Immobilization, of redox enzymes

Hiller, M., C. Kranz, J. Huber, P. Bauerle, and W. Schuhmann. 1996. Amperometric biosensors produced by immobilization of redox enzymes at polythiophene-modified electrodesurfaces. Adv Mater 8 219-222. 

Much more effectual and very often applied are polymer-coated electrodes. Especially electrochemical polymerization is an attractive method for the immobilization of redox enzymes at electrode surfaces, and/or accumulation of electroactive reactants. An approximative analytical treatment of the response of an amperometric enzymatic electrode leading to plots of fluxes and concentration profiles has been made in [14]. The electron transport through poly-4-vinylpyridine and polystyrene-sulfonate films (widely used for immobilization of redox centers on electrodes) has been studied in [15]. 

Biocatalytic fuel cells using isolated redox enzymes were first investigated in 1964 [4], These fuel cells represent a more realistic opportunity for provision of implantable power, given the exquisite selectivity of enzyme catalysts, their activity under physiological conditions, and the relative ease of immobilization of isolated enzymes,... 

Stabilization of activated oxidoreductases on time scales of months to years has historically been challenging, and the lack of success in this regard has limited the industrial implementation of redox enzymes to applications that do not require long lifetimes. However, as mentioned in the Introduction, some possibility of improved stability has arisen from immobilization of enzymes in hydrophilic cages formed by silica sol—gels and aerogels, primarily for sensor applications.The tradeoff of this approach is expected to be a lowering of current density because... 

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. 

For preparative applications, especially in the case of continuous processes, means for the recovery of the valuable enzymes, the cofactors, and the redox catalysts in the reactor must be developed. One attractive possibility is the immobilization of the enzyme and sometimes also the cofactor and the redox catalyst at the electrode surface. However, the formation of enzyme-modified electrodes has also some practical drawbacks ... 

The immobilization of the enzyme, the redox catalyst, and sometimes also the cofactor can also take place at a solid support different from the electrode so that the components can be recovered within a solid-bed reactor (a column filled with the enzyme-containing particles) or by a filter plate or membrane. The immobilization of enzymes at solid supports or by the foraiation of cross-linked enzyme crystals can sometimes also enhance the enzyme stability. This concept has the advantage of the ease of separation but the disadvantage of diffusional limitations due to the heterogeneity of the reactions between the enzyme and the substrate and the cofactor or the redox catalyst. Additionally, the number of available redox centers is usually limited. 

This chapter has addressed recent advances in the application biomolcules immobilized onto metal oxide nanoparticles for fabrication of biosensors. Electrochemical contacting of redox enzymes or proteins with electrode surfaces is a key step in construction of third generation reagent-free biosensors. We have described a variety of metal oxide nanoparticles... 

Eor instance, if one would like to design a suitable redox polymer for a certain enzymatic reaction, it helps to think about the following factors. Eirst, the redox polymer needs to create a three-dimensional network that allows secure immobilization of the enzyme with a reasonable pore size. In addition, fast diffusion of the analyte, products, or counter-ions and fast ET kinehcs need to be ensured. The polymer film deposited as sensing layer creates a diffusion barrier which often prolongs the response time, shifts the linear measuring range, and decreases the sensitivity of the sensor. Second, the redox polymer should create a local... 

By one method a relay-cofactor dyad is assembled on the electrode, and the respective apo-protein is reconstituted on the surface to yield an aligned protein that is linked to the conductive surface by the relay component. The second method involves the synthesis of the relay-cofactor unit and the reconstitution of the apo-protein in solution. The specific immobilization of the enzyme on the electrode by the relay unit provides the structurally organized enzyme electrodes. While the first method is technically easier, the second methodology that involves tedious synthetic and separation steps, permits the fundamental structural characterization of the reconstituted protein. In the two configurations, the redox enzymes are anticipated to be electrically contacted with the electrode by means of the relay, a conductive... 

Most work related to the covalent labeling of proteins with organometallic is related to the development of enzyme or antibody amperometric biosensors. For the majority of redox enzymes, the active center (or redox-aetive cofactors) are buried inside the protein and are therefore electrically inaccessible for direct electron transfer to the electrode surface of an amperometric biosensor. This problem has been resolved by (i) addition of a diffusional redox-active mediator, (ii) covalent tethering of the mediator to the protein, or (iii) immobilization of the protein in a redox-active polymer. Ferrocenyl derivatives have frequently been used in all three formats as mediators because of their almost ideal electrochemical properties. 

It should also be noted that many of the previously reported latex 1-b-l modifications have described the deposition of layers of redox enzymes [55, 84, 85], and hence these structures could also be used as labels. Hollow capsules have also been used to entrap enzymes [101]. While enzyme stability can sometimes be an issue, the sensitivity provided by enzymes is often very good. For example, 1-b-l deposition of alkaline phosphatase onto carbon nanotubes resulted in electrochemical DNA sensing down to 5.4 aM [132]. In comparison with a nanotube, a latex sphere of diameter 0.5 m presents a very much larger surface area for immobilization. Finally, virtually everything stated in this chapter regarding DNA labeling can equally be applied to the labeling of antibodies. 

Ion Transport Stability Adhesion Multienzyme and Multilayer Configurations Immobilization of Redox Mediators Derivatization of Monomers before Conducting Polymer Electrosynthesis Direct Electrical Modulation of Enzymes Electroactive Polymer Hydrogels... 

Fig. 6 Cyclic voltammetric analysis of the kinetics of an electrode coated with antigen-antibody immobilized monomolecular layer of redox enzyme with a one-electron reversible cosubstrate in the solution, (a) Cyclic voltammetry at saturation coverage (2.6 x 10 mol cm ) of glucose oxidase with 0.1 M glucose and 0.1 mM ferrocenemethanol in a pH 8 phosphate buffer (0.1 M ionic strength). The dotted and dashed lines represent the cyclic voltammogram (0.04 V sec ) in the absence and presence of glucose (0.1 M), respectively. The full line represents the catalytic contribution to the current,/ cat (see text), (b) Primary plots obtained under the same conditions with, from top to bottom, 0.01, 0.02, 0.05, and 0.1 M glucose, (c) Secondary plot derived from the intercepts of the primary plots in (b).

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