Fabrication of Multilayer Microfluidic Arrays for Passive, Efficient DNA Trapping and Profiling.

Trace amounts of cell-free DNA containing cancer-specific biomarkers can be found in blood plasma. Detection of these biomarkers holds tremendous potential for applications such as noninvasive cancer diagnostics and therapeutic monitoring. However, such DNA molecules are extremely rare, and a typical patient blood sample may only contain a few copies. Here we describe the fabrication and operation of a microfluidic device to efficiently trap single DNA molecules into chambers for detection of tumor-specific biomarkers through a passive, geometric manipulation strategy.

Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation.

Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).