Solid-Phase Synthesis of High Affinity Interleukin-6 Binders Made of Gelatin-Methacryloyl Via a Molecular Imprinting Technique

Natural polymers can be engineered into nanoscale “molecular traps”, the bioMIPs, by the technique of molecular imprinting, which is a template-directed synthesis to generate selective binding sites in a crosslinked matrix. To date, bioMIPs are synthesized in solution by nano-aggregation of the polymeric units around a template, yielding to selective meta-biomaterials. Yet, the in-solution process limits the uniformity and scale-up of the bioMIPs. Here we report the first solid-phase synthesis (SPS) of interleukin-6 (IL-6) selective bioMIPs made of Gelatin methacryloyl (GelMA) and establish design rules for producing monomodal bioMIPs from protein building blocks. SPS synthesis is based on the oriented immobilization of the template to a solid support. SPS nucleation was studied on a plasmonic surface permitting real-time monitoring of bioMIP genesis. Subsequently, it was translated into a prototype reactor for scaling up the production. Guided by Flory-Huggins-De Gennes theory, we mapped the synthetic space by varying GelMA concentration, density and architecture of the surface-immobilized templates, and nucleation-time. The formed SPS bioMIPs were structurally and functionally characterized. Compared with analogous bioMIPs prepared by in-solution imprinting, SPS bioMIPs displayed a low picomolar dissociation constant, surpassing the in-solution ones by 3 orders of magnitude; showed ∼1 ng/μg IL-6 uptake in human serum, i.e., ∼45-fold increase with respect to in-solution ones, underscoring robust selective recognition under physiologically relevant conditions. Overall, the SPS strategy enables template-free isolation of high-quality bioMIPs and provides a practical route toward manufacturing cytokine-targeting nanotraps for inflammation-modulating biomedical applications.