Pico-washing: simultaneous liquid addition and removal for continuous-flow washing of microdroplets

Droplet microfluidics is based on a toolbox of several established unit operations, including droplet generation, incubation, mixing, pico-injection, and sorting. In the last two decades, the development of droplet microfluidic systems, which incorporate these multiple unit operations into a workflow, has demonstrated unique capabilities in fields ranging from single-cell transcriptomic analyses to materials optimization. One unit operation that is sorely underdeveloped in droplet microfluidics is washing, exchange of the fluid in a droplet with a different fluid. Here, we demonstrate what we name the "pico-washer," a unit operation capable of simultaneously adding fluid to and removing fluid from droplets in flow while requiring only a small footprint on a microfluidic chip. We describe the fabrication strategy, device architecture, and process parameters required for stable operation of this technology, which is capable of operating with kHz droplet throughput. Furthermore, we provide an image processing workflow to characterize the washing process with microsecond and micrometer resolution. Finally, we demonstrate the potential for integrated droplet workflows by arranging two of these unit operations in series with a droplet generator, describe a design rule for stable operation of the pico-washer when integrated into a system, and validate this design rule experimentally. We anticipate that this technology will contribute to continued development of the droplet microfluidics toolbox and the realization of novel droplet-based, multistep biological and chemical assays.