Attachment redox enzymes

Homo- and copolymers of 1-alkyl-4-vinylpyridinium ions, 1, are well-known for their ability to form complexes with a wide variety of materials [1, 2]. For example, the interaction of poly(l-alkyl-4-vinylpyridiniums), 2, 3, and 4 with natural and synthetic polyanions has been used to form stable inter-polymer complexes [3-10]. Such complexes have been successfully used to attach redox enzymes or electroactive systems of small molecules to working electrodes. When 2 or 3 is used as a matrix for an anionic enzyme such as glucose oxidase, at pH > pKi the enzyme can be effectively wired to an electrode and can contribute to a variety of electroanalytical applications. Heller and associates [11] have utilized such interpolymer complexes to provide a glucose-specific... 

The large size of redox enzymes means that diffusion to an electrode surface will be prohibitively slow, and, for enzyme in solution, an electrochemical response is usually only observed if small, soluble electron transfer mediator molecules are added. In this chapter, discussion is limited to examples in which the enzyme of interest is attached to the electrode surface. Electrochemical experiments on enzymes can be very simple, involving direct adsorption of the protein onto a carbon or modified metal surface from dilute solution. Protein film voltammetry, a method in which a film of enzyme in direct... 

Figure 17.3 Anatomy of a redox enzyme representation of the X-ray crystallographic structure of Trametes versicolor laccase III (PDB file IKYA) [Bertrand et al., 2002]. The protein is represented in green lines and the Cu atoms are shown as gold spheres. Sugar moieties attached to the surface of the protein are shown in red. A molecule of 2,5-xyhdine that co-crystallized with the protein (shown in stick form in elemental colors) is thought to occupy the broad-specificity hydrophobic binding pocket where organic substrates ate oxidized by the enzyme. Electrons from substrate oxidation are passed to the mononuclear blue Cu center and then to the trinuclear Cu active site where O2 is reduced to H2O. (See color insert.)...

Schuman et al. have synthesized ferrocene-modified glucose oxidase with the ferrocene derivatives bound via long and flexible chains directly to the outer surface of the enzyme [17]. A peripherally attached redox mediator may accept electrons through either an intramolecular or through an intermo-lecular process. 

A further approach to electrically wire redox enzymes by means of supramolecular structures that include CNTs as conductive elements involved the wrapping of CNTs with water-soluble polymers, for example, polyethylene imine or polyacrylic acid.54 The polymer coating enhanced the solubility of the CNTs in aqueous media, and facilitated the covalent linkage of the enzymes to the functionalized CNTs (Fig. 12.9c). The polyethylene imine-coated CNTs were covalently modified with electroactive ferrocene units, and the enzyme glucose oxidase (GOx) was covalently linked to the polymer coating. The ferrocene relay units were electrically contacted with the electrode by means of the CNTs, and the oxidized relay mediated the electron transfer from the enzyme-active center to the electrode, a process that activated the bioelectrocatalytic functions of GOx. Similar results were observed upon tethering the ferrocene units to polyacrylic acid-coated CNTs, and the covalent attachment of GOx to the modifying polymer. 

It is well known that the flavin adenine dinucleotide redox centers of many oxidases are electrically inaccessible due to the insulating effect of the surrounding protein thus, direct electron transfer from the reduced enzyme to a conventional electrode is negligible. In the present work, a variety of polymeric materials have been developed which can facilitate a flow of electrons from the flavin redox centers of oxidases to an electrode. Highly flexible siloxane and ethylene oxide polymers containing covalently attached redox moieties, such as ferrocene, are shown to be capable of rapidly re-oxidizing the reduced flavoenzyme. 

Attaching the enzyme directly on the electrode surface is expected to improve elec-trocatalytic efficiency and response and improve the reproducibility of immobilization (147). Metallic (122, 144, 145) and carbonaceous (146) enzyme electrodes develop potentiometric responses to H2O2 produced by the enzymatic reaction. Unfortunately, the signal is markedly dependent on the redox surface of the electrode and thus on the electrode pretreatments (which are quite difficult to reproduce). 

The chemical modification of redox enzymes with electron relay groups permits the mediated electron transfer and the electrical wiring of the proteins [83-85] (Figure 5A). The covalent attachment of electron-relay units at the protein periphery, as well as inner sites, yields short inter-relay electron-transfer distances. Electron hopping or tunneling between the periphery and the active site allows electrical communication between the redox enzyme and its environment. The simplest systems of this kind involve electron relay-functionalized enzymes diffusionally communicating with electrodes [83], but more complex assemblies including immobilized enzymes have also been reported. 

The biomolecules are attached directly to the matrix by chemical/covalent linkage, which is not reversed by pH or changes in ionic strength. Carbodiimide coupling to form peptide bonds has been extensively used for the covalent coupling of the enzyme with polymers [126-128]. Since chemical modification is involved, this method results in the drastic loss of activity of the enzymes/biomolecules. Covalently attached redox biomolecules on polymers have been utilised as highly electron transfer mediators in flavin adenine dinucleotide (FAD) centres of oxidases [73]. 

Taking advantage of the steady increasing techniques for CNT functionalization [7], several routes were explored to attach redox molecules onto SWCNTs. Ferrocene was also attached to MWCNTs by, amide coupling, 7c-stacking interactions [8], aryldiazonium reduction [8] or 1,3 dipolar cycloaddition of azomethyne ylides [9] in order to establish electrical communication between the enzyme and the electrode (Fig. 3.5). 

In an analogous fashion, a flavin-type redox cofactor was attached to the nucleophilic thiol group in the active site of papain. By this means, a hydrolase was transformed into the artificial redox enzyme flavopapain [499-504]. 

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