Design of Synthetic Hydrogel Compositions for Noncovalent Protein Recognition

Multifunctional hydrogels composed of segments with ionizable, hydrophilic, and hydrophobic monomers have been optimized for sensing, bioseparation, and therapeutic applications. While the "biological identity" of bound proteins from biofluids underlies device performance in each context, design rules that predict protein binding outcomes from hydrogel design parameters are lacking. Uniquely, hydrogel designs that influence protein affinity (e.g., ionizable monomers, hydrophobic moieties, conjugated ligands, cross-linking) also affect physical properties (e.g., matrix stiffness, volumetric swelling). Here, we evaluated the influence of hydrophobic comonomer steric bulk and quantity on the protein recognition characteristics of ionizable microscale hydrogels (microgels) while controlling for swelling. Using a library synthesis approach, we identified compositions that balance the practical balance between protein-microgel affinity and the loaded mass at saturation. Intermediate quantities (10-30 mol %) of hydrophobic comonomer increased the equilibrium binding of certain model proteins (lysozyme, lactoferrin) in buffer conditions that favored complementary electrostatic interactions. Solvent-accessible surface area analysis of model proteins identified arginine content as highly predictive of model proteins' binding to our library of hydrogels containing acidic and hydrophobic comonomers. Taken together, we established an empirical framework for characterizing the molecular recognition properties of multifunctional hydrogels. Our study is the first to identify solvent-accessible arginine as an important predictor for protein binding to hydrogels containing both acidic and hydrophobic subunits.