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Ratiometric Molecular Probes Based on Dual Emission of a Blue Fluorescent Coumarin and a Red Phosphorescent Cationic Iridium(III) Complex for Intracellular Oxygen Sensing.

Yoshihara T, Murayama S, Tobita S - Sensors (Basel) (2015)

Bottom Line: The introduction of the cationic Ir (III) complex improved the cellular uptake efficiency compared to that of a neutral analogue with a tetraproline linker.The complex RP2 with an octaproline linker provided ratios comparable to the ratiometric measurements obtained using a microplate reader: the ratio of the R(I)) value of RP2 under hypoxia (2.5% O2) to that under normoxia (21% O2) was 1.5 and 1.7 for HeLa and MCF-7 cells, respectively.Thus, the intracellular oxygen levels of MCF-7 cells could be imaged by ratiometric emission measurements using the complex RP2.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. yoshihara@gunma-u.ac.jp.

ABSTRACT
Ratiometric molecular probes RP1 and RP2 consisting of a blue fluorescent coumarin and a red phosphorescent cationic iridium complex connected by a tetra- or octaproline linker, respectively, were designed and synthesized for sensing oxygen levels in living cells. These probes exhibited dual emission with good spectral separation in acetonitrile. The photorelaxation processes, including intramolecular energy transfer, were revealed by emission quantum yield and lifetime measurements. The ratios (R(I) = (I(p)/I(f))) between the phosphorescence (I(p)) and fluorescence (I(f)) intensities showed excellent oxygen responses; the ratio of R(I) under degassed and aerated conditions ( R(I)(0) was 20.3 and 19.6 for RP1 and RP2. The introduction of the cationic Ir (III) complex improved the cellular uptake efficiency compared to that of a neutral analogue with a tetraproline linker. The emission spectra of the ratiometric probes internalized into living HeLa or MCF-7 cells could be obtained using a conventional microplate reader. The complex RP2 with an octaproline linker provided ratios comparable to the ratiometric measurements obtained using a microplate reader: the ratio of the R(I)) value of RP2 under hypoxia (2.5% O2) to that under normoxia (21% O2) was 1.5 and 1.7 for HeLa and MCF-7 cells, respectively. Thus, the intracellular oxygen levels of MCF-7 cells could be imaged by ratiometric emission measurements using the complex RP2.

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Emission images of HeLa and MCF-7 cells incubated with RP1, RP2, or C343-Pro4-BTP (2 µM) for 4 h at 37 °C. Scale bar: 50 µm.
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sensors-15-13503-f004: Emission images of HeLa and MCF-7 cells incubated with RP1, RP2, or C343-Pro4-BTP (2 µM) for 4 h at 37 °C. Scale bar: 50 µm.

Mentions: We then examined the cellular uptake properties of RP1 and RP2, and that of C343-Pro4-BTP, which we previously synthesized [72]. Each probe was added to the medium of HeLa or MCF-7 cells at a final concentration of 2 µM, the cells were incubated for 4 h, then washed with medium without phenol red. The excitation wavelength was 400–410 nm. Figure 4 displays the emission images of the living cells observed in the wavelength range over 455 nm, i.e., the total emission of the probes. The emission images of RP1 and RP2 in both cell lines are much brighter than those of C343-Pro4-BTP, suggesting that the cellular uptake efficiencies of RP1 and RP2 are significantly enhanced by replacing the neutral BTP in C343-Pro4-BTP with a cationic BTQphen. This is consistent with our observation that the cellular uptake efficiencies of cationic iridium complexes with a 5-dialkylamino-1,10-phenanthroline ligand are much larger than those of the neutral analogues with an acetylacetonato ligand [46]. Very recently, Tanabe and co-workers have synthesized a water-soluble ratiometric oxygen probe comprising a fluorescent coumarin and a phosphorescent ruthenium complex [71]. The cellular uptake efficiency of this probe seems to be lower than those of our probes, because they have added a much higher concentration of probe (100 µM) into the culture medium compared with that (2 µM) of our probes.


