Guide to the Preparation of Molecularly Imprinted Polymer Nanoparticles for Protein Recognition by Solid-Phase...

Molecularly imprinted polymers (MIPs) are synthetic antibody mimics possessing specific cavities designed for a target molecule. Nowadays, molecular imprinting of proteins still remains a challenge as the generation of selective imprinted cavities is extremely difficult, due to their flexible structure and the presence of a multitude of functional sites. To overcome this difficulty, we propose a solid-phase synthesis strategy to prepare MIPs specific for any protein that can be immobilized in an oriented way on a solid support. Trypsin and kallikrein were used as model proteins. The solid-phase support consists of glass beads functionalized with two affinity ligands of the enzymes, the competitive inhibitor p-aminobenzamidine to orient the enzymes via their active site, or a Cu²⁺chelate to orient via the surface histidine residues of the enzyme. Thermoresponsive molecularly imprinted polymer nanoparticles (MIP-NPs) are then synthesized around the immobilized enzyme. The MIP-NPs are released by a simple temperature change, resulting in protein-free polymers endowed with improved binding site homogeneity since all binding sites have the same orientation. The MIP-NPs exhibit apparent dissociation constants between 0.02 and 2nM toward their target proteins, which is comparable to those of natural antibodies. Moreover, these water-compatible polymers, targeting different domains of the enzyme, can also function as protective agents (armor), hence preventing the target proteins from denaturation by heat or pH.