Article pubs.acs.org/ac
Ratiometric Iridium(III) Complex-Based Phosphorescent Chemodosimeter for Hg2+ Applicable in Time-Resolved Luminescence Assay and Live Cell Imaging Jiaxi Ru,† Xu Chen,‡ Liping Guan,‡ Xiaoliang Tang,*,† Chunming Wang,‡ Yue Meng,‡ Guolin Zhang,† and Weisheng Liu*,† †
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ‡ School of Life Sciences, Lanzhou University, Lanzhou 730000, China S Supporting Information *
ABSTRACT: A novel iridium(III) complex-based probe Ir4-1 has been designed and synthesized conveniently by incorporating the chemodosimeter into phosphorescent luminophor, which displayed ratiometric luminescence change from yellowish-green to reddish-yellow only toward Hg2+ ions in aqueous media via desulfurization and intramolecular cyclization with a broad pH range of 5−10. The phosphorescent chemodosimeter could eliminate effectively the signal interference from the short-lived fluorescent background, and the signal-to-noise ratio of the detection was improved distinctly by using time-resolved photoluminescence technique. Furthermore, the mechanism of phosphoresce change of the chemodosimeter was analyzed in detail by time-dependent density functional theory (TD-DFT) calculations, and the probe with long-wavelength emission could be applied to label cells and monitor intracellular Hg2+ effectively by luminescence ratio imaging.
A
presence and absence of analytes, which are independent of sensor concentration, sample environment, and light scattering. The detection results for the target analyte are more accurate and visual. Therefore, for the quantification of Hg2+, especially in a complicated analysis system, the development of ratiometric fluorescent sensing is highly desirable.14−16 On the other hand, phosphorescent sensors are considered to be excellent signal transduction materials due to larger Stokes shifts, long excited-state lifetimes, prominent color tenability, and suitable excitation in the visible region in comparison to most purely organic fluorophors.17 By using the time-resolved photoluminescent technique (TRPT), these long-lifetime luminescent complexes can effectively eliminate the undesirable background fluorescence or scattered light in biological samples by appropriate time delay.18 Among various phosphorescent materials, cyclometalated iridium(III) complexes have emerged as suitable candidates in luminescent regulation and biological labels because of their good solubility in most solvents and exceptional charge-transfer properties, which can be tuned over wide ranges by choosing an appropriate ligand.19,20 To date, cyclometalated iridium(III) complexes have been applied in a few chemosensors for specific amino acids,21−23
ccompanied with the rapid development of manufacturing, the diffusion of heavy metal ions from fossil fuel combustion, the burning of coal, metallurgy, mining, chemical industry, pharmaceuticals, and battery manufacturing has caught considerable attention.1 Thus, the signal transduction for detecting toxic heavy-metal ions is a significant issue in fields as diverse as chemistry, biology, and environmental and material sciences.2,3 As one of most widely used metals and dangerous toxins, mercury contamination even at low concentration can be bioaccumulated through the food chain, causing a variety of diseases such as neurological damage, prenatal brain damage, serious cognitive and motion disorders, and increased risk of myocardial infarction.4−6 Therefore, considerable efforts have been documented toward the design and synthesis of various fluorescent sensing systems to rapidly and accurately monitor Hg2+ in the environment in the last few decades.7−10 However, parts of fluorescent chemosensors act only in organic media,11 and some other Hg2+-selective fluorescent sensors are only involved in the fluorescence quench,12,13 which is easily influenced by many external factors including the detector sensitivity, excitation power efficiency, and sample environment (solvent polarity, temperature, pH, redox potential, and so forth). The sensitivity and accuracy of detection would face a severe challenge. It is worth noting that ratiometric fluorescent sensors allow simultaneous recording of two emission intensities at different wavelengths in the © 2015 American Chemical Society
Received: October 13, 2014 Accepted: February 19, 2015 Published: March 3, 2015 3255
DOI: 10.1021/ac503878s Anal. Chem. 2015, 87, 3255−3262
Article
