Enzyme immobilization, methods

Enzyme immobilization methods have been extensively reviewed and can be classifled into three categories ... 

Sakai-Kato, K., Kato, M., Ishihara, K. and Toyo-oka, T. (2004) An enzyme immobilization method for integration of biofunctions on a microchip using a water-soluble amphiphilic phospholipid polymer having a reacting group. Lab on a Chip, 4, 4—6. 

For various reasons, it was only in the 1970s that enzymes began to be used as bioelectrochemical catalysts. Some of the reasons were difficulty in preparation of pure enzymes, their instability, and the lack of multiple applications. These problems have been largely overcome, and better purification methods and enzyme immobilization methods on electrode surfaces have been developed. 

Research into new enzyme immobilization methods directly on the electrode surface is in progress. 

Enzyme immobilization methods other than using the hydrogels (A) Since appUcations of the redox hydrogels (A) to glucose sensors was first reported by Pishko et al. in 1991, various other methods have been reported to immobilize redox polymers to construct biosensors as indicated (see Section 3.3.3.2). Other recent examples are as follows. 

Enzyme immobilization methods are classified as chemical or physical. Chemical methods involve the formation of covalent bonds between functional groups on the... 

Figure 4.1. Enzyme immobilization methods, (a) Nonpolymerizing, (b) cross-linking, (c) adsorption, (d) entrapment, and (e) encapsulation.

Affinity ligands are immobilized through covalent binding to the support material. A dense, stable coverage of the support is desired, and most methods for immobilization use two steps support activation and coupling to the affinity ligand (see Section 4.2 regarding enzyme immobilization methods). 

In order to scale-up the method, however, both d-LDH and FDH have to be recycled to make the process economically feasible. While the starting material 6 could be prepared readily in large scale and only a catalytic amount of NAD is needed for the reaction, neither of the commercially available enzymes is inexpensive. Initial recycling efforts were directed to a batch process using either membrane-enclosed enzyme catalysis or enzyme immobilization methods [13] (Fig. 7). In our hands, these reactor systems were not ideal for scaling-up. 

Table 1 lists a few of the enzyme electrodes reported/ Both poten-tiometric and amperometric devices have been employed with these electrodes. Test results with enzyme electrodes have been reported as ranging from excellent to poor. Problems reported deal principally with lack of stability of the enzymes used, extended response times, and temperature sensitivity. There does not appear to be any pattern to the problems reported. It is apparent however, that (a) care must be taken in the choice of enzyme immobilization method, (b) the immobilization technique should provide a minimal barrier to reactant and product diffusivity, and (c) temperature stabilization is desirable. 

Conducting polymer-based immobilization or wired enzymes is a global enzyme immobilization method that differs in many respects from those just described. In one example, a redox polymer is formed on the surface by the oxidation of pyrrole molecules to pyrrole radical cations, which then polymerize on the surface to form conductive polypyrrole [60,68]. Other conducting polymers include polyvinylpyridine, polythiophene, polyaniline, and polyindole. If enzymes are present in the solution as polymerization takes place, they are entrapped within the polymer. When these polymers are cross-linked with redox mediators such as [Os(bpy)2Cl]+ 2 the resulting amperometric (or potentiomet-ric) biosensors are referred to as wired enzyme electrodes [5-7]. The distance between the redox centers of the polymer and the FADH2 centers of the reduced enzyme is reduced sufficiently for electrons to be transferred and, therefore, for the mediated electro-oxidation of glucose on conventional electrodes. These electrodes do not require diffusing redox mediators or membranes to contain the enzyme and the redox polymer. 

K. Nilsson, K. Mosbach, Tresyl chloride-activated supports for enzyme immobilization, Methods Enzymol. 135 (1987), 65-78. 

Medium Immobilized enzyme Immobilization method Strategy and performance application References... 

The enzyme activity rapidly degraded in water or PBS solution. Although TGG allowed the maintenance of activity in solution for several days, AChE dried in this mixture remained active for extended periods of time (i.e., 1 year) at 25° C or for short periods at 60 °C. Although the enzyme immobilization method and stabilization results described here are similar to those previously reported by Nguyen et al. (5), the demonstration of preservation of AChE activity over extended storage periods at 25 °C and at high temperature are unique to our study. 

Gutierrez-Sanchez, C. Jia, W. Beyl, Y. Pita, M. Schuhmann, W. De Lacey, A. L. Stoica, L. Enhanced direct electron transfer between laccase and hierarchical carbon microfibers/carbon nanotubes composite electrodes. Comparison of three enzyme immobilization methods. Electrochim. Acta 2012,82, 218-223. 

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