RESEARCH PAPER Screening of the binding properties of molecularly imprinted nanoparticles via capillary electrophoresis Giacomo Musile 1 & Lucia Cenci 2 & Erika Andreetto 2 & Emmanuele Ambrosi 3 & Franco Tagliaro 1 & Alessandra Maria Bossi 2 Received: 12 November 2015 /Revised: 2 February 2016 /Accepted: 11 February 2016 # Springer-Verlag Berlin Heidelberg 2016 Abstract In response to the need for straightforward analyt- ical methods to assess the affinity of molecularly imprinted nanoparticles (MIP NPs) for ligands, capillary electrophoresis (CE) was exploited using MIP NPs targeting the iron- regulating hormone hepcidin. In this work, MIP NPs were challenged with their template peptide, i.e., the N-terminal 5- mer of hepcidin, in comparison to unrelated ligand peptides. A CE separation method was developed ex novo achieving, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35 °C), the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed the estimation of a micromolar dissociation constant for the 5-mer template– MIP NPs complex, in agreement with independent measure- ments. The CE offered the advantages of a direct injection of the MIP NPs/ligand incubation mix, without preliminary frac- tionation steps, requiring only minimal sample volumes and short analysis times. In conclusion CE proved to be a valid technique for characterizing the interactions of MIP NP librar- ies for selected target compounds. Keywords Molecularly imprinted nanoparticles . Capillary electrophoresis . Binding isotherm . Affinity . Hepcidin Introduction Nanomaterials, in virtue of their peculiar physicochemical properties, such as sizes similar to macromolecules, limited and controllable number of binding sites, fast binding equilib- ria, and particular spectral properties, stand at the basis of the development of new products and processes, finding applica- tions in imaging, sensing, drug delivery, catalysis, assays, op- tics, physics, and electronics [1]. Among the strategies to pre- pare nanomaterials and nanoparticles (NPs) with suitable functionalities, molecularly imprinted polymers (MIP) display great versatility and potential [2–5]. MIPs are obtained by a template-assisted synthesis in which a target analyte acts as a molecular template around which functional monomers and cross-linkers are polymerized [6]. At the end of the polymer- ization, the template is removed and cavities complementary to the template are exposed on the polymer. To prepare MIPs in the form of NPs (MIP NPs) the synthesis is conducted under conditions confining the growth of the polymeric chains to a limited size (10–200 nm) by precipitation polymerization [7, 8], emulsion polymerization [9], and post-dilution methods [10]. Moreover, MIP NPs can be in the form of full polymeric or as core-shell materials [11–13]. To date, the MIP NPs have been successfully employed in sensing, catalysis, and assays [14–16]. In parallel to the advancement in MIP NP synthesis and applications, straightforward analytical methods suit- able for the evaluation of the binding properties of the generated MIP NP libraries become necessary. The anal- ysis of the MIP NP binding behavior often poses prob- lems associated with their nature (amphipathic, Electronic supplementary material The online version of this article (doi:10.1007/s00216-016-9418-y) contains supplementary material, which is available to authorized users. * Alessandra Maria Bossi alessandramaria.bossi@univr.it 1 Department of Diagnostics and Public Health, Unit of Forensic Medicine, University of Verona, Piazzale Ludovico Antonio Scuro 10, 37134 Verona, Italy 2 Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy 3 Department of Molecular Sciences and Nanosystems, University Ca’ Foscari Venezia, via Torino 155/b, 30173 Venice, Italy Anal Bioanal Chem DOI 10.1007/s00216-016-9418-y