Periodic Reporting for period 2 - INDEX (Integrated nanoparticle isolation and detection system for complete on-chip analysis of exosomes)
Okres sprawozdawczy: 2018-10-01 do 2021-03-31
Exosomes are biological nanoparticles known to mediate communication between cells; since they exert significant roles in various pathological conditions such as cancer, infectious and neurodegenerative diseases, their effective utilization holds a great promise of revolutionizing the standard of diagnostics and clinical care. However, the clinical breakthrough exosomes may present in healthcare could not be realized to date due their inherent characteristics such as high heterogeneity, small size and low refractive index. The Long-term vision of project INDEX was indeed to develop a tool for translational medicine towards use of exosomes as biomarkers of molecular diagnostics of cancer in this three-year project, we have pursued and achieved the following specific objectives:
1.Develop a novel sensor to detect and classify individual biological nanoparticles as small as 30nm
2.Develop a novel microfluidic device for efficient magnetic isolation of biological nanoparticles
3.Develop novel exosome immune-capture and release assays
4.Develop a novel phenotyping assay for exosomes
5.Integrate isolation and sensor modules within a prototype instrument to demonstrate complete on-chip
exosome analysis
6.Demonstrate the application of the system with clinical samples for lung cancer diagnostics
1.Develop a novel sensor to detect and classify individual biological nanoparticles as small as 30nm
2.Develop a novel microfluidic device for efficient magnetic isolation of biological nanoparticles
3.Develop novel exosome immune-capture and release assays
4.Develop a novel phenotyping assay for exosomes
5.Integrate isolation and sensor modules within a prototype instrument to demonstrate complete on-chip
exosome analysis
6.Demonstrate the application of the system with clinical samples for lung cancer diagnostics
The work plan is divided into 9 WPs, including 4 technical WPs (WP2-5), and those dedicated to integration (WP6), validation (WP7), exploitation and dissemination of results (WP8), and project management (WP9). The achievements are illustrated below:
WP1 was completed at month 3. The characteristics of the platform in terms of sensitivity, specificity and throughput, and the specifications for the microfluidic components such as pressure ranges, flow- rate ranges, and actuations for extraction (WP2) and detection modules (WP3) were defined.
WP2 (M3-M24). This WP was devoted to the development of a novel microfluidic device for efficient magnetic isolation of nanoparticles. A microfluidic component to extract and pre-concentrate exosomes selectively from serum or plasma using magnetic beads functionalized with specific antibodies (WP5) was developed in Task 2.1. Automation and optimization of sample introduction and output collection was achieved in Task 2.2.
WP3. A novel BNP sensor to detect and classify individual biological nanoparticles as small as 30nm was developed in WP3 (M3-M30). An optical instrument for interference-based detection of particle scattering was developed in Task 3.1. Electromagnetic model and image processing software on MATLAB platform have been developed (Task 3.2).
WP4. From M3 to M36. A microfluidic chamber for in-liquid real time measurements, subsequently adapted for in-air measurements, was developed and tested (Task 4.1).
WP5. The development of a novel exosome immune-capture and release assay was the aim of WP5 (M3-M18). Reversible capturing of exosomes was achieved in Task 5.1. a novel surface chemistry for antibody immobilization was successfully accomplished at M12 in Task 5.2. In Task 5.3 a bioassay for the microarray detection of EVs was developed.
WP6. From M7 to M36. An initial user interface for instrument control (Task 6.1) was coded in LabVIEW. The work on the development of the integrated instrument of Task 6.2 started earlier than planned (M12) allowing the installation of first prototypes at partner IC for initial testing of sample separation. In parallel, a fluid handling module was developed to perform recirculation in the detection chamber. For controlling both separation and detection modules, a custom piece of software developed in C#. A user interface was designed.
WP7. From M13 the WP was devoted to the validation of the integrated system with clinical samples. Due to Covid outbreack it was impossible assemble the instrument in a single laboratory. AUH has shared healthy and patient samples with partner IC . IC and AUH have sent purified EVs from the plasma of healthy people and patients to BU for microarray analysis on the SP-IRIS detection module. Despite each step was conducted separately, the conditions in terms of volumes, flow rates, and analysis time were integrable in a single process and a clinical demonstration on the potentiality of the prototype was successfully carried out.
According to activities foreseen in WP8, a final dissemination and exploitation plan report the actions envisaged by partners to exploit and/or disseminate projects results.
The project website (http://www.indexproject.eu) and social media accounts are online.
WP1 was completed at month 3. The characteristics of the platform in terms of sensitivity, specificity and throughput, and the specifications for the microfluidic components such as pressure ranges, flow- rate ranges, and actuations for extraction (WP2) and detection modules (WP3) were defined.
WP2 (M3-M24). This WP was devoted to the development of a novel microfluidic device for efficient magnetic isolation of nanoparticles. A microfluidic component to extract and pre-concentrate exosomes selectively from serum or plasma using magnetic beads functionalized with specific antibodies (WP5) was developed in Task 2.1. Automation and optimization of sample introduction and output collection was achieved in Task 2.2.