Ratiometric Molecular Probes Based on Dual Emission of a Blue Fluorescent Coumarin and a Red Phosphorescent Cationic Iridium(III) Complex for Intracellular Oxygen Sensing.

Yoshihara T, Murayama S, Tobita S - Sensors (Basel) (2015)

Emission images of HeLa and MCF-7 cells incubated with RP1, RP2, or C343-Pro4-BTP (2 µM) for 4 h at 37 °C. Scale bar: 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507661&req=5

sensors-15-13503-f004: Emission images of HeLa and MCF-7 cells incubated with RP1, RP2, or C343-Pro4-BTP (2 µM) for 4 h at 37 °C. Scale bar: 50 µm.
Mentions: We then examined the cellular uptake properties of RP1 and RP2, and that of C343-Pro4-BTP, which we previously synthesized [72]. Each probe was added to the medium of HeLa or MCF-7 cells at a final concentration of 2 µM, the cells were incubated for 4 h, then washed with medium without phenol red. The excitation wavelength was 400–410 nm. Figure 4 displays the emission images of the living cells observed in the wavelength range over 455 nm, i.e., the total emission of the probes. The emission images of RP1 and RP2 in both cell lines are much brighter than those of C343-Pro4-BTP, suggesting that the cellular uptake efficiencies of RP1 and RP2 are significantly enhanced by replacing the neutral BTP in C343-Pro4-BTP with a cationic BTQphen. This is consistent with our observation that the cellular uptake efficiencies of cationic iridium complexes with a 5-dialkylamino-1,10-phenanthroline ligand are much larger than those of the neutral analogues with an acetylacetonato ligand [46]. Very recently, Tanabe and co-workers have synthesized a water-soluble ratiometric oxygen probe comprising a fluorescent coumarin and a phosphorescent ruthenium complex [71]. The cellular uptake efficiency of this probe seems to be lower than those of our probes, because they have added a much higher concentration of probe (100 µM) into the culture medium compared with that (2 µM) of our probes.

Bottom Line: The introduction of the cationic Ir (III) complex improved the cellular uptake efficiency compared to that of a neutral analogue with a tetraproline linker.The complex RP2 with an octaproline linker provided ratios comparable to the ratiometric measurements obtained using a microplate reader: the ratio of the R(I)) value of RP2 under hypoxia (2.5% O2) to that under normoxia (21% O2) was 1.5 and 1.7 for HeLa and MCF-7 cells, respectively.Thus, the intracellular oxygen levels of MCF-7 cells could be imaged by ratiometric emission measurements using the complex RP2.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515, Japan. yoshihara@gunma-u.ac.jp.

ABSTRACT
Ratiometric molecular probes RP1 and RP2 consisting of a blue fluorescent coumarin and a red phosphorescent cationic iridium complex connected by a tetra- or octaproline linker, respectively, were designed and synthesized for sensing oxygen levels in living cells. These probes exhibited dual emission with good spectral separation in acetonitrile. The photorelaxation processes, including intramolecular energy transfer, were revealed by emission quantum yield and lifetime measurements. The ratios (R(I) = (I(p)/I(f))) between the phosphorescence (I(p)) and fluorescence (I(f)) intensities showed excellent oxygen responses; the ratio of R(I) under degassed and aerated conditions ( R(I)(0) was 20.3 and 19.6 for RP1 and RP2. The introduction of the cationic Ir (III) complex improved the cellular uptake efficiency compared to that of a neutral analogue with a tetraproline linker. The emission spectra of the ratiometric probes internalized into living HeLa or MCF-7 cells could be obtained using a conventional microplate reader. The complex RP2 with an octaproline linker provided ratios comparable to the ratiometric measurements obtained using a microplate reader: the ratio of the R(I)) value of RP2 under hypoxia (2.5% O2) to that under normoxia (21% O2) was 1.5 and 1.7 for HeLa and MCF-7 cells, respectively. Thus, the intracellular oxygen levels of MCF-7 cells could be imaged by ratiometric emission measurements using the complex RP2.

Show MeSH
Related in: MedlinePlus