Article Cite This: J. Am. Chem. Soc. 2018, 140, 7827−7834
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Dual-Phosphorescent Iridium(III) Complexes Extending Oxygen Sensing from Hypoxia to Hyperoxia Kenneth Yin Zhang,† Pengli Gao,† Guanglan Sun,† Taiwei Zhang,† Xiangling Li,† Shujuan Liu,† Qiang Zhao,*,† Kenneth Kam-Wing Lo,*,‡ and Wei Huang*,†,§
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Key Laboratory for Organic Electronics and Information Displays and Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, P. R. China ‡ Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, P. R. China § Key Laboratory of Flexible Electronics and Institute of Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, 30 South Puzhu Road, Nanjing 211816, P. R. China S Supporting Information *
ABSTRACT: Hypoxia and hyperoxia, referring to states of biological tissues in which oxygen supply is in sufficient and excessive, respectively, are often pathological conditions. Many luminescent oxygen probes have been developed for imaging intracellular and in vivo hypoxia, but their sensitivity toward hyperoxia becomes very low. Here we report a series of iridium(III) complexes in which limited internal conversion between two excited states results in dual phosphorescence from two different excited states upon excitation at a single wavelength. Structural manipulation of the complexes allows rational tuning of the dual-phosphorescence properties and the spectral profile response of the complexes toward oxygen. By manipulating the efficiency of internal conversion between the two emissive states, we obtained a complex exhibiting naked-eye distinguishable green, orange, and red emission in aqueous buffer solution under an atmosphere of N2, air, and O2, respectively. This complex is used for intracellular and in vivo oxygen sensing not only in the hypoxic region but also in normoxic and hyperoxic intervals. To the best of our knowledge, this is the first example of using a molecular probe for simultaneous bioimaging of hypoxia and hyperoxia.
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under hypoxic conditions giving rise to fluorescence change, while transition-metal complexes are used for detection and imaging of oxygen contents in cell and in vivo owing to the efficient phosphorescence quenching of the complexes in triplet excited states by oxygen via energy transfer. Both types of probes exhibit fast and sensitive luminescence response toward hypoxia, but the sensing sensitivity toward high oxygen content significantly drops. It is a great challenge to design probes that show sensitive responses toward both hypoxia and hyperoxia. The development of probes for hyperoxia has not been reported until now. Under hyperoxic conditions, the reductases are inactive so that fluorescent organic oxygen probes do not work.24−27 During the development of phosphorescent oxygen probes, most efforts have been put on increasing the sensing sensitivity, improving the biocompatibility, and red-shifting the phosphorescence wavelength of the probes for in vivo biological applications.29−35 For these turn-off type probes, high sensitivity ensures good sensing performance toward
INTRODUCTION Molecular oxygen is important and essential to sustaining life. It is consumed during cellular respiration and takes part in producing adenosine triphosphate (ATP) during oxidative phosphorylation in mitochondria.1,2 The oxygen contents in healthy organs, tissues, and cells are kept in a certain range. Hypoxia and hyperoxia, referring to states in which oxygen supply is insufficient and excessive, respectively, are often pathological conditions. The former is one of the most important features of many diseases including solid tumors,3−5 inflammatory diseases,6,7 and cardiac ischemia,7,8 whereas the latter can cause an increased level of reactive oxygen and nitrogen species (RONS)9−11 and oxygen toxicity syndrome,12,13 leading to damage to lungs,14−16 eyes,17−20 and the central nervous system.21 Therefore, the accurate detection of oxygen contents in biological environments is of great importance to early diagnosis of these diseases. Many efforts have been made to develop luminescent probes for intracellular oxygen.22,23 Current achievement mainly includes two families of probes, fluorescent organic dyes24−27 and phosphorescent transition-metal complexes.28−30 Organic dyes are designed to respond to specific reductases irreversibly © 2018 American Chemical Society
Received: March 4, 2018 Published: June 6, 2018 7827
DOI: 10.1021/jacs.8b02492 J. Am. Chem. Soc. 2018, 140, 7827−7834
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Journal of the American Chemical Society
Figure 1. Structures of the iridium(III) complexes and their photoluminescence spectra in PBS/CH3OH (9:1, v/v) under N2 (blue), air (green), and O2 (red) atmospheres. Insets are the corresponding photographs of complex 2b upon excitation by a UV lamp.
