Nanofluidics for label-free detection of exosomes and protein aggregates in neurodegenerative disease research

Project Information

EXO-CHIP

Grant agreement ID: 101064246

DOI

10.3030/101064246

Project closed

EC signature date 8 September 2022

Start date 1 January 2023

End date 31 December 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 210 911,04

Coordinated by

UNIVERSITETET I TROMSOE - NORGES ARKTISKE UNIVERSITET
Norway

Periodic Reporting for period 1 - EXO-CHIP (Nanofluidics for label-free detection of exosomes and protein aggregates in neurodegenerative disease research)

Reporting period: 2023-01-01 to 2024-12-31

Summary of the context and overall objectives of the project

The capability to observe single molecules is key for finding new tools in the complex space of biology and physics as our society strives for next-generation health diagnostics, intends to combat neurodegenerative diseases in an ageing population and wishes to improve medical treatment for cancer patients in hospitals. EXO-CHIP's aim was to develop nanofluidic mass producible chips for the analysis of biomarkers - e.g. Extracellular vesicles (small round particles circulating in human blood) or neurodegenerative protein aggregates e.g. Alpha-synuclein ( a protein affiliated with the development of Parkinson's and Alzheimer's disease).As these specimen are rather small (EVs = 40-120nm, A-syn=3-10nm) they are hard to detect with current assays without the use of chemistry to fluorescently label them. This chemical labelling can induce changes in the specimen and alter its biophysical behaviour - ultimately biasing any assay conducted on these so far. Therefore we have tremendeous need and motivation to develop label-free methods (preferentially optical methods) to observe their assembly and size without altering their physiological behaviour. Awareness of this bias is crucial for interpretation and criticism towards developments made in the field and policies in place for treatment of these disease but also legislation of drugs to treat those.
EXO-CHIP's objectives are (i) to train the grant recipient in the scientific disciplines needed, for (ii) the development of nanofluidic chips and the combination with two label-free microscopy techniques (iii) Quantitative phase microscopy (QPM) and (iv) deep-ultra-violet microscopy (DUVM).
Expected impact of successful developments during the action are: (i) a new method and scalable device for improved healthcare, (ii) reduction of precious biomaterial for development of next generation treatment of neurodegenerative diseases at (iii) reduced economic costs due to savings in material and time for hit-generation in the development of medicines.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Summarize WPs and Objectives 1-4
WP1(Training): The researcher obtained professional training in QPM, DUVM and Machine Learning for diffusional sizing of molecules in solution. At the hosting institution, the researcher joined experienced personal on collaborative imaging session with QPM, imaged with and built a DUV microscope supervised by an expert of the field and learnt how to analyse trajectories in imaging data using open-source machine laerning software which allows to estimate size, diffusional behaviour and their classification upon their movement and optical appearance. Outcome: Important knowledge and training acquired for successful conduction of technological WPs.
WP2(Nanofluidic chip fabrication): The researcher designed, built and applied a new cutting-edge 2-photon lithography system and used it to produce master wafers for nanofluidic chip fabrication at UiT. Outcome: The host institution gained expertise in micro-, nanofluidics amongst their staff and educational materials, as well as a new custom-built nanofabrication equipment capable of writing sub-micron features in 3D, able to produce also other optical components related to science conducted at this university.
WP3(Combination of nanofluidics and QPM):The researcher conducted QPM measurements with collaborators and found that QPM is not suitable for imaging EVs in solution under the experimentally intended conditions. Therefore, the researcher changed to another label-free microscopy technique (dark-field microscopy) and invented on the way a new simpler manufacturing process for nanofluidic confinement called NANOSPACER. Outcome: NANOSPACER in combination with dark-field became a powerful tool for various specimen and allowed sizing of EVs in solution down to 40 nm.
WP4(Combination of nanofluidic and DUVM): The researcher conducted DUVM measurements and found that DUVM is not optimal for imaging aggregation prone proteins in solution under the experimentally intended conditions. Therefore, the researcher changed to another label-free microscopy technique (dark-field microscopy) which made the observation of monomers, oligomers and fibrils in solution possible. Outcome: NANOSPACER in combination with dark-field became a powerful tool for detection of A-synuclein aggregates in solution and facilitates to run label-free aggregation assays in a scalable manner to test drugs and treatment against neurodegenerative diseases.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

EXO-CHIP resulted successfully in the development and establishmen of label-free nanofluidic sizing of molecules for life science applications and demonstrated label-free sizing of EVs and label-free detection of aggregates related to protein misfolding diseases with broadly available microscopy tools.
The impact on science is immense as the follow-up project NANOSPACER already found 30 new applications and collaborations around the globe (amongst them 5 hospitals in Norway) but would benefit from further collaborative research, commercialisation in form of a company, internationalisation via networks and the establishment as a new standardisation framework via policy makers to maximise impact on the scientific community.
Overview of the results beyond state of the art:
-Development of smallest footprint and light weight 2p-lithography system in the field with high positioning precision accuracy (approx. 1nm/ sub-nm with fine positioning mode) over whole travel range.
-Label-free detection and sizing of EVs with standard microscopes - replacing expensive sizing equipment with disposable broadly available microscope slides.
-Label-free detection and sizing of A-syn aggregates in solution with standard bench top microscopes - opening up unbiased label-free aggregation assays for protein misfolding disease research at single molecule level.
-Replacing expensive NTA equipment with disposable NANOSPACER microscope slides - providing an affordable tool for every standard biology lab to conduct single molecule science.