Continuous Generation of Droplets Encapsulating two Single Particles Using an on-Chip “Virtual Particle Valve” Integrated with Micropillar Arrays

We propose a novel microfluidic system for continuously generating droplets co-encapsulating two single particles (e.g., a cell and a bead) using a “virtual particle valve” integrated with micropillar arrays. This system features a cross-shaped microfluidic droplet generator (MFDG) composed of a rhombic-shaped reservoir connected to a constriction channel. This configuration enables the trapping and synchronized release of particles via controlled microvortex formation at the cross junction, functioning as a virtual valve. By fine-tuning flow rates, we achieved dynamic control over vortex formation, enabling precise manipulation of particle trapping and release. Additionally, we demonstrated size-based separation of cell-bead encapsulated droplets from predominantly empty ones using deterministic lateral displacement (DLD) micropillar arrays. The proposed system achieved a 3 % co-encapsulation rate, representing a ~ 1000- fold improvement over the theoretical Poisson limit (0.003%). Our platform offers a scalable and efficient solution for particle pairing in small droplets, overcoming a major limitation of conventional droplet microfluidics.