Analytical Chemistry cations,24−27 anions,28−31 and biomolecules.32,33 However, there are only a few designs for detecting toxic Hg2+. For example, Zhao et al. prepared a class of charge-neutral iridium(III) complexes containing sulfur atom that showed multisignaling changes or turn-off phosphorescent signaling behavior by Hg2+-induced decomposition of iridium(III) complex moieties to form a solvent complex.34−38 Hyun and co-workers synthesized aza crown ether appended iridium(III) complex, which exhibited notable phosphorescence quenching for Hg2+ in 50% acetonitrile aqueous solution.39 Nabeshima and co-workers synthesized cationic iridium(III) complexes based on a Hg2+-promoted thioacetal deprotection reaction that exhibited a spectral blue-shift response for Hg2+ in acetonitrile− water (98:2, v/v).40 Although these phosphorescent functional materials have shown potential and unique superiority, the further applications in time-resolved luminescence assay and biological imaging analysis are quite rare. Therefore, it is worthwhile to develop ratiometric phosphorescent chemosensors for Hg2+ detection and to effectively utilize TRPT. To improve the detection selectivity and accuracy, the approach making use of a chemodosimeter as a powerful tool through a specific chemical reaction between dosimeter molecule and target species should be introduced, which has attracted increasing attention and been applied in bioimaging and bioanalysis.41−43 For this kind of chemosensor based on an irreversible chemical reaction, the binding interaction or catalysis of the analyte in the reaction site of chemodosimeter is usually high sensitivity and results in the change of dosimeter in molecular structure and optical characteristics.7,9 Therefore, we envisioned that the incorporation of a chemodosimeter into an iridium(III) luminophor on the key position could trigger irreversible change of the electron-donating effect in donorbridge-acceptor ligand, thus leading to variation of the optical properties of the phosphorescent complex to produce an Hg2+ ratiometric phosphorescent probe. Here, we present the design and synthesis of an iridium(III) complex-based phosphorescent chemodosimeter, [Ir(ppy)2(L)]·PF6 (Ir4-1) (ppy = 2-phenylpyridine) (Scheme 1), which utilizes Hg2+-promoted desulfurization and intramolecular cyclic guanylation of thiourea reaction exploited by Liu and Tian44 and Kim and coworkers.45 In the presence of Hg2+, the chain-like group of
ligand is cyclized and the phosphorescence emission significantly shifts from yellowish-green to reddish-yellow. As far as we know, the ratiometric iridium(III) complex-based chemodosimeter with red-shifted response for Hg2+ in aqueous solution has not yet been systematically designed and reported. In addition, the mechanism of phosphorescence change is analyzed in detail by TD-DFT calculations, and the chemodosimeter has been applied to label cells and monitor intracellular Hg2+ effectively by luminescence ratio imaging.
■
EXPERIMENTAL SECTION General Information and Materials. All reagents and solvents were obtained commercially and used without further purification unless otherwise noted. 1H NMR and 13C NMR spectra were recorded on Bruker DRX400 and Varian INOVA 600 spectrometers and referenced to the solvent signals. Mass spectra (ESI) were performed on Bruker Daltonics Esquire6000 and Bruker MicroTOF ESI-TOF mass spectrometers. UV−vis spectra were recorded on a Shimadzu UV-2550 spectrophotometer. Fluorescence spectra were measured using FLS920 of Edinburgh Instruments equipped with a xenon lamp, 1.0 cm quartz cells. Luminescence quantum yields at room temperature were measured by the optically dilute method46 with an aerated aqueous solution of [Ru(bpy)3]Cl2 (Φem = 0.028, water) as the standard solution.35,47 All pH measurements were made with a pH-10C digital pH meter. Stock solutions (10 mM) of Hg2+ and various other cation ions were prepared by dissolving their perchlorate salts in acetonitrile. Stock solution of Ir4-1 (1 mM) was also prepared in acetonitrile. All tests and cell culture were carried out by diluting these stock solutions to known concentrations with appropriate solvents. Caution! Perchlorate salts are potentially explosive. All compounds containing perchlorates should be handled with great care and in small amounts. Synthesis. The synthetic route is shown in Scheme 1. Ligand 2 was first prepared in two steps in 67% yield. Ir4-1 as a yellow powder was synthesized via a simple one-step reaction of 2 with 0.5 equiv of [Ir2(ppy)4Cl2] in refluxing CH2Cl2/MeOH and further purified by column chromatography on silica gel in 84% yield. The purity of 2, as well as that of complexes Ir4-1 and Ir4-2, was fully confirmed by 1H, 13C NMR, and ESI mass spectrometry analysis (Supporting Information). Computational Details. Calculations were performed using the Gaussian 09 suite of programs.48 The ground-state structures of complexes were optimized using density functional theory (DFT) with Becke’s three-parameter hybrid functional with the Lee−Yang−Parr correlation functional (B3LYP)49 and 6-31+G(d)/LanL2DZ basis set. The LanL2DZ basis set was used to treat the iridium atom,50 whereas the 631+G(d) basis set was used to treat all other atoms.51 The excited-state calculations were carried out with the timedependent density functional theory (TD-DFT) with the optimized structure of the ground state.52 Fifteen singlet absorptions and three triplet emissions were obtained to determine the vertical excitation energies for Ir4-1 and Ir4-2 using TD-DFT calculations, respectively. There are no imaginary frequencies in the frequency analysis of all calculated structures, and each calculated structure expresses an energy minimum. The orbital analyses of the complexes were also performed, and the contours of the main highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) were plotted.
