Molecularly Imprinted Membranes: From Protein Recognition to Refolding Activity

Molecular imprinting is a powerful strategy for fabricating synthetic materials with selective recognition toward specific biomolecules. In this work, molecularly imprinted (MIM) membranes based on poly (ethylene-co-vinyl alcohol) (EVAL) were developed for selective protein recognition and conformational modulation using α-amylase as a model template. Membranes were prepared by phase inversion, generating porous structures suitable for mass transport and adsorption. Template extraction, measured using UV-Vis spectroscopy, showed a rapid and effective removal of α-amylase while preserving membrane morphology, as confirmed by SEM. FTIR-ATR and chemical imaging confirmed template removal from the membrane and a uniform surface distribution of rebound α-amylase after successive template incubation. Rebinding experiments showed a concentration-dependent uptake of α-amylase and an apparent saturation trend at higher concentrations. Selectivity tests using bovine serum albumin as an analog confirmed preferential recognition of α-amylase. Enzymatic assays showed partial recovery of catalytic activity after rebinding of thermally denatured α-amylase, indicating that imprinted cavities may promote protein conformational reorganization. These results highlight the potential of EVAL-based imprinted membranes as biomimetic platforms for selective protein recognition and functional modulation.