Optimised solution-phase synthesis of nanoMIPs for protein detection in electrochemical diagnostics.

Abstract

NanoMIPs are nanoscale molecularly imprinted polymers (MIPs) ranging in size between 30 to 300 nm offering a high affinity binding reagent as an alternative to antibodies. They are being extensively researched for applications in biological extraction, disease diagnostics and biosensors. Various methodologies for nanoMIP production have been reported demonstrating variable timescales required, sustainability, ease of synthesis and final yields. We report herein a fast (<2 h) method for one pot aqueous phase synthesis of nanoMIPs using an acrylamide-based monomer and N,N'-methylenebisacrylamide crosslinker. NanoMIPs were produced for a model protein template namely haemoglobin from bovine species. We demonstrate that nanoMIPs can be produced within 15 min. We investigated reaction quenching times between 5 and 20 min. Dynamic light scattering results demonstrate a distribution of particle sizes (30-900 nm) depending on reaction termination time, with hydrodynamic particle diameter increasing with increasing reaction time. We attribute this to not only particle growth due to polymer chain growth but based on AFM analysis, also a tendency (after reaction termination) for particles to agglomerate at longer reaction times. Batches of nanoMIPs ranging 400-800 nm, 200-400 nm and 100-200 nm were isolated using membrane filtration. The batches were captured serially on decreasing pore size microporous polycarbonate membranes (800-100 nm) and then released with sonication to isolate nanoMIP batches in the aforementioned ranges. Rebinding affinities of each batch were determined using electrochemical impedance spectroscopy, by first trapping nanoMIP particles within an electropolymerized thin layer. Binding constants determined for NanoMIPs using the E-MIP sensor approach are in good agreement with surface plasmon resonance results. We offer a rapid (<2 h) and scalable method for the mass production (40-80 mg per batch) of high affinity nanoMIPs.