Methods of Immobilisation

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. 

The crosslinking of the bioraolecules via bifunctional reagents, e.g., glutaraldehyde and bovine serum albumin, has been utilised to stabilise the biomolecules. In this case, a crosslinked network of enzyme is formed resulting in the formation of a bigger molecule on the polymer surface [122, 123]. Enzymes have also been immobilised on the surface of electrodeposited polymer by applying the bovine serum albumin and glutaraldehyde procedure followed by the electrodeposition of the polymers [1,124,125]. This method has certain limitations since it may cause a drastic loss in the activity of the biomolecules. 

The biomolecules are attached directly to the matrix by chemical/covalent linkage, which is not reversed by pH or changes in ionic strength. Carbodiimide coupling to form peptide bonds has been extensively used for the covalent coupling of the enzyme with polymers [126-128]. Since chemical modification is involved, this method results in the drastic loss of activity of the enzymes/biomolecules. Covalently attached redox biomolecules on polymers have been utilised as highly electron transfer mediators in flavin adenine dinucleotide (FAD) centres of oxidases [73]. 

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 

It has been observed that the surface of the conducting polymer plays an important role in the effective immobilisation of the desired enzyme. The Langmuir-Blodgett (LB) technique can be successfully applied to deposit a desired monolayer with the desired orientation of the biomolecules/enzymes [142-145]. Ramanathan and co-workers [146] have utilised the polyemeraldine base LB films for the immobilisation of GOD. These films have been shown to function as amperometric glucose biosensors and have a linear range from 5 to 50 mM. LB films of PT immobilised with GOD and urease have also been prepared for application to respective biosensors [147, 148]. 

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Immobilisation of biomolecules on the surface of an effective matrix with maximum retention of their biological recognition properties is a crucial problem for the commercial development of a biosensor. Different methods of immobilisation have been used. One such method is electrochemical entrapment. Several conducting polymers can be deposited electrochemically and, in the process, a biological molecule can be entrapped. This process is also useful in the fabrication of microsensors in preparation of a multilayered structure with one or more enzymes/biomolecules layered within a multilayered copolymer for analysis of multiple analytes [133-135]. A number of reports have appeared on immobilisation of biomolecules using electrochemical entrapment [130, 131, 136-143]. 

The wide chemical variation of radioactive wastes which are extremely complex in composition require different methods of immobilisation and containment. The waste forms range from Low Level Waste (LLW), Medium Level Waste (MLW) through to High Level Waste (HLW). MLW can be embedded in cement or bitumen whereas HLW is either reprocessing waste immobilised in borosilicate type glasses or is the spent fuel when declared as waste. 

To develop a continuous process, the immobilisation of aminoacylase of Aspergillus oryzae by a variety of methods was studied, for example ionic binding to DEAE-Sephadex, covalent binding to iodo-acetyl cellulose and entrapment in polyacrylamide gel. Ionic binding to DEAE-Sephadex was chosen because the method of preparation was easy, activity was high and stable, and regeneration was possible. 

B. Bucur, A.F. Danet, and J.L. Marty Versatile method of cholinesterase immobilisation via affinity bonds using concanavalin A applied to the construction of a screen-printed biosensor. Biosens. Bioelectron. 20, 217-225 (2004). 

The method of catalyst immobilisation appeared to affect its performance in catalysis. Catalyst obtained by method II showed a low selectivity in the hydroformylation of 1-octene (l b aldehyde ratio was even lower than 2) at a very high rate and high yields of isomerised alkenes (Table 3.2, entry 2), whereas procedure IV resulted in a catalyst that was highly selective for the linear aldehyde (with a l b ratio of 37) (entry 5). In accordance with examples from literature it is likely that procedure II gave rise to the ionic bonding of ligand-free rhodium cations on the slightly acidic silica surface [29],... 

Sakamoto [243] determined picomolar levels of cobalt in seawater by flow injection analysis with chemiluminescence detection. In this method flow injection analysis was used to automate the determination of cobalt in seawater by the cobalt-enhanced chemiluminescence oxidation of gallic acid in alkaline hydrogen peroxide. A preconcentration/separation step in the flow injection analysis manifold with an in-line column of immobilised 8-hydroxyquinoline was included to separate the cobalt from alkaline-earth ions. One sample analysis takes 8 min, including the 4-min sample load period. The detection limit is approximately 8 pM. The average standard deviation of replicate analyses at sea of 80 samples was 5%. The method was tested and inter calibrated on samples collected off the California coast. 

Effective syntheses, convergent as well as batch-splitting, are possible using these methods, which can provide both combinatorial arrays and fragment sets of new molecules (Fig. 2). The act of immobilisation can be accomplished in a variety of ways such as on beads, active... 

Other biocatalytic methods of producing D-p-hydroxyphenylglycine have not proved competitive, for instance transaminase based processes require glutamate to be supplied. Others include the hydrolysis of N-acyl derivatives by acylase and amides by aminopeptidase (DSM), the use of L-specrfic hydantoinases and immobilised subtilisin for the resolution of D,L-2-acetamido-/>-hydroxyphenylacetic acid methyl ester (Bayer). 

Jensen, V.J. and Rugli, S. (1987) Industrial scale production and application of immobilised glucose isomerase. Methods in Enzymology, 136, 356-370. 

Although at present their use has been restricted to redox-active sensors in solution, it should be possible to immobilise these receptors at an electrode, and we may then have a simple redox-active electrode whose behaviour in solution is modified by the presence of ions. The high sensitivity of electrochemical techniques would then give us a sensitive and selective method of anion detection. 

Overall there are benefits and limitations to all of the common methods for immobilising Lewis acids. This helps illustrate the apparently inescapable conclusion in heterogeneous acid catalysis that there are likely to be as many optimum catalysts as there are acid-catalysed processes. 

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