Microfluidic Device Senses Smaller DNA Fragments

Researchers from Japan have developed a microfluidic device based on isotachophoresis (ITP) and electrokinetic trapping of DNA that overcame the sensitivity limitations of cell-free DNA extraction and purifications methods. 

The findings could expand the diagnostic utility of liquid biopsies. Though preferred over invasive surgical biopsies, detecting diseases like cancer from blood or other bodily fluids is more challenging. Liquid biopsies primarily target a molecular marker called cell-free DNA (cfDNA), which provides information about the presence of pathogenic DNAs in a sample. To carry out any form of analysis, cfDNA has to be extracted and purified, a challenging task owing to their low abundance. The gold-standard cfDNA purification method, called solid-phase extraction, relies on the DNA’s affinity towards a solid phase, but it fails to yield DNA fragments with less than 200 base pairs. This lack of sensitivity is a major drawback, since circulating tumor DNA (ctDNA) or pathogenic DNAs are typically smaller than cfDNA.

Newer techniques such as liquid phase extraction (LPE), ITP, and electrokinetic trapping of DNA can improve this sensitivity. However, LPE is very labor-intensive and time-consuming, and ITP and electrokinetic trapping—despite their excellent abilities in providing quick and automated extraction and detection of pathogenic DNA—have not been explored for the selective purification of short cfDNA fragments.

To address this, researchers from Shibaura Institute of Technology (SIT) describe a novel extraction system that combines ITP and electrokinetic trapping. Led by Professor Nobuyuki Futai, the team designed an open microfluidic system that uses transient ITP to detect M. tuberculosis (MTB) from human plasma samples.

“The millimeter-scale fluidic device we developed consists of movable gel gates that allow precise extraction of separate species,” says Futai.  “It uses ITP for the purification of DNA, and the purified DNA can be easily extracted as a PCR-ready gel strip.” The fluidic system showed a high recovery rate, precise separation, and sensitivity towards short cfDNA fragments of 100-200 bp. It was also able to purify MTB DNA for further qPCR analysis.

Further investigation into its separation abilities revealed that treatment of the plasma with the enzyme proteinase K generated plasma peptides. These peptides acted as endogenous spacer molecules and improved the resolution of the extraction method.  

The team believes that their findings could be used to develop advanced purification systems for recovering nucleic acids from plasma or serum. “Most biopsy sample preparation techniques use marker dyes during DNA purification, which often leads to contamination and decrease in the qPCR signal level,” Futai adds. “The specific chemistries and sieving effects created by our movable gate design not only ensures the excellent recovery of purified DNA but also eliminates the need for marker dyes. This would, for instance, enable accurate diagnosis of diseases and infections from a small amount of blood sample.”

The findings were published recently in Analytica Chimica Acta.