A microfluidic method for controlled generation and trapping of membraneless water-in-water droplets

Aqueous two-phase systems (ATPS) provide a versatile platform for controlling microscale aqueous environments. Microfluidic devices are particularly valuable for studying such systems, as they allow precise control of geometry, flow, and composition. However, understanding the dynamic behavior of ATPS requires studying the time-dependent exchange between the two phases, which is difficult in existing, water-in-oil microfluidic setups, where the overall composition within each ATPS droplet remains fixed. Here we introduce an oil-free platform that creates ATPS droplets in situ via liquid-liquid phase separation in dead-end chambers, with time-dependent control of the chemical composition in the channel and tunable exchange kinetics set by the connecting geometry. The approach is remarkably robust, reliably forming stable droplets without surfactants or fine flow control. We validate the platform by demonstrating precise droplet control for the well-studied PEG-dextran ATPS, and use it to study an associative coacervate (PAA/PDMAEMA) system, where we observe reversible sub-compartment formation, highlighting the platform's potential for dynamic studies of multiphase systems.