A single microfluidic device approach to direct isolation, purification, and amplification of cfDNA from undiluted plasma

Circulating cell free DNA (cfDNA) is a valuable source of biomarkers for a range of medical applications including detection and monitoring of diseases. Currently, cfDNA sequence analysis must take place in a laboratory setting, due to the multiple steps required for processing including collection, purification, amplification, and analysis. Developing a point-of-care test system that combines these steps would simplify DNA processing thereby increasing diagnostic screening accessibility and enabling real-time monitoring for individual patients. Here, we have developed a system that combines multiple cfDNA processing steps into a single microfluidic-based device. This includes cfDNA collection directly from undiluted human plasma followed by purification and on chip amplification. A microelectrode array embedded within the microfluidic chip collected cfDNA through the creation of dielectrophoretic (DEP) forces followed by a wash to achieve purification. DEP utilizes differences in dielectric properties between cfDNA and plasma to preferentially induce a force on cfDNA. We then achieved on-chip amplification of collected DNA by designing a thermal cycling system to enable polymerase chain reaction (PCR) directly on the chip. This successfully consolidated the most labor-intensive steps of collection, purification, and amplification into a single device. Compared to elution of cfDNA for off-chip amplification, our on-chip PCR method improved the lower limit of detection by 3-fold and improved the total DNA yield by 5-fold. Furthermore, we demonstrate its clinical diagnostic potential by detecting KRAS mutations from a pancreatic ductal adenocarcinoma patient using only 60 µL of plasma. This paves the way for future development of a fully self-contained system facilitating the rapid detection of mutations in cfDNA.