A precise diagnosis has been taken as one of the initial conditions for efficiently treating cancer, for which a technique called biopsy that used to remove a piece of solid tissue or a sample of cells for laboratory testing. Though traditional tumor biopsies can informatively identify diseases when patients are experiencing certain signs and symptoms in an area of concern, they are not satisfying enough due to their invasive nature, which could pose the risk of bleeding and infection to patients. Alternatively, the recent fluid biopsy technique combined with microfluidics can detect disease through blood or other body fluids rather than solid tissue, which increasingly receives attention in terms of its non-invasiveness and simplicity.
DNA Fragments for Cancer Diagnosis
The primary target of a liquid biopsy in cancer diagnosis is molecular markers known as cell-free DNA (cfDNA). cfDNA includes all non-encapsulated DNA, and these nucleic acid fragments enter the bloodstream during apoptosis or necrosis. Some cell-free DNA originating from a tumor clone is named circulating tumor DNA (ctDNA). Usually, macrophages will clean up cfDNA, but when it comes to the case that cells are overproduced in cancer, some ctDNA could be left behind in blood. Therefore, a liquid biopsy in this condition can indicate the presence of pathogenic DNAs in a sample.
Microfluidics for DNA analysis
Obviously, cfDNA needs to be extracted and purified from the sample for a more accurate analytical result. However, this is a challenging procedure because cfDNA exists with a low abundance in the sample, and the pathogenic DNAs typically have a smaller size than other cfDNA, meaning it's difficult for existing techniques to sense and extract these smaller fragments.
A recent study published in Analytica Chimica Acta disclosed a microfluidic device developed by scientists from Japan and the USA for the purification of tuberculosis genomic DNA fragments from human-plasma samples. This is a novel cell-free DNA extraction system based on an open microfluidic system that uses transient isotachophoresis (ITP) to detect M. tuberculosis (MTB) from human plasma samples. ITP and electrokinetic trapping as new techniques can efficaciously provide quick and automated DNA detection and extractions of pathogenic DNA.
Considering that these new techniques have not been explored for the selective purification of short cfDNA fragments, this research led by professor Nobuyuki Futai from Shibaura Institute of Technology (SIT), Japan could be a breakthrough in detecting diagnostic DNA. This microfluidic DNA purification device consists of movable gel gates, allowing precise extraction of different species. And purified DNA from this microfluidic gated device can be easily extracted as a PCR-ready gel strip.
The research team then purified and enriched MTB-genomic DNA fragments from spiked human plasma on the microfluidic gated device for cell-free DNA extraction. The microfluidic blood plasma analysis shows a high recovery rate, accurate separation, and sensitivity towards short cfDNA fragments, as well as a powerful ability to purify MTB DNA for further qPCR analysis.
Different from most sample preparation techniques for biopsies using marker dyes during DNA purification, this novel microfluidic device for DNA purification is designed with movable gates, which can achieve specific chemistries and sieving effects. It means less contamination and a more accurate qPCR signal level, ensuring the excellent recovery of purified DNA. This system for cfDNA extraction and purification indicates how microfluidic-assisted diagnosis could help diagnose cancer, disease, or infections from a small amount of blood sample.
Indeed, microfluidics has numerous important applications for DNA analysis because microfluidic devices can integrate all the complex procedures required for DNA analysis on-chip and drastically reduce the analysis time. But due to higher expense than other regular methods, the technology of microfluidic-based DNA analysis is more applied in laboratories for high-throughput analysis or extremely sensitive assays.
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