Deterministic Co-encapsulation of Microparticles in Droplets via Synchronized Merging for Single-Cell Genomics

Droplet encapsulation of microparticles, such as hydrogel beads and cells, is fundamental to high-throughput bioanalytical assays, including single-cell genomics. However, co-encapsulation of multiple particles at defined ratios remains a technical challenge. Here, we present a simple and robust strategy for deterministic co-encapsulation, combining particle-triggered droplet generation with synchronized droplet merging. Close-packed hydrogel particles initiate droplet formation, and the resulting droplets are synchronized and merged within a microfluidic channel, enabling the efficient encapsulation of microparticles at user-defined ratios. We systematically characterized the process and demonstrated reliable coloading of 55 and 80 μm hydrogel particles at 1:1 and 2:1 ratios. Using this platform, we developed a single-cell 16S rRNA gene sequencing workflow by co-encapsulating hydrogel capsules containing amplified microbial genomes with barcode beads. Applied to a 1:1 mixture of Escherichia coli and Bacillus subtilis, the method enabled accurate, high-throughput single-cell taxonomic profiling. This co-encapsulation approach offers broad applicability for single-cell genomics, digital assays, and interaction-based studies requiring precise particle pairing.