Surface reconstitution

Zayats M, Katz E, Willner I. Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor. Journal of the American Chemical Society 2002, 124, 2120-2121. 

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 3-4. (A) Surface reconstitution of apo-glucose oxidase on a PQQ-FAD monolayer associated with an Au-electrode. (B) Cyclic voltammograms corresponding to (a) the reconstituted glucose oxidase monolayer in the absence of glucose, and (b) in the presence of glucose, 80 mM. Data recorded in 10 mM phosphate buffer, pH 7.0, at 35°C, scan rate 5 mV s . Inset Calibration curve corresponding to the chronoamperometric currents atE = 0.2 V at different glucose concentrations. Reproduced with permission from ref. 31. Copyright 1996 American Chemical Society.

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.

The surface reconstitution of apo-HRP on a hemin-fimctionalized monolayer was reported to yield a surface-oriented HRP, exhibiting direct ET with the electrode. A mercaptobutanoic acid monolayer was assembled on an Au-electrode, followed by the covalent coupling of 1,12-diaminododecane to the base monolayer. Hemin was then covalently linked to the surface, and apo-HRP was reconstituted on the hemin sites. Fig. 3-15. The resulting surface-reconstituted HRP revealed direct ET with the electrode, and the bioelectrocatalyzed reduction of H2O2 was activated by the reconstituted HRP. 

Figure 3-15. The surface-reconstitution of apo-horseradish peroxidase on a hemin monolayer...

Fig. 12 On-surface reconstitution of immobilized membrane proteins with lipid bUayers by injection of mixed micelles and rapid detergent elution...

On-surface reconstitution (OSR) as an alternative to liposome-mediated coupling When working with membrane-spanning or membrane-embedded proteins that require a hydrophobic microenvironment for their biological activity... 

The roughness was assessed by means of a laser interferometer apparatus (Alti-surf 500 ). Among all the available information deduced from roughness analyses, the parameter determined from surface reconstitution [Eq. 1, where z is the hight difference of a given point having x,y coordinates], was used instead of the classical R. Indeed, seems to be more representative of such rough anisotropic surfaces. 

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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