title: Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).
abstract: Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 μM, 44 ± 3 μM, 17 ± 2 μM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface.
authors.affiliation: Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK. s.reddy@surrey.ac.uk.
authors.forename: Hazim F
authors.initials: HF
authors.lastname: EL-Sharif
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Hawkins
authors.forename: Derek
authors.initials: D
authors.lastname: Stevenson
authors.forename: Subrayal M
authors.initials: SM
authors.lastname: Reddy
mesh_terms: D000818:Animals; D002374:Catalase / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D054802:Molecular Imprinting; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D058105:Polymerization; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D006454:Hemoglobins; D009211:Myoglobin; D011108:Polymers; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D002374:Catalase