Institute of Process Engineering in Life Sciences, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
BioEcho Life Sciences GmbH, BioCampus Cologne, Köln, Germany.
Biotechnol J. 2024 Sep;19(9):e2400394. doi: 10.1002/biot.202400394.
The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.
液体活检作为一种微创技术用于肿瘤分析，这对从体液中高效提取生物标志物提出了要求。循环无细胞 DNA（cfDNA）的分析尤其有前景，但在血浆中发现的 cfDNA 含量低且高度碎片化，这对其分离造成了挑战。虽然两相水溶液系统（ATPS）在各种生物分子的提取和纯化方面的潜力已经得到了成功的证实，但关于这些发现对短 cfDNA 样片段的适用性的文献却很有限。本研究介绍了在 pH 值为 7.4 的聚乙二醇（PEG）/盐 ATPS 中 160bp 的 DNA 片段的分配行为。评估了 PEG 分子量、系线长度、中性盐添加剂和相体积比的影响，以实现 DNA 的最大回收。对含有合成血浆溶液的 ATPS 进行了测试，该溶液中添加了人血清白蛋白和免疫球蛋白 G，以确定 DNA 片段与主要血浆蛋白部分的分离情况。在 17.7%（w/w）PEG 400/17.3%（w/w）磷酸盐 ATPS 中添加 1.5%（w/w）NaCl，可以在富含盐的下相中实现 88%的 DNA 回收率，而超过 99%的蛋白质被去除。
