Physical Methods

1.2.1. Adsorption

Physical adsorption of an enzyme onto a solid matrix is probably the simplest and fastest way to prepare immobilized enzymes. The method relies on a nonspecific physical interaction—based on weak forces, such as van der Waals or dispersion forces—between the enzyme and the surface of the matrix which is brought about by mixing a concentrated solution of enzyme with the solid (3). The active site is normally unaffected and a nearly full activity is observed.

Numerous enzymes have been immobilized though adsorption procedures. The main advantage of adsorption as a general method for insoluble enzymes is that usually no reagents, and only a minimum of activation steps, are required. As a result, adsorption is cheap, easily carried out, and tends to be less disruptive to the enzyme protein than chemical means of attachment. Because of the weak bonds involved, desorption of the protein (resulting from changes in temperature, pH, and ionic strength) appears to be the main problem. Another disadvantage is nonspecific adsorption of other proteins or substances (1,3).

1.2.2. Microencapsulation (Outer Membrane Entrapment)

This method is based on confinement of the enzyme in a membrane that is placed on an electrode surface. This membrane retains the enzyme and presents controlled porosity in order to allow free diffusion of substrate and reaction products through it (i.e., semipermeability of the membrane). Numerous membranes have been used, such as nylon, cellulose nitrate, cellulose acetate, epoxy resins, collagen, polysulfones, polyacrylates, and polycarbonates. Many disadvantages are associated with the mass transfer phenomena of substrates, reaction products, and inhibitors through the membrane, as well as with microorganisms growing on the membrane surface, leading to inhibition of the enzyme layer and causing the sensor to behave erroneously (3).

Charcoal Microencapsulation

Table 1

Comparison of the Four Basic Enzyme Immobilization Methods

Characteristics (A) Adsorption

(B) Entrapment

(C) Covalent coupling (D) Crossl-inking

Matrix material Inorganic supports: ion exchange resins, active charcoal, silica gel, clay, aluminum oxide, titanium, diatomaceous earth, hydroxyapatite, ceramic, celite, treated porous glass. Organic supports: starch, collagen, modified sepharose, CM cellulose.

Bonding nature Reversible; changes in pH, temperature, and ionic strength may detach the enzyme.

Enzyme loading Low Enzyme leakage Some Loss of enzyme Negligible activity Cost Inexpensive

Operational Simple and non-destructive technique;

some instability (attributable to enzyme desorption).

Alginate, carageenan, collagen, polyacrylamide, gelatin, silicon rubber, polyurethane, polyvinyl alcohol with styrylpyrid-inium groups.

Physical entrapment

Low Some Negligible

Inexpensive Easy control and nondestructive technique, possible diffusion barriers; some enzyme losses.

Agarose, cellulose, PVC, ion exchange resins, porous glass.

Chemical bonding (diazotation, peptic bond, alkylation, bonding with poly-functional agents). High Very low Significant

Expensive

High stability; absence of diffusion barriers; low response time; high enzymatic charge; low reproducibility.

Cross-linking reagents: glutaraldehyde, bis-isocyanate, bis-diazobenzidine, diazonium salts. Functionally inert proteins, such as ovoalbumin and BSA, are often used as binder elements. Entrapment

High Low Small

Inexpensive

Higher enzyme activity loss; operational facility; high enzymatic charge.

1.2.3. Occlusion Methods

Methods based on confinement of the enzyme within the lattices of a polymerized matrix or into intersticial spaces of a gel have also been used for enzyme immobilization. Such methods allow for free diffusion of low-molecular-weight substrates and reaction products. Numerous materials have been used for the enzyme occlusion, such as polyacrylamide gels, polyvinyl alcohols (PVAs), charged polymers, and cationic and anionic groups (3).

Generally, this immobilization method is useful for all types of enzymes (e.g., dehydrogenases, alcohol oxidase, cholinesterase, choline oxidase, glucose oxidase, tyrosinase). Moreover, it is simple, inexpensive, and results in relatively stable systems. The main disadvantages associated with this method are losses in enzyme activity by denaturation resulting from the presence of free radicals and/or by the ultraviolet radiation applied on the electrodes after enzyme immobilization in order to accomplish polymerization. These problems can affect the biosensor response and decrease the electrode stability.

In the last few years, immobilization methods based on sol-gel matrices have been used extensively for the construction of biosensors, mainly because gel materials present specific properties that are particularly interesting for biosensor construction (i.e., rigidity, thermal and photochemical stabilities, chemical inertness, and functionality in aqueous and organic media) (4). Figure 1 exemplifies the enzyme occlusion by polymerization in PVA with styrylpyridinium groups (PVA-SbQ) and by using a sol-gel matrix.

1.2.4. Electro-Polymerization

Enzymes can be also physically immobilized into conductor polymers such as polypyrrole, polyaniline, polyphenols, and polythiophenes. Electrochemical potentiostatic and galvanostatic polymerization of the monomers in the presence of an enzyme produces a very sensitive and thin layer. The advantages associated to this technique are: an immobilization process that is entirely controlled, the use of several commercially available electrode materials (e.g., platinum, gold, carbon), and the possibility of miniaturization. Despite its operational simplicity, this procedure has one main disadvantage—it is difficult to determine the exact amount of the immobilized enzyme (5).

1.2.5. Electrode Modification

Enzyme immobilization can be performed also by simply adding the enzyme to a commercial activated graphite or epoxy—graphite containing some mediator. This is done in order to obtain a sensitive paste that will be incorporated into the working electrode surface. Obtained electrodes have excellent operational stability and high half-life (6).

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