Oxidase-type enzymes, immobilization

During the last decade, immobilization of oxidase type enzymes by physical entrapment in conducting or ionic polymers has gained in interest, particularly in the biosensor field. This was related to the possibility for direct electron tranfer between the redox enzyme and the electroconducting polymers such as polypyrrole (1,2), poly-N-methyl pyrrole (3), polyindole (4) and polyaniline (5) or by the possibility to incorporate by ion-exchange in polymer such as Nafion (6) soluble redox mediators that can act as electron shuttle between the enzyme and the electrode. 

The enzyme ascorbate oxidase (AOD) is immobilized over Clark-type oxygen electrodes. The biocatalyst is attached to polyamide nets with GA (252), cross-linked with collagen-glutaraldehyde (251) or albumin-GA (253), or linked to cellulose acetate membranes (253). 

Edwards et al. (1999a) reported that during removal of polyphenols in an enzyme-immobilized membrane reactor, in order to remove the colored quinone-type by-products, which passed through the membrane, a chitosan-packed post-adsorption column was required to be integrated into the system. In another study Edwards et al. (1999b) utilized chito-san gel as an immobilization matrix for polyphenol oxidase on polysul-fone capillary membranes. They reported that during the treatment of... 

For sensing glucose in the blood, glucose oxidase (GOx) enzyme is immobilized onto the membrane and attached on the Clark electrode s surface. GOx is a type of... 

The bioelectrocatalytic oxidation or reduction of substrates at biocatalyst electrodes can be accelerated by the presence of a small molecule which functions as an electron transfer mediator between the electrode and the prosthetic group of the immobilized enzyme " . Two types of the enzyme-modified electrode with entrapped mediator have been designed" , a carbon paste electrode and a porous electrode, both modified with enzyme and a reservoir of mediator. In the former type of electrode , where glucose oxidase (GOD) was immobilized on the surface of a carbon paste electrode along with p-benzoquinone (BQ) by coating the enzyme-loaded surface with a semipermeable membrane, BQ was dissolved into the immobilized enzyme layer and retained there effectively to serve as an electron transfer mediator between the carbon paste electrode and the immobilized enzyme. It has been shown " that the kinetics of the bioelectrocatalytic oxidation of D-glucose (Glc) at the membrane-coated GOD-modified carbon paste electrode with mixed-in BQ can be explained by theoretical equations in which the diffusion enzyme reaction of the substrate and mediator in the immobilized-enzyme layer and the diffusion of the substrate (and mediators) in the coating membrane are taken into account - . In this study, a number of quinone derivatives and a few ferrocene derivatives were examined as electron transfer mediators in the GOD electrode, and the mediator activity of the compounds was evaluated on the basis of theoretical equations. 

In 1989, to the best of our knowledge, the first report appeared on the use of an avidin-biotin system for the immobilization of enzymes in the preparation of biosensors. Walt et al. have immobilized biotin-modified enzymes (urease, esterase, and penicillinase) on the surface of biotin-modified optical fiber using avidin as a binder (Figure 5a). They have demonstrated the general use of this procedure in immobilizing several types of enzymes. At nearly the same time, Gunaratna and Wilson used an enzyme column in which choline esterase and choline oxidase were immobilized through avidin-biotin complexation for the determination of acetyl-... 

Therefore, another type of planar glucose biosensor with Pt electrodes on a sihcon substrate has therefore been developed for in vivo measurements [61]. The enzyme glucose oxidase was immobilized by the well known GDA-BSA method and the whole sensor was covered subsequently by a polyurethane membrane. This sihcon chip has to be sawed and assembled on a flexible carrier for in vivo application, the assembled catheter was successfully evaluated in rats [79]. This sensor gives encouraging results in aqueous solutions and subcutaneous apphcations. Drawbacks of this include the complicated mounting and assembling procedures which are difficult and cumbersome. 

This study on the immobilization of glucose oxidase and the characterization of its activity has demonstrated that a bioactive interface material may be prepared from derivatized plasma polymerized films. UV/Visible spectrophotometric analysis indicated that washed GOx-PPNVP/PEUU (2.4 cm2) had activity approximately equivalent to that of 13.4 nM GOx in 50 mM sodium acetate with a specific activity of 32.0 U/mg at pH 5.1 and room temperature. A sandwich-type thin-layer electrochemical cell was also used to qualitatively demonstrate the activity of 13.4 nM glucose oxidase under the same conditions. A quantitatively low specific activity value of 4.34 U/mg was obtained for the same enzyme solution by monitoring the hydrogen peroxide oxidation current using cyclic voltammetry. Incorporation of GOx-PPNVP/PEUU into the thin-layer allowed for the detection of immobilized enzyme activity in 0.2 M sodium phosphate (pH 5.2) at room temperature. 

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