WP3. A novel BNP sensor to detect and classify individual biological nanoparticles as small as 30nm was developed in WP3 (M3-M30). An optical instrument for interference-based detection of particle scattering was developed in Task 3.1. Electromagnetic model and image processing software on MATLAB platform have been developed (Task 3.2).
WP4. From M3 to M36. A microfluidic chamber for in-liquid real time measurements, subsequently adapted for in-air measurements, was developed and tested (Task 4.1).
WP5. The development of a novel exosome immune-capture and release assay was the aim of WP5 (M3-M18). Reversible capturing of exosomes was achieved in Task 5.1. a novel surface chemistry for antibody immobilization was successfully accomplished at M12 in Task 5.2. In Task 5.3 a bioassay for the microarray detection of EVs was developed.
WP6. From M7 to M36. An initial user interface for instrument control (Task 6.1) was coded in LabVIEW. The work on the development of the integrated instrument of Task 6.2 started earlier than planned (M12) allowing the installation of first prototypes at partner IC for initial testing of sample separation. In parallel, a fluid handling module was developed to perform recirculation in the detection chamber. For controlling both separation and detection modules, a custom piece of software developed in C#. A user interface was designed.
WP7. From M13 the WP was devoted to the validation of the integrated system with clinical samples. Due to Covid outbreack it was impossible assemble the instrument in a single laboratory. AUH has shared healthy and patient samples with partner IC . IC and AUH have sent purified EVs from the plasma of healthy people and patients to BU for microarray analysis on the SP-IRIS detection module. Despite each step was conducted separately, the conditions in terms of volumes, flow rates, and analysis time were integrable in a single process and a clinical demonstration on the potentiality of the prototype was successfully carried out.
According to activities foreseen in WP8, a final dissemination and exploitation plan report the actions envisaged by partners to exploit and/or disseminate projects results.
The project website (http://www.indexproject.eu) and social media accounts are online.
We have demonstrated a complete system for extraction, detection, quantification, and in-depth characterization of exosomes present in biofluids from cancer patients. The innovative solutions we propose addresses the following challenges:
1) Lack of efficient technologies for high-purity extraction of exosomes.
The current immunological approaches for isolation of exosomes do not provide intact vesicles and are difficult to automize.
We have implemented a magnetic based microfluidic immune-capture device for in-line purification of exosomes. A novel immobilization approach has allowed the release of intact exosomes after separation for size and count determination and characterization.
INDEX technology provides a substantial advancement beyond conventional magnetic-based isolation as it provides higher purity and requires substantially less magnetic material. The microfluidic separation device can replace ultracentrifugation methods, improving the current state-of-the-art for exosome purification.
2) Lack of methods to detect, quantitate, and characterize exosomes.
A major ongoing challenge is identification and characterize exosomes. Existing technologies face the following hurdles: 1) proper recognition of EVs, 2) accurate measurement of size, 3) phenotype determination.
INDEX has developed a beyond the state-of-the-art optical sensor for biological nanoparticles capable of detecting and sizing nanoparticles down to 30nm diameter.
In summary, project INDEX has delivered an integrated, robust, quantitative, high-throughput, and low-cost technology with unprecedented capabilities for exosome analysis for effortless adaptation to clinical workflow. This technology was demonstrated in exosome-based cancer diagnostics, exemplified with lung cancer. The modular character of this system will enable seamless adaptation to a wide range of nanoparticle based applications beyond exosome detection such as viral load measurements for diagnostics, nanoparticle based drug delivery for therapy, or analysis of nano-structures for material science. Therefore, INDEX has foundational value towards enabling new research directions and technologies.
1) Lack of efficient technologies for high-purity extraction of exosomes.
The current immunological approaches for isolation of exosomes do not provide intact vesicles and are difficult to automize.
We have implemented a magnetic based microfluidic immune-capture device for in-line purification of exosomes. A novel immobilization approach has allowed the release of intact exosomes after separation for size and count determination and characterization.
INDEX technology provides a substantial advancement beyond conventional magnetic-based isolation as it provides higher purity and requires substantially less magnetic material. The microfluidic separation device can replace ultracentrifugation methods, improving the current state-of-the-art for exosome purification.
2) Lack of methods to detect, quantitate, and characterize exosomes.
A major ongoing challenge is identification and characterize exosomes. Existing technologies face the following hurdles: 1) proper recognition of EVs, 2) accurate measurement of size, 3) phenotype determination.
INDEX has developed a beyond the state-of-the-art optical sensor for biological nanoparticles capable of detecting and sizing nanoparticles down to 30nm diameter.
In summary, project INDEX has delivered an integrated, robust, quantitative, high-throughput, and low-cost technology with unprecedented capabilities for exosome analysis for effortless adaptation to clinical workflow. This technology was demonstrated in exosome-based cancer diagnostics, exemplified with lung cancer. The modular character of this system will enable seamless adaptation to a wide range of nanoparticle based applications beyond exosome detection such as viral load measurements for diagnostics, nanoparticle based drug delivery for therapy, or analysis of nano-structures for material science. Therefore, INDEX has foundational value towards enabling new research directions and technologies.