Formation of molecularly imprinted polymers: Strategies applied for the removal of protein template (review).

The key step in the entire molecularly imprinted polymer (MIP) preparation process is the formation of the complementary cavities in the polymer matrix through the template removal process. The template is removed using chemical treatments, leaving behind selective binding sites for target molecules within the polymer matrix. Other MIP preparation steps include mixing monomers and template molecules in the appropriate solvent(s), monomer-template complex equilibration, and polymerisation of the monomers around the template. However, template removal is the most important among all the preparation steps because the final structure, which can be accepted and recognised as the MIP, is obtained only after the template removal. A thorough analysis of the studies dedicated to MIP applications demonstrates that this MIP preparation step, namely the template removal, is relatively understudied. MIP template removal is especially challenging in the synthesis, where the molecular template is a macromolecule such as a protein. This review aims to provide a deliberate, systematic, and consistent overview of protein removal as the MIP template molecules. The most prevalent template removal methods are outlined for removing protein templates from electrochemically synthesised MIPs, particularly thin layers on electrodes used in electrochemical sensors. Five protein template removal approaches involving chemical treatment are highlighted, which include the utilisation of (i) chaotropic agents, (ii) salt, (iii) acidic cleavage, (iv) alkaline, and finally, (v) proteolytic treatment focusing on studies conducted over the past decade. In addition, we discuss the interactions driving the removal of protein templates in each approach and associated challenges. This review provides insights into MIPs protein template removal strategies while highlighting the prevalent issue of this understudied step of template removal.