The stepwise modification of carbon electrodes using electrochemical and solid phase synthesis for covalent binding of proteins

Abstract

The chemical and electrochemical functionalisation of surfaces is of great importance in the construction of electrodes modified with biomolecules. Besides fundamental investigations, research is driven by numerous important applications including biosensing, biofuel cells or molecular electronics. While significant developments have been made some key problems remain, particularly the stability, orientation and electrical communication of the immobilised biomolecule with the conducting support. In order to address these issues a modular approach to electrode modification has been developed, allowing simple variation of the key elements of the tether.

After electrochemical attachment of a mono-Boc-protected diamine linker, deprotection followed by amide coupling can be used to introduce a spacer to control the length of the structure. A reactive group may then be introduced and the enzyme coupled. In this work the reactive group chosen was maleimide. A maleimide group will react 1000 times faster with a thiol than an amine at neutral pH, allowing selective attachment to a free cysteine residue in a biomolecule.

The initial modification of the electrode surface with linker has been modified to create a partial coverage of the linker on the electrode surface The oxidation of a mixture of amines, using a Boc-protected diamine and a capping group allowed the Boc amine to be spaced out on the surface for optimal enzyme attachment. The developed methods have been used to successfully couple Cytochrome C and two engineered variants of glucose dehydrogenase from Glomerella cingulate to modified electrodes.

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Identifiers

ORCID for Philip N. Bartlett: orcid.org/0000-0002-7300-6900

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