Construction of FRET‐Based Off‐On Fluorescent Nanoprobes for Sensitive Detection of Intracellular Singlet Oxygen

Abstract Singlet oxygen (¹O₂) detection remains an active area of research in the field of biological and biomedical science. In this work, we reported a novel Off‐On type FRET‐based fluorescent ¹O₂ nanoprobe comprising the energy‐donating conjugated polymer, poly(9,9‐dioctylfluorenyl‐2,7‐diyl) (PFO), and the energy‐accepting ¹O₂ indicator, 3‐diphenylisobenzofuran (DPBF). Prior to the reaction with ¹O₂, the nanoprobe was in Off‐state because fluorescence of PFO (λ_em=441 nm) was completely quenched by loaded DPBF; in the presence of ¹O₂, it switched to On‐state due to the depletion of DPBF. The as‐prepared ¹O₂ nanoprobe had a particle size of ∼59 nm, good biocompatibility, high specificity and sensitivity with a limit of detection of 353 nM. Importantly, the fluorescent nanoprobes were highly sensitive to ¹O₂ under two‐photon excitation (λ_ex=800 nm). Using a multiphoton confocal microscope, generation of intracellular ¹O₂ during photodynamic therapy was sensitively detected by recording fluorescence intensity of the nanoprobes.