Abstract
Cell-free DNA (cfDNA) has been implicated as an important biomarker in cancer management. Thus, efficient techniques for cfDNA extraction are necessary for precision medicine. We developed a centrifugation-free cfDNA extraction microfluidic chip capable of extracting cfDNA from plasma samples through microfluidic circuits within 15 min under vacuum pressure using an immiscible solvent. The microfluidic chip had excellent performance that was comparable to the most widely used commercial product (QIAamp kit) in terms of extraction efficiency, purity, and quality of DNA samples. The microfluidic chip was validated for the continuous monitoring of HER-2 type breast cancer and was able to successfully detect a point mutation in phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA) during severe liver metastasis. The chip effectively eliminates the repetitive centrifugation processes and dramatically shortened the sample preparation time. The proposed platform could facilitate the development of a sample-to-answer system for use in liquid biopsy of cancers.
Highlights
Mineral oil was chosen as a proper immiscible solvent, since it does not mix with the elution buffer and does not interact with DNA amplification during polymerase chain reaction (PCR)
Accumulating evidence supports the existence of tumor DNA circulating in the blood stream, which can be detected in small sample volumes using state-of-the-art technology.[38,44,45]
The advantage of liquid biopsy in terms of its ease of sampling of blood compared to tissue has attracted research and clinical attention for the longitudinal monitoring of cancer and response to treatments.[45]
Summary
Technical advances in molecular diagnostics based on nucleic acids have advanced the use precision medicine in clinical applications.[1,2,3] Among nucleic acids, DNA that freely circulates in the bloodstream (cell-free DNA, cfDNA) is a non-invasive and real-time biomarker of cancer that is useful for diagnosis, prognosis, treatment selection, and monitoring of tumor burden.[3,4,5,6] Liquid biopsy, which detects circulating tumor DNA (ctDNA) in blood, has been investigated as an alternative to tissue biopsy that can overcome limitations such as sampling bias, intratumoral heterogeneity, and difficulty in repetitive sample extraction.[7,8,9]. CfDNA extraction is done using columns or magnetic beads, a phenol-chloroform-based method, and a filtration-based method.[6] Among these methods, commercial spin column kits containing silica membranes have been widely used in clinical applications.[22,23,24] In the silica-based solid phase extraction methods, nucleic acids can bind to the silica surface under high chaotropic salt conditions and can be detached at low salt concentration.[25,26] the spin column method provides high yield and high purity of DNA, it requires a benchtop centrifuge operating at an extremely high centrifugal force and has an inconsistent workflow, with the use of various solvents or drying of the washing reagent remaining inside the silica membrane being required This method involves manual handling, like frequent sample transfer to other tubes and removing and replacing spin-columns for centrifugation.
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