Droplet microfluidics is utilized in a wide range of applications in biomedicine and biology. Applications include rapid biochemical analysis, materials generation, biochemical assays, and point-of-care medicine. The integration of aqueous two-phase systems (ATPSs) into droplet microfluidic platforms has potential utility in oil-free biological and biomedical applications, namely, reducing cytotoxicity and preserving the native form and function of costly biomolecular reagents. In this review, we present a design manual for the chemist, biologist, and engineer to design experiments in the context of their biological applications using all-in-water droplet microfluidic systems. We describe the studies achievable using these systems and the corresponding fabrication and stabilization methods. With this information, readers may apply the fundamental principles and recent advancements in ATPS droplet microfluidics to their research. Finally, we propose a development roadmap of opportunities to utilize ATPS droplet microfluidics in applications that remain underexplored.
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