Hepcidin determination: on the construction of assays and sensors made with molecularly imprinted polymers (MIPs) and HDB domain.

Iron homeostasis is regulated by the 25 aminoacid peptide hormone Hepcidin (Hepc), whereas truncated forms of the hormone (22 and 20) lack in physiological effect. The urinary and blood levels of Hepc are associated with dis-regulation of iron metabolism, leading to iron overload or iron deficiencies pathologies, both with high incidence in the population. Measures of Hepc based on the implementation of classical immunological assays found inherent difficulties in getting antibodies against Hepc which limits seriously its. The alternative methods developed are heavily based on mass spectrometry technologies (MS). Notwithstanding the very last progresses, a method for blood and urine Hepc measurement having characteristics of accuracy, ease of implementation, and of large distribution is still very highly welcomed. For this reasons we attempted the measurement of Hepc in a label free assay based on the affinity between Hepc and recently described Hepc binding domain (HDB), exploiting the SPR technique. The synthetic HBD was immobilised as ligand on the gold surface of CM5 chips and the binding of Hepc was tested, indicating a linear range of sensitivity in the interval 0.05-1.25 ng. These results fits with concentrations of physiological interest. Furthermore, dissociation constants and kinetic constants of the process were extrapolated from the fitting of the binding curves. Truncated forms of the hormone bound to the HBD-chip by a negligible extent. The SPR-Hepc sensor hold promises at a preliminary study and therefore will be further investigated for its suitability in real samples and finally in clinical measures. In parallel, we attempted the strategy of creating synthetic receptors for Hepc by exploiting molecularly imprinted polymer (MIP) technology. MIP technology offers a mean to the synthesis of material with entailed recognition properties, by exploiting a template mediated synthesis, where the biomarker is the template. During the polymerisation the functional monomers assemble around the template according to thermodynamic; such pre-assembly is further fixed by crosslinking, forming a material with embedded depressions complementary to the template. The monomers for the MIP were chosen with a computational modelling approach, the polymers were synthesised in thin polymeric films. The binding of hepcidin on MIP and control was evaluated spectrofluorimetrically. Parameters influencing the binding, such as pH and saline concentration were optimised. Results indicate IF = 3.5, binding time of 10 min. Reusability of the MIP resulted in a loss of approximately the 30% of the original binding ability. Experiment outcomes were extremely encouraging.