Single Molecule Free Solution Hydrodynamic Separation for Size Profiling of Serum Cell-Free DNA

Circulating cell-free DNA (cfDNA) has accumulated significant interest as a noninvasive biomarker. One important accessible parameter, the cfDNA size distribution, appears to be of significant value. Here, we introduce our single molecule free solution hydrodynamic separation platform (SML-FSHS) for robust characterization of the cfDNA size profile from human serum. The platform utilizes hydrodynamic separation for DNA sizing and cylindrical illumination confocal spectroscopy (CICS) for highly sensitive single molecule detection and quantification of DNA molecules. We first used a custom wide dynamic range DNA ladder (50 bp-23 kbp) stained with an intercalating dye to create a calibration curve relating DNA retention time to DNA size in base pairs. From this, we demonstrate that the system can profile the cfDNA size distributions in a clinical sample with wide sizing dynamic range and high resolution ~5% of fragment length) all in a single run. The method consumes negligible (3pL) volume of precious sample and minimizes sample processing bias that affects other methods such as PCR and sequencing. We also compare the cfDNA size distributions from stage 1V cancer patients with normal patient samples and demonstrate that specific cfDNA size ranges may correlate with disease state. In addition, we can absolutely quantify cfDNA and gate out the contributions of cellular contamination by size. With these properties, we believe that this platform can help to accelerate future circulating DNA research.