In the war against cancer, the first step in treatment is to accurately diagnose the problem, and one technique that helps plan a better treatment plan is biopsy.
In recent years, liquid biopsies, which are able to detect disease through blood or other body fluids rather than solid tissue, have become more popular than surgical biopsies due to their non-invasive and immediate nature.
Liquid biopsies primarily target a molecular marker called "free DNA" (cfDNA), which provides information about the presence of disease-causing DNA in the sample.
To be able to proceed to the next step of analysis, cfDNA must be extracted and purified, which is a challenging task due to its low abundance.
A cfDNA purification method called "solid phase extraction" is the gold standard for extraction and purification, which relies on the affinity of DNA to the solid phase. However, this method cannot extract and purify DNA fragments smaller than 200 base pairs (bp), but this is the basic unit of DNA.
Why is sensitivity to smaller fragments necessary?
It has been reported in the literature that circulating tumor DNA (ctDNA), or pathogenic DNA, is usually smaller than cfDNA. Thus, sensitivity to DNA fragments smaller than 200 bp allows for better detection of disease.
Some relatively new technologies, such as liquid phase extraction (LPE), isotope extraction (ITP), and electrodynamic capture of DNA, can facilitate this size-independent extraction and purification.
However, LPE is a labor-intensive experiment and very time-consuming.
Although ITP and motorized capture have a good ability to provide rapid and automated extraction and detection of pathogen DNA, such as Mycobacterium tuberculosis (MTB), research has not yet begun on the selective purification of short cfDNA fragments.
Now, in a recent study published in Analytica Chimica Acta, researchers from Japan and the United States are demonstrating a novel extraction system that combines the advantages of ITP and electrodynamic capture.
Led by Professor Nobuyuki Futai of the Shibaura Institute of Technology (SIT) in Japan, the team designed an open microfluidic system that uses instantaneous ITP to detect TB bacteria in human plasma samples.
This millimeter-scale microfluidic device consists of a movable gel gate that can precisely extract different species. It uses ITP for DNA purification, which is enriched by gel strips and can be easily used for subsequent PCR detection.
To test the device's performance, the R&D team used the device to purify and enrich MTB genomic DNA fragments from injected human plasma. This microfluidic device shows high recovery, precise isolation, and high sensitivity to cfDNA fragments of 100-200 bp.
It also purifies MTB DNA for further qPCR analysis. Further investigation of its isolation capacity revealed that treating plasma with proteinase K yielded plasma peptides. These peptides act as endogenous spacer molecules, improving the resolution of the extraction method.
This reconfigurable open microfluidic device creates a versatile sample preparation platform for analytical techniques such as PCR and deep sequencing. The R&D team believes their findings could be used to develop advanced purification systems for recovering nucleic acids from plasma or serum.
Most liquid biopsy sample preparation techniques use labeled dyes during DNA purification, which often leads to contamination and a decrease in qPCR signal levels.
But this problem does not exist in this device, and the specific chemical and screening effects produced by the movable door design not only ensure excellent recovery of purified DNA, but also eliminate the need for labeled dyes. This will enable people to accurately diagnose diseases and infections from small blood samples.
Exciting!
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Futai N, Fukazawa Y, Kashiwagi T, et al. A modular and reconfigurable open-channel gated device for the electrokinetic extraction of cell-free DNA assays. Anal. Chim. Acta. 2022:339435. doi: 10.1016/j.aca.2022.339435
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