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Integrated nanoparticle isolation and detection system for complete on-chip analysis of exosomes

Periodic Reporting for period 1 - INDEX (Integrated nanoparticle isolation and detection system for complete on-chip analysis of exosomes)

Reporting period: 2017-10-01 to 2018-09-30

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 is indeed to develop a tool for translational medicine towards use of exosomes as biomarkers of molecular diagnostics of cancerIn this three-year project, we will pursue the following specific objectives:
1. Develop a novel sensor to detect and classify individual biological nanoparticles as small as 30nm (30%)
2. Develop a novel microfluidic device for efficient magnetic isolation of biological nanoparticles (80%)
3. Develop novel exosome immune-capture and release assays (80%)
4. Develop a novel phenotyping assay for exosomes (50%)
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
Within the First Reporting Period (month 12) the partners have carried out their activity in the different WPs without significant deviations from the original plan. Specifically:

The characteristics of the platform in terms of sensitivity, specificity and throughput, and the specifications for the microfluidic components have been defined. Flow control sensors and actuators have been selected, and needs for automation identified as well as the global layout of the instrument.
The work in WP2 proceeded according to the plan. A microfluidic component to extract and pre-concentrate exosomes selectively from serum or plasma using magnetic beads functionalized with specific antibodies (WP5) was developed. The geometry of the fluidized bed as well as the magnetic field distribution was investigated and an efficient and high throughput capture of exosomes was achieved. Work on automation and optimization of sample introduction and output collection by means of fluid handling (valve, pressure & flow control) units is in progress .
The activities towards the development of the detection system (WP3) proceeded in line with the initial plan. Specifically, the development of the optical instrument for interference based detection of particles scattering was successfully accomplished. The work towards the implementation of advanced optical techniques for detection of nanoparticles down to 30 nm is ongoing with demonstration of first results on the detection of 50 nm silica particles.
The activities of WP4 on the microfluidic chamber for in-liquid real time measurements has been performed and first tests are ongoing The work on the validation of sensor with exosomes standards will start soon.
Work towards exosome capturing and release (WP5) (successfully achieved capturing on chip surface of exosomes by DNA-modified antibodies and release of the biological nanoparticles. The adaptation of this protocol to magnetic nanoparticles is ongoing. The Task on the development of surface chemistry for antibody immobilization has been successfully accomplished at month 12, according to the plans. The work on the bioassay development is ongoing
An initial user interface for instrument control (WP6) was coded in LabView. The work on the development of the integrated instrument started earlier than planned allowing the installation of first prototypes for initial testing..
According to activities foreseen in WP8, an intermediate dissemination and exploitation plan reports the plan envisaged by partners to exploit and/or disseminate projects results.
The project website (http://www.indexproject.eu) and social media accounts are online
We aim to demonstrate a new paradigm in diagnostics through extraction of critical information from clinical samples inaccessible by the state-of-the-art methodologies. Towards this ambition, we propose a complete system with extraction, detection, quantification, and in-depth characterization capability for exosomes present in biofluids from cancer patients We aim to provide innovative solutions to address the following challenges:
1) Lack of efficient technologies for high-purity extraction of exosomes.
Improvement of extraction methods for exosome isolationis one of the main challenges in the field. Currently, the most popular approach for isolation of exosomes is based on differential ultracentrifugation or immune-adsorption by targeting a protein of interest. These methods are often very lengthy, difficult, and unreliable leading to damages of isolated exosomes.
In INDEX will implement a magnetic based microfluidic immune-capture device for in-line purification of exosomes. Magnetic beads will be functionalized with capture agents and allowed to interact with exosomes in multiple rounds within the device to increase capture efficiency. Novel surface chemistry will allow release of intact exosomes after isolation for size and count determination and characterization.
INDEX technology will provide a substantial advancement beyond conventional magnetic-based isolation as it presents higher purity and requires substantially less magnetic material. The device will replace ultracentrifugation methods, altering the current state-of-the-art for exosome purification.
2) Lack of methods to detect, quantitate, and characterize exosomes.
A major ongoing challenge is to identify and characterize exosomes. Main hurdles existing technologies face are: 1) properly recognizing exosomes, 2) accurately measuring size, 3) determining phenotype.
Size discrimination of extracellular vesicles is crucial as size distribution of the population may be indicative of sample heterogeneity and disease state
INDEX will develop a beyond the state-of-the-art optical sensor for biological nanoparticles capable of detecting and sizing nanoparticles down to 30nm diameter. Exosomes will be phenotyped through specific immune-capture by antibody microarrays.
In summary, project INDEX will deliver an integrated, robust, quantitative, high-throughput, and low-cost technology with unprecedented capabilities for exosome analysis for effortless adaptation to clinical workflow. To accomplish this task, multiple advanced methods that each pushes the limits of gold standards will be developed by partners, and integrated to finally present a radically new and groundbreaking nanoparticle analysis tool. This technology will first be applied to exosome-based cancer diagnosis, exemplified with lung cancer. In addition, the modular aspect 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.