Effect of droplet size on the encapsulation efficiency of microparticles in passive microfluidic systems

Droplet microfluidic technologies are widely used in single-cell analysis, in which passive encapsulation methods are commonly used to isolate individual cells or particles. In these systems, the number of particles per droplet is assumed to follow a Poisson distribution. However, the extent to which droplet size affects encapsulation behavior under passive conditions is poorly understood. In this study, we systematically investigated the effect of droplet diameter on single-particle encapsulation fidelity by generating agarose droplets 30, 50, and 100 μm using a flow-focusing microfluidic device. Fluorescent beads were encapsulated at a concentration corresponding to a mean occupancy of 1, and encapsulation statistics were analyzed across droplet sizes. The 30 and 50 μm droplets exhibited encapsulation distributions closely aligned with the Poisson model, whereas 100 μm droplets showed a large excess of empty droplets and substantial deviation from the expected profile. To examine whether comparable size-dependent trends are observed in a biological sample, green fluorescent protein-expressing Escherichia coli were encapsulated in droplets of the same sizes. The 30 μm droplets consistently showed the closest agreement with Poisson statistics, whereas the 50 and 100 μm droplets showed comparable encapsulation fidelity, with moderate deviations and a modest increase in empty droplets at the largest diameter. These findings indicate that smaller droplets provide robust stochastic encapsulation in passive systems, while larger droplets may exhibit reduced fidelity depending on particle type. Our results suggest that droplet size is an important factor to consider when designing simplified and reliable microfluidic systems for biological applications.