Bio-orthogonal conjugation for “wiring” redox-active proteins/enzymes to any conductive surface

This thesis first illustrates the utility and limitations of protein film electrochemistry of adsorbed species via the analysis of a fungal Lytic Polysaccharide Monooxygenase enzyme. While a useful enzyme assay of H2O2 reduction is developed, detailed analysis of the underpinning reversible enzyme electron transfer processes is limited by background redox contributions from the carbon nanotubes which are required to stabilise the enzyme in an electroactive configuration. The rest of this thesis describes the development of methodologies for achieving electroactive immobilisation of redox proteins/enzymes onto electrode surfaces. Electroactive immobilisation of redox proteins/enzymes via covalent bonds has applications in the fabrication of biosensors and the development of green technologies/biochemical synthetic approaches, yet most published methodologies for achieving covalent immobilisation of proteins have relied on performing ligations between amine-reactive motifs on electrode surfaces and protein lysine residues. This results in many redox proteins/enzymes becoming immobilised in non-electroactive orientations or in orientations that otherwise hinder the redox enzyme’s ability to perform catalysis. In an effort to address this, methodologies for the incorporation of bio-orthogonal aldehyde motifs into proteins were investigated, and a protein immobilisation approach was developed in which hydroxylamine-functionalised electrode surfaces undergo bio-orthogonal ligation to aldehyde-functionalised proteins. As characterising and controlling surface chemistries is notoriously difficult, a new method is also under development that aims to enable covalent orientation-selective electroactive redox protein/enzyme immobilisation via the direct electro-grafting of redox proteins/enzymes that have been site-specifically labelled with aryl diazonium cations. I report the first ever usage of triazabutadienes as photocaged sources of aryl diazonium cations for use as electrode derivatisation agents in aqueous solutions at near-neutral pH, and, to the best of my knowledge, the first site-specific installations of triazabutadienes/diazonium cations onto the surfaces of proteins. I hope that further development of this method will enable the orientation-selective electroactive immobilisation of redox proteins/enzymes.