Design, synthesis and characterisation of protein based molecularly imprinted polymers: advancing protein diagnostics

Abstract

Molecularly imprinted polymers (MIPs) are becoming realistic alternatives to antibodies in the diagnosis and treatment of disease. Their recognition properties combined with low-cost manufacturing, robustness and stability make them attractive for use in biosensors, assays and separation applications. Atomistic level knowledge of the pre- polymerization process is limited, hampering the rational design of MIPs. Using computation and experimentation, protein-monomer binding interactions, that may influence specificity, were investigated. Using SiteMap, the potential binding sites were predicted for the protein (myoglobin) and binding free energies of a library of acrylamide-based monomers (capable of forming MIP hydrogels) were calculated for each site, using MM-GBSA. A complex interplay between different protein-monomer binding effects and MIP efficacy was observed. These findings corroborated MIP binding events determined using FTIR spectroscopy of thin-film MIPs. FTIR exhibited C=O peaks (for the hydrogels) at around 1700 cm-1 for MIP and NIP. Characteristic changes in hydrogen-bonding interactions occurred upon MIP and target protein complexation.
Selective binding of the target protein to the MIP, produced a decrease in the C=O amide peak; non-target proteins did not elicit this change in the MIP. All proteins studied did not elicit this change in the NIP, suggesting FTIR could be used for rapid prototyping of MIP affinity. Previously unreported, a rapid (20 minutes) microwave-assisted synthesis of aldehyde-functionalized SPIONs was developed. The nanoparticles have an average size of 4.6 ± 1.2 nm, with a saturation magnetization of 38 emu g-1 and near-closed magnetization loops, confirming their superparamagnetic
properties. Protein (myoglobin) was subsequently tethered, with 0.25 ± 0.013 mg of myoglobin adsorbed on to 20 mg of SPION. Using protein-bound SPIONS and a MIP (fluorescently tagged) capture molecule, a non-competitive assay was developed to detect myoglobin levels at a low LOD of 60pg mL-1. The assay protocol could be extended to the determination of other disease biomarkers.