Size-independent and automated single-colony-resolution microdroplet dispensing

Abstract

Droplet microfluidics-based high-throughput screening (HTS) has proven to be an effective method for rapidly analyzing broad ranges of biological samples, such as cell libraries. However, the final step of the droplet microfluidics-based screening workflow, which is to dispense the sorted “hit” droplets to off-chip, ideally one “hit” droplet at a time, is prone to high error, especially when droplet size variation is present. Such size variations, often unavoidable in complex multi-step droplet microfluidics assay, lead to variations in droplet transition speed as they flow towards the dispensing tip, where smaller droplets move faster than larger droplets within a microchannel. Such difference in transit speed causes simultaneous dispensing of multiple droplets at a time instead of single-droplet dispensing, resulting in “hit” cells in a dispensed droplet being mixed with those from another “hit” droplet. To address this challenge, an approach that uses blank spacing droplets as physical barriers between “hit” droplets so that “hit” droplet-to-droplet distance remains the same throughout the droplet transition process is proposed here. These blank droplets (up to 1000) and a single “hit” droplet flow out of the droplet dispensing tip, forming a “drip”, which is then dispensed one “drip” at a time into a well of a well plate or on an agar plate with sufficient drip-to-drip distance. This method of mixing empty droplets with “hit” droplets, combined with an off-the-shelf distance sensor that detects the formation of a “drip”, which then moves the dispensing plate upward using a linear motor for the formed “drip” to be dispensed, enables precise and automated single-drip (i.e., one “hit” droplet) dispensing even with polydisperse droplets. The developed system demonstrated a droplet dispensing accuracy of 99.9%, with a throughput of up to 8640 single drips (i.e., single “hit” droplets) per hour. The system's effectiveness was demonstrated through a droplet microfluidics-based antimicrobial susceptibility test (AST) assay, where four resistant strains from a mixture of 11 strains could be successfully identified. By smoothly transitioning droplet-encapsulated samples of interest identified through high-throughput droplet microfluidics assays to traditional biological assay workflow, this system offers a highly efficient, accurate, and cost-effective solution for conducting complex droplet microfluidics-based assays followed by further off-chip assays.