Enzyme surface-reconstituted

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... 

Scheme 12 Surface reconstitution of apo-glucose oxidase on a mixed monolayer associated with an electrode consisting of an FAD cofactor and photoisomerizable nitrospiropyran units, and reversible photoswitching of the bioelectrocatalytic functions of the enzyme electrode.

The need to improve the electrical communication between redox proteins and electrodes, and the understanding that the structural orientation at the molecular level of redox proteins and electroactive relay units on the conductive surfaces is a key element to facilitate ET, introduced tremendous research efforts to nano-engineer enzyme electrodes with improved ET functionalities. The present chapter addresses recent advances in the assembly of structurally aligned enzyme layers on electrodes by means of surface reconstitution and surface crosslinking of structurally oriented enzyme/cofactor complexes on electrodes. The ET properties of the nano-structured interfaces is discussed, as well as the possible application of the systems in bioelectronic devices such as biosensors, biofuel cell elements or optical and electrical switches. 

Figure 3-2. Methods for the assembly of reconstituted relay-cofactor enzyme assembhes (A) by the surface reconstitution of the apo-enzyme (B) by the attachment of the prereconstituted enzyme on the electrode.

Integrated electrically-contacted enzyme electrodes were prepared by the surface reconstitution of different apo-enzymes on electrode surfaces. The pyrroloquinoline quinone, PQQ, (7), was covalently linked to a cystamine monolayer associated with an Au-electrode, and A -(2-aminoethyl-FAD), (8), was covalently attached to the PQQ units. Fig. 3-4A. The integrated enzyme-electrode was then prepared by the reconstitution of apo-GOx on the FAD units. The surface coverage of the PQQ-FAD units was estimated to be 5.5x10 ° mole cnr. whereas the surface coverage of the reconstituted... 

Figure 3-10. The assembly of an Au-nanoparticle (1.4 nm) electrically contacted glucose oxidase electrode by (a) the primary reconstitution of apo-GOx on the FAD-functionalized Au-nanoparticle, and the immobilization of the enzyme-nanoparticle hybrid on an electrode surface (b) the primary immobilization of the FAD-modified Au-nanoparticle on tire electrode surface and the surface reconstitution of apo-GOx on the functionalized electrode. Reproduced with permission from ref. 41. Copyright 2003, AAAS.

Co-immobihzation of mediator and enzyme may be achieved using a novel reconstitution approach. For example, Katz et al. [15] have developed a biocatalytic anode functionahzed by a surface reconstitution of apo-GOx onto FAD that was previously coupled to a pyrrolo-quinohne quinine (PQQ) relay conjugated to a self-assembled monolayer of cysteamine on gold. The CV study of this assembly in the presence of glucose yields an electrocatalytic current for glucose oxidation commencing at... 

Electrical Contacting of Enzymes by Surface-reconstitution of Apo-flavoenzymes on Relay-FAD-functionalized Electrodes... 

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