Enzymes immobilisation methods

Immobilisation methods are treated in detail in chapter 6. Most enzyme immobilisation methods used in coimection with non-conventional media rely on noncovalent interactions between the support and the enzyme. The reason why this works well in many cases is that enzymes normally have a low tendency to dissolve in the reaction media used. Adsorption or deposition on porous supports are often used methods. It is important to remember that other substances (for example salts and other polar substances) are often immobilised on the support because they are present during the immobilisation procedure and not soluble in the reaction medium. Those substances influence the microenviromnent of the enzyme and thereby its catalytic activity. 

Ref. Matrix Enzyme/immobilisation method Electrode configuration/ applied potential Mediator... 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

The enzyme immobilisation was carried out on 7,7,8,8-tetra-cyanoquinodimethane (TCNQ)-modified screen-printed electrodes. TCNQ allows the electrochemical oxidation of thiocholine, the product of the reaction between acetylthiocholine and the enzyme, at +100 mV (vs. Ag/AgCl) (Fig. 16.6). The enzymatic activity of the acetylcholinesterase can thus be monitored by electrochemical methods. 

New reactor designs and immobilisation methods have been used to extend the lifetime of lipases in scCC>2 (Lozano et al., 2004). Ceramic membranes have been coated with hydrophilic polymers and the enzyme covalently attached to these. In SCCO2, activities and selectivities were excellent and the half-life of the catalyst was enhanced. It is thought the hydrophilic layer of the membrane protected the enzyme. Operational stability of enzymes has also been increased by using ionic liquid/scC02 biphasic systems (Lozano et al., 2002 Reetz et al., 2003). 

Tlie electrochemically-derived kinetic constants can then be compared to those obtained by conventional methods, to judge how the activity of the enzyme immobilised on the electrode compares with that observed in solution. Even when the population of electroactive enzyme is too small to measure reliably (generally below 2 pmol cm for one-electron signals on a graphite electrode) a catalytic wave can be observed whose current... 

Despite the previous examples very little fundamental understanding exists about the nature of biological interactions in the gas phase. More thorough investigations are needed to determine binding affinities, association and dissociation constants, and rates of the antigen/antibody interaction. These then need to be compared to the parameters in the aqueous phase. The activity of the enzyme or antibody could be affected by many factors such as accessibility and reactivity. Orientation of the bio-component, which is affected by its immobilisation method, will probably differ significantly when in gas and liquid phase. 

Another case of heterogeneous systems refers to immobilized enzymes. The kinetic behaviour of a bound enzyme can differ significantly from that of the same enzyme in free solution. The properties of an enzyme can be modified by suitable choice of the immobilisation protocol, whereas the same method may have appreciably different effects on different enzymes. These changes may be due to conformational alterations within the enzyme, immobilisation procedure, the presence and nature of the immobilisation support. The advantages of immobilised enzymes are for instance in reusability and possibility to use continuous mode. 

The biocatalysts obtained were evaluated with respect to the composition, morphology, activity and stability of the immobilised enzyme in the starch hydrolysis reaction. In general, two alternative methods can be used, considering the bioartificial matrix as a substrate for the enzyme (this method is used for example to drive drug release into erosion control devices), or alternatively, as in the case of this work, after blending the enzyme with a polymer, and investigating its activity against an external substrate. The apparent kinetic parameters of the reaction catalyzed by the immobilised and native enzymes were determined and compared. 

However, the main goal was to study the behavior and performance of a novel bioartificial material (obtained with an innovative method involving prefreezing and inversion steps) as a suitable matrix for the entrapment of proteins or enzymes in a stable manner. The tests performed (determination of enzyme activity, determination of and V ax kinetic parameters, repeatability test) confirmed both that the enzyme immobilised in the bioartificial polymeric matrix maintained its catalytic activity unchanged and that the catalytic reaction rate was comparable with that of the free a-amylase reaction (used as control). 

The goal of the present review is an analysis of the electrochemical sensor approaches used for the detection of hydrogen peroxide. Due to similarity of the subjects the present review discusses both, the development of the biosensors where hydrogen peroxide is a product/substrate of enzynutic reaction and also the development of chemical sensors/biosensors specifically designed for the detection of this analyte. Several rechnical aspects of the development of sensors for hydrogen peroxide are reviewed in the following chapters the choice of physical transducer, the choice of enzyme and immobilisation method, and the performance of sensors with respect to response time, sensitivity, linear range, detection limit and operational stability. 

The factors that affect the performance of H2O2 biosensor are (i) the type of enzyme, (ii) the immol sation method, and (iii) the thickness of the created enzyme layer. Immobilisation processes are quite an important Eictor for the development of biosensors. This step has been smdied extensively, in order to achieve easy operation, quick measurement and reduce the cost of the analysis. The methods of enzymes immobilisation used for the development of hydrogen peroxide sensors can be divided into five major groups (Fig. 3) ... 

Figure 10.5 Different methods of enzyme immobilisation on conducting polymers (a) physical adsorption (b) crosslinking by bifunctional reagents (c) covalent bonding to the matrix (d) entrapment within the polymer matrix (e) immobilisation in a conducting polymer polyvinyl carbazole (PVCZ)/stearic acid (StA) monolayer using...

A number of biomolecules have been physically immobilised on conducting polymers [66,112, 116-119]. This is the simplest method of enzyme immobilisation. Since the binding forces involved are hydrogen bonds, van der Waals forces, etc., porous conducting polymer surfaces are most commonly used. The pre-adsorption of an enzyme monolayer prior to the electrodeposition of the polymer, [120] and two-step enzyme adsorption on the bare electrode surface and then on PPy film [121] have also been investigated. 

Polymerisation based on the electrochemical oxidation of a given monomer from a solution containing the enzyme is the simplest method of enzyme immobilisation in a polymer at the working electrode surface and results in the formation of conducting or non conducting... 

Biosensors require highly active enzymes/biomolecules therefore, the immobilisation methods must be chosen in such a way that they can achieve a high sensitivity and functional stability. This is important for economic reasons also. The measurable activity gives an idea about the biocatalytic efficiency of an immobilised enzyme. The rate of substrate conversion should rise linearly with enzyme concentration. The measured reaction rates depend not only on the substrate concentration and the kinetic constants (Michaelis Menten constant) and (maximum velocity of the reaction) but also on the immobilisation effects. The following effects have been observed [157] due to the immobilisation process ... 

Potentiometry is a rarely used detection method employed in biosensors, with enzymes immobilised in an electrodeposited polymer layer, although certain advantages over... 

A general design of a DNA biosensor and the steps involved in the analyte determination are presented in Fig. 18. Initially, an electrodic surface is modified with a ssDNA probe by a convenient immobilisation method. After that, the DNA biosensor is immersed in a solution containing the target complementary DNA for the occurrence of the specific hybridisation. The hybridisation event is usually detected directly through the change of an electric signal i.e. current), or indirectly with the help of an enzyme or redox labels. There are three types of DNA... 

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