Periodic Reporting for period 2 - NEXUS (automated in-line separatioN and dEtection of eXtracellular vesicles for liqUid biopsy applicationS)
Reporting period: 2023-06-01 to 2025-05-31
New modalities for non-invasive diagnostics, namely Liquid Biopsy, are considered a breakthrough innovation poised to have a relevant impact on timely and efficient cancer prevention and treatment. Extracellular Vesicles (EV) have the potential to outperform other liquid biopsy biomarkers such as circulating tumour cells (CTC), circulating tumour DNA (ctDNA), and traditional circulating protein biomarkers. EV is a collective term for a heterogeneous group of cell-released membranous vesicles abundantly present in the circulation and can be secured from blood, and from other body fluids in a non-invasive manner. They are natural multiplex biomarkers, enclosing different molecular species such as proteins, nucleic acids, glycans, and lipids that can be simultaneously investigated to provide real-time information on all tissue homeostasis alterations. However, despite their apparent abundancy, the heterogeneity and complexity of both EVs and the biofluids that contain them remain a huge challenge that is poorly addressed by state-of-art technologies for EV purification and characterization, which are still at research grade and don’t have clinical and commercial maturity.
The project NEXUS aims at industrializing a platform that integrates affinity isolation and in situ enrichment of target (cancer) sEVs from plasma to obtain pure, integral, and concentrated vesicles and Multiparameter analysis giving the simultaneous information on vesicle size, number, and phenotype. The aim of the project is to raise the TRL of the individual components and integrate them into a full-fledged analytical tool, characterized by an appealing “sample in – result out” design and featuring a real-time in-liquid measurement of scalable sample volumes as well as automatized and software-controlled flow. The most challenging goal of the project is the validation of the instrument with clinically relevant sample to demonstrate its usefulness in prostate cancer detection.
The project NEXUS aims at industrializing a platform that integrates affinity isolation and in situ enrichment of target (cancer) sEVs from plasma to obtain pure, integral, and concentrated vesicles and Multiparameter analysis giving the simultaneous information on vesicle size, number, and phenotype. The aim of the project is to raise the TRL of the individual components and integrate them into a full-fledged analytical tool, characterized by an appealing “sample in – result out” design and featuring a real-time in-liquid measurement of scalable sample volumes as well as automatized and software-controlled flow. The most challenging goal of the project is the validation of the instrument with clinically relevant sample to demonstrate its usefulness in prostate cancer detection.
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.
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.
The NEXUS platform combines separation and detection of EVs using a unified set of tagged antibodies, enhancing detection accuracy. This is beyon the state of the art as there are no existing platforms of reagents that allows capture and analysis of EV using the same set of reagents
The prototype detector is highly innovative as it allows the detection of single nanoparticles in microarray format. The technology and the prototype incorporate several innovations that simplify data analysis and support practical, real-world application.
The project’s holistic approach, from technology development to stakeholder engagement and business planning, sets a new standard for EV-based diagnostic tools.
Preparation for clinical validation and regulatory compliance ensures readiness for market adoption and investment attraction.
The prototype detector is highly innovative as it allows the detection of single nanoparticles in microarray format. The technology and the prototype incorporate several innovations that simplify data analysis and support practical, real-world application.
The project’s holistic approach, from technology development to stakeholder engagement and business planning, sets a new standard for EV-based diagnostic tools.
Preparation for clinical validation and regulatory compliance ensures readiness for market adoption and investment attraction.