Probing the Interplay of Protein Self‐Assembly and Covalent Bond Formation in Photo‐Crosslinked Silk Fibroin Hydrogels

Abstract Covalent crosslinking of silk fibroin via native tyrosine residues has been extensively explored; however, while these materials are very promising for biomedical, optical, soft robotics, and sensor applications, their structure and mechanical properties are unstable over time. This instability results in spontaneous silk self‐assembly and stiffening over time, a process that is poorly understood. This study investigates the interplay between self‐assembly and di‐tyrosine bond formation in silk hydrogels photo‐crosslinked using ruthenium (Ru) and sodium persulfate (SPS) with visible light. The effects of silk concentration, molecular weight, Ru/SPS concentration, and solvent conditions are examined. The Ru/SPS system enables rapid crosslinking, achieving gelation within seconds and incorporating over 90% of silk into the network, even at very low protein concentrations (≥0.75% wt/v). A model emerges where silk self‐assembly both before and after crosslinking affects protein phase separation, mesoscale structure, and dynamic changes in the hydrogel network over time. Silk concentration has the greatest impact on hydrogel properties, with higher silk concentration hydrogels experiencing two orders of magnitude increase in stiffness within 1 week. This new understanding and ability to tune hydrogel properties and dynamic stiffening aids in developing advanced materials for 4D biofabrication, sensing, 3D cancer models, drug delivery, and soft robotics.