Protein immobilization methods adsorption

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... 

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. 

The avidin-biotin immobilization method maintains the biological activity of the receptor molecules. Besides the biocompatibility of the procedure, the surface geometry of these films provides high accessibility of the immobilized biomolecules. In addition, the avidin molecules form a passivation layer on the transducer surface that prevents nonspecific adsorption of proteins on the surface. In contrast with conventional grafting or affinity binding, this step-by-step approach can also be applied to the preparation of assemblies containing multilayers of biological molecules [42],... 

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 fractionation and purification of deteriorated proteins is undoubtedly one of the least successful techniques. This is simply because all of the methods that have been developed, with very few exceptions, are directed toward purifying the undeteriorated protein. The methods available are usually based on some particular biochemical activity of the protein, usually enzyme activity, and sometimes an affinity column or affinity adsorbent could be used to separate the native protein from the deteriorated one. Quite often a good affinity adsorbent is unavailable. This procedure, however, does not always work properly even when an adsorbent is available, because the deteriorated protein may possess some activity or an affinity for the adsorbent even though it has lost its natural enzyme activity (see Figure 24). The antigen-antibody reaction can also be used by means of precipitation with antibodies against the native proteins or adsorption on the immobilized antibodies. But here again, the specific antibody must be available, and the deteriorated protein may retain so much affinity for the antibody that differential separations will be impractical in some cases. 

Another problem in model applications is the adsorption on a nonuniform adsorbent. The immobilization of polyclonal antibodies will lead to different populations of binding sites, and the measurements will only give an apparent adsorption rate constant [22], The properties of the adsorbent surface are also greatly affected by the procedure used for protein immobilization. It may be important to select coupling methods that orient the covalently attached protein... 

It was shown I8 that the binding process is the rate-limiting step, with an adsorption rate constant of kd = 4.8 x 104 dm mol s. The calculated mass transfer coefficient for the diffusion into the pores of the support contributes 11% to the overall adsorption process. The value of ka is an order of magnitude lower than that reported in Table 2. The binding properties of the polyclonal immunoadsorbents used in these two studies may differ because of the different methods employed for protein immobilization. Another possible explanation may be an underestimation of the contribution for the diffusion rate-limiting step as the polyclonal anti-HSA antibody was attached to a silica matrix of large pores [18]. 

Physical immobilization methods do not involve covalent bond formation with the enzyme, so that the native composition of the enzyme remains unaltered. Physical immobilization methods are subclassified as adsorption, entrapment, and encapsulation methods. Adsorption of proteins to the surface of a carrier is, in principle, reversible, but careful selection of the carrier material and the immobilization conditions can render desorption negligible. Entrapment of enzymes in a cross-linked polymer is accomplished by carrying out the polymerization reaction in the presence of enzyme the enzyme becomes trapped in interstitial spaces in the polymer matrix. Encapsulation of enzymes results in regions of high enzyme concentration being separated from the bulk solvent system by a semipermeable membrane, through which substrate, but not enzyme, may diffuse. Physical immobilization methods are represented in Figure 4.1 (c-e). 

The adsorption method is the simplest one and is often used in bioelectrocatalysis research. Essentially it involves the incubation of protein in the carrier suspension with the subsequent washing of the nonadsorbed protein. Adsorption of proteins on different types of surfaces is effected due to electrostatic, hydrophobic, and dispersion interactions. The most popular carriers are carbon, soot, clays, aluminum oxide, silica gel, and glass. The optimal inert carrier is glass. It has recently been shown that porous glass with calibrated pore size can be used for immobilization of enzymes by adsorption. An interesting method of immobilization by adsorption has been proposed in which lipid is first adsorbed on carbon or silica gel and then the enzyme is adsorbed on the so-called soft surface of the lipid. 

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. 

The third item to consider in the development of an affinity technique is the way in which the ligand is attached to the solid support, or the immobilization method. There are many ways immobilization can be accomplished. These approaches include simple adsorption to a solid support, bioselective adsorption to a secondary ligand (e.g., the noncovalent binding of antibodies to immobilized protein A), entrapment, imprinting, and covalent attachment. 

---