Bioaffinity-based immobilization

While the interest in irreversible and covalent methods of immobilization continues, bioaffinity-based procedures are gaining appreciable attention, especially for analytical applications [42]. Compared to other methods of immobilization, those based on bioaffinity offer several distinct advantages. These include ... 

An interesting but underutilized approach to screening combinatorial libraries combines bioaffinity-based isolation and mass spectrometric identification. This approach requires the characterization of a drug to a macromolecular receptor such as an enzyme. This immobilized receptor can be conveniently packed into a chromatographic column [41], although configurations that do not use a column have also been reported [42]. An example of the latter, pulsed ultra-filtmtion (PUF) has been reported [43] and is illustrated in Fig. 12. 

These preliminary developments have relied on either metabolic enzyme reactions (viz. those where the substrate is consumed and a product is formed as a result) and bioaffinity reactions (viz. those that are followed via electron density changes). This Section discusses sensors based on immobilized enzymes and both types of reaction metabolic and bioaffinity. 

Immobilization provides great flexibility, particularly in the design of enzyme-based bioanalytical systems. Some relatively recent developments in protein immobilization methodology which can be broadly classified as reversible immobilization methods, have resulted in novel analytical approaches such as bioaffinity sensors (7) and flow injection binding reactions (8). 

A urea sensor has conventionally consisted of a urease-immobilized membrane and electrode for the determination of the metabolites electrodes for ammonium ion (20), pH (27), ammonia gas (22), and carbon dioxide (23) have been used. These sensors were all potentiometric ones to give Nernstian responses, the linear concentration range of which was typically between 10" and 10 M of urea. If one wishes to detect urea with using the ion- or gas-selective electrodes, urease is indispensable not only for the specific recognition based on the bioaffinity interaction but also for chemical conversion of urea to detectable species, since urea is essentially electro-inactive. On the contrary, the polymer-modified electrode described here does not rely upon enzymes the concentration of urea can be determined directly. 

An electrode for the determination of vitamin H (biotin) is based on the competitive affinity between biotin and its homologue HABA for the same protein, avidin [84]. The catalase-labelled HABA-avidin complex is immobilized on a p02 electrode to produce a biotin-sensitive sensor [255]. Biotin dissociares some of the HABA-avidin complexes by its strong affinity for avidin (Figure 6.1), leading to a reduction in enzymatic activity at the pC>2 electrode which can be related to the concentration ctf biotin. The coupling between biotin and avidin occurs more from bioaffinity than from an irmnunological process. 

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