Investigation and Characterisation of Protein-Specific Hydrogel-Based Molecularly Imprinted Polymers (Hydromips).

This work describes the experimental optimisation, characterisation and potential application of protein-specific molecularly imprinted polymers (MIPs), with a view to employing the resulting hydrogel based smart-material (HydroMIPs) as the selective recognition element of a biosensor strategy. Molecular imprinting (MI) involves the production of polymeric recognition materials that exhibit a molecular memory towards a pre-determined template molecule. We have tailored a methodology where polyacrylamide is employed as the material within which proteins are entrapped. The subsequent removal of the template leaves an imprinted cavity that shows a selective and specific affinity towards the original template molecule. We have extensively optimised the production of HydroMIPs, having investigated key variables involved in producing the localised architecture around which the imprinted sites are formed. We also report the optimisation of the template removal process, and in all cases demonstrate a distinctive recognition effect in relation to non-imprinted controls. A high degree of selectivity towards the template molecule is exhibited by the HydroMIPs (in relation to proteins analogous to that of the template). As a result, the proposal of a generic imprinting methodology, which can be applied to the imprinting of a variety of biomolecules is made. Characterisation using a series of high-powered imaging techniques has also been performed. Confocal microscopy of FITC-albumin imprinted HydroMIPs has detailed the HydroMIP structure in its natural form, with the fluorescently labelled template-protein detailing the orientation of imprinted cavities within the polymer. Images of the in situ real-time imaging of template removal studies are also presented. Transmission electron microscopy (TEM), has been used in conjunction with a novel cryogenic preparation technique, freeze etching and immunohistochemical procedures. Electron micrographs are presented that detail imprinted cavities within the HydroMIP matrix that are 5. 5nm in diameter - the size of the original template molecule (bovine haemoglobin -BHb). Cavities were also observed that were up to 20nm in diameter, which suggests that imprinted sites formed to an agglomeration of template molecules. Such structural features were not observed in images of the HydroNIP controls prepared in an identical manner (in the absence of template). Atomic force microscopy (AFM) was also used to interrogate the BHb-HydroMIPs. Topographic images acquired in contact mode further corroborated the data obtained following TEM analysis. Force curve measurements were taken using bio-modified AFM probes. A significantly greater force was required to withdraw the template-modified probe from the cavity containing HydroMIP sample compared to that of the HydroNIP control (23. 08nN ± 0. 31 and 18. 90nN ± 0. 31 respectively). Substantial forces were also required to withdraw an AFM probe (modified with an antibody raised against the template) from template containing samples compared to that of samples prepared (largely) in the absence of template (before template removal = 14. 87nN ± 0. 33nN; after template removal = 11. 22nN ± 0. 18nN; after template rebinding = 14. 77nN ± 0. 53nN & HydroNIP control = 9. 60nN ± 1. 78nN). The BHb-HydroMIP was incorporated as the selective recognition element of an optical biosensor system with Dual Polarisation Interferometry (DPI) employed as the method of analysis. The template molecule was deposited upon the sensor surface with the HydroMIP and HydroNIP gels flowed across the surface. Changes in the density and orientation of bound protein upon the sensor surface allowed the quantification of an imprinting effect. The cavity containing HydroMIP removed a significantly greater amount of template protein (layer thickness and mass decrease of 1. 17nm and 0. 22ng/mm2 respectively) in relation to HydroNIP control gels, where no template stripping effect was observed. This study makes a substantial contribution to the understanding of aqueous-phase molecular imprinting strategies and holds much promise for the future imprinting of biomolecules of clinical significance