Ratiometric Nanoparticle Probe Based on FRET-Amplified Phosphorescence for Oxygen Sensing with Minimal Phototoxicity

Luminescent oxygen probes enable direct imaging of hypoxic conditions in cells and tissues, which are associated with a variety of diseases, including cancer. Here, a nanoparticle probe that addresses key challenges in the field is developed, it: i) strongly amplifies room temperature phosphorescence of encapsulated oxygen-sensitive dyes; ii) provides ratiometric response to oxygen; and iii) solves the fundamental problem of phototoxicity of phosphorescent sensors. The nanoprobe is based on 40 nm polymeric nanoparticles, encapsulating ≈2000 blue-emitting cyanine dyes with fluorinated tetraphenylborate counterions, which are as bright as 70 quantum dots (QD525). It functions as a light-harvesting nanoantenna that undergoes efficient Förster resonance energy transfer to ≈20 phosphorescent oxygen-sensitive platinum octaethylporphyrin (PtOEP) acceptor dyes. The obtained nanoprobe emits stable blue fluorescence and oxygen-sensitive red phosphorescence, providing ratiometric response to dissolved oxygen. The light harvesting leads to ≈60-fold phosphorescence amplification and makes the single nanoprobe particle as bright as ≈1200 PtOEP dyes. This high brightness enables oxygen detection at a single-particle level and in cells at ultra-low nanoprobe concentration with no sign of phototoxicity, in contrast to PtOEP dye. The developed nanoprobe is successfully applied to the imaging of a microfluidics-generated oxygen gradient in cancer cells. It constitutes a promising tool for bioimaging of hypoxia.