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

A flexible method for the stable, covalent immobilization of enzymes at electrode surfaces
A flexible method for the stable, covalent immobilization of enzymes at electrode surfaces
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.
Enzyme immobilization, Site-specific immobilization, Maleimide, solid-phase synthesis, Cellobiose dehydrogenase
1528-1534
Al-Lolage, Firas A.
83184275-ce0e-4887-aee4-4b4c81bf7c06
Meneghello, Marta
60e2bbbb-2ac7-4ba1-b856-ce8feb4104ab
Ma, Su
50815c21-9ec5-424e-a241-c5e3e98f71bf
Ludwig, Roland
9485312f-f31f-412f-85f0-cda0e34fb1aa
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Al-Lolage, Firas A., Meneghello, Marta, Ma, Su, Ludwig, Roland and Bartlett, Philip N. (2017) A flexible method for the stable, covalent immobilization of enzymes at electrode surfaces ChemElectroChem, 4, (6), pp. 1528-1534.

Al-Lolage, Firas A., Meneghello, Marta, Ma, Su, Ludwig, Roland and Bartlett, Philip N. (2017) A flexible method for the stable, covalent immobilization of enzymes at electrode surfaces ChemElectroChem, 4, (6), pp. 1528-1534.

Record type: Article

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.

PDF Accepted Article - Accepted Manuscript
Restricted to Repository staff only until 12 April 2018.

More information

Accepted/In Press date: 24 March 2017
e-pub ahead of print date: 12 April 2017
Published date: June 2017
Keywords: Enzyme immobilization, Site-specific immobilization, Maleimide, solid-phase synthesis, Cellobiose dehydrogenase
Organisations: Chemistry, Electrochemistry

Identifiers

Local EPrints ID: 411115
URI: http://eprints.soton.ac.uk/id/eprint/411115
PURE UUID: 2113e846-ac46-44f8-816e-f3138e794bdb
ORCID for Philip N. Bartlett: ORCID iD orcid.org/0000-0002-7300-6900

Catalogue record

Date deposited: 14 Jun 2017 16:31
Last modified: 17 Jul 2017 13:29

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Contributors

Author: Firas A. Al-Lolage
Author: Marta Meneghello
Author: Su Ma
Author: Roland Ludwig

University divisions

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