MOLECULARLY IMPRINTED NANOPARTICLES FOR THE MEASUREMENT OF THE IRON REGULATOR HORMONE HEPCIDIN IN SERUM SAMPLES

Hepcidin is a peptide hormone, mainly excreted by the liver, that was identified as the central systemic iron-regulator. The quantification of hepcidin gained significant importance in the past years for diagnostic and prognostic purposes concerning human diseases related to iron homeostasis. However the development of a reliable and broadly applicable assay for the assessment of hepcidin levels in biological fluids has proved to be very challenging and a general consensus solution for hepcidin determination is still missing. In the present thesis, a method for hepcidin 25 quantitation based on synthetic recognition nanoparticles (NPs) prepared via Molecularly Imprinted Polymer (MIP) technology is proposed. Molecularly imprinted polymers (MIPs) are biomimetic materials capable of specific recognition towards an analyte. Their molecular recognition properties, combined with their high stability, robustness, and easy synthesis, make MIPs extremely attractive as alternative to antibodies. For this reason, imprinted nanoparticles (MIP NPs) were synthesized for the selective capturing of hepcidin. During my PhD the synthesis of a library of MIP NPs for hepcidin 25 was performed, exploiting a non-covalent imprinting precipitation polymerization strategy. The NPs library was physically characterized using: Dynamic Light Scattering (DLS), to collect information about the size distribution and the sample monodispersity; Static Light Scattering (SLS), for measuring the averaged molecular weight (Mn) of the NPs; and Atomic Force Microscopy (AFM) in air, to investigate the morphology of our NPs batches. In a subsequent part of the work, the NP library was functionally characterized by Surface Plasmon Resonance (SPR). In fact, good affinity for human hepcidin 25 of MIP respect to control non-imprinted (NIP) polymer was estimated for some NP formulations by Surface Plasmon Resonance (SPR) analysis. In particular the most significant SPR responses were found for three MIP batches exhibiting Kd values in the nanomolar range, in similarity with the Kds of natural monoclonal antibodies. In the last part of the PhD thesis, the development of an ELISA like MIP-based assay for the assessment of the hepcidin 25 levels in serum was approached. Variables, such as the quantity of NPs deposited per microtiter plate well, the most suitable solution to quench the aspecific binding, the competitive or direct format of assay, the time of response of the assay, were evaluated. The response of the ELISA like assay with model hepcidin solutions was evaluated in the range 1-100nM. In order to diminish the background noise of the assay, a protocol for real sample pre-treatment was set up. In particular, a filtration strategy to avoid hepcidin depletion from samples after filtration was optimized. This method allowed the clean-up of real samples in order to minimize the interference of complex matrixes during the analyte quantification. Finally, the developed ELISA like MIP-based assay for hepcidin 25 level assessments in serum was approached with resulting responses within the range of concentrations of physiological interest, i.e. ≈ 1-100 nM. The possibility of using MIPs as plastic antibodies in ELISA-like assays has the advantage of a potential widespread diffusion thanks to their high stability, selectivity, low cost and simple manufacture procedure.