Chemical Methods

1.3.1. Covalent Bonding

The most extensively studied of the insolubilization techniques is the formation of covalent bonds between the enzymes and the support matrix. This is the retention of the enzyme on support surfaces by covalent bonding between functional groups on the enzyme and those on the support surface. The active site of the

Immobilization Support Encapsulation
Fig. 1. Schematic representation of two occlusion methods for enzyme immobilization: polymerization in PVA with styrilpirydinium groups (PVA-SbQ) (A) and enzyme entrapment in a sol-gel matrix (B).

enzyme must not participate in covalent bonding so that enzyme inhibitors may be added to the enzyme solution during covalent bonding treatment (3). When trying to select the type of reaction by which a given protein should be insolubilized, the choice is limited by the fact that the binding reaction must be performed under conditions that do not cause loss of enzymatic activity, and as previously mentioned, the active site of the enzyme must be unaffected by the reagents used.

The functional groups of proteins suitable for covalent binding under mild conditions include: the a-amino groups of the chain and the e-amino groups of lysine and arginine; the a-carboxyl group of the chain; the P- and y-carboxyl groups of aspartic and glutamic acids; the phenol ring of tyrosine; the thiol group of cysteine; the hydroxyl groups of serine and threonine; the imidazol group of histidine, and the indol group of tryptophane.

A small number of reactions have been designed to couple with functional groups on the protein other than the amino and phenolic residues. Amino ethyl cellulose has been coupled to the carboxylic acid residues of enzyme protein in the presence of carbodiimide, and thiol residues of a protein have been oxidatively coupled to the thiol groups of a cross-linking copolymer of acrylamide and N-acryloyl-cysteine. It is possible in some cases to increase the number of reactive residues of an enzyme in order to increase the yield of insolubilized enzyme and provide alternative reaction sites to those essential for enzyme activity. As with cross-linking, covalent bonding should provide stable and insolubilized enzyme derivatives that do not leach enzyme into the surrounding solution. The wide variety of binding reactions and insoluble carriers with functional groups capable of covalent coupling (or of being activated to give such groups) makes this a reasonably applicable method of insolubilization, even if very little is known about the protein structure or active site of the enzyme to be coupled.

There are many commercially available pre-activated membranes that have been used as supports for covalent enzyme fixation (7). Nevertheless, this enzyme immobilization method is generally the most complicated and difficult to reproduce. Moreover, the quantity of enzyme to be immobilized is usually higher.

1.3.2. Reticulation

Glutaraldehyde can also lead to the reticulation of enzyme molecules, creating an insoluble, reticulated, and rigid net. Reticulation can be performed in the presence of an inert protein (e.g., bovine serum albumin [BSA]) in order to increase the stabilization of the immobilized enzyme. This immobilization procedure is called reticulation and offers advantages such as solidity of enzyme-enzyme and enzyme-protein couplings. Unfortunately, reticulation can induce the formation of diffusion barriers, which can result in a higher biosensor response time.

1.3.3. Enzyme Fixationin Self-Assembled Monolayers

More recently, enzyme fixation of biomolecules on self-assembled monolayers (SAMs) has been used for biosensor construction. This method allows for controlled and oriented enzyme immobilization, thus being preferable for application in immobilization of enzymes on metallic electrodes such as gold, platinum, and silver (8).

In practice, certain molecules (e.g., thiolalkanes and silanes) are used for electrode surface activation, and monolayer formation takes place directly on it. Such monolayers utilize bifunctional molecules for the enzyme immobilization through covalent bonding or specific linking (e.g., enzyme affinity or recognition protein-ligand process). In this mode, acid functions can be activated by succinimide or carbodiimide groups and amine groups can be activated by glutaraldehyde (3,8).

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