Towards a high-throughput/high-sensitivity homogeneous assay for the detection of cfDNA using parallel single-molecule detection - 11th International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS)

Xavier Michalet ¹ Doron Gerber ² Yuval Ebenstein ³ Angelo Gulinatti ⁴ Roger Kornberg ^5,6 Yuval Dor ⁷ Shimon Weiss ^1,2

¹Chemistry & Biochemistry, UCLA, Los Angeles, California, USA
²Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat Gan, Israel
³Department of Chemical Physics, Tel Aviv University, Tel Aviv, Israel
⁴Dipartimento Di Elettronica, Informazione E Bioingegneria, Politecnico Di Milano, Milan, Italy
⁵Department of Structural Biology, Stanford University, Stanford, California, USA
⁶Israel Institute for Advanced Studies, The Hebrew University of Jerusalem, Jerusalem, Israel
⁷Institute for Medical Research, The Hebrew University of Jerusalem, Jerusalem, Israel

Recent approaches that go beyond sequencing and PCR are focusing on epigenetic information embedded in cfDNA, such as fragment length, tissue-specific nucleosome positioning and tissue-specific patterns of DNA methylation. These allow to infer the tissue origins of cfDNA and hence to assess the rate of cell death in specific tissues, and even to determine gene expression programs in the source tissues of cfDNA. Genetic and epigenetic analysis of cfDNA opens a broad, minimally-invasive window into human biology and pathology including any condition involving cellular turnover, with or without genetic alterations: cancer, transplantation biology, cardiometabolic diseases, neurodegeneration, immunity and infections, trauma, exercise physiology and more.

Current technology puts stringent limitations on the effectiveness of liquid biopsies. For instance, early detection of cancer and other diseases such as Alzheimer’s and type 1 diabetes will require a significant enhancement of assay sensitivity. In addition, liquid biopsies are currently based on time consuming methods (e.g. overnight next-generation sequencing), such that applications of real-time and emergency medicine are limited. Moreover, the cost of reagents limits the number of biomarkers that can be tested in parallel, and PCR nonlinearity makes liquid biopsies susceptible to sequence bias and hence high false negative rates.

Here, we present our project to achieve high sensitivity (attomolar concentration), high-throughput (100-fold multiplexing) detection of circulating DNA molecules based on the combination of three rapidly evolving key technologies: single-molecule detection in solution using FRET (smFRET), parallel excitation/detection optics based on novel single-photon avalanche diode (SPAD) arrays, and integrated microfluidic devices handling sample and probe in an automated fashion. Preliminary data demonstrating the feasibility of our approach and challenges yet to overcome are discussed.