Methods immobilization

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

Bianalytical Chemistry, by Susan R. Mikkelsen and Eduardo Corton ISBN 0-471-54447-7 Copyright 2004 John Wiley Sons, Inc. 

The following sections consider each immobilization method in detail. 

As we saw in Chapters 2 and 3, the fluorescence emitted from single molecules can be collected by two distinct methods. In diffusion experiments the fluorescence emitted by a single molecule is collected as it passes through a small ( 0.1 fl) detection volume. This is repeated many thousands of times and burst analyses (such as FRET) and correlation techniques (FCS) can be used to extract both equilibrium and kinetic parameters of interest. The advantage of this technique lies in its relative simplicity, however the time over which a single molecule can be observed is limited to the time 

Simultaneous optical and electrical recording of single gramicidin channels. Biophysical Journal84 (2003) 612-622. 

Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 3197-3202. 

Many ready-to-use resins are available commercially. Newer proprietary ligands resulting from the use of combinatorial library sources pose their own problems. The immobilization method influences the properties of the final resin significantly and the production of ligand may require a high level of technical competence. Thus, a basic knowledge of immobilization chemistry is required by the customer. 

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Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

The choice of a suitable immobilization method for a given enzyme and appHcation is based on a number of considerations including previous experience, new experiments, enzyme cost and productivity, process demands, chemical and physical stabiHty of the support, approval and safety issues regarding support, and chemicals used. Enzyme characteristics that greatly influence the approach include intra- or extraceUular location size surface properties, eg, charge/pl, lysine content, polarity, and carbohydrate and active site, eg, amino acids or cofactors. The size, charge, and polarity of the substrate should also be considered. 

In the case of solid supports, the immobilization methods can be classified according to the support-complex interaction. However, this is not a simple classification given that more than one interaction can be present and it is often difficult to discern which interaction is the most significant in terms of immobilization. In this review, however, we have distinguished three categories of interaction [4] (Fig. 1) ... 

As a general rule, the optimal immobilization method is found empirically by a process of trial and error, where the selectivity, activity, and operational stability of the enzyme after immobilization are taken into account. The immobilization process is very sensitive to different parameters and is treated as a kind of art [16]. 

Prepolymer method for entrapment of biocatalyst developed by Fukui and Tanaka is considered to be one of the best immobilization methods. Characteristic features and advantages of the prepolymer method can be summarized as follows ... 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

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

Biocatalyst preparation Immobilization method Catalyzed reaction Diphasic system Characteristics Reference... 

In this stoichiometric forerunner, the use of a polymeric support demonstrated the concept of using an immobilization method to prevent reagents from reacting with each other in an undesired manner, permitting a reaction to occur that is not normally possible. By analogy, there are possibilities where various immobilization methods (in this case, we are interested in nanoencapsulation methods) are used to enable two incompatible catalysts to work concomitantly in an otherwise impossible reaction. 

Vorlop et al. described a novel cross-linked and subsequently poly(vinyl alcohol-entrapped PaHNL for synthesis of (//(-cyanohydrins. These immobilized lens-shaped biocatalysts have a well-defined macroscopic size in the millimeter range, show no catalyst leaching, and can be recycled efficiently. Furthermore, this immobilization method is cheap and the entrapped (/ )-oxynitrilases gave similar good results compared with those of free enzymes. The (//(-cyanohydrin was obtained in good yields and with high enantioselectivity of up to >99% ee [55],... 

Supported palladium catalysts for fine chemicals synthesis are generally based on metal particles. Nevertheless, a few examples are reported of the use of supported complexes as catalysts for the Heck reaction (see Chapter 9.6). Nearly all the possible immobilization methods have been tested for this reaction. 

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. 

In comparison to the other immobilization methods, this process is disadvantageous since generally the support must be modified which is time consuming and... 

The adsorption of proteins onto surfaces is the oldest and easiest immobilization method. Adsorbing forces can be of different types Van der Waals interactions, ionic, hydrophobic or hydrogen bonding. The main advantages of this procedure are the simplicity of preparation and the little... 

Immobilization methods used in pesticide biosensors design... 

S. Andreescu, L. Barthelmebs, and J.L. Marty, Immobilization of acetylcholinesterase on screen-printed electrodes comparative study between three immobilization methods and applications to the detection of organophosphorus insecticides. Anal. Chim. Acta 464, 171—180 (2002). 

H. Wang, J.S. Li, Y.J. Ding, C.X. Lei, G.L. Shen, and R.Q. Yu, Novel immunoassay for Toxoplasma gondii-specific immunoglobulin G using a silica nanoparticle-based biomolecular immobilization method. Anal. Chim. Acta 501, 37-43 (2004). 

The physical adsorption of protein onto the surface of an electrode is a simple immobilization method. The adsorption is obtained by volatilizing the buffers containing proteins. The physical adsorption needs no chemical reagent, seldom activation and rinse, so that the bioactivities of the immobilized proteins can be retained well. However, the immobilized proteins are easy to break off from the electrode, which restrict broad applications of this method. Below are some examples of the physical adsorption of proteins immobilized on electrodes. 

Jung, Y. Jeong, J. Y. Chung, B. H., Recent advances in immobilization methods of antibodies on solid supports, Analyst 2008, 133, 697 701... 

Scheme 39.5 Hydrogenation of styrene as a test reaction for immobilization methods, using scC02 as the mobile phase.

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