Scheme 1. Synthetic Routes of Chemodosimeter Ir4-1 and Its Cyclized Product Ir4-2
3256
DOI: 10.1021/ac503878s Anal. Chem. 2015, 87, 3255−3262
Article
Analytical Chemistry Time-Resolved Emission Spectra Experiments. Timeresolved emission spectra experiments (TRES) were performed through a time-correlated single photon counting (TCSPC) technique by using a FLS920 instrument (Edinburgh, U.K.) with an LED laser (360 nm) as the excitation source. The luminescence signal from 450 to 780 nm was collected and recorded with a R928-P instrument at a step size of 5 nm. The TRES experiment was performed in duplicate using freshly prepared samples. To exhibit the emission peaks of the complex and Rhodamine 6G (as a typical fluorescent interference) simultaneously, different concentrations of Ir4-1 (50 μM) and Rhodamine 6G (1 μM) were used and mixed in acetonitrile (air-equilibrated) because of the stronger fluorescence of Rhodamine 6G. Photoluminescence spectra at 0 and 100 ns delay were chosen and compared. Cell Culture and Cellular Imaging Experiments. SMMC-7721 cells (human liver cancer cells) were maintained in RPMI-1640 medium supplemented with 10% heatinactivated fetal calf serum, 100 units per mL penicillin, and 100 μg mL−1 streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. HBL cells (human breast epithelial cells) and L929 cells (mouse fibrosarcoma cells) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (10% heat-inactivated fetal calf serum) at 37 °C in 5% CO2. Cells (5 × 108 L−1) were plated on 18 mm glass coverslips and allowed to adhere for 24 h, treated with Ir4-1 (10 μM in cell culture medium), and incubated for 30 min. Subsequently, the cells were treated with Hg(ClO4)2 (100 μM in cell culture medium). Cells were incubated for 30 min and rinsed with PBS three times to remove free compound and ions before analysis. Cells incubated with only 10 μM Ir4-1 for 30 min acted as a control. Subsequent confocal luminescence images of cells were carried out on an Olympus FV1000 laser scanning confocal microscope and a 100× oil-immersion objective lens. The cytotoxic activity experiment of the complex against SMMC-7721 cells was tested according to 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay procedures.
Figure 1. UV−vis absorption change (a) and phosphorescent emission change (b) of Ir4-1 in the presence of Hg2+ ions (10 equiv) in aprotic acetonitrile within 20 min. λex = 360 nm, [Ir4-1] = 10 μM.
(Figure 1b), and the final quantum yield was measured to be 0.018. The changes in both UV−vis and luminescence wavelengths illustrate that the chemical transformation from the chain thiourea unit to an imidazoline moiety modulates the intramolecular charge-transfer (ICT) process of ligand and thereby results in the change of energy levels of iridium(III) complex and the emergence of the ratiometric phosphorescence response. Furthermore, the spectral response could not be found for other metal ions. Sensor Mechanism. ESI mass spectral changes of Ir4-1 in the absence and presence of Hg2+ ion were employed to provide direct evidence of Hg2+-induced desulfurization and cyclization process. The chemodosimeter Ir4-1 displayed a characteristic peak of [Ir4-1−PF6]+ at m/z 874.1733. However, after the addition of Hg2+, the peak completely disappeared and a new peak arose at m/z 840.1265 assigned to [Ir4-2−PF6]+. By reacting Ir4-1 with Hg2+ in acetonitrile at room temperature, 1H NMR spectra of Ir4-1 and the isolated product Ir4-2 were compared. Although there is significant overlap in the aromatic region of the 1H NMR spectra, rendering the exact assignment difficult for both complexes, the changes of the characteristic peaks of N−H groups in ligand could be identified easily. Compared with Ir4-1, two peaks corresponding to N−H groups in Ir4-1 disappeared and another N−H group in the thiourea unit at δ 9.71 ppm shifted downfield in Ir4-2. In addition, the chemical shifts of two methylene groups changed; especially one −CH2− obviously shifted downfield and was split, supporting the cyclization of ligand and the formation of a rigid five-membered ring structure (Figures S1− S3, Supporting Information). Response Time Experiments of Chemodosimeter. For chemodosimeter, luminescence response time depends on the rate of reaction between dosimeter molecule and target species, and the quick response time is an important index in practical application. Thus, efforts were then made to shorten the reaction time and improve the detection efficiency. As shown in Figure S4 (Supporting Information), the time needed for the
