Enzyme fixation

A number of important processes depend on the permanence of particle attachment to surfaces by Van der Waal forces in the presence of flowing fluids. These include enzyme fixation, particle filtration, oil production, nuclear reaction excursions, migration of surface contaminants, etc. The release of particles attached to a surface plays an important role in these processes. 

Even though efficient enzyme-recovery and enzyme-fixation systems are available, they must be used under carefully controlled conditions in order to prevent loss of enzyme activity by accidental deviation from defined temperature and pH conditions and by bacterial contamination. 

The direct fixation of the biocatalyst to the sensitive surface of the transducer permits the omission of the inactive semipermeable membranes. However, the advantages of the membrane technology are also lost, such as the specificity of permselective layers and the possibility of affecting the dynamic range by variation of the diffusion resistance. Furthermore, the membrane technology has proved to be useful for reloading reusable sensors with enzyme. In contrast, direct enzyme fixation is mainly suited to disposable sensors. This is especially valid for carbon-based electrodes, metal thin layer electrodes printed on ceramic supports, and mass-produced optoelectronic sensors. Field effect transistors may also be envisaged as basic elements of disposable biosensors. 

Foulds and Lowe (1986) combined mass production of the base sensor and enzyme immobilization as follows. Using gold or platinum ink, a working and counter electrode were deposited on a ceramic substrate. After thermal treatment of the electrode material a solution containing GOD and a pyrrole derivative of ferrocene was electrochemically polymerized at the electrode. The pyrrole component forms a conducting polymer and the immobilized ferrocene acts as electron acceptor for GOD. The structured immobilization permits this technique to be used for successive enzyme fixation to multiparameter sensors. 

When macromolecular substrates are involved in the transformation under study, concentration polarization phenomena affect the EMR performance more severely. Diffusion limitations of macromolecular substrates hamper the use of immobilized enzymes in the hydrolysis of high-molecu-lar-weight substrates. By selecting membranes with an appropriate molecular weight cut-off, both enzyme and substrate are retained in an EMR in touch with each other, and hydrolysis products and/or inhibitors are continuously removed from the system. Soluble enzymes can then act directly on substrate macromolecules without diffusion limitations and steric hindrance imposed by enzyme fixation to a solid support. The stirring features of CST EMRs moreover assures that substrates and/or inhibitors within the reactor vessel are maintained at the lowest possible concentration level. Such reactor configuration is then extremely useful when substrate inhibited reaction patterns are involved, or when inhibiting species are assumed to exist in the feed stream. 

Prior to the development of polystyrene resins, phenol-formaldehyde (P-F) condensates were used as matrices, but they have now been replaced. A few weak-base types still exist (e.g., Duolite ES562 of Rohm and Haas), which are made by adding an amine during polycondensation. These P-F condensates are used for enzyme fixation. 

The activity of an enzyme immobilized by one of the chemical methods is determined by numerous factors the properties of the enzyme, the properties of the carrier, the method of formation of the covalent bond with the carrier, etc. Therefore, it is difficult to predict the properties of the enzyme in every given case. The methods of immobilization, as well as specific examples of enzyme fixation, are discussed elsewhere. ... 

Ordinary ELISA system conformational changes of the surface-fixed antigen and nonspecific adsorption of enzyme fixation antibody appears. 

It is said that the first attempt at enzyme fixation to make better utilization was done Grubhofer and Schleith when they covalently bonded carboxypeptidase and diazotase to diazopolyaminostjnjene [1]. 

Materials that exhibit an isoelectric point in the acid (e.g., glucosidase) can be fixed by aminated, superfine fibers. On the other hand, tiiose with an alkaline isoelectric point can be fixed by sulfonated superfine fibers (see Table 1) [5]. When the nitrogen content in tire fiber increased, tire amount of enzyme fixation increased linearly proportionately and was almost saturated at 1 wt%. 

The adsorptive fixation rate of enzymes by superfine fibers is much faster than ordinary PVA fibers. When the amount of added enzyme is 2000U/g-fiber, 93% of the enzyme is adsorbed 5 min after the superfine fibers are added. By contrast, merely 9% is adsorbed during the same time with ordinary PVA fibers, and even after 4h, only 26% is adsorbed. Accordingly, enzyme fixation ability improves drastically by increasing the surface area [4]. 

In order to develop high value-added glucose sensors with long-term stability and biocompatibility, systems with polymer mediators with redox active groups have recently been studied. For example, long-term stability was improved as follows. The mediator molecules can be chemically fixed to the polymer substrate, which traditionally has been used as the enzyme-fixation matrix. This polymer functions as a polymer matrix and a mediator, allowing the preparation of an enzyme electrode without leakage of the enzyme and mediator. If a hydrophilic polymer mediator that swells... 

Naturally, the first enzyme fixation methods [2] involved physical retention, and it was attempted to preserve, as far as possible, the integrity of the biological system. It was later realized that covalent fixation would prevent enzyme loss, and ensure long-lasting immobilization. The immobilization of enzymes is of prime importance. So long as the enzymes remain active, their immobilization enables repetitive and multiple determination. Conventional enzymatic methods discard the enzyme after each separate sampling. Enzymes are expensive because of the various extraction and purification stages they require, and we can immediately understand the economic interest of the new procedure, and its impact on the cost of sample analysis in automated systems. 

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