state.42 In principle, dual-emissive probes should fulfill two basic requirements, rich excited-state characters and limited electronic communication between the states, to allow possible radiative decays occurring from two different states. Fraser reported luminescent polymers that displayed fluorescence and phosphorescence simultaneously under hypoxic conditions at room temperature.43 The phosphorescence was sensitively quenched by O2, rendering ratiometric response toward trace oxygen. Transition-metal complexes display long-lived phosphorescence involving both metal- and ligand-centered orbitals, resulting in a diversity of emissive-state characters.44 Phosphorescent iridium(III) polypyridine complexes of a general formula [Ir(N∧C)2(N∧N)](PF6) coordinated with two types of ligands exhibit intense phosphorescence from triplet metal/ligand-to-ligand charge transfer (3CT) or intraligand (3IL) excited states depending on the electronic structures of the ligands.45,46 Incorporation of functional groups baring unconjugated lone pair electrons, such as amino groups, into the structure of the complexes provides occupied lone pair orbitals, giving rise to additional electronic transitions to unoccupied π* orbitals, which may interrupt the internal conversion of the excited complex to the lowest excited states, resulting in dual phosphorescence from two independent states.47−49 However, a direct link between the chemical structures of the iridium(III) complexes and the occurrence of dual phosphorescence has not yet been established, which is essential to further the rational design of single component dual-emissive small molecular probes that
hypoxia but, in the meantime, indicates reduced sensing ability toward hyperoxia because the phosphorescence becomes weak under normoxic (normal oxygen level) conditions. Increased excitation power is usually required to obtain trustworthy phosphorescence signals under hyperoxic conditions. In this respect, wavelength-ratiometric probes are good candidates for hyperoxia sensing. The intensity ratio of two wavelengths is used to reflect the oxygen content. Thus, the influence from the inherent variability in incident laser power and the uncertainty of intracellular probe concentration is minimized.36 Current design strategy of ratiometric probes relies on incorporation of an additional nonsensitive fluorescent compound into phosphorescent probes as an internal standard, and the phosphorescence/fluorescence ratio is used for oxygen sensing.37−41 Even so, the utilization of probes based on the covalent or noncovalent assembly of phosphorescent and fluorescent constituents for oxygen sensing is still focused on the hypoxia region, while there is no study extending to hyperoxia sensing because of lack of suitable probes. Compared to the bicomponent probes, single component small molecular dual-emissive luminophores are easy to synthesize. Their small molecular size also facilitates cellular internalization. Additionally, the exclusion of uncontrollable intramolecular energy/electron transfer will improve the accuracy of sensing. The development of single-component dual-emissive smallmolecular luminophores is slow because, as stated by Kasha’s rule, photon emission occurs only from the lowest excited 7828
DOI: 10.1021/jacs.8b02492 J. Am. Chem. Soc. 2018, 140, 7827−7834
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Journal of the American Chemical Society
Figure 2. Selected TD-DFT-calculated energy levels, isosurface plots, and triplet transitions of complexes 1a−1d, 2a, and 2b.
