Click'n ROPISA: One-Pot Synthesis of Ultrabright Fluorescent Nanoparticles Made of Polypeptides

Fluorescent nanoparticles are widely used in bioimaging and sensing, yet their preparation relies on multistep synthetic routes that limit scalability and structural control. Here we introduce a one-pot strategy that couples aqueous ring-opening polymerization-induced self-assembly (ROPISA) of γ-propargyl-L-glutamate N-carboxyanhydride with copper-catalyzed azide-alkyne cycloaddition (CuAAC) to produce fluorescent polypeptide nanoparticles. This one-pot "Click'n ROPISA" process enables the simultaneous formation of polypeptide nano-objects and covalent incorporation of diverse azido-functionalized fluorophores. The resulting assemblies exhibit tunable emission across the visible spectrum and are accessible at high solids content while maintaining high brightness and structural uniformity. Dense fluorophore incorporation enables efficient Förster resonance energy transfer (FRET) both within the assemblies and with dye-labeled proteins, supporting sensing applications. The intense brightness of the red-emitting nanoparticles further enables single-particle tracking in microscopy toward advanced bioimaging.