Quantitative Analysis of Enzyme Partitioning and Interfacial Stabilization in Polyelectrolyte Coacervates

Polyelectrolyte complex coacervate dispersions represent a versatile platform for the realization of aqueous colloidal encapsulants and microreactors. These membraneless droplets, formed via liquid–liquid phase separation, establish a distinct and unstable water–water interface with supernatant, often resulting in coalescence and eventual macro-phase separation. Previously, we demonstrated that the incorporation of comb polyelectrolytes (cPEs) can effectively stabilize these coacervate droplets. Such stabilization confers unique properties, including enhanced salt resistance, an expanded two-phase coexistence region, prolonged compartmentalization stability, and improved robustness across a range of temperature, pH, and ionic strength.

In this work, we employ confocal laser scanning microscopy (CLSM) to quantify the stabilization of the droplets and the co-encapsulation of enzymes within them. Fluorescently labeled cPE and PEs reveal a pronounced droplet formation, where cPE localizes at the liquid-liquid interface, creating a corona-like structure preventing coalescence. Varying enzyme stoichiometry and reducing intermediate product transport, we establish quantitative relationships between enzyme partitioning and overall reaction kinetics in the enzymatic cascades. Overall, these results provide evidence for the interfacial stabilization mechanism of cPE-stabilized droplets and establish a strong foundation for designer microreactors.