Drop-by-drop Addition of Reagents to a Double Emulsion

Abstract

Developments in droplet microfluidic assays 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, identification of rare variants, and more. Double emulsions (DEs) have seen new interest in recent years for their potential to enable new droplet assays and 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, addressing inherent limitations of SEs. Despite these strengths, DEs lack important technical functions that exist in SEs such as picoinjection or any other method for adding reagents to droplets on demand. Consequently, DEs cannot be used for multistep workflows which has limited their adoption in assay development. Here, we report a simple device achieving picoinjection of DEs. We developed strategies to enable active manipulations on DEs by converting DE inputs to SEs on chip. The released aqueous cores of the DE can be manipulated using existing SE techniques, such as reagent addition, before reforming a DE at the outlet. We identified device designs and operation conditions achieving drop-by-drop reagent addition to DEs and used it as part of a muti-step aptamer screening assay performed entirely in DE drops. This work enables the further development of multistep DE droplet assays.