Optimization of Fluorescent Nanoparticles Architecture for Efficient Brightness, FRET Efficiency and Biosensing

Precise control over fluorophore localization within polymer nanoparticles remains a major challenge, limiting the rational optimization of brightness, Förster Resonance Energy Transfer (FRET), and biosensing performance. Here, we report architecturally controlled donor core-shell-crown fluorescent polymer nanoparticles synthesized via a controlled radical polymerization in miniemulsion through a one-pot process performed entirely in water at high solid content. The polymerizable BODIPY monomers are covalently incorporated into the polymer backbone, preventing dye leakage while enabling spatial confinement within defined compartments. By selectively positioning the dye in the core, the shell, or both, we decouple brightness and donor-acceptor distance effects. Core-confined dyes maximize brightness by reducing water-induced quenching, whereas shell-localized dyes enhance FRET efficiency (up to 75%) by minimizing donor-acceptor separation, revealing a brightness/distance trade-off analogous to that observed in inorganic nanomaterials. Exciton migration and multivalent surface functionalization further amplify energy transfer within each nano-object. In a competitive biotin-streptavidin proof of concept assay, nanoparticle brightness emerges as the dominant parameter controlling analytical sensitivity. The optimized sensor achieves a limit of detection of ∼4-8 nM in different buffers or artificial fluids, and using both a spectrofluorometer and a low-cost LED-based device, highlighting the potential of these scalable, metal-free nanotransducers for point-of-care biosensing.