■
RESULTS AND DISCUSSION Photophysical Properties. Initial spectroscopic studies of chemodosimeter Ir4-1 were carried out in the aprotic CH3CN to avoid the disturbance from external solvent factors. Ir4-1 (10 μM) exhibited a broad emission at ca. 560 nm at ambient temperature with luminescence quantum yield (Φ) ca. 0.013. The UV−vis absorption spectrum of Ir4-1 displayed a broad energy absorption band centered at 320−470 nm with extinction coefficients (ε) of ∼104 mol−1·L−1·cm−1 (Figure 1a), which could be assigned to an overlap of the spin-allowed metal-to-ligand charge-transfer transition (1MLCT, dπ(Ir) → π*(ppy and L)), ligand-to-ligand charge-transfer transition (1LLCT, π(ppy) → π*(L) or π(L) → π*(ppy)), and intraligand charge-transfer transition (1ILCT, π(L) → π*(L)), as confirmed by TD-DFT calculations (vide infra). Upon addition of Hg2+ (10 equiv) within 20 min, the absorption band of Ir4-1 at 350 nm decreased prominently with a concomitant slight increase in the broad energy absorption from 265 to 310 nm. A well-defined isosbestic point occurs at 319 nm, indicating that only a new species is produced. Also, the luminescence intensity of Ir4-1 at 560 nm (yellowish-green) underwent a significant decrease while a new broad emission at 620 nm (reddish-yellow) increased gradually. A distinct red-shift with an isoemissive point at 600 nm was readily detected as the time went on after addition of Hg2+ 3257
DOI: 10.1021/ac503878s Anal. Chem. 2015, 87, 3255−3262
Article
Analytical Chemistry luminescence intensity of Ir4-1 (10 μM) to gradually reach a stable state at 620 nm in acetonitrile was obviously shortened with the increase of the ratio of [Hg2+]/[Ir4-1]. The progress curve with 20 equiv of Hg2+ clearly demonstrated fast response within 5 min, indicating that the reaction rate of chemodosimeter Ir4-1 with Hg2+ could be efficiently accelerated. In addition, it is worth noting that the luminescence response of Ir4-1 for Hg2+ was markedly quickened with the increase of the water volume fraction, although the luminescence intensity slightly decreases to some extent and the maximum emission peak slightly blue-shifted from 620 to 610 nm because of the solvent effect. The ratiometric phosphorescent change of Ir4-1 for Hg2+ remained relatively stable when the water volume fractions in acetonitrile were >20% and 1.0 with only the positive logarithm value in the presence of Hg2+ ion (Figure S6, Supporting Information). The other metal ions were relatively nonresponsive to the emission peak position of Ir4-1, keeping a negative logarithm value of I610 nm/I560 nm (Figure 2b). To deeply examine the selectivity and anti-interference of Ir4-1, the luminescence sensing behavior for Hg2+ was tested in the presence of various competing ions. Cation-competitive experiments (Figures S7−S9, Supporting Information) also clearly showed that the sensing for Hg2+ was hardly interfered by commonly coexistent metal ions and there was only positive logarithm value of I610 nm/I560 nm when Hg2+ ions exist. Thus, a red-shift of ∼50 nm in the emission spectrum makes Ir4-1 have the potential to serve as a good luminescent probe in ratiometric bioimaging. Further, the emission spectral changes of Ir4-1 (10 μM) were measured with different concentrations of Hg2+ (0−20 μM) in CH3CN−H2O mixture (4/6 v/v) to understand the detection ability of Ir4-1 in lower concentrations of Hg2+. As shown in Figure 3, the luminescence intensity ratios (I610 nm/ I560 nm) of Ir4-1 increased linearly with the amount of Hg2+ in the range 0−10 μM and then achieved a maximum due to the exhaustion of chemodosimeter Ir4-1. The mercury-promoted cyclic guanylation of the chemodosimeter has 1:1 stoichiometry, and a new species Ir4-2 is only produced during the titration of Hg2+. From the linear equation (Figure S10, Supporting Information), the detection limit of probe Ir4-1 for
Figure 2. (a) Luminescent spectra of Ir4-1 upon the addition of various metal ions (Li+, Na+, K+, Mg2+, Ca2+, Al3+, Fe3+, Co2+, Ni2+, Cu2+, Cr3+, Pb2+, Cd2+, Ag+, Mn2+, Zn2+, Fe2+, and Hg2+) within 5 min in CH3CN−H2O mixture solutions (4/6 v/v); (b) the logarithm value of luminescence intensity ratio of Ir4-1 at 610 and 560 nm (I610 nm/ I560 nm) for various metal cations. λex = 360 nm, [Ir4-1] = 10 μM, [Mn+] = 100 μM.
