Attachment of electron-transfer relays

FIGURE 33 Electrical contacting of a flavoenzyme by its reconstitution with a relay-FAD semisynthetic cofactor. 

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The covalent attachment of electron transfer mediators to siloxane or ethylene oxide polymers produces highly efficient relay systems for use in amperometric sensors based on flavin-containing oxidases. It is clear from the response curves that the biosensors can be optimized through systematic changes in the polymeric backbone. The results discussed above, as well as those described previously (25-32), show that the mediating ability of these flexible polymers is quite general and that it is possible to systematically tailor these systems in order to enhance this mediating ability. 

Fig. 1.12. Schematb drawing of the glucose oxidase molecule, showing the electron-transfer distances involved in the various steps of moving an electron from its two flavin adenine dinucleotide/reduced flavin adenine dinucleotide (FAD/FADHg) centers to a metal electrode. Left The enzyme before modification. Right The modified enzyme, after chemical attachment of an array of electron transfer relays ( R ). (Reprinted from Y. Degani and A. Heller, J. Phys. Chem. 91 1286, 1987.)...

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. 

When ferrocene-containing polysiloxane proved to be an efficient electron-transfer relay system, further modification of this type redox pol3uner was investigated to develop optimal enzyme biosensors. Attempts were made to synthesize redox polymers with different mediators and/or different polymer backbones and/or different side chains through which mediators are attached to the polymer backbone. Resulting redox poisoners were tested to construct different types of enzyme sensors. 

Some of the materials highlighted in this review offer novel redox-active cavities, which are candidates for studies on chemistry within cavities, especially processes which involve molecular recognition by donor-acceptor ii-Jt interactions, or by electron transfer mechanisms, e.g. coordination of a lone pair to a metal center, or formation of radical cation/radical anion pairs by charge transfer. The attachment of redox-active dendrimers to electrode surfaces (by chemical bonding, physical deposition, or screen printing) to form modified electrodes should provide interesting novel electron relay systems. 

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. 

Spectroscopic methods can be used to specify the position of donors and acceptors before photoexcitation [50]. This spatial arrangement can obviously influence the equilibrium eomplexation in charge transfer complexes, and hence, the optical transitions accessible to such species [51]. This ordered environment also allows for effective separation of a sensitizing dye from the location of subsequent chemical reactions [52], For example, the efficiency of cis-trans isomerization of A -methyl-4-(p-styryl)pyridinium halides via electron transfer sensitization by Ru(bpy) + was markedly enhanced in the presence of anionic surfactants (about 100-fold) [53], The authors postulate the operation of an electron-relay chain on the anionic surface for the sensitization of ions attached electrostatically. High adsorptivity of the salt on the anionic micelle could also be adduced from salt effects [53, 54]. The micellar order also influenced the attainable electron transfer rates for intramolecular and intermolecular reactions of analogous molecules (pyrene-viologen and pyrene-ferrocene) solubilized within a cationic micelle because the difference in location of the solubilized substances affects the effective distance separating the units [55]. 

Glucose Sensors. Siloxane polymers are known to be extremely flexible. This flexibility will, of course, be sensitive to the amount of side-chain substitution present along the polymer backbone. For instance, in the homopolymer used in these studies (polymer A), the presence of a ferrocenylethyl moiety bound to each silicon subunit should provide an additional degree of steric hindrance, and thus a barrier to rotation about the siloxane backbone, in comparison with the copolymers, which have ferrocene relays attached to only a fraction of the Si atoms. Because these siloxane polymers are insoluble in water, their flexibility is an important factor in their ability to facilitate electron transfer from the reduced enzyme. Relays contained within more rigid redox polymers, such as poly(vinylferrocene), cannot achieve close contact with the enzyme s redox centers and are thus less effective as electron transfer mediators (25,34). The importance of this feature can be seen quite clearly by comparing the mediating ability of the homopolymer A with that of copolymers B-D, as shown in Figures 4 and 5. 

The ratio of ferrocene-modified siloxane subunits to unsubstituted siloxane subunits rrv.n ratio) was varied as was the length (a ) of the alkyl side chain onto which the ferrocene moiety was attached as shown in Fig. 3.3. The electrode containing co-polymer with m n ratio of 1 1 or 1 2 was the more efficient electron relay systems. The ferrocene-modified homopolymer on the other hand loses flexibility due to steric hindrance caused by the side chain substitution by ferrocene, preventing efficient electron transfer from the enzyme to the electrode. The length of the alkyl side chain onto which the ferrocene moiety is attached was also found to influence the electron transfer efficiency of the electron relay system. Maximal current density was measured... 

A novel principle for accelerating the electron transfer has been the direct chemical modification of GOD by electron-mediating groups such as ferrocene derivatives (Heller and Degani, 1987). The distance between the mediator molecules was at most 1 nm and the relays had to be attached in the vicinity of the prosthetic group. The binding of ferrocene to GOD was therefore conducted in 2 mol/1 urea. After refold-... 

FIG. 11 Schematic view of the designed photocatalytic systems with (a) transmembrane and (b) interfacial electron transfer, which is photosensitized by the CdS nanoparticles attached to the lipid membrane surface. Menaquinone (MQ) and heteropolyanions (HPA, SiWi20jo) are lipophilic molecular electron relays. Palladium particles are attached to CdS and operate as dark catalysts of hydrogen evolution from water. MV + methylviologen bication Gl glucose. 

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