Procedures for immobilization

Surface chemistry is a key technology for protein microarray development. The supports used for protein immobilization have to fulfil some important requirements they must provide good quality spots, low background, simplicity of manipulation and compatibility with detection systems. An ideal surface or immobilization procedure for all proteins and applications does not exist however current methods are more than adequate for many applications. Basic strategies for protein immobilization consider covalent versus non-covalent and oriented versus random attachment, as well as the nature of the surface itself [106]. It has been demonstrated that the specific orientation of immobilized antibody ( capture agents ) consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvement over surfaces with randomly oriented capture agents [107]. 

Dybko, A. et al., Efficient reagent immobilization procedure for ion-sensitive optomembranes, Sens. Actuat. B Chem., 39, 207,1997. 

Immobilized enzymes are defined as biocatalysts that are restrained or localized in a microenvironment yet retain their catalytic properties. Immobilization often increases stability and makes the reuse of the enzyme preparation very simple. The repeated analysis that can be performed by the use of enzymes in an immobilized form reduces the cost of the analysis. The ideal immobilization procedure for a given enzyme is one which permits a high turnover rate of the enzyme yet also retains a high catalytic activity over time. There are two major applications for immobilized enzymes in analytical systems. In one, the enzyme is immobilized onto a particulate solid support matrix, which is then packed into a small column and incorporated into a flow system. The other involves immobilization within or on the surface of an electrode, whereby the electrochemical transduction of enzymatic product is monitored. 

Table 8.2. Immobilization procedures for receptor reagents at optodes...

The ligand has to be immobilized on a sensor chip surface by either chemical cross-linking or with an affinity interaction like biotin-streptavidin (sensor chip SA), nickel chelate-His-Tag (sensor chip NTA) or antibody-antigen (sensor chip CMS). To ensure the measurement of accurate data the immobilization procedure should not interfere with the ligand-analyte interaction. The most common immobilization procedure for proteins is chemical cross-linking, which can reduce the activity or affinity of the interaction due to the modification of the protein or by blocking the binding site. 

Canofeni S, Disario S, Mela J, Pilloton R (1994) Comparison of immobilization procedures for development of an electroehemical Ppo-based biosemsm for on line monitoring of a depuration process. Anal Lett 27(9) 1659-1669... 

The immobilization of the enzymes on various solid carriers gives perfect possibilities to develop not only sensitive and selective, but also rapid and simple test procedures for different compounds determination with visual detection of the analytical signal. 

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

A procedure for immobilization of a P. stutzeri UP-1 strain using sodium alginate was reported [133], This strain does not perform sulfur-specific desulfurization, but degrades DBT via the Kodama pathway. Nevertheless, the report discussed immobilization of the biocatalyst cells in alginate beads with successful biocatalyst recovery and regeneration for a period of 600 h. However, the immobilized biocatalyst did decrease in specific activity, although the extent of loss was not discussed. The biocatalyst was separated after every 100 h of treatment, washed with saline and a boric acid solution and reused in subsequent experiment. The non-immobilized cells were shown to loose activity gradually with complete loss of activity after four repeat runs of 20 hour each. The report does not mention any control runs, which leaves the question of DBT disappearance via adsorption on immobilized beads unanswered and likewise the claim of a better immobilized biocatalyst. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

Depending on the immobilization procedure the enzyme microenvironment can also be modified significantly and the biocatalyst properties such as selectivity, pH and temperature dependence may be altered for the better or the worse. Mass-transfer limitations should also be accounted for particularly when the increase in the local concentration of the reaction product can be harmful to the enzyme activity. For instance H2O2, the reaction product of the enzyme glucose oxidase, is able to deactivate it. Operationally, this problem can be overcome sometimes by co-immobilizing a second enzyme able to decompose such product (e.g. catalase to destroy H202). 

Enzymes can be immobilized by enclosing them within semi-permeable polymer membranes. The preparation of the microcapsules requires extremely well controlled conditions and it is possible to use different procedures for their preparation ... 

Since immunosensors usually measure the signals resulting from the specific immu-noreactions between the analytes and the antibodies or antigens immobilized, it is clear that the immobilization procedures of the antibodies (antigens) on the surfaces of base transducers should play an important role in the construction of immunosensors. Numerous immobilization procedures have been employed for diverse immunosensors, such as electrostatic adsorption, entrapment, cross-linking, and covalent bonding procedures. They may be appropriately divided into two kinds of non-covalent interaction-based and covalent interaction-based immobilization procedures. 

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