Microfluidic extraction and digital quantification of circulating cell-free DNA from serum

Abstract

Miniaturized devices for the extraction of DNA have been used for assessing genetic material in biological, forensic and environmental samples. However, the ability to isolate trace amounts of highly fragmented DNA from biological fluids remains a challenge. The current work reports a microfluidic approach that combines on line a dynamic magnetic extraction procedure with droplet-based digital PCR (ddPCR). This strategy maximizes the surface area for DNA binding within the chip, in order to capture short DNA fragments, with the possibility of recovering the purified samples into picoliter volumes for high sensitivity mutation detection. The application of this technology to capture circulating cell-free DNA (ccfDNA) from serum samples of cancer patients is demonstrated herein, with efficiencies comparable to standard column-based DNA extraction methods. This technology uses lesser amounts of required material and reagents, and has a higher potential for automation and multiplex DNA analysis. Furthermore, this approach can also be extended for the detection of other circulating biomarkers, such as nucleic acid sequences with aberrant methylation patterns or miRNA.

An Overview on Microfluidic Systems for Nucleic Acids Extraction from Human Raw Samples

“…Influenza A virus [69,72] Mucosal lining or Nasopharyngeal fluids DNA Bacillus anthracis [25] Bordetella pertussis [44] Mycobacterium tuberculosis [53,63] Staphylococcus aureus [56,68] Bacillus cereus [66] RNA Human immunodeficiency virus [66] Parainfluenza virus, Rhinovirus A, Metapneumovirus [30] Respiratory-syncytial virus [30,70] Multiple respiratory RNA viruses [71] SARS-CoV-2 Although more often used for detection of proteins, metabolites or other small molecules, extraction LOCs can also be helpful in genetic disease detection [104], usually performed through DNA offline extraction followed by sequencing or genotyping approaches. More recently, LOCs were designed to capture circulating cell-free DNA (ccfDNA) from serum samples of cancer patients [105][106][107]: ccfDNA capture requires increased ability in adsorbing short fragments, thus implying customized isolation strategies [19]. Another interesting application of DNA extracted with LOCs is represented by forensic evaluations, especially when available sample volumes are extremely low [9,24,29,75].…”

The Growing Impact of Micro/Nanomaterial‐Based Systems in Precision Oncology: Translating “Multiomics” Technologies

“…[36] Toralla et al developed an integrated microfluidic magnetic extraction platform (METRO) for cfDNA isolation from biological samples such as undiluted serum. [37] The capture domain of the microfluidic platform was equipped with paramagnetic microparticles which can be expanded by flowing binding buffer (pH < 6) through the channel. Furthermore, the orientation and porosity of the microparticles were tunable by an external magnetic field and fluid flow rate.…”

Microfluidic Technologies for cfDNA Isolation and Analysis

“…Such DNA extraction method in a segmented flow microfluidic system successfully removed 90% or even 95% of the original sample volume. More recently, magnetic bead-based approach has been applied to cfDNA isolation (Figure 2), with a constant position of external magnetic field and without the need for an incubation step, which had comparable efficiencies with standard column-based method [38,39]. The emergence of new technology, like 3D-printing, makes magnetic bead-based microdevices more diversified [40].…”