Redox enzyme assemblies

REDOX ENZYME ASSEMBLIES WITH ELECTRON TRANSFER ON ELECTRODE SURFACE... 

Redox enzymes have been assembled in a monolayer on the solid surface by a potential-assisted self-assembling method as well as a thiol-gold selfassembling method. These enzymes are electronically communicated with the solid substrate through a molecular interface of conducting polymer and a covalently bound mediator. Electron transfer type of enzyme sensors have been fabricated by the self-assembling methods. 

Two different types of self-assembling processes are described. The one is a potential-assisted self-assembly of redox enzymes on the surface of an electrode. The potential-assisted self-assembly is followed by electrochemical... 

One of the key technologies required for fabricating biomolecular electronic devices concerns with molecular assembly of electronic proteins such as redox enzymes in monolayer scale on the electrode surface. Furthermore the molecularly assembled electronic proteins are required to be electronically communicated with the electrode. Individual protein molecules on the electrode surface should be electronically accessed through the electrode. 

Self-Assembly of Mediator-Modified Redox Enzymes on the Porous Gold Electrode Surface... 

In contrast to the molecular wire of molecular interface, electron mediators are covalently bound to a redox enzyme in such a manner as an electron tunneling pathway is formed within the enzyme molecule. Therefore, enzyme-bound mediators work as molecular interface between an enzyme and an electrode. Degani et al. proposed the intramolecular electron pathway of ferrocene molecules which were covalently bound to glucose oxidase [ 4 ]. However, few fabrication methods have been developed to form a monolayer of mediator-modified enzymes on the electrode surface. We have succeeded in development of a novel preparation of the electron transfer system of mediator-modified enzyme by self-assembly in a porous gold-black electrode as schematically shown in Fig.12 [14]. 

Several protein assemblies have successfully been fabricated on the solid surfaces sifter the bioinformation transduction. These include the following molecular systems molecularly interfaced redox enzymes on the electrode surfaces, calmodulin / protein hybrides, and ordered antibody array on protein A. These protein assemblies find a wider application in various fields such as biosensors, bioreactors, and intelligent materials. 

Electrical Wiring of Redox Enzymes by Relay-Functionalized Monolayer Assemblies... 

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... 

Table 2 summarizes different possible applications of photoswitchable biomaterials, while detailing the nature of the biomaterial, the area of application, and, when possible, specific examples. Reversible light-induced activation and deactivation of redox proteins (enzymes) corresponds to write - read - erase functions. The photonic activation of the biomaterial corresponds to the write function, whereas the amperometric transduction of the recorded optical information represents the read function of the systems. Switching off of the redox functions of the proteins erases the stored photonic information and regenerates the photosensory biomaterial. These integrated, photoswitchable redox enzyme electrode assemblies mimic logic functions of computers, and may be considered as first step into the era of biocomputers. 

Fig. 42. Photoindueed reduction of N03 to N02 in an organized assembly composed of NitraR immobilized in an acrylamide-bipyridinium redox polymer. ET-communication between the redox polymer and the enzyme active site takes place in the redox polymer-enzyme assembly...

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. 

By the second approach, the enzyme is immobilized in a redox polymer assembly (Figure 39B). Electron-transfer quenching of the photosensitizer by the polymer matrix generates an electron pool for the activation of the enzyme. Photoreduction of nitrate to nitrite was accomplished by the physical encapsulation of NitraR in a redox-functionalized 4,4 -bipyridinium acrylamide copolymer [234]. In this photosystem, Ru(bpy)3 + was used as a photosensitizer and EDTA as a sacrificial electron donor. Oxidation of the excited photosensitizer results in electron transfer to the redox polymer, and the redox sites on the polymer mediate further electron transfer to the enzyme redox center, where the biocatalyzed transformation occurs. The rate constant for the MET from the redox polymer functionalities to the enzyme active site is — (9 + 3) x 10 s. Similarly, the enzyme glutathione reductase was electrically wired by interacting the enzyme with a redox polymer composed of polylysine modified with A-methyl-A -carboxyalkyl-4,4 -bipyridinium. The photosensitized reduction of oxidized glutathione (GSSG) (Eq. 21) ... 

There appear to be two classes of redox enzymes intrinsic and extrinsic (5). With the former, the catalytic reaction between an enzyme and its substrates takes place within a highly localized assembly of redox-active sites. There need be no electron transfer pathways from these sites to the surface of the enzyme, where, it is presumed, it would interact with an electrode. For such intrinsic redox enzymes, electrode reactions may require (1) that the sites of the catalytic reaction be close to the protein surface, (2) that the enzyme can deform without loss of activity, (3) that the electrode surface projects into the enzyme. 

Several spiropyran-assembled Au electrodes were prepared and used to control the activities of redox enzymes by photostimulation. Willner etal.44 organized a mixed self-assembled monolayer consisting of native cofactor pyrroloquinolinoquinone (PQQ) and spiropyran units in Au electrodes. PQQ monolayers have been reported to act as an electrocatalytic interface for the oxidation ofNAD(P)H in the presence of Ca2+. With the mixed monolayer, in the... 

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