Immobilisation methods

This process has been operated successfully by the Tanabe Seiyaku Co. in Japan since 1973. Similar processes have since been commercialised by other companies, such as the Kyowa Hakko Co., often using different immobilisation methods such as polyurethane. The same iimnobilized cell approach has also been used by Tanabe since 1974 in their coimnercial process for the production of L-malic acid from fumarate using the hydratase activity of Brevibacterium ammoniagenes cells. 

Compare the two immobilisation methods with respect to the parameters listed in section 6.3. 

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. 

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. 

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

S. Andreescu, L. Barthelmebs and J.-L. Marty, Immobilisation of acetylcholinesterase on screen-printed electrodes comparative study between three immobilisation methods and applications to the detection of organophosphorus pesticides, Anal. Chim. Acta, 464 (2002) 171-180. 

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). 

Method 1 is known as the method of incipient wetness, because the ionic liquid is added to the support until the mixture starts to lose the appearance of an dry powder. This is the most simple of the presented methods, allowing the immobilisation of high amounts of chloroaluminate liquids on any given silica support. Unfortunately, during the immobilisation step HC1 is created which leads to a decomposition of zeolites and MCM 41 type supports. This problem could be overcome by a modification of the immobilisation method. The supported ILs synthesised this way show a high catalytic activity in Friedel-Crafts reactions. 

The type of immobilisation dictates the ability to regenerate the sensor surface, or the antibody surface, for reuse as well as the stability and storage conditions needed to extend the lifetime of the sensor. The importance of immobilisation is reflected in the fact that a significant percentage of Pz immunosensor publications deal with the development of novel or the improvement of existing immobilisation methods. 

Biomolecular immobilisation remains the primary challenge to commercialisation of immunosensors. Many successful sensors have been produced for numerous analytes, incorporating many different immobilisation methods, but most never make it past a laboratory, with very few ever making it to manufacturing prototypes. The transfer of a laboratory-based sensor to a mass-produced product while still retaining stabiUty, reUabil-ity and sensitivity requires simple and reproducible procedures. Complex multistep immobilisation procedures that require a trained operator to perform in a laboratory will not be easy to transfer to a manufacturing situation and would probably result in a poor quality product when compared to its laboratory-based prototype. 

Their ease of use and the high sensitivity usually obtained means that these are the most common immobilisation method used in Pz immimosen-sor work, with protein A most often used. 

Duan et al. used a monolayer of thioctic acid to covalently immobihse monoclonal antibodies to a gold electrode while performing amperometric analysis [28]. Frey et al. demonstrated potential biosensor application of this immobilisation method by binding polylysine to gold surface plasmon resonance (SPR) electrodes via a SAM of 11-mercaptoundecanoic acid [29]. 

Protein engineering to tag protein A molecules with cysteine residues and to self-assemble these onto gold surfaces has been performed [37]. This method would incorporate the simplicity, reproducibility and covalent advantages of self-assembly with the orientational advantages of protein immobilisation and potentially lead to the most useful immobilisation method. Cleaving of antibodies to break the disulfide bridges and self-assembling these portions onto electrodes has also been attempted by many authors. 

Erom these reports and many other investigations it seems the choice of immobilisation methods is dependent on the specific situation and cannot be predicted. The orientation of protein A-immobilised antibodies is far superior to most other methods and an improvement in sensitivity is often evident, but the formation of reproducible stable layers and the inability to regenerate the antibody surface is a problem. Despite the results reported, the protein layers are only adsorbed to the surface and will eventually leach from the surface. 

Combining immobilisation methods seems to be the best option, such as incorporating the stability and reproducibility of covalent methods such as SAMs with the orientated binding of protein A/G. The first example of this was in 1987 by Muramatsu et al. [4]. The authors cross-linked protein A to a layer of APTES, although it is unhkely that it was done to combine the advantages of both immobihsation methods since this was one of the earhest Pz immunosensors and more basic fundamental problems were being overcome at this stage. 

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. 

A Pz immunosensor for the detection of Listeria monocytogenes was published by Jacobs et al. [87]. The bacteria could be measured to 10 cellsmL using dip and dry methods of detection. Analysis carried out directly in solution allowed detection to 5 x 10 cells mL. Antibody coated crystals were stored for 17 days without detectable loss in activity. Protein A was used as the immobilisation method but not compared to others. Later the group published details of a displacement assay for the detection of this species [88]. This assay could detect the antigen from 2.5 x 10 to 2.5 x 10 cells/crystal directly in solution monitoring the response in real time. The assay was also performed in milk. Assays in milk were also shown to be specific for... 

KOnig and Gratzel published details of a sensor for the detection of diarrhoea-causing bacteria and viruses [9]. Immunosensors were described for the detection of rotavirus and adenovirus. Both viruses were measured linearly from lO to 10 ° virions. As explained earher, protein A was shown to be the most favourable immobilisation method but immobilisation using silane and polyethyleneimine showed a 40% increase in response when detecting rotavirus in stool samples. 

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