Cross-linking

This type of immobilisation is support free and involves joining cells (or enzymes) to each other to form a large three-dimensional complex structure and it can be achieved by chemical or by physical methods . Chemical methods of cross-linking normally involve covalent bond formation between proteins or cells by means of a bi- or multifunctional reagent such as glutaraldehyde and toluene diisocyanate.
 However, the toxicity of such reagents is a limiting factor in applying this method to living cells and many enzymes. Both albumin and gelatin have been used to provide additional protein molecules as spacers to minimise the close proximity or crowding problems that can
be caused by cross-linking an enzyme.
Physical cross-linking of cells by flocculation is well known in the biotechnology industry and does lead to high cell densities. Flocculating agents such as polyamines, polyethylenimine, polystyrene sulfonates and various phosphates have been used extensively and are well characterised.
Cross-linking is rarely used as the only means of immobilisation because absence of mechanical properties and poor stability are severe limitations. Cross-linking is most often used to enhance other methods of immobilisation by assisting the reduction of cell or enzyme
leakage in other systems.
A potentially useful development is the production of cross-linked enzyme crystals (CLECs).Production of enzyme crystals has long been seen as a final step in enzyme purification procedures as it signifies high purity. The principal use of enzyme crystals per se has been in X-ray crystallography to study enzyme structures and recent recognition that some enzyme crystals were catalytically active has led to the development of CLECs. 

Indications are that CLECs have superior stability characteristics and may enable novel enzyme-catalysed biotransformations to occur in difficult environments such as in organic solvents, in a gas phase or in supercritical fluids. Enzyme crystals might be more easily incorporated into microelectronic devices such as a CHEMFET (chemically sensitive field effect transistor) and ISFET (ion-selective field effect transistor) to produce novel ‘bio-chips’ for the next generation of biosensors.