Design of Maleimide‐Functionalised Electrodes for Covalent Attachment of Proteins through Free Surface Cysteine Groups

Wright, Emma J.; Sosna, Maciej; Bloodworth, Sally; Kilburn, Jeremy D.; Bartlett, Philip N.

doi:10.1002/chem.201400246

Cited by 10 publications

(12 citation statements)

References 25 publications

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“…These results confirm that proteins are really present on the surface of glassy carbon, but there is no clear evidence of enzyme attachment via the cysteine tag. Indeed, in the absence of cysteine tag, any cysteine residues from the protein 41,42 could react with the vinyl moieties in a comparable way as reported for cytochrome c with maleimide 43. The structure of DSDH is shown in Supporting Information(Fig.…”

mentioning

confidence: 80%

Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol–Ene Click Chemistry

Lin

Vilà

Klein

et al. 2016

ACS Appl. Mater. Interfaces

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Thiol-ene click chemistry can be exploited for the immobilization of cysteine-tagged dehydrogenases in an active form onto carbon electrodes (glassy carbon and carbon felt). The electrode surfaces have been first modified with vinylphenyl groups by electrochemical reduction of the corresponding diazonium salts generated in situ from 4-vinylaniline. The grafting process has been optimized in order to not hinder the electrochemical regeneration of NAD(+)/NADH cofactor and soluble mediators such as ferrocenedimethanol and [Cp*Rh(bpy)Cl](+). Having demonstrated the feasibility of thiol-ene click chemistry for attaching ferrocene moieties onto those carbon surfaces, the same approach was then applied to the immobilization of d-sorbitol dehydrogenases with cysteine tag. These proteins can be effectively immobilized (as pointed out by XPS), and the cysteine tag (either 1 or 2 cysteine moieties at the N terminus of the polypeptide chain) was proven to maintain the enzymatic activity of the dehydrogenase upon grafting. The bioelectrode was applied to electroenzymatic enantioselective reduction of d-fructose to d-sorbitol, as a case study.

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mentioning

confidence: 80%

Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol–Ene Click Chemistry

Lin

Vilà

Klein

et al. 2016

ACS Appl. Mater. Interfaces

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“…18 Alternatively, mixed monolayers of diamines could be obtained by simultaneous electrografting of two amines in solution in varying relative ratios. 19 The use of mixed monolayers with two or more components presents many advantages in the development of biosensors: enzymes can be anchored to the surface and surrounded by redox mediators to improve the mediated electron transfer and their orientation, density and distance to the surface could be regulated, leading an optimal response from the system. Moreover the approach could be used to create models that mimic the environment of the active site of enzymes by surrounding redox mediators with functionalities that could affect their physical and electrochemical properties.…”

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confidence: 99%

Toward the Control of the Creation of Mixed Monolayers on Glassy Carbon Surfaces by Amine Oxidation

Groppi

Bartlett

Kilburn

2015

Chemistry A European J

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A versatile and simple methodology for the creation of mixed monolayers on glassy carbon (GC) surfaces was developed, using an osmium bipyridyl complex and anthraquinone as model redox probes. The work consisted in the electrochemical grafting on GC of a mixture of mono-protected diamine linkers in varying ratios which, after attachment to the surface, allowed orthogonal deprotection. After optimisation of the deprotection conditions, it was possible to remove one of the protecting groups selectively, couple a suitable osmium complex and cap the residual free amines. The removal of the second protecting group allowed the coupling of anthraquinone. The characterisation of the resulting surfaces by cyclic voltammetry showed the variation of the surface coverage of the two redox centres in relation to the initial ratio of the linking amine in solution.

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“…[15] A key aspect of our procedure is the creation of a modified surface on the electrode in a stepwise manner, allowing the single elements of the con-struction to be independently varied. These were modified with maleimide groups following a method developed in previous work by our group using electrochemical surface attachment and solid phase synthesis methodology.…”

Section: Resultsmentioning

confidence: 99%

“…[18] A significant aspect of the design is the dilution of the reactive group, maleimide in our case, at the surface and the length of the linkage. [15] Cellobiose dehydrogenase (CDH) [19] was chosen as the model redox enzyme system in this work. Dilution of the reactive group on the surface of the electrode and the choice of a suitable length of linkage allows the maleimide to react with the cysteine at the enzyme surface without steric restrictions.…”

Section: Resultsmentioning

confidence: 99%

A Flexible Method for the Stable, Covalent Immobilization of Enzymes at Electrode Surfaces

Al‐Lolage

Meneghello

et al. 2017

ChemElectroChem

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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 immobilize redox proteins and enzymes in a specific orientation, but still with some flexibility to optimize reaction kinetics. In this work, we establish such an approach by 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 immobilize the mutated enzymes. Using cellobiose dehydrogen-ase (CDH) as a model system, carbon nanotube 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 immobilization.

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Design of Maleimide‐Functionalised Electrodes for Covalent Attachment of Proteins through Free Surface Cysteine Groups

Cited by 10 publications

References 25 publications

Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol–Ene Click Chemistry

Immobilization of Cysteine-Tagged Proteins on Electrode Surfaces by Thiol–Ene Click Chemistry

Toward the Control of the Creation of Mixed Monolayers on Glassy Carbon Surfaces by Amine Oxidation

A Flexible Method for the Stable, Covalent Immobilization of Enzymes at Electrode Surfaces

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