Mediator-modified enzymes, entrapment

Fig. 8. Entrapment of mediator-modified enzymes within a conductive polymer film where ( ) represents the mediator ferrocene and (B) the active site...

Entrapment of Mediator-Modified Enzymes within Conducting-Polymer FUms. 

Future work will be directed towards the entrapment of mediator-modified enzymes into mediator-modified polymer layers. As a final goal, reagentless am-perometric enzyme electrodes with low working potential, decreas influence of the oxygen partial pressure and interfering compounds should be envisaged, simultaneously taking into account the need for mass production and miniaturization. 

Ferrocene modified flexible polymeric electron transfer systems Ferrocene and its derivatives are readily available and commonly used organometalUc redox mediators, so it is quite natural that they were selected first to synthesize mediator modified polymeric electron transfer systems. Siloxane pol5uners are flexible but aqueous insoluble pol3nmers. As previously indicated, a flexible polymer backbone allows close contact between the redox center(s) of the enzyme and the mediator, and the water insoluble property of the polymer prevents not only redox polymer from leaching into bulk media but also prevents enzyme diffusion away fi-om the electrode surface by entrapping it in the polymer/carbon paste matrix. Therefore, ferrocene and... 

A direct electron transfer from entrapped quinohemoprotein alcohol dehydrogenase (QH-ADH) to a Pt electrode, via chains of the polypyrrole, acting as immobilization matrix, was demonstrated [152]. QH-ADH is able to translocate in a fast inner-enzymatic reaction, the electrons primarily accepted by PQQ to heme units located close to the outer protein shell, from where they can be transferred on the conducting-polymer chains (Fig. 13). A similarity between the electron-transfer pathway in multicofactor proteins and that of mediator-modified electroenzymes is apparent, if one considers that a multicofactor enzyme can be regarded as a combination of a primary redox site and protein-integrated electron-transfer relays. 

Thicker membranes from polymers are usually cast as solution (drop, spin, and spray coating), and the membrane forms after evaporation of the solvent. Alternate routes are application of monomers, direct polymerization on the electrode surface, and mechanical attachment of the ready-to-use membranes. Usually, modifiers (mediators, catalysts, enzymes, etc.) are dissolved in the membraneforming solution. Membranes can be generated also by electropolymerization, most commonly from aniline, pyrrole, or thiophene [117-119] the resulting 2D structure can entrap active molecules (e.g., enzymes) or serve as anchors for the actual modifier. Attachment of active molecules to polymeric structures can... 

Figure 17.4 Cartoon representation of strategies for studying and exploiting enzymes on electrodes that have been used in electrocatalysis for fuel cells, (a) Attachment or physisorption of an enzyme on an electrode such that redox centers in the protein are in direct electronic contact with the surface, (b) Specific attachment of an enzyme to an electrode modified with a substrate, cofactor, or analog that contacts the protein close to a redox center. Examples include attachment of the modifier via a conductive linker, (c) Entrapment of an enzyme within a polymer containing redox mediator molecules that transfer electrons to/from centers in the protein, (d) Attachment of an enzyme onto carbon nanotubes prepared on an electrode, giving a large surface area conducting network with direct electron transfer to each enzyme molecule.

Cenas and Kulys (1981) described an elegant method for modifying electrode surfaces. A layer of TCNQ was deposited on a glassy carbon electrode by anodic oxidation of Li+TCNQ. Similarly, a layer of Li+TCNQ may be formed by cathodic reduction of TCNQ. An enzyme solution, e.g. GOD, was entrapped on this mediator layer by a dialysis... 

Mediator-chemically modified electrodes have been coupled either with ADH membranes to give enzyme electrodes (Cenas et al. 1984) or with ADH reactors, e.g., in an FIA device (Huck et al. 1984). Quinoidic groups, Meldola s Blue, and Nile Blue have been used as mediators. Albery et al. (1987b) employed an electrode containing NMP+ and TCNQ" in a PVC carrier for NADH oxidation. ADH was entrapped on the sensor surface by a dialysis membrane. 

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