escence lifetimes of the HE and LE bands of complex 1c allowed ratiometric luminescence response toward O2. The phosphorescence spectra of complex 1c were recorded in aqueous phosphate buffered saline (PBS)/methanol (9:1, v/v) under an atmosphere of N 2 , air, and O 2 , and the phosphorescence lifetimes of the HE and LE were measured. To highlight the spectrum-profile response toward O2, the luminescence spectra were normalized by dividing by the number of total photons emitted, namely, the integral of the spectrum, and they are shown in Figure 2. Complex 1c exhibited a HE emission band at ca. 520 nm with a LE shoulder at ca. 620 nm under N2. The emission lifetimes at the two wavelengths were determined to be 2.5 μs and 24 ns, respectively, indicating that their sensitivities toward oxygen quenching were different from each other by more than 100 times. As expected, the HE emission was significantly quenched in aerated solution, resulting in predominance of a broad LE band. The HE emission lifetime was shortened to 0.45 μs, while that of the LE luminescence remained 24 ns. Bubbling neat O2 into the solution led to further shortening of the lifetime of HE emission to 0.14 μs, while the LE emission lifetime was slightly shortened to 20 ns. However, the spectral difference was not significant because the HE band was embedded into the LE emission even under ambient air. We aim to tune the dual phosphorescence properties of the complex via structural manipulation to enable target complexes to exhibit remarkable spectral response toward both hyperoxia and hypoxia. To investigate the effect of the amino groups in the cyclometalating ligands on the dual phosphorescence properties and thus the spectral response toward O2, we first changed the number of substituents bound to the nitrogen atom of the amino group, synthesizing three complexes 1a, 1b, and 1d, which do not contain an amino group or contain primary and tertiary amino groups, respectively (Figure 1). All these complexes have been characterized by 1H NMR, 13C NMR, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS), electrospray ionization (ESI) high-resolution MS, IR, and UV−vis absorption spectroscopy (see Supporting Information). Normalized luminescence spectra of these complexes in PBS/methanol (9:1, v/v) were recorded under an atmosphere of N2, air, and O2 as shown in Figure 1. The photoluminescence quantum
display wavelength-ratiometric response toward specific analytes. In this work, we designed and synthesized a series of iridium(III) complexes containing aminomethyl substituted phenylpyridine ligands and investigated their dual-phosphorescence properties. The large difference in emission wavelengths and lifetimes of the two phosphorescence bands rendered these complexes single-component small-moleculebased ratiometric sensors for molecular oxygen. Structural manipulation of the complexes allowed rational tuning of the internal-conversion efficiency and thus the dual-phosphorescence properties and the spectral profile response of the complexes toward oxygen. Interestingly, one of the piperidinomethyl substituted complexes exhibited naked-eye distinguishable green, orange, and red emission in aqueous buffer solution under an atmosphere of N2, air, and O2, respectively. This complex was further used for simultaneously imaging both hypoxia and hyperoxia in living cells, zebrafish, and mice with distinct spectral responses.
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RESULTS AND DISCUSSION We have recently unexpectedly developed a series of iridium(III) complexes, including complex 1c (Figure 1), that exhibit spectrum-resolved dual phosphorescence.46,48 The high energy (HE) and low energy (LE) phosphorescence bands have been assigned to 3IL and 3CT excited states, respectively. Herein we aim to establish a relationship between the chemical structures of the iridium(III) complexes and the occurrence of dual phosphorescence and to develop novel ratiometric probes for both hypoxia and hyperoxia based on the dual-phosphorescent iridium(III) complexes. According to Stern−Volmer equation,50 I0 τ = 0 = 1 + kqτ0[O2 ] I τ
where I0 and I are phosphorescence intensities in the absence and presence of O2, respectively, τ0 and τ are lifetimes of the emissive excited state in the absence and presence of O2, kq is the quenching rate coefficient, and the quenching efficiency (KSV = kqτ0, Stern−Volmer constant) is proportional to the phosphorescence lifetime. A longer excited-state lifetime favors the nonradiative quenching. Hence, the different phosphor7829
DOI: 10.1021/jacs.8b02492 J. Am. Chem. Soc. 2018, 140, 7827−7834
