Magnetic DIagnostic Assay for neurodegenerative diseases

Periodic Reporting for period 2 - MADIA (Magnetic DIagnostic Assay for neurodegenerative diseases)

Berichtszeitraum: 2018-07-01 bis 2020-03-31

MADIA’s objective was to develop an innovative cost-effective in-vitro diagnostic device (IVD) for the early diagnosis of Neurodegenerative diseases, mainly Alzheimer’s (AD) and Parkinson’s diseases (PD), combining nano- and micro-scale technologies.
The core concept was the capacity to detect very low concentrations of biomarkers through a disruptive magnetic sensor technology coupled with microfluidic system. The innovation lies in a new architecture of arrays of magnetic sensors integrated into a microfluidic component, capable of recognising biomarkers from cerebrospinal fluid or blood samples, conjugated with ultra-small magnetic nanoparticles.
MADIA achieved paved a new way towards the early and accurate diagnosis of AD PD, which is fundamental for a better pharmacological treatment and development of new and improved therapies.
Two SMEs, Orthokey and BioSys, have already agreed to invest their own resources and collaborate with CNR (ISMN) to further develop the MADIA prototypes to pre-market levels in the IVDs area. Furthermore, CNR, SCRIBA and SERGAS will undertake the modernisation of the Aggregation chamber (in-vitro mixing tool), to reach a TRL which is more attractive for additional funding from other EU initiatives, (e.g. FTI), and other investors.
The knowledge developed in MADIA will be made available into a reference open platform promoted by the European Commission to boost the innovation in the smart bioelectronics’ domain.

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.

1) The optimization of specially designed Planar Hall effect sensors allowed to achieve the limit of 5x103 superparamagnetic nanoparticles detectable by one sensor. In terms of accuracy this amounts for Αβ 1-42 biomarker to about 40x10-18 g. Considering the need of statistical data analysis for in vitro detection, this accuracy allows to operate at levels as low as femtograms/ml concentration of biomarker, which is approximately two orders of magnitude better than best available ELISA parameters.

2) MADIA has developed an innovative magnetic tool for mixing and attaching magnetic labels and biomarkers via magnetophoresis (called Aggregation Chamber), employing ultrahigh magnetic field gradients, able to control statistical ensembles of ultra-small magnetic nanoparticles (superparamagnetic at room temperature). The control of such objects by standard commercial magnetic solutions is prevented by the Brownian motion of the nanoparticles, which thermal energy exceeds the magnetic capturing energy in standard, even high field, solenoids. The developed tool allows the detection of extremely diluted biomarkers from the biological fluids with the efficiency of about 90%. This outstanding achievement has a double Impact value, (1) by enabling the operation of MADIA technology at record low concentrations and 2) by allowing to enhance the concentration of biomarkers in fluids of patient samples, bringing thus the concentrations to levels detectable by standard equipment.

3) MADIA produce scaled up batches of aptamer- functionalised superparamagnetic iron oxide nanoparticles capable of capturing Alzhemer’s Disease (AD) biomarkers. Four aptamers with the highest affinity for clinically-recognised AD biomarkers (i.e. A amyloids and tau protein) were identified by computer modelling as described in WP2 and then experimentally validated after their synthesis, characterisation, grafting on silica-coated super paramagnetic nanoparticles and testing with biomarkers. Noticeably, the obtained functionalised magnetic nanoparticles MNPs were showed to be able to concentrate amyloids in mock biological samples enhancing the sensitivity limits of 3 commercially-available Enzyme-Linked Immuno Assays (ELISA) kits for Abeta amyloids.

Core-shell structure with high degree of complexity based on an incremental engineering of the NPs s

Apparatus for complexation of Biomarkers and Magnetic Nanoparticles

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Superparamagnetic NP detection and film stack for Planar Hall Effect-sensors with Oersted field line

Periodic Reporting for period 2 - MADIA (Magnetic DIagnostic Assay for neurodegenerative diseases)

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