Nickel chelating functionalization of graphene composite for metal affinity membrane isolation of lysozyme

A novel functionalized graphene-based composite is prepared by successive modification of graphene oxide (GO) with epichlorohydrin (ECH), iminodiacetic acid (IDA) and 1-phenylboronic acid (1-PBA) through covalent functionalization and then chelating with nickel ions. Characterizations by FT-IR, XRD, SEM, TGA and ICP-MS demonstrate the successful modification of the graphene surface, resulting in a GO-PBA-IDA-Ni composite with a Ni²⁺ immobilization amount of 3.01 × 10^-3 mol g^-1. The composite shows favorable selectivity for the adsorption of lysozyme (Lys). In practice, a homogeneous GO-PBA-IDA-Ni film with a thickness of ca. 1.0 μm is fabricated by filtering the composite through a cellulose membrane with a pore aperture of 1.2 μm. The GO-PBA-IDA-Ni film is subsequently fastened into a rounded membrane cartridge and incorporated into a sequential injection system for on-line selective isolation of Lys. An adsorption efficiency of ca. 96% is achieved for 25 μg ml^-1 Lys in 500 μl of sample solution at a loading rate of 5 μl s^-1. Metal affinity and electrostatic interactions are the main driving forces for governing the protein adsorption behaviors. The retained Lys is readily recovered by a borate buffer (pH 10) containing 1.0 mol l^-1 NaCl and 20 mmol l^-1 imidazole, giving rise to a recovery of ca. 90%. The practical applicability of the composites is demonstrated by selective isolation of Lys from chicken egg white, and SDS-PAGE assay indicates that Lys is selectively isolated with high purity from complex matrices.