Liquid-Liquid Phase Separation to Fabricate Microgels of Recombinantly Expressed Proteins

Abstract

Microgels, a microscale variant of hydrogels (1–100 µm), exhibit high surface area and responsiveness to external stimuli while retaining the soft, viscoelastic nature of their macroscale counterparts. While microgels can be derived from both synthetic and natural polymers, protein-based microgels offer significant advantages due to their diverse function and activities. However, traditional fabrication methods, such as microfluidics and emulsion-based techniques, often involve trade-offs between scalability, structural integrity, and functionality. To overcome these limitations, liquid-liquid phase separation is leveraged to fabricate microgels using globular supercharged fluorescent protein and a terminal epoxy derivative of PEG polymer – poly(ethylene glycol)diglycidyl ether (PEGDE). The presence of terminal epoxy groups on PEGDE facilitates internal crosslinking with lysine residues of supercharged proteins, resulting in stable microgels. The microgels are characterized with fluorescence microscopy, SEM, and FTIR. Fluorescence recovery after photobleaching experiments suggest the encapsulation of the polymers within the dense phase and are dependent on the polymer chain length. The results are further supported by coarse-grained MD simulations providing mechanistic insights. Finally, the utility of the microgels in dye and nanoparticle adsorption, along with biomineralization of fluorinated calcium phosphate, is shown. These highlight the ability of microgels to potentially open avenues for biomimetic material synthesis. © 2025 Elsevier B.V., All rights reserved.