Bioinspired materials for enzyme immobilisation and transport of biomolecules

• Main Author: Sousa, Ana M. L.
• Published: University of Strathclyde 2017
• Subjects: 572
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.725152

Protein and enzyme immobilisation on synthetic material surfaces enables a range of applications from biosensing to industrial biocatalysis. There are several immobilisation techniques, but common methods need multiple preparation steps or are material-dependent, which reduce the effectiveness and success of biosensing/industrial applications. In this thesis, the possibility of using plant-based polyphenol coatings to immobilise a range of proteins and enzymes (avidin, immunoglobulin G, acid phosphatase, chymotrypsin, lactate dehydrogenase, horseradish peroxidase) on polymeric or oxide materials (cellulose, polyester, silica, alumina and stainless steel) was shown for the first time. Polyphenols are in abundance in nature and less costly than dopamine (common immobilisation agent). Polyphenols were more effective than physisorption and polydopamine coatings for certain combinations of materials and proteins. Several parameters that can influence the immobilisation procedure were evaluated showing that there is a dependence on the coating and immobilisation pH as well as the type of coating and material. Polyphenol coatings were also used to functionalise nanoporous anodic aluminium oxide (AAO) membranes in order to measure molecular transport through nanopores. Inspired by the biological nanopores that enable the highly specific and efficient separation of a range of biomolecules, we used AAO membranes with a pore size matching the biological nuclear pore complex for controlling the diffusion of molecules through the pores. AAO membranes also match the requirements for optical waveguide spectroscopy (OWS) that was used to characterise and differentiate processes that occurred inside and above the nanoporous membranes. In a second approach, a nanoporous membrane was placed between two gaskets to be suspended on the flow cell. This work brings a new concept of how the molecular diffusion can be characterised, which is important for controlling the transport of biomolecules. It was possible to monitor in situ biocatalytic reactions as well as nanopore transport control by using a responsive polymer that was able to allow and restrict the transport of molecules.