Horseradish immobilization

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

Tatsumi, K., Wada, S., and Ichikawa, H., Removal of chlorophenols from wastewater by immobilized horseradish peroxidase, Biotechnol. Bioeng., 51, 126-130, 1996. 

Mohan SV, Prasad KK, Rao NC, Sarma PN (2005) Acid azo dye degradation by free and immobilized horseradish peroxidase (HRP) catalysed process. Chemosphere 58 1097-1105... 

It should be pointed out that the addition of substances, which could improve the biocompatibility of sol-gel processing and the functional characteristics of the silica matrix, is practiced rather widely. Polyethylene glycol) is one of such additives [110— 113]. Enzyme stabilization was favored by formation of polyelectrolyte complexes with polymers. For example, an increase in the lactate oxidase and glycolate oxidase activity and lifetime took place when they were combined with poly(N-vinylimida-zole) and poly(ethyleneimine), respectively, prior to their immobilization [87,114]. To improve the functional efficiency of entrapped horseradish peroxidase, a graft copolymer of polyvinylimidazole and polyvinylpyridine was added [115,116]. As shown in Refs. [117,118], the denaturation of calcium-binding proteins, cod III parvalbumin and oncomodulin, in the course of sol-gel processing could be decreased by complexation with calcium cations. 

Except the physical entrapment of horseradish peroxidase in a polyacrylamide gel used by Freeman and Seitz23, immobilization of the... 

Horak, D., Karpisek, M., Turkova, J., and Benes, M. (1999) Hydrazide-functionalized poly(2-hydroxye-thyl methacrylate) microspheres for immobilization of horseradish Peroxidase. Biotechnol. prog. 15, 208-215. 

J. Yu and H.X. Ju, Preparation of porous titania sol-gel matrix for immobilization of horseradish peroxidase by a vapor deposition method. Anal. Chem. 74, 3579-3583 (2002). 

A.N. Diaz, F.G. Sanchez, M.C. Ramos, and M.C. Torijas, Horseradish peroxidase sol-gel immobilized for chemiluminescence measurements of alkaline-phosphatase. Sens. Actuat. B 82, 176-179 (2002). 

A.L. Crumbliss, J.Z. Stonehuerner, R.W. Henkens, J. Zhao, and J.P. O Daly, A carrageenan hydrogel stabilized colloidal gold multi-enzyme biosensor electrode utilizing immobilized horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol in serum and whole blood. Biosens. Bioelectron. 8, 331-337 (1993). 

Y. Zhang, P.L. He, and N.F. Hu, Horseradish peroxidase immobilized in Ti02 nanoparticle films on pyrolytic graphite electrodes direct electrochemistry and bioelectrocatalysis. Electrochim. Acta 49, 1981-1988 (2004). 

Y. Xiao, H.X. Ju, and H.Y. Chen, Hydrogen peroxide sensor based on horseradish peroxidase-labeled Au colloids immobilized on gold electrode surface by cysteamine monolayer. Anal. Chim. Acta. 391,... 

J.L. Tang, B.Q. Wang, Z.Y. Wu, X.J. Han, S.J. Dong, and E.K. Wang, Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide. Biosens. Bioelectron. 18, 867-872 (2003). 

Table 2. Immobilization of a laccase (from Trametes versicolor) and a peroxidase (from horseradish) on different supports (Gianfreda and Bollag 1994)...

However, an important problem arises during the peroxidative removal of phenols from aqueous solutions PX is inactivated by free radicals, as well as by oligomeric and polymeric products formed in the reaction, which attach themselves to the enzyme (Nazari and others 2007). This suicide peroxide inactivation has been shown to reduce the sensitivity and efficiency of PX. Several techniques have been introduced to reduce the extent of suicide inactivation and to improve the lifetime of the active enzyme, such as immobilization. Moreover, Nazari and others (2007) reported a mechanism to prevent and control the suicide peroxide inactivation of horseradish PX by means of the activation and stabilization effects of Ni2+ ion, which was found to be useful in processes such as phenol removal and peroxidative conversion of reducing substrates, in which a high concentration of hydrogen peroxide may lead to irreversible enzyme inactivation. 

Advances have been achieved in recent years, such as the use of CL reagents as labels to derivatize and sensitively determine analytes containing amine, carboxyl, hydroxy, thiol, and other functional groups and their application in HPLC and CE [35, 36], the synthesis and application of new acridinium esters [37], the development of enhanced CL detection of horseradish peroxidase (HRP) labels [38], the use of immobilization techniques for developing CL-based sensors [39-42], some developments of luminol-based CL in relation to its application to time-resolved or solid-surface analysis [43], and the analytical application of electrogenerated CL (ECL) [44-47], among others. 

Freeman and Seitz [6] developed one of the first enzyme-based CL sensors with convincing performance. They immobilized horseradish peroxidase (HRP) at the end of an optical fiber and achieved a detection limit of 2 X 10 4 6 mol/L H202. Preuschoff et al. [23] developed a fiberoptic flow cell for H202 detection with long-term stability, suitable for fast FTA. Different peroxidases were covalently... 

Fig. 8. Schematic presentation of a enzyme linked immunosorbent assay (ELISA). An antigen ( ) is immobilized on the surface of a microtiter plate and incubated with its antibody (abl). A second antibody (ab2) with a covalently linked enzyme ( , e.g., horseradish peroxidase) binds to the primary one and catalyzes a color reaction with its enzyme. All incubations are separated by washing steps...

A very interesting approach was presented recently by Niemeyer et al. [113]. They prepared covalent DNA-streptavidin conjugates to which biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase, as well as biotinylated anti-mouse and anti-rabbit immunoglobulins, were coupled. Immobilization of DNA-streptavidin conjugates was performed by hybridization with the complementary oligonucleotides, bound to the surface. It was demonstrated... 

The immobilization proceeded as follows horseradish peroxidase (HRP) was biotinilated with biotinamidocaproate AT-hydroxysuccinimide ester to obtain biotinylated HRP with two biotin molecules per enzyme molecule. Avidin was immobilized on polystyrene support beads using the carbodiimide method. This procedure was followed by an attachment of the disubstituted biotinylated HRP... 

Fig. 1. Schematic presentation of the principle of multilayer immobilization. Biotinilated horseradish peroxidase (HRP) is linked via avidin. (Reprinted with permission from [ 120])...

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