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2b were 0.36 μs and 36 ns, respectively, under ambient air. Bubbling pure N2 and O2 into the solution tuned the phosphorescence colors sharply to green and red, respectively (Figure 1). The lifetime of HE phosphorescence was correspondingly elongated and shortened while that of the LE one was negligibly altered. Taken together, we tentatively conclude that, in the series of iridium(III) complexes we studied, the nonconjugated amino groups give rise to additional electronic transitions from the lone pairs to the π* orbitals of the diimine ligands, leading to new 3NLCT excited states. Once the energy of the new state stands between those of the traditional emissive states (3IL and 3 CT), the internal conversion from the HE state to the LE one is inhibited, resulting in occurrence of dual phosphorescence from two independent emissive states (Figure 3). This is
yields are listed in Table S1. Interestingly, only the tertiaryamine-containing complex 1d, similar to complex 1c, displayed dual phosphorescence upon photoexcitation and O2 quenching-induced luminescence spectral response. Both the aminefree and the primary-amine-containing complexes 1a and 1b exhibited intensity quenching of the single broad LE band in the presence of O2 without wavelength shift. Theoretical timedependent density functional theory (TD-DFT) calculation revealed that the frontier orbitals contributable to the emissive states are quite similar in energy for all four complexes (Figure 2 and S1). Since both triplet states TIL and TCT originating from HOMO to LUMO + 2 and LUMO, respectively, are present with similar energy for all the complexes 1a−1d, the occurrence of dual phosphorescence depends on the efficiency of internal conversion from TIL to TCT. The most significant difference among these complexes is the occupied orbital localized at the lone pair of the amino groups, which is absent for complex 1a, occurs at a low-energy level (−6.59 eV, HOMO − 5) for complex 1b, becomes higher in energy for complex 1c (−6.02 eV, HOMO − 1), and moderately contributes to the HOMO for complex 1d (Figure 2). The high energy levels of the amine lone pairs of complexes 1c and 1d give rise to additional 3NLCT (n → π*) transitions (Figure 2), which are probably the cause of limited electronic communications between TIL and TCT states. Although complexes 1c and 1d exhibit sensitive and efficient spectral response toward hypoxia, the huge difference in luminescence lifetimes between HE TIL and LE TCT states rendering highly efficient oxygen quenching of the HE TIL emissive state limits the sensitivity of the luminescence spectrum profile toward hyperoxia. To reduce the quenching sensitivity of the HE phosphorescence by oxygen, complexes 2a and 2b (Figure 1) were designed, synthesized, and characterized. We expected that the additional electronwithdrawing amide substituent in the diimine ligand should stabilize the π* orbital of the diimine ligand, hence facilitating internal conversion from the TIL to the TCT states, shortening the excited-state lifetime of the HE phosphorescence, and thus reducing the quenching efficiency. Theoretical TD-DFT calculation confirmed a lower π*-orbital (LUMO) energy of the diimine ligands (about −2.70 eV) for both complexes compared to complexes 1a−1d (about −2.50 eV, Figure 2). The energy of π orbitals of the cyclometalating ligands is also slightly reduced owing to the inductive effect along the trans N−Ir−C bonds. Interestingly, the secondary-amine-substituted complex 2a only exhibited a broad LE emission band at 620 nm even under 100% N2 atmosphere upon photoexcitation (Figure 1). The LE phosphorescence was slightly quenched by O2 owing to the short lifetime (τ0 = 31 ns). In contrast, the tertiary-amine-substituted complex 2b exhibited dual phosphorescence with comparable HE and LE intensities in PBS under ambient air, giving an orange phosphorescence color (Figure 1). The occurrence of dual phosphorescence of complex 2b is probably due to the presence of a 3NLCT (n → π*) (T2, 2.12 eV) state inhibiting the communication between the 3IL (T6, 2.65 eV) and 3CT (T1, 1.79 eV) states (Figure 2). For complex 2a, the 3NLCT (T7, 2.78 eV) is much lower in energy than both 3IL (T4, 2.67 eV) and 3CT (T1, 1.76 eV) states and thus does not disturb their communication. The comparable intensity of the HE and LE luminescence bands of complex 2b under normoxic conditions renders the complex a potential candidate for both hyperoxia and hypoxia sensing. The lifetimes of the HE and LE phosphorescence of complex
Figure 3. (A) Structure skeleton of a potential dual phosphorescent iridium(III) complex. (B) Energy diagrams showing molecular orbitals involved in IL, NLCT, and CT transitions. (C) Energy diagrams of ground and excited states showing that the NLCT state interrupts the internal conversion, resulting in dual phosphorescence from both 3CT and 3IL states.
