1 in Hela cells with different delayed time.
To demonstrate the attractive advantage of the long phosphorescence lifetime of iridium(III) complexes in bioimaging, photoluminescence lifetime imaging and time-gated luminescence imaging experiments were carried out to isolate the phosphorescence signal from other contributions to the total photoluminescence. As shown in photoluminescence lifetime images (Fig. 4(c)), the emission lifetime signal of τ = 120 ns was observed, which is long enough to be distinguishable from the short-lived interference signals of background fluorescence (<10 ns) [39–41]. In addition, photoluminescence intensity images were collected via TGLI by exerting different delayed time (50 and 300 ns), which effectively eliminate the background interference signals. As shown in Fig. 4(d-f), the evident luminescence could be clearly observed when 50 ns or even 300 ns delay was exerted to collect signals. The delay time for signals collection was significantly longer than the lifetime of the short-lived background fluorescence. So the luminescence collected in Fig. 4(e) and (f) was assigned to iridium(III) complex, which further demonstrated the advantage of long-lived phosphorescent signal in biosensing and bioimaging.
Based on the above sensing results of C1, an ion-paired iridium(III) complex A1C1 from cationic complex C1 and anionic complex A1 was designed and synthesized (Fig. 5(a)). A1 was prepared according to the previous report [14]. Next, ion-paired iridium(III) complex A1C1 was obtained through a metathesis reaction by mixing two oppositely charged iridium(III) complexes in water and then washed for several times. The structure of A1C1 has been characterized via NMR and MALDI-TOF-MS spectroscopy.
Fig. 5 (a) Chemical structure and sensing mechanism of ion-paired complex A1C1. (b) Changes in phosphorescence emission spectra of A1C1 in CH3CN/H2O (3:2, v/v) with various amounts of Cys. Inset in (b): Emission image of A1C1 in the absence (left) and presence (right) of Cys. (c) Titration curve plotted with phosphorescence emission intensity at 560 nm over that at 485 nm as a function of Cys equivalent.
Then, the photophysical properties and sensing performances of A1C1 have been studied. In the CH3CN/H2O (3:2, v/v) solution, A1C1 exhibits evident green emission with two peaks at 485 and 505 nm and lifetime of 1560.0 ns, which are attributed to the emission of A1. The emission from C1 at 560 nm can be barely observed. With the addition of Cys to the solution of A1C1, the yellow phosphorescence emission from C1 at 560 nm with lifetime of 355.0 ns enhanced significantly, whereas only the slight variation in blue-green emission from A1 was observed (Fig. 5(b)). Thus, the ratiometric detection based on two phosphorescent signals with the green emission as the internal standard was realized. More importantly, Fig. 5(c) shows an excellent linear relationship from 0 to 25 equiv. of Cys, which is very suitable for Cys detection and quantification, and can increase the sensitivity and minimize the external and environment influences.
We then tested the ability of A1C1 to detect biothiols within living cells by confocal luminescence imaging and lifetime imaging. The living cell imaging experiment was carried out on the Hela cells, and the results are shown in Fig. 6. The optical windows at the blue-green channel (450-520 nm) and yellow channel (530-630 nm) were chosen as two signal outputs for imaging. As shown in Figs. 6(a) and 6(b), obvious luminescence signal was collected for both blue-green and yellow channel under excitation at 405 nm, and an emission intensity ratio of >1.5 at yellow channel to blue-green channel was obtained (Fig. 6(c)). When the Hela cells were preincubated with 200 μM N-ethylmaleimide at 37 °C for 30 min and then incubated with A1C1, the phosphorescence intensity of the blue-green channel remains unchanged while the yellow channel decreased dramatically (Figs. 6(e) and 6(f)), and the emission intensity ratio was reduced to <0.25 (Fig. 6(g)). Furthermore, the photoluminescence lifetime imaging of living cells incubated with A1C1 before and after treatment of NEM was also measured. Before treatment of NEM, the intracellular average lifetime was measured to be 340 ns (Fig. 6(d)). However, after treatment with NEM, the average lifetime was decreased to 160 ns (Fig. 6(h)). Thus, the detection of the changes of Hcy/Cys concentration within living cells could be achieved through ratiometric luminescence and lifetime imaging by A1C1.
Fig. 6 Luminescence images of Hela cells incubated with A1C1 before and after treatment of NEM observed at emission channels of (a,e) 450–520 nm and (b,f) 530–630 nm (λex = 405 nm). (c,g) Ratio of emission intensity at 530–630 nm to that at 450–520 nm. (d,h) Photoluminescence lifetime images (λex = 405 nm).
4. Conclusion
In conclusion, an effective design strategy has been demonstrated for ratiometric and time-resolved luminescence bioprobes based on phosphorescent ion-paired iridium(III) complex. Based on this design concept, an excellent phosphorescent biothiol probe was synthesized and characterized. The utilization of this probe to detect intracellular biothiols has been achieved by ratiometric and time-resolved luminescence imaging with excellent sensing performance. As far as we know, this is the first report about molecular probe for both ratiometric and time-resolved luminescence imaging of intracellular biothiols. We believe that ion-paired phosphorescent complex will be a good platform for further design of various ratiometric and time-resolved luminescence bioprobes.
Funding
National Natural Science Foundation of China (21671108), National Program for Support of Top-Notch Young Professionals, Scientific and Technological Innovation Teams of Colleges and Universities in Jiangsu Province (TJ215006); Natural Science Foundation of Jiangsu Province of China (BK20130038, BK20141422 and BK20160885); Priority Academic Program Development of Jiangsu Higher Education Institutions (YX03001).
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