Immobilization of Enzymes on Electrodes

Gilvanda Silva Nunes and Jean-Louis Marty

Summary

This chapter focuses on the four main immobilization techniques used in the development of enzyme electrodes: adsorption, entrapment, covalent coupling, and cross-linking. The performance of the immobilization method depends on the enzyme and each method has both advantages and drawbacks. Loss of the activity results from the change in enzyme conformation depending on the method of immobilization as well as modification of the microenvironment (i.e., local pH of the enzyme). The activity of the immobilized enzyme decreases as a result of barrier of diffusion, which slows down or prevents the substrate from reaching the active site of the enzyme. The development and the characteristics of an acetylcholinesterase biosensor are described

Key Words: Enzyme immobilization; conformation; microenvironment; acetylcholinesterase; biosensor.

1. Introduction

Enzymes can be operated in the liquid form or immobilized on various supports. In enzymatic biosensor engineering, the term immobilized means "unable to move" or "stationary," but the enzyme activity remains still in the analytical device, although in a generally minor extension when compared with free enzymes (1).

The main advantages that accompany immobilized enzymes and many methods for immobilization are reduced cost of operation (as compared with free enzyme systems, which require additional separation and purification steps); the higher enzyme stability in immobilized form; the ability to stop the reaction rapidly by simply removing the enzyme from the reaction solution (or vice versa); the fact that products are not contaminated with the enzyme (and vice versa); the elimination of either multiple or repetitive use of a single batch of enzymes; and the possibility of establishing a model system to study enzyme action with variable applications. Among the disadvantages are the greater expense as compared to preparing free enzymes (depending on the immobilization method and on the

From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ

reagents used), the lower activity exhibited by many immobilized enzymes when compared with free ones, and limitations in mass transfer because of the chosen immobilization method.

The use of biosensors based in immobilized enzymes for analytical purposes is ever increasing. The longer mean-life of the obtained enzymatic systems, the predictable decay rates, and the possibility of the elimination of sample preparation have made the enzymatic biosensors preferable and particularly useful in the food and pharmaceutical industries (2).

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