Size-independent and Automated Single-Colony-Resolution Microdroplet Dispensing

Droplet microfluidics-based high-throughput screening (HTS) enables rapid analysis of complex biological libraries, but accurate off-chip recovery of sorted "hit" droplets remains a key bottleneck. In particular, polydispersity in droplet size-common in multi-step assays-causes variation in flow velocities, where smaller droplets travel faster than larger ones. This results in the unintended co-dispensing of multiple droplets, leading to cross-contamination of hits. To address this challenge, we use blank spacing droplets as physical barriers between "hit" droplets to maintain consistent spacing during droplet transition. Each unit, or “drip,” consists of a single "hit" droplet buffered by up to 1,000 blank droplets and is dispensed individually into a well or onto agar. A distance sensor detects each drip's formation and triggers a linear motor to raise the collection plate, enabling precise and automated single-drip dispensing—even with polydisperse droplets. This approach achieves single-drip dispensing precision with 99.9% accuracy and a throughput of up to 8,640 drips per hour. We validated this system using an antimicrobial susceptibility test (AST) assay, where four resistant bacterial strains were identified from a mixed population of eleven. Our method ensures reliable and automated transfer of microfluidic hits to conventional biological assays, overcoming a key limitation in droplet HTS workflows. This system offers a scalable, cost-effective, and accurate solution for integrating microfluidic screening with downstream phenotypic analysis and paves the way for more complex droplet-based biological applications requiring precise hit recovery.