Nile red-doped fluorescent semiconducting polymer dots as a highly sensitive hydrophobicity probe: protein conformational changes detection and plasma membrane imaging

One-pot synthesis of a photostable, hydrophobic and green fluorescent probe remains a challenge to develop luminescent biomaterials as well as biomedia technology. Semiconducting polymer dots (PDs) hold a great promise as fluorescence nanoprobes due to their photostability, biocompatibility and high quantum yield. Here, the synthesis and characterization of Nile red-doped PFBT PDs as a highly sensitive hydrophobicity probe has been reported. Nile red, a phenoxazone dye, fluoresces intensely in organic solvents and hydrophobic lipids and in varying color; however its fluorescence is totally quenched in water. First, Nile red-doped poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1′,3)-thiadiazole)] (PFBT) PDs were synthesized through a nano-precipitation technique. Energy transfer from PFBT to Nile red happens through FRET mechanism. The PDs exhibited a red fluorescence with a peak centered at 620 nm (photo-excited at 460 nm). In aqueous media, the fluorescence strongly quenched, but by increasing the hydrophobicity the fluorescence of PDs significantly enhances as the polarity of the medium decreases. This can happen by adding organic solvents or nonpolar species to the medium. The Nile red@PFBT PDs have remarkable specification over Nile red dye. Compared to Nile red, this probe is soluble in water, more photostable, and more brightness due to light-harvesting ability of PDs and less wavelength shift in different medium polarity. We used this probe to investigate polarity of different organic solvents, to study surface hydrophobicity changes of BSA and also Ca²⁺ determination due to conformational changes of calmodulin. Also we used this probe for imaging of lipid bilayers and cellular membrane.