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Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics

Periodic Reporting for period 1 - CATCH-U-DNA (Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics)

Reporting period: 2017-06-01 to 2018-05-31

The project suggests replacing the labor-intensive, occasionally biased and costly PCR method currently used for the detection of genetic markers with a simple, non-PCR based DNA quantification method. The suggested system will exploit the ability of acoustic waves to probe the hydrodynamic shape of surface-bound molecules, rather than mass. The scientific challenge to be addressed is to push the limit of detection to the zM range obviating the need to use a polymerase for DNA amplification. The technological challenge is to fabricate ultra-sensitive acoustic devices and after capturing with high efficiency very low numbers of DNA present in a complex medium detecting them on the device surface. These ambitious goals will be achieved by developing novel probes and nanoparticles of tailor-made sizes and shapes for enhanced acoustic response; exploiting high frequency acoustic devices up to the GHz range; and, employing magnetic beads with microfluidics for specific target-capturing and enrichment. The proof-of-principle will be demonstrated during the detection of circulating-tumor DNA (ctDNA), currently an area perceived by cancer researchers as the “Holy Grail” of future cancer diagnosis, prognosis and treatment. We intend to validate our integrated acoustic platform towards the detection of common mutations occurring in colorectal and lung cancers, i.e. KRAS, EGFR and BRAF in serum. We anticipate that the “CATCH-U-DNA” concept will set the foundations for a simpler, more sensitive and affordable diagnostic method, from which patients in both the developed and developing countries will greatly benefit.
The reporting period concerns the first twelve months of the project and specifically, the period between 1/6/17 till 31/5/18. During the first year, work carried out was in line with tasks described within each WP of the proposal. Briefly, within WP1, a first prototype of an array of acoustic wave sensors has been manufactured and a novel characterization method has been proposed, developed and simulated with good results. Within WP2, progress was made regarding molecular simulations of oscillating surface-bound proteins and surface-suspended liposomes, as well as the development and characterization of liposomes of various compositions and sizes. Within the former, the hydrodynamic nature of acoustic sensing has been theoretically verified and preliminary good fit was obtained with experimental data. Protocols regarding DNA capturing and extraction from serum as well as enrichment using the Ligase Chain Reaction (LCR) were developed within WP3, focusing initially on BRAF mutation. Within WP4, progress was made towards the development of an ultra-sensitive acoustic assay for DNA detection by using the liposomes characterized within WP2 together with the LCR, also developed within WP3. A limit of detection (LOD) of less than 1000000 molecules is now reported through the combined use of LCR and liposomes amplification. This is 6 times lower to the one reported in the initial proposal. Clinical partners are reporting activities related to the collection and storage of patient samples and their subsequent sequencing for the detection of colorectal cancer mutations (WP5); these samples will be used for future testing with the final platform. So far, 75 eligible samples have been identified and sequenced. The first design of a microfluidic module to be used with the acoustic sensor array together with studies on applying high shear flow for potentially enhancing target binding to the surface are also reported within WP6. Some dissemination activities have been taken place and are listed under WP7, while the overall management plan is presented within WP8. Last, a detailed plan on ethics is reported within WP9.
CATCH-U-DNA project is expected to have substantial impact in a number of areas, namely technology, clinical practice and healthcare.

As anticipated and described within the DoA, the project’s impact on the development of new technologies is clearly demonstrated within the first twelve months. Novel technologies related to the fabrication and testing of new acoustic devices (HFF-QCM) and arrays is shown (WP1), never employed before to biosensing applications. Experimental work on the use of the new micro-nano-bio fluidized bed technology with established methodologies (ligation-based DNA enrichment) is currently under development (WP3 and WP4). This combined approach will enrich target sequences for ultra-selective and sensitive detection impacting further biomedical research and bioanalytical sciences

Moreover, the highly interdisciplinary nature of the work carried out within the project is still relevant and clearly brings added value to the project. The merging of molecular biology with biophysics, engineering and theoretical physics is shown to be a very effective way to push the limits of knowledge (and detection) even further. The theoretical understanding of the resonator response to different types of loads together with the development of new computational tools to simulate this behavior (WP2) is under way and expected to assist in proposing new structures for signal enhancement. Moreover, with more than 20 people working in the S&T parts of the work, the project is having a substantial impact in training young researchers, students and post-docs and in transferring knowledge across disciplines and countries.

Regarding CATCH-U-DNA’s impact to clinical practice, healthcare and the society in general, the application-area of the new concept, i.e. the development of a DNA-based diagnostic system for cancer in serum, is still relevant and of immense significance to citizens’ well-being and healthcare industry’s interests. The market for clinical diagnostic and point-of-care tests is still worldwide one of the fastest-growing sectors, currently dominated by the USA; the area of integrated DNA analyzers alone is valued 35 billion € in 2015. CATCH-U-DNA concept is a very attractive one for healthcare industry since it proposes to replace PCR-based diagnostic approaches with a non-polymerase DNA amplification method. The elimination of cumbersome, expensive, time consuming and occasionally scientifically-biased procedures are some of our proposed method’s strong competitive advantages; these advantages are still relevant and some already demonstrated. Indeed, our work until today indicates that our ambitious goal to reach the necessary sensitivity of 100-1000 molecules in 1 ml of sample is feasible (WP4). Moreover, demonstrating the operation of the full system by the end of the project will be critical for the future commercial uptake and exploitation of CATCH-U-DNA. Specifically, bringing the system after three years of work to a TRL level 4 or even 5 through the contribution of the clinical partner to real-world validation will enhance significantly the commercial potential of the work. In this case, we hope our project to become a clinical as well as a commercial success. Overall, the project’s initial aim to contribute to improved healthcare, bringing at the same time an economic gain to involved partners is still very relevant.
CATCH-U-DNA concept