Mediated electron transfer immobilization

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. 

In addition to self-generated shuttles, synthetic compounds with similar chemical structures also show the same function. Changing the molecular structure of phenazine-type redox mediators by artidcial synthesis can signid-cantiy induence microbial extracellular electron transfer. Immobilization... 

The RDE is a particularly good system for the measurement of the electrode kinetics of electrocatalytic films (modified electrodes), in which mediated electron transfer to substrate species in solution occurs via a surface-immobilized redox... 

The best method of enzyme and mediator immobilization seems to be an electrochemical polymerization and deposition. For example, a 50 mmol/1 solution of monomer, either pyrrole or N-methylpyrrole, in an aqueous buffer containing the enzyme(s) can be pulse-electrolyzed under potentiostatic or galvanostatic control. A conducting polymer film then grows on the metal anode. When ferrocene-modified pyrrole polymer (e.g., based on [(ferrocenyl)amidopropyl]pyrrole) is used [135], then the polymer also works as the electron-acceptor and mediates electron transfer from the reduced form of an enzyme to the metal electrode. 

In this section we consider the process of mediated electron transfer via a surface-immobilized redox couple. The latter is assumed to be covalently attached to a support electrode surface. In the absence of the surface-immobilized redox couple, the solution phase substrate is assumed to display rather sluggish electron transfer kinetics. The situation is shown in Fig. 2.1. 

The concept of a redox-modified hydrogel has also been successfully expanded to covalently immobilized ferrocene onto linear poly-(ethyl-enimine) (LPEI)." Ferrocene-modified LPEI has been shown to effectively mediate electron transfer for a wide range of enzymes including glucose oxidase, fructose dehydrogenase, and horseradish peroxidase to produce current densities as high as 13... 

In order to screen mutants with improved direct electron transfer, it is necessary to use an electrochemical screening system. Currently, only a few electrochemical screening methods were described in literature such as the system developed by the Bartlett group used to screen NADH electro-oxidation. This system uses a multichannel potentiostat with sixty electrodes to screen zinc(n) or ruthenium(ii) complexes bearing the redox phenidione as a mediator for NADH oxidation. It allows the complete evaluation of the electrochemical kinetic constants of the mediators and the immobilization procedure. Unfortunately, this system could only be used with a single electrolyte solution for all the electrodes (e.g., when a single reaction condition or enzyme is assayed), and it requires mL-scale reaction volumes. Recently, another system was described which makes it possible to screen bioelectrocatalytic reactions on 96 independent electrodes screen-printed onto a printed-circuit-board. It showed the possibility to screen direct or mediated electron transfer between oxidoreductases and electrode by intermittent pulse amperometry at the pL-scale (Fig. 6). The direct electron transfer assay was validated with laccase and unmodified electrodes.As an example of the mediated electron transfer assay, the 96 carbon electrodes were modified by phenazines to sereen libraries of a formate dehydrogenase obtained by directed evolution. ... 

To explore the utility of DNA-mediated electron-transfer events for biosensing applications, an electrochemical assay was developed [23-25]. Electrochemical sensors are typically more portable and inexpensive than those using fluorescence-based detection, hence a system suitable for monitoring electron transfer through DNA duplexes immobilized on the surface of an electrode was sought. 

The next generation of amperomethc enzyme electrodes may weU be based on immobilization techniques that are compatible with microelectronic mass-production processes and are easy to miniaturize (42). Integration of enzymes and mediators simultaneously should improve the electron-transfer pathway from the active site of the enzyme to the electrode. 

The choice of immobilization strategy obviously depends on the enzyme, electrode surface, and fuel properties, and on whether a mediator is required, and a wide range of strategies have been employed. Some general examples are represented in Fig. 17.4. Key goals are to stabilize the enzyme under fuel cell operating conditions and to optimize both electron transfer and the efficiency of fuel/oxidant mass transport. Here, we highlight a few approaches that have been particularly useful in electrocatalysis directed towards fuel cell applications. 

Fig. 5.32 Scheme of the reduction of a solution species Sox within the polymer film. Electron transfer is mediated by the immobilized redox active sites Red/Ox... 

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