DEVELOPMENT OF BIOFUNCTIONAL MEMBRANES FOR ORIENTED ANTIBODY IMMOBILIZATION

The immobilization of biomolecules, such as antibodies, is an essential technique in several biotechnological applications, including immunosensors1. A key step in the development of biofunctional surfaces is the design of experimental and operational procedures for biomolecules immobilization. Thus, it is important to investigate the boundary conditions (including the choice of a suitable surface, the way of immobilization and the proteins bulk properties) to control proteins surface coverage, distribution, orientation and related bioactivity. Among the different existing immobilization methods, the covalent immobilization provides high surface loading and low protein loss. In this work, the covalent immobilization of three different proteins (BSA, protein G and the enzyme lipase from C. Rugosa) on regenerated cellulose membranes was studied, with the aim to investigate the influence of the proteins bulk properties (effective size, aggregation phenomena) on the resulting protein surface coverage and distribution, for the development of biofunctional membranes with covalently bounded protein G, for the oriented immobilization of antibodies2. The membrane was initially modified for the introduction of aldehyde functional groups able to bind proteins covalently. The individual proteins size and aggregation phenomena were studied by Dynamic Light Scattering by simulating the operating parameters used during the immobilization step. Atomic force microscopy was used to monitor proteins surface coverage and distribution. The maximum surface coverage achieved with BSA and protein G was 2.40 ± 0.03 mg/g and 2.65 ± 0.07 mg/g, respectively, leading to the formation of a protein monolayer on the membrane surface. On the contrary, the immobilized amount of lipase was high, leading to the formation of protein multilayer. The proteins binding kinetic was also studied. A good correlation between the proteins behavior in solution and the membrane surface coverage was found. Finally, protein G-immobilized membrane was used for antibody oriented immobilization with high efficiency.