We developed a three-dimensional (3D) polyglycerol–poly(ethylene glycol)-based hydrogel as a new biosensing matrix for affinity analysis by surface plasmon resonance to enable a high loading of ligands for small molecule analysis while lacking a carbohydrate structure to reduce nonspecific binding. The hydrogel was synthesized by cross-linking a polyglycerol functionalized with carboxylate and maleimide groups with a dithiolated poly(ethylene glycol) by thiol-click chemistry. We demonstrated that the hydrogel coating enabled a high immobilization capacity of biomolecules and led to less nonspecific binding. Here, the degree of loading with carbonic anhydrase II and the resulting binding signal of acetazolamide were increased by a factor of 5 compared to standard CMD sensors (CM5), and the loading was comparable to CMD sensors specialized for maximum loading (CM7). This high loading capacity, combined with the reduced nonspecific binding due to the missing carbohydrate structure, presents an innovative matrix for a broad application range of surface plasmon resonance (SPR) experiments since no current commercial SPR biosensor combines these two key features.
