Methods of protein immobilization

Lee Y, Lee EK, Cho YW, et al. (2003) Pro-teoChip a highly sensitive protein microarray prepared by a novel method of protein immobilization for application of protein-protein interaction studies. Proteomics 3, 2289-304. 

Over the last 40 yr, an increasing number of researchers studied various methods of protein immobilization and have found widespread application for these methods in many biotechnology areas such as clinical analysis, therapeutic medicine, and the production of biomaterials (7). Among these techniques, adsorption of proteins is very simple, mild, and reversible, permitting reuse of both enzyme and the support (8). Applications of immobilized or adsorbed enzymes as specific catalysts have gained new routes in modern applied chemistry (9). 

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. 

Covalent linking of protein to insoluble polymers is unsuitable as a method of enzyme immobilization... 

The structure and physicochemical properties of the enzymes which have been used to date to promote electrochemical reactions are briefly outlined. Methods of their immobilization are described. The status of research on redox transformations of proteins and enzymes at the electrode-electrolyte interface is discussed. Current concepts on the ways of conjugation of enzymatic and electrochemical reactions are summarized. Examples of bioelectrocatalysis in some electrochemical reactions are described. Electrocatalysis by enzymes under conditions of direct mediatorless transport of electrons between the electrode and the enzyme active center is considered in detail. Lastly, an analysis of the status of work pertaining to the field of sensors with enzymatic electrodes and to biofuel cells is provided. 

Immobilization through inclusion into a gel, similarly to immobilization by adsorption, is a physical method of protein fixation. Advantages of such a method are its simplicity and the absence of chemical modification of the enzyme molecules. Polyacrylamide, polyethylene glycol methacrylate, polysaccharide, polyionite, as well as various inorganic gels are used. 

As described before, there are many methods for protein immobilization, but some of them require chemical modification of the matrix, which may result in material degradation, especially when biodegradable polymers are used. In addition, these modifications, necessary to attach the enzyme to the matrix, often result in the loss of enzyme activity as well as the inclusion of toxic organic... 

Luo, K., Hurley, T., and Sefton, B. M. (1991) Cyanogen bromide cleavage and proteolytic peptide mapping of proteins immobilized to membranes. In Methods in Enzymology (T. Hunter and B. M. Sefton, eds.), Vol. 201, pp. 149-152. Academic Press, San Diego. 

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