Microfluidic Technologies for cfDNA Isolation and Analysis

Abstract

Cell-free DNA (cfDNA), which promotes precision oncology, has received extensive concern because of its abilities to inform genomic mutations, tumor burden and drug resistance. The absolute quantification of cfDNA concentration has been proved as an independent prognostic biomarker of overall survival. However, the properties of low abundance and high fragmentation hinder the isolation and further analysis of cfDNA. Microfluidic technologies and lab-on-a-chip (LOC) devices provide an opportunity to deal with cfDNA sample at a micrometer scale, which reduces required sample volume and makes rapid isolation possible. Microfluidic platform also allow for high degree of automation and high-throughput screening without liquid transfer, where rapid and precise examination and quantification could be performed at the same time. Microfluidic technologies applied in cfDNA isolation and analysis are limited and remains to be further explored. This paper reviewed the existing and potential applications of microfluidic technologies in collection and enrichment of cfDNA, quantification, mutation detection and sequencing library construction, followed by discussion of future perspectives.

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].…”

“…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 ].…”

“…LOCs, POC tests and microfluidic systems are recurrent topics in technology advancement, and these concepts have been widely treated in the literature. Recent examples include the following: Ayoib et al [ 15 ] and Reinholt et al [ 16 ], who focus on the most common NA isolation techniques; Park et al [ 17 ], who provide a detailed explanation of chip structures and sample motion strategies; Jayamohan et al [ 2 ], who describe the most recurrent post-processing techniques; Kong et al [ 18 ], who provide details regarding production and use of lab-on-a-disk (LOD) systems; Xu et al [ 19 ], who describe microfluidic approaches to isolate cell-free DNA; Cui et al [ 20 ], who summarize the major microfluidic techniques for preparation of clinically relevant samples (whole blood, urine, saliva); Bruijns et al [ 21 ], who describe forensic applications of LOCs. Nevertheless, an exhaustive overview of NA extraction approaches from human raw samples enabled on LOCs can be useful for scientists approaching the POC theme.…”

Emerging Lab-on-a-Chip Approaches for Liquid Biopsy in Lung Cancer: Status in CTCs and ctDNA Research and Clinical Validation

“…Thus, several lab-on-a-chip platforms designed to standardize ctDNA detection and characterization have been reported [ 101 , 102 , 103 ]. An overview on current microfluidic technologies for cfDNA isolation and analysis has been reported by Xu et al [ 104 ].…”

“…A microfluidic chip assists in sample preparation by water-oil emulsion and partition of the PCR reaction into thousands of individual nanoliter-sized droplets so that amplification occurs in each individual droplet. This technology disclosed a lower limit of detection (LOD: 0.001%) than any other PCR-based methods and more precise and reproducible results [ 104 , 134 ].…”