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 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.

Controlling Redox Enzyme Orientation at Planar Electrodes

“…As a recent illustration, cellobiose dehydrogenase from Myriococcum thermophilum has been shown to be an ideal candidate for site-directed mutagenesis. Having no surface cysteine residues, this amino acid was introduced at specific surface locations in view of the oriented immobilization [130]. "Thiol-ene" click chemistry between the thiol group available on the cysteine moieties and vinyl groups grafted on the electrode was successfully exploited for site-specific covalent linkage.…”

“…In case of chelation between a histidine moiety on the modified protein and Cu-NTA ligands on the electrode, stability can be affected by dissociation of the Cu ion from the NTA-modified electrodes induced by competitive binding with the His-tag on the enzyme [135]. In comparison, the operational stability of bioelectrodes prepared by covalent conjunction using click chemistry between cysteine-modified protein and vinyl groups of the electrode lasts up to a few days [130].…”

Methodologies for “Wiring” Redox Proteins/Enzymes to Electrode Surfaces

“…Thus, it can be relatively trivial to use site‐directed mutagenesis and chemical biology conjugation methods to engineer proteins and enzymes with single, covalently modified surface‐cysteine residues Such strategies are of enormous value in the development of new biopharmaceutical therapies . Surface‐attachment strategies have been developed along similar lines, with the added consideration that for direct electron transfer between a conducting surface and a redox protein or enzyme, the cysteine residue must serve as a tethering site that holds the redox protein/enzyme in an electroactive orientation …”

“…In the field of electrode ligation, surfaces have been functionalized with maleimide groups, the most reactive of the commonly available vinyl Michael acceptors . Between pH 6.5 and 7.5, maleimide groups react selectively with thiols, as within this pH range amines remain protonated and are thus not of a high enough nucleophilicity to partake in competing side reactions (Scheme ) …”

“…Maleimide groups can be introduced onto electrode surfaces using a variety of techniques, including the use of specially designed alkanethiol SAMs, diazonium cation electrografting (Table , entry f), and sequential electrochemical and solid‐phase preparation . The reaction between surface‐maleimide groups and one of the two thiol groups that naturally occur near the heme cofactor of cytochrome c results in the immobilization of this protein in a near‐site‐specific orientation that is suitable for direct electron transfer (Scheme ) …”