A flexible method for the stable, covalent immobilization of enzymes at electrode surfaces

Abstract

Stable, site-specific immobilization of redox proteins and
enzymes is of interest for the development of biosensors and biofuel
cells, where the long-term stability of enzymatic electrodes as well
as the possibility of controlling the orientation of the biomolecules at
the electrode surface have a great importance. Ideally, it would be
desirable to immobilise redox proteins and enzymes in a specific
orientation, but still with some flexibility to optimise reaction kinetics.
In this work, we establish such an approach using site-directed
mutagenesis to introduce cysteine residues at specific locations on
the protein surface and the reaction between the free thiol group and
maleimide groups attached to the electrode surface to immobilise
the mutated enzymes. Using cellobiose dehydrogenase (CDH) as a
model system, carbon nanotubes electrodes were first covalently
modified with maleimide groups following a modular approach based
on electrografting of primary amines at the carbon surface and solid
phase synthesis methodology to elaborate the surface modified
electrode. The CDH-modified electrodes were tested for direct
electron transfer (DET), showing high catalytic currents as well as
excellent long-term storage stability. The key advantage of this
method is its great flexibility, as the main components of the
modification can be independently varied to change the local
environment at the electrode surface and a wide range of redox
proteins or enzymes can be specifically engineered to present
cysteine residues at their surface for oriented immobilisation.

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Identifiers

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

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