Figure 3. Luminescence spectra change of Ir4-1 upon titration with Hg2+ (0−20 μM) in CH3CN−H2O mixture solutions (4/6 v/v). Inset: Change trend of luminescence intensity ratio at 610 and 560 nm (I610 nm/I560 nm) with the increase of [Hg2+]/[Ir4-1]. λex = 360 nm, [Ir4-1] = 10 μM.
Hg2+ was calculated to be 1.73 × 10−7 M at the signal-to-noise ratio (S/N) = 3, exhibiting that chemodosimeter Ir4-1 was potentially useful for quantitative determination of Hg2+ in solution. 3258
DOI: 10.1021/ac503878s Anal. Chem. 2015, 87, 3255−3262
Article
Analytical Chemistry
energy level is higher. The calculated luminescent emission corresponding to triplet excitation is at 602 nm, which is less than that of Ir4-1 and could be attributed to an admixture of 3 MLCT/3LLCT/3ILCT based on HOMO−1 → LUMO+1 (85.9%). The calculation results strongly confirmed the trend of red-shift in phosphorescent emission of Ir4-1 after addition of Hg2+, which is helpful for designing and predicting ratiometric luminescent molecular probes based on iridium(III) complex in future work (Figure 4 and Tables S7−S10 and Figures S15 and S16, Supporting Information).
The effect of pH on the luminescence of Ir4-1 and the Ir41−Hg2+ system was also investigated. As seen from Figure S11 (Supporting Information), chemodosimeter Ir4-1 is stable within the wide pH range and there appears to be no dramatic shift on the phosphorescent emission spectra. In the presence of Hg2+, the ratiometric phosphorescent response (I610 nm/ I560 nm) also comes up and remains relatively stable within the pH range of 5−10, containing the biologically relevant pH range (5.25−8.93). However, when the pH value was 10.9, Hg2+-triggered desulfurization was influenced and ratiometric response could not happen, which might be due to the protonation of 1,10-phenanthroline ligand at lower pH and the hydrolysis of metal ion at higher pH. Considering the excellent sensing performances of chemodosimeter Ir4-1 in solution, it is possible that Ir4-1 could be used in living cells without interference. Theoretical Calculations of Spectral Changes. To understand the photophysical property and explain the ratiometric phosphorescent response of Ir4-1 in the presence of Hg2+, TD-DFT calculations were performed to estimate the corresponding transition energies of Ir4-1 and Ir4-2 based on their ground-state and excited-state geometries, respectively. The ground-state geometry of Ir4-1 was first optimized by DFT calculation, which exhibits that the chain thiourea unit of the ligand extends outward and a N−H···S hydrogen bond between S atom and NH group in 1,10-phenanthroline is formed. The hydrogen bond length between S and H atoms is 2.482 Å, and the bond angle of N−H···S is 156.834° closing to the line (Figure S12 and Tables S1 and S2, Supporting Information). The UV−vis absorption of Ir4-1 in the visible region was further calculated with TD-DFT, and singlet transitions reveal that the excitations of Ir4-1 are mainly assigned to an overlap of 1LLCT/1MLCT/1ILCT, supported by UV−vis spectral profiles around 320−470 nm. The calculated lowest transition and strongest absorption are at 488 nm (HOMO → LUMO) and 411 nm (HOMO → LUMO+1), respectively, which are partially contributed to by the ICT process in L (Tables S3 and S4 and Figure S13, Supporting Information). The same ground-state geometry and absorption calculation for the product Ir4-2 were handled. The chain thiourea unit is desulfurized and cyclized, showing a rigid molecule skeleton. The singlet transitions are also assigned to an overlap of 1 LLCT/1MLCT/1ILCT. However, the lowest-lying singlet transitions of Ir4-2 corresponding to HOMO → LUMO at 503 nm and HOMO → LUMO+1 at 491 nm are mainly assigned to 1ILCT, which leads more effectively to the ICT process in L. Moreover, the strongest absorption is shifted to 442 nm compared with that of Ir4-1. The discrepancy between the calculated absorption wavelength and the experimental results is in line with the known fact that the calculations usually underestimate the excitation energy for the transitions with ICT character (Tables S5 and S6 and Figure S14, Supporting Information).53,54 The geometry of the lowest-lying triplet excited state of Ir4-1 was optimized to study the emission. The lowest-lying triplet transition of Ir4-1 in the visible region corresponding to triplet excitation is at 573 nm, which is very close to the experimental data and could be assigned to an overlap of 3 MLCT/3LLCT/3ILCT from HOMO to LUMO+1 (65.9%) and 3MLCT/3LLCT from HOMO−1 to LUMO (23.1%). For the emission of Ir4-2, the LUMO+1, LUMO, and HOMO energy levels are lower than those of Ir4-1, but the HOMO−1
Figure 4. Molecular orbital (MO) distributions of complexes Ir4-1 and Ir4-2 corresponding to calculated emission transition. The emission-related calculations were based on the optimized lowestlying triplet excited state.
