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Synthesis of peptide imprinted nanoparticles and their integration to ELISA-like assay for the quantification of hepcidin

Final Report Summary - PINP (Synthesis of peptide imprinted nanoparticles and their integration to ELISA-like assay for the quantification of hepcidin)

More sustainable healthcare services and personalized medicine, to monitor health, aging and wellbeing of the population is a priority of European health program 2014-2020 and is actuated with policies that support the uptake of innovative solutions to improve healthcare provisions such as early diagnosis, individualized therapies and constant monitoring of the patient conditions. With the extended life expectancy of the population, these points are in fact keys to contrast chronic and long-term diseases and cardiovascular events, placing the conditions for an "active and healthy ageing".
The European Community responds to the major health-related socio-economic and societal challenges sustaining frontiers in nanobiomedicine and nanomaterials. Practical outcomes of the EU supporting actions span over a wide range, including the development of diagnostic systems (assays and bio/sensors) based on novel nanomaterials for an efficient measure of those biomarkers that are particularly important for diagnostic and prognostic information. In assays the efficient measurement of many biomarkers of clinical relevance is given by molecular recognition. In nature the recognition is mainly given from antibody/enzyme that selectively binds a specific target molecule. In the past years the necessity to find an effective route for the development of protein and peptides receptor-like became essential especially in the field of biotechnology and medicine. Up to date, materials and method employed for producing synthetic receptors span over a wide range. One of the most innovative strategies for mimicking receptors consists of nanoparticles produced by molecularly imprinted polymer (MIP) technology. MIPs are prepared by a template-assisted synthesis that creates complementary cavities exposed in the polymer. MIPs show selectivity similar to natural receptor and can be used as recognition elements.
PINP project is envisioned as a combination of disciplines such as peptide synthesis, polymer chemistry, analytical chemistry, biochemistry, medicine, nanotechnology, nanomaterials and physics merged together to propose advancements in the field of nanomedical assays. In particular, in PINP project molecularly imprinted nanoparticles (MIP-NPs) were designed to target and quantify hepcidin, a peptide hormone that regulates iron homeostasis and plays a pivotal role in highly prevalent human diseases, such as iron overload disorders and iron deficiency anemia; cardiovascular events, kidneys diseases.
Despite the interest for the dosage of hepcidin, this hormone of only 25 residues possesses a highly defined 3D structure and is extremely conserved through evolution, seriously hampering the production of antibodies for its recognition through classical assays. The development of a quantification assay for the hormone that would be of great benefit for a proper clinical evaluation of the patho-physiological conditions of patients, both for the diagnosis of the diseases and for defining individualized therapies.
The PINP project main objective was to develop an assay for the determination of serum hepcidin, based on MIP-NPs arranged in a pseudo-immuno assay format, further extended in sensitivity by the addition of fluorescent tags. Given the structural complexity of hepcidin, only two crucial portions of it were synthesized to be used for imprinting: N-terminal region and the loop region. These peptides were used as template in the synthesis of imprinted nanoparticles.
Physical and functional characterization of MIP-NPs showed a good homogeneity of the nanomaterial, low nanomolar affinity and high specificity for hepcidin. The integration of MIP-NPs to sandwich ELISA-like assay was established employing only NPs imprinted for different epitopes of the target analyte without the support of enzymes or antibodies. The assay responded in a linear manner in the range between 0,05 and 5 fmol of hepcidin. Since the range of linearity was very small the analysis of biological samples of clinical relevance was strongly conditioned by that point. Thanks to the knowledge gained in PINP project and further optimization of the imprinting, the proposed project could be brought to the next technology readiness level.