Controlled Assembly and In Situ Reconfiguration of Multiphase DNA Droplets in a Vibration-Induced Local Vortex Microfluidic Platform

Liquid–liquid phase separation (LLPS) of sequence-designed DNA nanostructures offers a powerful route toward dynamic artificial cells and intelligent molecular robots. However, conventional bulk synthesis suffers from stochastic polydispersity in terms of droplet size and topology, and physical compartmentalization restricts continuous chemical exchange with the external environment. This study reports an upgraded vibration-induced local vortex (VILV) microfluidic platform featuring secondary trapping pillars that decouple a localized microvortex-driven assembly from a global convective flow. This architecture enables highthroughput parallel manufacturing of uniform multiphase DNA droplets and their anchoring within micropillar arrays without washout. By systematically tuning the stoichiometry of the interphase hexavalent DNA linker, precise array-wide programming of the surface valence of the multiphase droplets is achieved, driving predictable structural transitions from monovalent Janus artificial cells to highly connected multivalent patchy networks. Notably, under continuous open perfusion, introducing invader strands rationally engineered to mimic clinically diagnostic microRNA (miRNA) biomarker sequences triggers toehold-mediated strand displacement (TMSD) across phase boundaries. This non-invasive tracking demonstrates linker-density-dependent morphological alterations. This study establishes a versatile foundation for mass-producing synthetic organelles and one-pot molecular diagnostic devices by bridging uniform multiphase artificial cell manufacturing with sequence-specific miRNA responsiveness under continuous fluidic flow.