Tethering of biomolecules  to transducer surfaces (often but not exclusively proteins like enzymes and antibodies) represents a critical step in the fabrication of any biosensor where the aim is usually to achieve an optimum loading of the sensing molecule without compromising its biological activity. Initial procedures concentrated on chemical modification typically with reagents such as glutaraldehyde as amine to amine crosslinker, or the use of carbodiimide mediated peptide bond formation between surface carboxy groups and transducer amines (or vice versa).  However, this often caused loss of activity and newer affinity methodologies represent a much cleaner and more targeted approach to biomolecule immobilisation.

Non-specific affinity procedures  rely on the multi-point electrostatic interaction between antibody/enzyme and typically anionic polymer coated transducer surface. Biosensor fabrication is accomplished merely by dip coating the transducer into a solution of the protein and the procedure is very gentle and essentially irreversible. In addition, we have recently developed a procedure for simultaneous construction of a polyanionic surface and deposition of Prussian Blue mediator nanocrystals.  There are also a wide range of specific affinity procedures. Tagging of the proteins with biotin allows cross linking with avidin to biotin tagged surfaces with very high efficiency and although the biomolecule must be chemically modified its activity can at least be checked prior to biosensor fabrication. Alternatively, polyhistidine tagged recombinant proteins, can be immobilised onto Ni²⁺ loaded NTA groups. This has the advantage that the transducer surface can be stripped of his_n - tagged protein using imidazole and reloaded with fresh sensing biomolecule. Finally, with antibodies (IgG), specific cleavage with 2-mercaptoethanolamine produces a "half-antibody" with unique cys groups on the Fc domain.  The cys thiol then provides a unique target for the  hetero-bifunctional crosslinker sulfoSMCC which has -NHS functionality for  an amine loaded transducer surface, and maleimido functionality specific for the thiol groups, thereby allowing oriented attachment of the half antibody.

For both specific and non-specific approaches examples will be presented from our work on amperometric and impedimetric electrochemical biosensors and from nanoparticulate optical biosensor systems. I will also describe data using sugar binding biosensors that hint at the underlying mechanism of impedimetric biosensor operation.

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1. Electrostatic immobilisation strategies for attachment of enzymes and antibodies to biosensor surfaces.
2. Mixed self-assembled monlayers (SAMs) for immunosensor construction
3. Designed affinity surfaces for biomolecule immobilisation and biosensor construction