important and helpful to furthering the rational design of single component dual-emissive small molecular probes that display wavelength-ratiometric response toward specific analytes. Owing to the naked-eye distinguishable emission colors under different atmospheres, we further record the phosphorescence spectra of complex 2b under different oxygen contents in detail. The normalized luminescence spectra highlighting the spectral response and the lifetimes of HE and LE phosphorescence are shown in Figure 4. A good and exciting spectral sensitivity was observed in both hypoxia and hyperoxia regions and has been ascribed to the different quenching efficiencies of the HE and LE phosphorescence by triplet
Figure 4. Phosphorescence spectral response and phosphorescence lifetimes of complex 2b in PBS/CH3OH (9:1, v/v) toward 0 to 100% O2 and Stern−Volmer plots of the HE (green) and LE (red) phosphorescence toward oxygen quenching. 7830
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Journal of the American Chemical Society molecular oxygen. Linear Stern−Volmer plots were obtained (Figure 4), indicating that the internal conversion efficiency depends on the chemical structure of the complex only and that it is independent of the oxygen contents. The quenching sensitivities, that is, the KSV values, of the HE and LE phosphorescence were 0.15 and 0.0031%−1, respectively (Figure 4). The 48 times gap in the KSV values was in the same order of magnitude as the 44 times difference in phosphorescence lifetimes in the absence of O2. To further confirm that the ratiometric spectral response of complex 2b toward O2 was due to the different quenching efficiency of the two excited emissive states, we synthesized two model complexes, 2c and 2d, in which the diimine and cyclometalating ligands were replaced by acetylacetone and 2-(4methylphenyl)pyridine, respectively (Figure 1). Upon photoexcitation, complexes 2c and 2d, respectively, exhibited single HE and LE phosphorescence at 516 and 608 nm with lifetimes of 0.72 μs and 33.4 ns. The oxygen quenching efficiency of complex 2c was remarkably higher than that of complex 2d (Figure 1). The KSV values for O2 quenching of complexes 2c and 2d were determined to be 0.13 and 0.0042%−1, respectively, which were similar to the values for the HE and LE phosphorescence of the dual-emissive complex 2b. The highly sensitive phosphorescence spectra of complex 2b toward O2 encouraged us to use it for detection of both hypoxia and hyperoxia in living cells. The 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed that the complex did not cause any viability loss in our experimental conditions during the following experiments (Figure S2). Phosphorescence pH titration ensured that the spectral response of complex 2b maintained high sensitivity in physiology pH range (Figure S3). The phosphorescence spectra recorded at different temperatures (10−50 °C) showed that increasing temperature reduced the phosphorescence intensity but did not change the spectral profile much (Figure S4). The stability of the complex in serum solution has also been evaluated. In the presence of fetal bovine serum (FBS), the phosphorescence spectral profile of complex 2b in PBS buffer did not exhibit noticeable change, and the sensitive profile response toward both hypoxia and hyperoxia was maintained (Figure S5). HeLa, HepG2, and 3T3 cells incubated with complex 2b (5 μM) for 2 h showed substantial cytoplasmic staining (Figure 5). The phosphorescence signals collected from the green (520 ± 20 nm) and red (620 ± 20 nm) channels were similar in intensity. Interestingly, when the
cells were cultured under low (5%) and high (50%) oxygen environments, the green phosphorescence was much brighter or dimmer than the red one, respectively (Figure 5). It is mentionable that in order to highlight the spectral-profile response, the images of cells under different culture atmospheres were taken under most suitable but different excitation laser powers. Thus, we cannot analyze the signals from the green or red channels independently. It is also an important and attractive advantage of dual emissive probes that the spectral profile or the intensity ratio of two luminescence bands is independent of the excitation laser power and probe concentration. Additionally, the phosphorescence spectra of the internalized complex 2b were recorded via a λ-scanning mode of photoluminescence confocal microscopy. As shown in Figure 5, the cytoplasmic complex displayed a broad emission band covering 480−680 nm when the three types of cells were cultured under ambient atmosphere. The green and red phosphorescence became predominant when the cells were cultured in hypoxic and hyperoxic conditions. This is the first demonstration that a small molecular probe showed sensitive spectral response toward both intracellular hypoxia and hyperoxia. To demonstrate real-time intracellular oxygen sensing, we designed the following experiment. A coverslip was used to cover the complex 2b-loaded HepG2 cells, and 50% O2 was sustainably and slowly bubbled into the culture dish (Figure 6). Thus, the oxygen content became higher and higher in the dish but became lower and lower under the coverslip because of consumption by cellular respiration and limited gas circulation. Before bubbling oxygen, the cells display green
Figure 6. Laser-scanning luminescence microscopy images of complex 2b loaded HepG2 cells covered at the top right-hand corner by a coverslip upon bubbling gas mixture of 50% O2, 45% N2, and 5% CO2 for different times and intracellular phosphorescence spectra of cells in selected areas after bubbling of the gas mixture for 30 min. Scale bar: 300 μm.
