|dc.description.abstract||The ability to immobilise biomolecules on solid surfaces whilst retaining their function is the foundation of microarrays, biosensors and other miniaturised assays that provide convenient ways of analysing binding reactions of molecules with their ligands. The stability, sensitivity, cost and ease of use are important considerations when designing these systems, with the immobilisation of the biomolecule to a surface being the key to success. Miniaturised assays can allow multiplex analysis of various samples from protein interactions, carbohydrate recognition events, DNA hybridisation and cell immobilisation. They can enable fast, high-throughput and efficient testing for diagnostics, environmental testing and research purposes, as well as enabling production of whole-cell arrays to tissue engineering. The drive to miniaturisation and integration has advanced the need for effective and stable attachment of biomolecules to solid surfaces which allow high probe density and high signal to noise ratio whilst maintaining the activity of the molecule.
KODE™ technology is a surface engineering technology that enables modification of a cell or virion surface without causing harm. This is achieved with Function-Spacer-Lipid (FSL) constructs that embed into the membrane. They contain a functional head group that conveys a desirable attribute to the cell or virion, which include carbohydrate or peptide antigens, fluorescent markers, whole proteins or useful molecules for attachment, e.g. biotin. This research set out to determine if FSL constructs could modify non-biological surfaces as well as biological ones.
The aims of this research were to establish whether FSL constructs could be attached to non-biological surfaces, and if so what the mechanisms of attachment and the limitations might be and what methods could be used to deliver the molecules to the surface. Inkjet printing was investigated as the primary delivery method, as it is becoming a popular approach to apply biological molecules to surfaces. It is a non-contact, precise, flexible and fast technique which lends itself well to this field where precise patterning, sample and waste volume and cost are important factors. The water soluble advantages of FSL constructs make their use as a “bioink” attractive and this method proved to be successful at applying FSLs to surfaces.
FSL constructs were successfully applied to various surfaces including papers, polymers, metals, modified cellulose materials and natural fibres. All surfaces were able to attach the FSLs. The stability of the attachment was shown to be unchanged after 8 months at room temperature. Some detergents and solvents were able to remove the FSLs from the surface, indicating particular limitations of the attachment. Variations of the FSL structure gave insights into the attachment mechanisms which indicated that the amphiphilic nature of these constructs is important, with hydrophobic forces causing adsorption of these constructs onto solid surfaces.
Investigations into possible applications of FSL modified surfaces included mapping monoclonal antibodies against different blood group antigens, assessing disease markers in solid phase and attaching recombinant proteins to surfaces. The surface selected as optimal for bioassays was paper due to low background staining and high sensitivity when used in immunoassays. FSL constructs appear to allow orientated immobilisation of biomolecules, spaced away from the surface. Good correlation to serological results using the same constructs was observed, indicating the solid phase FSL assays can provide accurate results.
Cells and kodecytes were also shown to be immobilised onto solid surfaces through FSL attachment. Through linker molecules, red blood cells were attached to microspheres, paper, plastic and other surfaces which themselves were modified with FSLs. This is a potential novel method for producing cellular arrays, cellular bandages and for other applications needing precise attachment of cells. Further research will reveal other types of cells able to be used in this way.
In summary it was discovered that FSL constructs were able to modify non-biological surfaces with carbohydrate antigens, peptide markers and biotin, adding functionality to a wide variety of surfaces. This now opens up the potential to use FSLs as a universal surface modification technology by providing a fast, simple and easily controlled approach for biomolecule immobilisation.||en_NZ