Inhibitor-assisted synthesis of silica-core microbeads with pepsin-imprinted nanoshells

Abstract

A novel approach for molecularly imprinting proteins, i.e. inhibitor-assisted imprinting, onto silica microspheres is discussed, which provides advanced functional materials addressing prevalent challenges in the field of protein purification and isolation from biotechnologically relevant media. Pepstatin-assisted surface-imprinted core-shell microbeads for the acidic protease pepsin were synthesized serving as selective sorbent materials for solid phase extraction (SPE) applications. The inorganic core, i.e. amino-functionalized silica spheres (AFSS), is prepared by the co-condensation of tetraethylorthosilicate (TEOS) and (3-aminopropyl) trimethoxysilane (APTMS) in water-in-oil (W/O) emulsion, which is then reacted with pepstatin, a selective inhibitor of pepsin, onto the surface of the AFSS via an amide bond. 3-Aminophenylboronic acid (APBA) serves as the functional monomer for establishing nanothin imprinted polymer films, i.e. poly(3-aminophenylboronic acid) (pAPBA) at the surface of the pepstatin-immobilized AFSS via oxidation by ammonium persulfate in aqueous solution in the presence (molecularly imprinted polymer, MIP) and absence (non-imprinted polymer; NIP) of pepsin. Thus obtained core-shell microbeads are packaged into SPE cartridges for evaluating the selectivity for pepsin. Each individual synthesis step is thoroughly characterized using x-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and BET methods. Finally, the imprinted core-shell microbeads indeed provide specific binding.