The main technological results obtained by the project are:
- Fabrication of high-quality superparamagnetic nanoparticles (SPNP) with high magnetisation values and well-defined diameter dispersion;
- Development and functionalisation of SPNP for specific recognition and binding to selected biomarkers;
- Development of protocols and tools for the in-vitro mixing of functionalised SPNP and biomarkers, for the assembly of nano-aggregates with hydrodynamic radii different from those of bare SPNP;
- Development of magnetic sensors capable of detecting the dynamics of the nano-aggregates with ultra-high sensitivity, thus advancing the state-of-the-art in bio-marker detection sensitivity to femto-gram (10-15gram) per ml level;
- Testing the technology in clinical environment with established laboratory techniques;
- Testing the assembled IVD in clinical environment using ad-hoc protocols (PARTLY FULFILLED).
MADIA research was a breakthrough in the area of bio-functionalization of nanoparticles: A number of novel peptidic aptamers was identified by computer modelling which showed a high binding affinity. The simulation data were confirmed by lab work using colorimetric methods and biosensing. The affinity was not lost when the aptamers were grafted on the surface of silica-coated superparamagnetic nanoparticles, but rather increased thanks to a novel hyperbranched molecular design that enhanced the nanoparticles binding capacity 16-fold. When applied to the detection of A1-42 amyloids, the results unveiled the ability of the aptamer-functionalised nanoparticles to capture more than 95% of the biomarker in solution. These results showed the potential of the technology as a stand-alone concentration kit compatible with established detection methods such as commercially-available enzyme-linked immunoassays (ELISA). The exploitation potential of this technology has exceeded expectations and made it an attractive proposition in combination with the Aggregation chambers developed by joint efforts of various partners.
We showed that the aptamer-functionalised magnetic nanoparticles formed complexes with the amyloids. Those could be detected by the MADIA biosensors as they formed discrete aggregates with a different diffusion rate along the sensor microchannels.
MADIA developed important knowledge about the novel biomarkers for AD and PD, such as NGF and pro-NGF. The availability of antibody fragments against specific biomarkers, of relatively low molecular weight, enabled their grafting on the nanoparticles without affecting significantly their physico-chemical properties. The role of NGF and its precursor proNGF as a therapeutic and diagnostic target in AD is recognized. Therefore, NGF and proNGF in CSF represent both a therapeutic target and a biomarker to be validated for diagnostic purpose in different pathologies.
EBRI has developed a new method to measure proNGF in CSF that exploits the differences in molecular weight between proNGF and NGF. This method, based on capillary electrophoresis, is robust, sensitive and automated and does not suffer from the limitation of NGF/proNGF interference. To further validate the assay and the innovative biomarkers investigated within this project, a wider number of CSF samples from patients are expected to be measured post-project.
Overall, MADIA generated 15 exploitable results. The IVD main components are the Aggregation Chamber and the Detection Unit, with their own independently exploitable sub-component, plus all the models (software, in-vitro and in-vivo) that have been developed to validate them. In addition, 3 results have reached a TRL level of 8 or beyond, and are currently being marketed as services by the respective owners.
