Aqueous Two-Phase Submicron Droplets Catalyze DNA Nanostructure Assembly for Confined Fluorescent Biosensing

Membraneless organelles (MLOs) are fundamental to cellular organization, enabling biochemical processes by concentrating biomolecules and regulating reactions within confined environments. While micrometer-scale synthetic droplets are extensively studied as models of MLOs, submicron droplets remain largely unexplored despite their potential to uniquely regulate biomolecular processes. Here, submicron droplets are generated by a polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS) as a model to investigate their effect on DNA assembly in crowded environments. The findings reveal that submicron droplets exhibit distinct advantages over microdroplets by acting as submicron catalytic centers that concentrate DNA and accelerate assembly kinetics. This enhancement is driven by a cooperative mechanism wherein global crowding from PEG induces an excluded volume effect, while local crowding from dextran provides weak but nonspecific interactions with DNA. By exploiting both the confinement and phase properties of submicron droplets, a rapid and sensitive assay is developed for miRNA detection using confined fluorescent readouts. These findings highlight the unique ability of submicron droplets to amplify biomolecular assembly processes, provide new insights into the interplay between global and local crowding effects in cellular-like environments, and present a platform for biomarker detection and visualization.