Bio-hybrid Nanostructured Cellulose Membranes as Functional Tools for Bio-separation and Bio-recognition Applications

The conjugation of biomolecules with synthetic materials is an advanced technique to date used in a wide range of biotechnological applications, e.g. diagnostic, separation, bioprocess applications. This 'hybrid' combination of a biological entity and a support material confers advanced 'biological' functionality to the support material and, at the same time, promotes the stabilization and permits the reuse of the costly biological molecule, giving also the possibility to tailor the properties of the solid surface. Today, a great interest is devoted to nanostructured materials as solid supports, among which membranes are increasingly attractive because of their unique properties: they allow to combine separation with molecular recognition in a single operation unit. The development of a bio-hybrid surface need consideration of different parameters. Indeed, the immobilization of a protein and the related bio-activity and stability are dependent on several factors including the method used for the bio-conjugation, the properties of the surface, the properties of the protein and the immobilization conditions. After the immobilization, biomolecules have to maintain their function, to retain their biological activity, to remain closely bound to the surface. The choice of the appropriate membrane material and immobilization method are of primary importance. Also, a deep knowledge of factors affecting the immobilization process is required in order to tune the degree of bio-functionalization. In this work, the development of bio-hybrid cellulose membranes for application in bio-recognition and bio-separation processes is presented. In particular, the study on the influence of protein bulk properties on the immobilization process and the development of bio-hybrid membranes able to recognize and capture target molecules of diagnostic interest on the base of immuno-affinity interactions is illustrated. Cellulose membranes were chemically activated in order to introduce reactive aldehyde groups on its surface for proteins attachment. The immobilization process was studied by using three model proteins having different properties and behavior: BSA, protein G, the enzyme lipase form C. Rugosa [1]. The effect of the proteins bulk properties (concentration, size and aggregation phenomena) on the i) kinetics of binding, ii) surface coverage, iii) structural rearrangement and the proteins bio-activity after immobilization were studied. The bio-functionalized membrane with protein G was employed to develop immuno-affinity membranes, with immobilized specific antibody, for the capture and recognition of interleukin 6 (IL-6), a cytokine involved in inflammatory processes. In particular, the ability of protein G to bind antibody with spatial orientation was exploited for the site specific and oriented immobilization of the antibody to IL-6. Two different strategies were used: 1) the immuno-affinity membrane was directly used for the IL-6 capture; 2) the specific antibody was stabilized by chemical cross-linking before IL-6 capture [2]. Several aspects and parameters were studied and optimized: the ability of protein G to bind the antibody; the bio-recognition properties of both immuno-affinity membranes; the stability and selectivity of the antibody and the possibility of reuse; the improvement of the specificity of the system. The results demonstrated that the aggregation behaviour of a protein has a significant influence on the bio-layer formation on the membrane surface (including surface coverage, protein distribution and rearrangement and protein bio-activity). Thus, a close correlation between the protein properties in solution and during immobilization was found. The bio-functionalized membrane with protein G was successfully used for the oriented immobilization of the antibody to IL-6 showing a high antibody binding efficiency (88 %). The immuno-affinity membranes, prepared by the two different strategies, efficiently captured the IL-6 antigen with a capture efficiency up to 91 %, also revealing a linear relationship between the amount of the captured IL-6 and the initial IL-6 concentration. Thanks to the cross-linking, the second strategy permitted the antibody stabilization and the regeneration and efficient reuse of the immuno-affinity system with similar performances of the first use