High-throughput, combinatorial droplet generation by sequential spraying

Advancements in bulk and microfluidic emulsion methodologies have enabled highly efficient, high-throughput implementations of biochemical assays. Spray-based techniques offer rapid generation, droplet immobilization, and accessibility, but remain relatively underutilized, likely because they result in random and polydisperse droplets. However, the polydisperse characteristic can be leveraged; at sufficiently high droplet numbers, sequential sprays will generate mixed droplets which effectively populate a combinatorial space. In this paper, we present a method involving the sequential spraying and mixing of solutions encoded with fluorophores. This generates combinatorial droplets with quantifiable concentrations that can be imaged over time. To demonstrate the method's performance and utility, we use it to investigate synergistic and antagonistic pairwise antibiotic interactions.