Immobilization Of The Anti-Thrombin DNA Aptamer: Attachment, Optimal Packing Density And Kinetics

Dissertation (Ph.D.)-- University of Kansas, Chemistry, 2005.Aptamers have been touted as viable complements to antibodies as molecular recognition elements in diagnostic assays. Several studies have demonstrated aptamer applicability in both homogeneous and heterogeneous assay formats. However, very few studies have been performed to investigate the parameters involving effect immobilization of aptamers, the analyte-binding kinetics of attached aptamers, and the optimal assay format for aptamers. These studies utilized the well-characterized anti-thrombin DNA aptamer. Investigations were conducted in order to ascertain effective methods of immobilization of the aptamer to silica and gold, the maximum packing density for which 100% of the aptamer activity is retained, and the binding kinetics of the immobilized aptamer. The attachment studies found that a short linker extending from the aptamer is essential for covalent attachment, the aptamer tertiary structure and cation dependence can influence the effectiveness of a covalent attachment, and affinity attachment methodologies are reliable for aptamer immobilization. In addition, the optimal packing density for active aptamers was determined to be similar to the reported optimal packing density of singlestranded oligonucleotides used for hybridization assays. The kinetic studies for the binding of the immobilized aptamer to its cognate ligand showed that the binding kinetics of the aptamer are unaffected by either covalent or affinity attachment to a substrate, the association and dissociation binding rates for this aptamer are very fast, and the association rate is diffusion-limited. These results indicate that aptamers may be well-suited to use in miniaturized, real-time, continuous-monitoring biosensors