Detection of lipopolysaccharide pyrogens by molecularly imprinted polymers
Bowen, Jenna Louise
Lipopolysaccharide (LPS) is commonly implicated in the development and rapid progression of sepsis however no efficient diagnostic assay currently exists. The over-arching aim of this project was therefore to develop a novel biomimetic peptide-polymer hybrid system capable of recognising and binding LPS in a variety of biologically relevant environments. Target selective peptides (both commercially available and synthesised) have been used as high affinity 'functional monomers' in a molecular imprinting approach. To reduce the concept to practice, a bi-functionalised resin was prepared so as to allow the use of two independent surface attachment strategies. Controlled polymer growth was initiated from surface bound iniferter groups whilst the attachment of the peptide was achieved through amme-amine imidoester linkages or via azide-alkyne 'click' chemistry. Polymyxin, a small, conformationally constrained cyclic peptide that possesses high affinity for lipopolysaccharide (LPS) was used to provide proof-of-principle. Polymyxin resins, produced via the immobilisation of alkyne derivitised polymyxin B on the surface of azidomethyl polystyrene via 'click' chemistry, were able to efficiently bind LPS from aqueous solutions with an apparent Ka of 0.2 μM. Although the development of the peptide-polymer hybrid system using these resins appeared somewhat unsuccessful, whether the observed reduction in binding is due to changes in the Bmax or the Kd of the resin remains to be elucidated. The assay performed with the polymerisation samples produced using resin displaying polymyxin immobilised via a dimethyl adipimidate linker, suggest that the hypothesised approach is feasible but that optimisation of a number of variables is needed before definitive results can be obtained.
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