Magnetic nanoparticle-facilitated rapid mass production of high affinity polymeric materials (nanoMIPs) for protein recognition and biosensing.

Abstract

Molecularly imprinted polymers (MIPs) have been investigated extensively for broad applications in diagnostics, imaging and therapeutics due to their antibody-like specificity, high stability, and low-cost and rapid production when compared with biological antibodies. Yet, their wide-scale adoption and commercial viability are limited due to low yields and relatively lengthy preparations of current methods. We report the novel application of protein-functionalised magnetic nanoparticles (MNPs) to enable the rapid mass production of nanoMIPs for protein recognition. An aldehyde-functionalised MNP (MNP@CHO) precursor was synthesised using a one-pot microwave method in less than 20 minutes, resulting in 330 mg yield for a 30 mL reaction volume. The MNP@CHO precursor (10 mg) was subsequently functionalised with 600 μg of a target template protein, giving MNP@protein. In the presence of an N-hydroxymethylacrylamide (NHMA) functional monomer and N,N'-methylene bisacrylamide as a crosslinker, the MNP@protein particles served as nucleants for the mass production of nanoMIPs in a 20-30 minute synthesis process. Subsequently, the nanoMIPs could be harvested with sonication and then retrieved using a magnet, leaving the MNP@protein particles to be recycled and re-used at least 5 times for further nanoMIP production cycles. In general, 10 mg of MNP@protein produced 10 mg of nanoMIP with a 20% decrease in the yield over the 5 synthesis cycles. For the bovine haemoglobin nanoMIP, the KD was determined to be 3.47 × 10-11 M, a binding affinity rivalling values found for monoclonal antibodies. We also demonstrate that the methodology is generic by producing high-affinity nanoMIPs for other proteins including albumin, lysozyme and SARS-CoV-2 recombinant protein. We therefore present a facile route to produce nanoMIPs in large industrially relevant quantities (hundreds of mg) and at short timescales (within a day). Our method offers realistic opportunities for the industry to adopt such materials as an antibody replacement technology in diagnostics, biological extraction and therapeutics.