The project was organized around three core technical work packages—WP2, WP3, and WP4—dedicated respectively to the development of the separation module, the assay, and the detection module along with its integration into the platform. WP5 and WP6 focused on the analytical and clinical validation of the integrated instrument. A key method, developed within the project, enabled the isolation of intact and concentrated vesicles. This approach was essential for the design and development of both the separation and detection modules addressed in WP2 and WP4. It relied on oligonucleotide–antibody conjugates designed to selectively target specific EV subpopulations.
A detailed description of the activities carried out within each work package, along with the corresponding achievements, is provided below.
• Microfluidic Isolation (WP2):
Partner INOREVIA developed a magnetic tweezers (MT)-based EV isolation system integrated into the Magelia platform, replacing the original fluidized bed plan. While performance issues—such as plasma viscosity—were addressed through automation and software improvements, the recovery yield remained insufficient to support reliable biomarker detection. Ultimately, two external robots (OT-2 and Nucleocube) enabled more effective EV recovery and reduced processing time from 2.5 hours to 30 minutes.
• Reversible Capture Chemistry (WP3):
CNR developed DNA-tagged antibody reagents and gold nanoparticle (GNP)-based EV labeling and enabled multiplexed detection via oligo–antibody conjugates using barcoded DNA for reversible capture and release. The technology was successfully transferred to other WPs
• Instrument Design & Integration (WP4):
A functional prototype detector for nanoparticle detection, incorporating innovative features that simplify analysis and enhance usability was developed in WP4. Full hardware integration of this module with the separation module was not realized yet, but data flow between modules worked seamlessly. Software, optics, and on-board computing were finalized and tested.
• Validation & Testing (WP5 & WP6):
Reference EV materials and antibodies were produced and validated. Key assay parameters (specificity, accuracy, reproducibility) were defined. Clinical testing using prostate cancer samples showed promising results. Detection in liquid with SP-iRiS was less reliable than expected; however, dry detection using the ExoView platform proved more effective, demonstrating that the NEXUS approach—based on the separation and analysis of EVs using a common set of DNA-tagged antibodies—holds strong potential. This result validates the core concept of using reversible capture chemistry across modules and suggests that, with further refinement, the platform could support robust and clinically meaningful EV biomarker detection..
• Economic Evaluation & Business Development (WP7):
A hybrid business model was formulated: initial RUO (research use only) products leading to clinical diagnostics. A full business plan and stakeholder engagement strategy were created, paving the way for a dedicated NEXUS enterprise.
Communications, dissemination, and IP planning were executed with multiple stakeholder and scientific engagements.
