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Microfluidic-based solid phase extraction of cell free DNA†
Lab on a Chip ( IF 6.1 ) Pub Date : 2018-10-04 00:00:00 , DOI: 10.1039/c8lc00716k
Camila D. M. Campos 1, 2, 3, 4, 5 , Sachindra S. T. Gamage 1, 2, 3, 4, 5 , Joshua M. Jackson 1, 2, 3, 4, 5 , Malgorzata A. Witek 1, 2, 3, 4, 5 , Daniel S. Park 4, 5, 6, 7, 8 , Michael C. Murphy 4, 5, 6, 7, 8 , Andrew K. Godwin 4, 9, 10, 11 , Steven A. Soper 1, 2, 3, 4, 5
Affiliation  

Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 → 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (μSPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the μSPE of cfDNA. The μSPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The μSPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire μSPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The μSPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100–700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the μSPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. μSPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.

中文翻译:

基于微流体的无细胞DNA固相萃取

无细胞DNA(cfDNA)是一种液体活检标记物,可以带有与某些病理状况相关的特征(突变)。因此,从多种临床样品中提取cfDNA可以成为疾病检测和后续管理的有效而微创的标记来源。在肿瘤疾病中,循环肿瘤DNA(ctDNA)是cfDNA的一个子类,可以携带临床上可操作的突变,并与下一代测序或其他突变检测方法相结合,为有效的体外诊断提供了场所。但是,cfDNA突变分析需要高质量的输入。这就要求提取平台能够在整个ctDNA大小范围(50→150 bp)内提供高回收率,并且干扰最小(例如,共提取基因组DNA)和简单的工作流程,具有很高的重现性。本文中,我们介绍了一种新型的微流体固相萃取装置(μSPE),该装置由被UV / O 3激活的塑料芯片组成为cfDNA的μSPE生成表面受限的羧酸官能团。μSPE使用固定化缓冲液(IB),该固定化缓冲液由聚乙二醇和盐组成,该盐会导致cfDNA缩合到活化的塑料微流体表面上。μSPE由一系列微柱组成,以增加提取床的负荷(可扩展至cfDNA的负荷> 700 ng),并且可以使用基于复制的技术以低成本生产。整个μSPE可以在单个成型步骤中制造,无需在设备上添加额外的提取载体,从而简化了生产过程,并使设备和测定成本保持较低。μSPE可以在各种大小(100–700 bp)范围内回收超过cfDNA模型片段的90%,甚至可以有效地提取短cfDNA片段(50 bp,> 70%)。此外,IB的组成可以减少共提取基因组DNA的干扰。我们通过定量健康供体以及非小细胞肺癌和结肠直肠癌患者中cfDNA的水平证明了μSPE的临床实用性。还将从血浆样品中提取的μSPE提取的cfDNA进行连接酶检测反应(LDR),以确定是否存在突变。大肠癌和非小细胞肺癌患者的KRAS基因。
更新日期:2018-10-04
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