Application in Time-Resolved Luminescence Assay. Because of the long emission lifetime, iridium(III) complex exhibited promising application in time-resolved luminescent detection, which could remove inevitable interference from undesirable background with short-lived fluorescence and has a significant advantage in real applications compared with fluorescent probes.18,20 In acetonitrile and at ambient temperature, the average emission lifetimes monitored at 560 nm for Ir4-1 and at 620 nm for cyclization product Ir4-2 were 68.76 and 95.80 ns, respectively, exhibiting long enough phosphorescent characters. To demonstrate the advantage of Ir4-1, fluorescent dye Rhodamine 6G with a short emission lifetime of 3.66 ns (1 × 10−4 mol·L−1) in acetonitrile was selected as a typical signal interference,55 whose emission could significantly overlap with the phosphorescence emission of Ir4-1 in the steady-state photoluminescence spectroscopy (Figure S17, Supporting Information). The time-resolved emission spectra of the mixture of Ir4-1 (50 μM) and Rhodamine 6G dye (1 μM) were measured before and after addition of Hg2+ (Figure 5a), which visually displayed the red-shift and the attenuation rate of the emission peaks after delaying a period of time. As shown in Figure 5b, under the excitation at 360 nm, the obvious maximum emission at 550 nm was assigned to the strong fluorescent Rhodamine 6G and the phosphorescence emission of probe Ir4-1 was covered up completely in the absence of Hg2+. There was no obvious change in emission wavelength over time. When 5 equiv of Hg2+ was added, the fluorescence interference at 550 nm from dye still existed and the emission intensity changed to some degree. Moreover, a shoulder peak at ∼620 nm appeared that could be attributed to the phosphorescence emission of Ir4-2. However, time-gated acquisition of the spectrum after 100 ns delay removed 3259
DOI: 10.1021/ac503878s Anal. Chem. 2015, 87, 3255−3262
Article
Analytical Chemistry
Figure 6. Confocal luminescence (a), bright-field (b), and overlay (c) images of SMMC-7721 cells incubated with Ir4-1 (10 μM) in RPMI1640 medium for 30 min at 37 °C (λex = 405 nm). (d) Overlap Z-scan confocal image of the living SMMC-7721 cells incubated with Ir4-1 (10 μM). (e) Luminescence intensity profile and luminescence image (across the line in d) of SMMC-7721 cells.
and the nucleus in stained living cells revealed that Ir4-1 was internalized into the living cells rather than merely staining the membrane surface and nucleus (Figure 6d and e), and the same results also could be observed in different kinds of cells (Figure S20, Supporting Information). Thus, these confocal luminescence images demonstrated that the chemodosimeter had good cell membrane permeability and exclusively stained the cytoplasm. Their facile internalization by cells may be correlative with the positive charge and lipophilicity of the cationic complexes. In addition, the cytotoxic activity of Ir4-1 against SMMC7721 cells has been tested by using MTT assay (Figure S21, Supporting Information). Although Ir4-1 complex at higher concentration would kill the cell, the cell viability of complex for SMMC-7721 cells remained above 80% upon incubation with Ir4-1 at the concentration (10 μM) of the cell imaging experiment. When the concentration of Ir4-1 was
