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Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics

Periodic Reporting for period 2 - CATCH-U-DNA (Capturing non-Amplified Tumor Circulating DNA with Ultrasound Hydrodynamics)

Reporting period: 2018-06-01 to 2020-11-30

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.
During the project, allele-specific PCR (AS-PCR) was employed as a method to amplify a genetic target with extremely high specificity and without bias towards specific sequences. This allowed the efficient acoustic detection of as little as one target-copy with a specificity of 0.01%. The verification of the protocol during the BRAF and KRAS mutations-detection was demonstrated using patients’ samples (UOC) together with the acoustic protocol (FORTH) and novel biochip array (AWS). The methodology was expanded as well towards tissue biopsy, a new approach not foreseen in the initial proposal.

Regarding the technology, CATCH-U-DNA project produced a new detection platform (AWS) for the simultaneous real-time probing of a several samples using a 24-array biochip. Moreover, the implementation of a new generation of microfluidic fluidized bed in plasma and serum was demonstrated (Curie), although further development is necessary to reach the desirable detection limits with the acoustic-based detection platform for direct implementation in liquid-biopsy. Finally, the new microfluidic chip developed by JOBST-partner has been tested successfully with the new array biochip during immuno-detection. The chip is incorporated in the final platform and used for multisample analysis in cancer-diagnostics and beyond.

New knowledge was produced in acoustic biosensing, both technologically and conceptually. We showed that acoustic energy dissipation was more sensitive than frequency and could be exploited for ultra-sensitive DNA detection (FORTH). Systematic study of a huge range of liposomes (FORTH, BGU) revealed the ability of acoustic sensors to experimentally determine a quantitative correlation between the acoustic ratio and the liposomes’ mechanical properties (FORTH). Modeling/theory produced two new approaches for the study of physics underlying the acoustic detection mechanisms: one has extended/verified an older systematic study (FORTH) and the other revealing a completely new one (UAM); these works shed light on the major importance of hydrodynamics in acoustic sensor measurements, a parameter whose role and potential contributions is poorly debated (but largely ignored) in the bioacoustics community. Last, the development of colorimetric dendrimers and polydiacetylene nanoparticles (BGU) has contributed to the production of novel biomimetic nano-structures of potential interest to the screening of membrane-active peptides.

Regarding innovation, a novel detection platform and acoustic 24-array biochip is a significant technological output.A novel molecular diagnostic device for the point of care was further developed by FORTH partner. Other notable innovations include a reusable microfluidic chip for QCM-device multisensing (JOBST). Curie partner also advanced considerably their microfluidic fluidized bed for crude-sample pretreatment application, a technology currently exploited by a spin off company. Last, innovation has been produced related to the delivery of a software predictive tool based on UAM-partner’s software for QCM analysis of discrete soft matter analytes.

Finally, the project has a significant output in terms of dissemination activities and training of your students. We report, so far, 11 scientific publications in peer reviewed journals with several more under submission/preparation (>6), 2 new patents and over 30 activities for disseminations of the project results to the wide audience and specific target groups. We are also proud to produce a leading to products very close or close to the market, depending on future investment.
CATCH-U-DNA project is expected to have substantial impact in a number of areas, namely technology, clinical practice and healthcare.

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.

In addition to the above, the new tools and technologies developed within the project are expected to have an impact in the development of molecular diagnostic platforms for the point of care and for the detection of infectious diseases and as a companion diagnostic tool (caner mutations, pharmacogenomics).
CATCH-U-DNA concept