Redox enzyme electrical wiring

Figure 13. A redox enzyme electrically wired to an electrode surface by flexible polymer chains functionalized with redox-mediator groups and surrounding the enzyme at the electrode surface.

Figure 3.15 [63]. The table in Figure 3.15 also shows enhanced electron transfer rates for ferrocene attatched on aligned SWNTs as compared to ferrocene attatched on randomly dispersed SWNTs [124]. Moreover, such vertically aligned SWNTs act as molecular wires that allow efficient electrical communication between the underlying electrode and the redox enzymes [45, 123, 127[.

Willner and coworkers have extended this approach to electron relay systems where core-based materials facilitate the electron transfer from redox enzymes in the bulk solution to the electrode.56 Enzymes usually lack direct electrical communication with electrodes due to the fact that the active centers of enzymes are surrounded by a thick insulating protein shell that blocks electron transfer. Metallic NPs act as electron mediators or wires that enhance electrical communication between enzyme and electrode due to their inherent conductive properties.47 Bridging redox enzymes with electrodes by electron relay systems provides enzyme electrode hybrid systems that have bioelectronic applications, such as biosensors and biofuel cell elements.57... 

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

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. 

Methods to electrically wire redox proteins with electrodes by the reconstitution of apo-proteins on relay-cofactor units were discussed. Similarly, the application of conductive nanoelements, such as metallic nanoparticles or carbon nanotubes, provided an effective means to communicate the redox centers of proteins with electrodes, and to electrically activate their biocatalytic functions. These fundamental paradigms for the electrical contact of redox enzymes with electrodes were used to develop amperometric sensors and biofuel cells as bioelectronic devices. 

Figure 2. The electrical wiring of a redox enzyme via a diffusionally mobile electron-transfer mediator shuttling between an enzyme reaction center and an electrode.

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. 

Figure 39. Electrical communication between an enzyme redox center and a photoexcited species attaining light-induced biocatalyzed transformations (A) direct electrical wiring of the protein by its chemical modification with tethered electron-relay units (B) electrical communication by the immobilization of the protein into a redox-functionalized polymer matrix.

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) ... 

Scheme 11. Idealized sketch showing the electroen matic oxidation of L-lactate at gold modified electrode surfaces, (a) Lactate dehydrogenase bound to CB-terminated alkylthiol SAMs prepared by covalent attachment of CB to 3-mercaptopropionic acid SAM derivatized with 1,4-diaminobutane. The electroenzymatic oxidation of lactate is observed only in the presence of soluble coenzyme (NAD" ") and a redox mediator (phenazine methosulfate) [215]. (b) Lactate deh3tdrogenase bound to NAD-terminated alkylthiol SAMs prepared by covalent attachment of Af -(2-aminoethyl)-NAD to a cystamine SAM derivatized with pjrrroloquinoline quinone. The reconstituted enzyme is electrically wired to the electrode surface via two NAD" -binding pockets involved in the affinity-binding surface reaction [242].

Electrical wiring of redox enzymes with redox polymers... 

Relevant issues still to be addressed in constructing amperometric enzyme sensors either using the electrical wiring of enzymes with redox polymers or with flexible polymeric electron mediators are sensor efficiency, accuracy, reproducibility, selectivity, insensitivity to partial pressure of oxygen, detectivity (signal-to-noise ratio) as well as sensor hfetime and biocompatibility [47]. Then we can address manufacturability and the cost of use of either in vitro or in vivo sensors. 

Heller A 1990 Electrical wiring of redox enzymes Accounts Chem. Res. 23 1280034 Janata J 1989 Principles of Chemical Sensors (New York Plenum) p 317 Fischer U, Rebin K, v Woedtke T and Abel P 1994 Clinical usefulness of the glucose concentration in the subcutaneous tissue—properties and pitfalls of electrochemical sensors Horm. Metab. Res. 26 515-22... 

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