Glucose oxidase bioelectrocatalytic activation

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. 

To optimize the photoswitchable bioelectrocatalytic features of the protein, site-specific functionalization or mutation of the active site microenvironment is essential. This was accomplished by a semisynthetic approach involving the reconstitution of the flavoenzyme-glucose oxidase with a semisynthetic photoisomerizable FAD cofactor (Scheme 9).1511 The photoisomerizable nitrospiropyran carboxylic acid (24) was covalently coupled to N6-(2-aminoethyl)-FAD (25), to yield the synthetic photoisomerizable nitrospiropyran-FAD cofactor 26a (Scheme 9(A)). The native FAD cofactor was removed from glucose oxidase, and the synthetic photoisomeriz-able-FAD cofactor 26a was reconstituted into the apo-glucose oxidase (apo-GOx), to yield the photoisomerizable enzyme 27a (Scheme 9(B)). This reconstituted protein... 

A further approach to controlling electrical communication between redox proteins and their electrode support through a photo-command interface includes photo stimulated electrostatic control over the electrical contact between the redox enzyme and the electrode in the presence of a diffusional electron mediator (Scheme 12).[58] A mixed monolayer, consisting of the photoisomerizable thiolated nitrospiropyran units 30 and the semi-synthetic FAD cofactor 25, was assembled on an Au electrode. Apo-glucose oxidase was reconstituted onto the surface FAD sites to yield an aligned enzyme-layered electrode. The surface-reconstituted enzyme (2 x 10-12 mole cm-2) by itself lacked electrical communication with the electrode. In the presence of the positively charged, protonated diffusional electron mediator l-[l-(dimethylamino)ethyl]ferrocene 29, however, the bioelectrocatalytic functions of the enzyme-layered electrode could be activated and controlled by the photoisomerizable component co-immobilized in the monolayer assembly (Figure 12). In the... 

The electrostatic control of the electrical contact between redox-proteins and electrodes by means of command interfaces was further demonstrated by the photochemical switching of the bioelectrocatalytic properties of glucose oxidase (Figure 7.22). Ferrocene units were tethered to the protein backbone of glucose oxidase to yield an electrically wired enzyme that is activated for the bioelectrocatalyzed oxidation of glucose. The enzyme is negatively charged at neutral pH values (pIc,o 4.2 ) and, hence, could be... 

A photoswitchable bioelectrocatalytic device based on a similar azo-SAM with a PAA-g-CD coating was designed, able to catalyze the oxidation of glucose by glucose oxidase upon inclusion of ferrocene-methanol (Fc), as electron mediator, into the available free CD units of the PAA-g-CD film. Photoreversible activation and deactivation of the enzyme could be obtained by UV/Vis light irradiation. The immobilization and release of the redox polymer was driven by the trans-cis photoisomerization of the azobenzene units in the SAM. ... 

The most usual choice for anodic enzyme has been glucose oxidase (GOx) [53], which, when under mediated ET conditions, can effectively electro-oxidize glucose. The enzyme carries a flavin (flavin adenine dinucleotide (FAD)) buried deep within the enzyme, which makes DET difficult. Although DET with GOx has been reported in many different studies [54-57], there is an ongoing debate as to whether true DET is achieved, or whether the observed bioelectrocatalytic currents are due to naturally mediated glucose oxidation by free FAD, which has diffused out firom the active centers of some partly denatured enzyme molecules. 

The bioelectrocatalytic oxidation or reduction of substrates at biocatalyst electrodes can be accelerated by the presence of a small molecule which functions as an electron transfer mediator between the electrode and the prosthetic group of the immobilized enzyme " . Two types of the enzyme-modified electrode with entrapped mediator have been designed" , a carbon paste electrode and a porous electrode, both modified with enzyme and a reservoir of mediator. In the former type of electrode , where glucose oxidase (GOD) was immobilized on the surface of a carbon paste electrode along with p-benzoquinone (BQ) by coating the enzyme-loaded surface with a semipermeable membrane, BQ was dissolved into the immobilized enzyme layer and retained there effectively to serve as an electron transfer mediator between the carbon paste electrode and the immobilized enzyme. It has been shown " that the kinetics of the bioelectrocatalytic oxidation of D-glucose (Glc) at the membrane-coated GOD-modified carbon paste electrode with mixed-in BQ can be explained by theoretical equations in which the diffusion enzyme reaction of the substrate and mediator in the immobilized-enzyme layer and the diffusion of the substrate (and mediators) in the coating membrane are taken into account - . In this study, a number of quinone derivatives and a few ferrocene derivatives were examined as electron transfer mediators in the GOD electrode, and the mediator activity of the compounds was evaluated on the basis of theoretical equations. 

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