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Microfluidic-based Solid Phase Extraction of Cell Free DNA

Abstract

Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the isolation 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 the need for extraction platforms that provide high recovery over the entire ctDNA size range (50 bp – 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 light to generate surface-confined carboxylic acid functionalities for the solid phase extraction (SPE) 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 extraction beds 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 bp – 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.

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Supplementary files

Publication details

The article was received on 09 Jul 2018, accepted on 27 Sep 2018 and first published on 04 Oct 2018


Article type: Paper
DOI: 10.1039/C8LC00716K
Citation: Lab Chip, 2018, Accepted Manuscript
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    Microfluidic-based Solid Phase Extraction of Cell Free DNA

    S. A. Soper, C. Campos, S. Thippalagamage, M. Jackson, M. A. Witek, D. S. Park, M. C. Murphy and A. K. Godwin, Lab Chip, 2018, Accepted Manuscript , DOI: 10.1039/C8LC00716K

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