Biphasic enzymatic reaction in coacervate-containing droplets

Abstract Spatiotemporal compartmentalization is a powerful cellular strategy for concentrating biomolecules, buffering noise, and creating local niches, that enable robust information-processing and reaction cascades. While compartments formed by coacervation are known to modulate catalytic activity, how reaction kinetics and interphase mass transfer between the coacervate and the surrounding phase jointly regulate catalysis within these dynamically evolving compartments remains poorly understood. Here we introduce synthetic coacervate-containing droplets (CCDs)—microfluidically generated arrays of single droplets containing single coacervates in equilibrium with the dilute phase—to investigate coupled reaction-diffusion dynamics under confinement. Using CCDs formed by complex coacervation of oppositely charged polyelectrolytes, we quantify guest molecular partitioning and enzymatic dephosphorylation reactions within single droplets. By integrating spatiotemporal imaging, bulk kinetics, and mathematical modeling, we quantitatively link enzymatic activity to dynamic molecular redistributions across phases, revealing how reaction-driven fluxes regulate enzymatic reactions within biphasic droplets and demonstrating the noise buffering capability of the coacervate phase.