Figure 5. Laser-scanning luminescence microscopy images and intracellular phosphorescence spectra of HepG2, HeLa, and 3T3 cells incubated with complex 2b (5 μM, 2 h, 37 °C) cultured under 5% (blue), 21% (green), and 50% (red) oxygen conditions. Scale bar: 30 μm. 7831
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similar when the mouse was in ambient conditions (Figure 8). The LE/HE ratio remained almost unchanged for at least 4 h.
and red phosphorescence with comparable intensities. As bubbling proceeded, the HE phosphorescence of the cells under the coverslip became dominant while the uncovered cells exhibited predominant red phosphorescence (Figure 6). The cells at the edge of the coverslip maintained green and red dual-phosphorescence. The phosphorescence spectra of the covered and uncovered cells and those at the edge of the coverslip have also been recorded and shown in Figure 6, demonstrating remarkable spectral response of complex 2b toward intracellular hypoxia and hyperoxia. Complex 2b has further been used for oxygen monitoring in zebrafish. The complex (10 μM, 50 pL) was injected into 4day-old zebrafish at the head. Upon photoexcitation, the whole zebrafish was emissive intensely (Figure 7). Photolumines-
Figure 8. In vivo images of the complex 2b-injected mouse under ambient conditions for 4 h (A) and after breathing pure oxygen (B) and 10% oxygen (C) for 4 h. In vivo images of the HepG2 tumor bearing mouse injected with complex 2b into the tumor (circled) and normal tissue (uncircled) (D). Bar chart showing the intensity analysis of signals from images A−D (E). Error bars represent the standard deviations of six independent measurements.
When the 2b-injected mouse was allowed to breathe in a pure oxygen atmosphere for 4 h, the HE intensity became 60% of the LE intensity, and the intensity ratio of LE/HE was remarkably increased to about 1.68 (Figure 8), indicative of a high sensitivity of complex 2b toward in vivo hyperoxia. After the mouse was allowed to breathe a nonlethal low-oxygen atmosphere (10%), the LE/HE ratio became as low as 0.63, demonstrating the sensitivity of complex 2b toward in vivo hypoxia. Since hypoxia is one of the most important characteristics of solid tumors, complex 2b was used to distinguish the tumor from normal tissue owing to its spectral response toward hypoxia. The complex was intratumorally injected into the intravenous HepG2 tumor of a male nude mouse and intravenously injected into normal tissue of the same mouse. In the normal tissue, the HE and LE intensities of the complex were similar (Figure 8). In the solid tumor, both the HE and LE emission was reduced in intensity due to the deeper injection depth. Interestingly, the HE intensity is 1.62 fold of the LE intensity in the solid tumor (Figure 8), demonstrating the high sensitivity of complex 2b toward in vivo hypoxia. It is noteworthy that errors in the luminescence ratio (blue bars) are obviously smaller than those in luminescence intensity (green and red bars), demonstrating the advantage of ratiometric sensing.
