Enzyme electrodes immobilization strategies

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. 

The modification of electrodes with enzymes and other biological macromolecules was well underway before 1978, and a detailed history of this field is beyond the scope of the present paper. A brief discussion of biological systems is given, however, to place them in context with other modification layers. A recent review by Frew and Hill (121) discusses past and future strategies for design of electrochemical biosensors. Topics discussed were enzyme electrodes, electron transfer mediators, conducting salts, electrochemical immunoassay, enzyme labels, and cell-based biosensors. In general, the bioactive molecule or cell is immobilized in proximity to an electrochemical transducer and exposed to the analyte solution for real-time analysis. 

The degree of enzyme purity will ultimately affect fuel cell performance, particularly when enzyme preparations are used to form immobilized films on electrode surfaces in DET reactions. Contaminating proteins that do not provide electron transfer effectively foul the electrode. When enzyme immobilization techniques are specific to the enzyme, then enzyme purity may not be as much as an issue, but rarely the immobilization technique is absolutely specific to the cathodic or anodic enzyme. For example, an attractive immobilization strategy is to link a particular enzyme to an electrode via its cofactor (e.g., flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), etc.) [59]. The cofactor is linked to the electrode material first and then the apoenzyme is allowed to naturally bind to the cofactor all other proteins in the enzyme preparation that cannot bind the cofactor remain unbound and can be removed. Enzymes used in fuel cells are not so unique, and proteins in the immobilizing preparation may use the same cofactor but not the same fuel during fuel cell analysis or operation. 

A novel application for ionic hquids in the preparation of functional materials has also served as a pre-immobilization strategy for a robust laccase electrode. In this case, the enzyme was first adsorbed to an ioiuc liquid-functionalized cellulose acetate that was then incorporated into a carbon paste electrode. The approach served to produce a device with acceptable levels of accuracy for methyldopa determination in pharmaceutical samples. In a similar manner, microencapsulation of laccase from T. versicolor in polyethylenimine (PEI) was demonstrated as a prior treatment for creating a coating on a paper support. Although first attempts showed a deleterious effect of PEI on laccase because of negative conformafional changes that reduced the activity of the encapsulated enzyme [28], optimized microencapsulation conditions resulted in superior stabihty compared with free enzyme [27]. 

An alternative strategy for co-immobilization of mediator and GOx is based on adsorption of enzyme, cross-linked, as was described for the laccase-based biocatalytic cathodes [30, 37 42], to an osmium-based redox polymer film, on carbon electrodes [1-3, 54],... 

A remaining crucial technological milestone to pass for an implanted device remains the stability of the biocatalytic fuel cell, which should be expressed in months or years rather than days or weeks. Recent reports on the use of BOD biocatalytic electrodes in serum have, for example, highlighted instabilities associated with the presence of 02, urate or metal ions [99, 100], and enzyme deactivation in its oxidized state [101]. Strategies to be considered include the use of new biocatalysts with improved thermal properties, or stability towards interferences and inhibitors, the use of nanostructured electrode surfaces and chemical coupling of films to such surfaces, to improve film stability, and the design of redox mediator libraries tailored towards both mediation and immobilization. 

Several strategies for immobilizing proteins on CNTs modified electrodes have been proposed. The step of immobilization is critical, since the enzyme has to remain as much active as possible in order to perform an efficient biorecognition of the substrate. The other aspect to consider is that the transducer where the protein will be immobilized has to allow a fast charge transfer to ensure a rapid and sensitive response. Therefore, it is important to take into account that the noncovalent functionalization of the sidewalls of SWCNTs is the best way to preserve the sp nanotube structure and their electronic characteristics. 

The preparation of dendrimer biocomposite-modified electrode is the primary step in the development of biosensors. Appropriate strategies have been formulated to prepare stable and highly reproducible dendrimer-modified surfaces. Immobilization of biomolecules like enzymes, proteins and other suitable ligands on the dendrimer-modified electrode with extended lifetime is very important. Some general procedures adopted for the preparation of dendrimer biocomposite-modified surfaces for electrochemical biosensing are described below. Some of the unique procedures developed by various authors are elaborated later during the discussion of the performance of biosensors. 

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