Cyclic Electro-Hydrodynamic Stacking Enables fg/μL-Level DNA Sizing for Ultra-Sensitive Cell-Free Nucleosome Analysis in Liquid Biopsy

Abstract Circulating cell-free DNA (cfDNA) analysis in blood plasma provides valuable diagnostic information for cancer detection, transplant rejection monitoring, and prenatal screening. However, the low abundance of cfDNA remains a major analytical challenge that can limit sensitivity and reproducibility across studies. Here, we present an improved BIAbooster platform for DNA sizing that incorporates up to 10 cycles of sample loading and electrohydrodynamic stacking, achieving a limit of detection of 3 fg/μL, corresponding to a 15−20-fold lower detection limit than highly sensitive commercial platforms such as FemtoPulse. This enhanced sensitivity, achieved through repeated loading/electrohydrodynamic stacking cycles that enable processing of ∼8 μL in a single run, allows direct quantification of immunocaptured epigenetically modified cfDNA. Using this approach, we show that immunocaptured fractions are enriched in higher-molecular-weight fragments, suggesting that enrichment workflows may deplete diagnostically relevant cfDNA populations. These findings demonstrate the value of high-sensitivity DNA sizing for cf-nucleosome analysis and establish its utility for liquid biopsy workflows.