Figure 7. Laser-scanning luminescence microscopy images, photoluminescence lifetime image, and time-gated photoluminescence image (gated time = 5 ns) of zebrafish injected with complex 2b (1 μM, 50 pL) at the head area, and laser-scanning luminescence microscopy images and in vivo luminescence spectra of the complex 2b-injected zebrafish under normoxia, hyperoxia, and hypoxia conditions.
cence lifetime imaging indicated that the long-lived phosphorescence signals were only localized in the head area, while intense short-lived autofluorescence was observed from the belly (Figure 7). Owing to the long-lived phosphorescence of complex 2b, the short-lived autofluorescence was completely filtered off when a time-gate of 5 ns was applied between photoexcitation and signal collection (Figure 7). The response of the phosphorescence signals from the head area toward O2 was analyzed. Bubbling 5% and 50% O2 into the culture media of the zebrafish did not cause noticeable effect on the red phosphorescence (620 ± 20 nm) but enhanced and quenched, respectively, the green phosphorescence (520 ± 20 nm). The phosphorescence spectra clearly showed the sensitive profile response toward in vivo hypoxia and hyperoxia (Figure 7). Furthermore, complex 2b was used to sense hypoxia and hyperoxia in living mice. The complex (50 μM, 50 μL) was intravenously injected into a male nude mouse. Imaging was performed under excitation at 420 nm, and the luminescence at HE (520 ± 20 nm) and LE (620 ± 20 nm) was extracted from autofluorescence via spectra unmixing for intensity analysis. The HE and LE intensities of the complex were
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CONCLUSION Although the photophysical properties of transition-metal complexes have been extensively studied and their applications in biological sensing and imaging have been widely investigated, the knowledge of dual phosphorescence has not been systematically built up and the application of dualphosphorescent complexes in biosensing and bioimaging is still rare. In this work, we investigated the dual phosphorescence properties of a series of iridium(III) complexes of general formula [Ir(N∧C)2(N∧N)](PF6). The internal conversion from TIL to TCT states can be inhibited by additional 7832
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nonconjugate amino groups in the cyclometalating ligands with high lone-pair energy levels but facilitated by a low-energy π*(N∧N) orbital. Simultaneous occurrence of phosphorescence from the two states is possible by rational structure manipulation tuning the energy levels of related molecular orbitals to allow the energy of 3NLCT to lie between those of 3 IL and 3CT. The small-molecular dual-phosphorescent complexes are attractive candidates for development of wavelength-ratiometric probes, which display distinct spectrum-profile response toward a variety of analytes, especially disease related factors. Sensitive spectral response of the dualemissive probes allows a naked-eye recognizable color change, which is independent of probe concentration, excitation power, or tissue depth. By tuning the sensitivity of the two excited states, we can broaden the response interval toward the specific analyte. For example, we demonstrated the utilization of rationally designed dual phosphorescent complex 2b for intracellular and in vivo oxygen sensing not only in hypoxia region but also in normoxia and hyperoxia ranges. To the best of our knowledge, this is the first example that a small molecular probe showed sensitive spectral response toward both hypoxia and hyperoxia. The development of dual-emissive complexes with two red or infrared phosphorescence bands for deep hypoxia and hyperoxia sensing is underway. Apart from what has been demonstrated in this work, incorporation of two recognition units into the dual phosphorescent skeleton is possible to allow the two emissive states to be sensitive toward multiple analytes simultaneously. We anticipate wide application prospects for the small-molecular dual-phosphorescent probes in bioimaging, biosensing, and disease diagnosis.
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ASSOCIATED CONTENT
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b02492. Synthesis, characterization, computational details, experimental information, and additional figures (PDF)
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AUTHOR INFORMATION
Corresponding Authors
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[email protected] ORCID
Qiang Zhao: 0000-0002-3788-4757 Kenneth Kam-Wing Lo: 0000-0002-2470-5916 Wei Huang: 0000-0001-7004-6408 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Juan Song, Qi Wu, and Xuecheng Wang for their help and valuable discussions. We thank National Natural Science Foundation of China (Grants 61775104, 21501098, 51473078, and 21671108), National Program for Support of Top-Notch Young Professionals, Natural Science Foundation of Jiangsu Province of China (Grant BK20150833), Scientific and Technological Innovation Teams of Colleges and Universities in Jiangsu Province (Grant TJ215006), and Priority Academic Program Development of Jiangsu Higher Education Institutions (Grant YX03001) for financial support. 7833
DOI: 10.1021/jacs.8b02492 J. Am. Chem. Soc. 2018, 140, 7827−7834
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