Drop‐by‐Drop Addition of Reagents to a Double Emulsion

Developments in droplet microfluidics have facilitated an era of high-throughput, sensitive single-cell, or single-molecule measurements capable of tackling the heterogeneity present in biological systems. Relying on single emulsion (SE) compartments, droplet assays achieve absolute quantification of nucleic acids, massively parallel single-cell profiling, and more. Double emulsions (DEs) have seen recent interest for their potential to build upon SE techniques. DEs are compatible with flow cytometry enabling high-throughput multi-parameter drop screening and eliminate content mixing due to coalescence during lengthy workflows. Despite these strengths, DEs lack important technical functions that exist in SEs such as methods for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, strategies to enable reagent addition and other active manipulations on DEs are reported by converting DE inputs to SEs on chip. After conversion, drops are manipulated using existing SE techniques, including reagent addition, before reforming a DE at the outlet. Device designs and operation conditions achieving drop-by-drop reagent addition to DEs are identified and used as part of a multi-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.