Integrated microfluidic platform for inertial separation and encapsulation of single cells in droplets

The ability to sort and separate large cellular subpopulations based on their intrinsic properties underpins a wide range of biological, diagnostic, and therapeutic applications. For many of these applications, preserving cellular homogeneity while achieving uniform single-cell encapsulation within droplets is essential for accurate quantitative analysis and downstream processing. Although microfluidic platforms have successfully enabled the separation of cellular subpopulations from heterogeneous samples, the lack of droplet-based encapsulation post separation remains a major bottleneck for achieving high-throughput single-cell analysis. Here, we address this limitation by developing an integrated microfluidic device that enables size-based cell separation and simultaneously encapsulating single cells into picolitre droplets. The device overcomes unstable encapsulation of cells by uniformly spacing cells prior to the encapsulation process. Proof-of-concept experiments achieved a single-particle encapsulation efficiency of 60% for 15 μm polystyrene beads, exceeding the Poisson limit of ∼35% single occupancy. Size-based separation of 15 μm particles from 10 μm particles yielded a separation efficiency of 94.39%, with nearly 60% of the separated particles successfully encapsulated as single particles in droplets. Validation experiments using MDA-MB-231 cancer cells dispersed in white blood cells (WBCs) demonstrated a 92.74% separation efficiency, with approximately 28% of cancer cells encapsulated as single cells within droplets. In contrast to existing microfluidic systems, which are limited to bulk collection of sorted cells or particles, our platform uniquely integrates high-purity size-based separation, cell ordering, and single-cell droplet encapsulation within a unified device, offering a powerful tool for high-throughput single-cell analysis and downstream molecular profiling.