A magnetic molecularly imprinted nanoparticle assay (MINA) for detection of pepsin

A fully abiotic Molecularly Imprinted Nanoparticle Assay (MINA) is developed for detection of the model protein pepsin. The format of the pepsin assay is based on binding of fluorescent pepsin-specific molecularly imprinted polymer nanoparticles (nanoMIPs) to the magnetic pepsin nanoparticles (MPN) immobilised on magnetic microtiter plate inserts. Competition between free pepsin and immobilised pepsin results in a decrease in nanoMIPs binding to the magnetic inserts, resulting in an increase in fluorescence. Pepsin-specific are prepared using a solid phase synthesis protocol. In order to increase the sensitivity of MINA, two labelling approaches are performed. The first approach uses a fluorescein acrylate included in the monomeric mixture during polymerisation, and the second approach is based on a post-imprinting modification of nanoparticles using the commercial fluorophore AlexaFluor® 647 NHS ester. It is observed that upon increase of free pepsin concentration from 1 μM to 100 μM, fluorescence is increased by 68%. No cross-reactivity in the presence of non-specific protein trypsin is detected. The results show that AlexaFluor-labelled nanoMIPs demonstrate higher performance towards pepsin than fluorescein-labelled nanoMIPs. The novel assay reduces the time and cost of analysis and does not uses any antibodies, eliminating the need for animal-derived reagents. The portfolio of the optimised protocols can potentially be applied for the detection of any other proteins of clinical and environmental interest.