Aqueous liquid–liquid phase separation (AqLLPS) droplet microreactors for biocatalysis

Abstract

Droplet microreactors utilize micro- to nanoscale droplets as discrete reaction compartments, effectively overcoming thermodynamic limitations inherent in bulk-phase systems and facilitating entropically unfavorable reactions. These systems have significantly advanced biocatalytic applications in enzyme engineering and biomanufacturing. However, conventional water–organic solvent platforms suffer from critical drawbacks, including interfacial instability and enzyme denaturation, which limit their practical utility. Aqueous liquid–liquid phase separation (AqLLPS) has emerged as a promising solution, generating fully aqueous, biomimetic microenvironments that maintain enzyme stability while enhancing catalytic efficiency through molecular crowding effects. This review summarizes AqLLPS mechanisms (segregative/associative types), design strategies, and applications in enzymatic synthesis, biosensing, and therapeutics. Current challenges and future directions for advancing biocatalysis are discussed.