Journal
FREE RADICAL RESEARCH
Volume 56, Issue 3-4, Pages 258-272Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1080/10715762.2022.2077202
Keywords
Fluorescent probe; time-resolved spectroscopy; reactive oxygen species; pyrene; triphenylphosphonium salt
Categories
Funding
- Occitanie region
- Julius-Maximilians-Universitat Wurzburg
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To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, researchers developed new probes by connecting pyrene derivatives to a triphenylphosphonium salt as a mitochondrial vector. The fluorescence lifetime of pyrene was used to monitor the variation of cellular free radicals and oxygen in real-time. The new probes showed comparable results to previously published probes and had high cellular uptake at a low loading concentration.
To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, we connected pyrene derivatives (PB) to a triphenylphosphonium salt (TPP+) as a mitochondrial vector forming PB-TPP+ probes. Two pyrene isomers with the n-butyltriphenylphosphonium moieties attached at their 1- or 2- positions were synthesized and characterized. Using the long fluorescence lifetime of pyrene, it was possible to monitor the variation of cellular free radicals and oxygen and to follow the reversibility of both quenchers in real-time. We compared the behavior of these new probes to the previously published pyrene-probes, functionalized by a mitochondrial-penetrating peptide, allowing their transfer to the mitochondria (Mito-PB) or to the cytosolic membrane for pyrene butyric acid (PBA). The high cellular uptake of the new probes allows cells to be loaded with an initial concentration 40 times lower than that for Mito-PB probes, without inducing perturbations in cell growth. The variation in free radicals and oxygen levels was monitored within cells under different stress conditions through the fluorescence lifetime of the new TPP+-based probes giving comparable results to those obtained for MPP-based probes. However, at a loading concentration as low as 25 nM, our technique allows the detection of increased production of free radicals in the mitochondria in the presence of the TPP+ vector, a warning to the user of this well-known vector.
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