Extraction and purification of short DNA fragments from plasma using aqueous two-phase systems for liquid biopsy

• Aqueous two-phase systems enable efficient and selective DNA capture from plasma. • Addition of solid PEG and salts increases plasma input and maintains high DNA recovery. • Robust DNA recovery is achieved despite plasma and collection tube variability. • Combined DNA purification and concentration can be achieved with reverse elution. • Foundation for novel cfDNA extraction process for liquid biopsy is established. Liquid biopsy is a minimally invasive method for tumor profiling, relying on detecting and analyzing tumor-derived biomarkers present in bodily fluids. Key biomarkers, such as circulating cell-free DNA (cfDNA), are often present at very low concentrations in plasma. Efficient extraction and purification of cfDNA are critical, as impurities such as proteins and salts can interfere with downstream applications like next-generation sequencing. This study demonstrates the potential of aqueous two-phase systems (ATPS) as a capture step for short DNA fragments from human plasma, building on prior findings with synthetic systems. DNA partitioning, recovery, and separation from plasma proteins were evaluated in selected polyethylene glycol (PEG)/phosphate/plasma ATPS. PEG 1000 systems proved to be more effective than PEG 400 systems, achieving up to 90 % DNA recovery in the bottom phase. Total protein concentration was reduced to 3 mg/ml, while most of the protein was removed as reversible aggregates forming an interphase. System robustness was confirmed using plasma from various donors and blood collection tube types, showing consistent DNA recovery and phase separation behavior. To address dilution caused by liquid phase-forming components, a modified ATPS design using solid PEG and phosphate was implemented. This approach increased plasma input from 37.7 % (w/w) to 66.7 % (w/w) without altering DNA or protein partitioning, effectively maximizing the plasma load for extraction. Lastly, a reverse elution protocol using purification plates containing a desalting matrix was introduced to concentrate the DNA-containing phase while removing salts and residual proteins, improving its suitability for downstream sequencing-based applications.