Capillary electrophoresis screening of the binding affinity of Molecularly Imprinted Nanoparticles.

The fast paced advancement in molecularly imprinted nanoparticles (MIP NPs) synthesis and applications [1] requires straightforward analytical methods suitable for the evaluation of the binding properties of the generated MIP NP libraries. Often the analysis of the MIP NP binding behaviour cannot be easily performed by classical means, because of their nature, i.e. amphipathic, polymeric and their dimensions (10-200 nm), which are greater than biological macromolecules, but smaller than most of the micromaterials. In response to these needs, capillary electrophoresis (CE) was here exploited to screen the binding affinities of MIP NPs targeting the iron regulating hormone hepcidin towards their template peptide, i.e. the N terminal 6-mer of hepcidin [2- 4]. The CE separation method was developed ex novo, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35°C), achieving the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed estimating a micromolar dissociation constant for the 6-mer template/MIP NP complex. The results were found in agreement with independent measures. The CE offers the advantages of a direct analysis of the MIP NP/ligand incubation mix without preliminary fractionation steps, requiring only minimal sample consumption and short analysis times, thus in conclusion, it appeared to be a valid technique for characterizing the interaction of MIP NP libraries for selected target compounds.