BIABooster: Online DNA Concentration and Size Profiling with a Limit of Detection of 10 fg/µL and Application to High-Sensitivity Characterization of Circulating Cell-Free DNA.

We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/µL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of ∼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.

DNA separation and enrichment using electro-hydrodynamic bidirectional flows in viscoelastic liquids.

DNA size separation followed by purification and enrichment constitute essential operations for genetic engineering. These processes are mostly carried out using DNA electrophoresis in gels or in polymer solutions, a well-established yet lengthy technique which has been notably improved using Lab-on-Chip technologies. So far, innovations for DNA separation or enrichment have been mostly undertaken separately, and we present an approach that allows us to perform these two processes simultaneously for DNA fragments spanning 0.2-50 kilo base pairs (kbp) in length. Our technology involves an electric field and a counter hydrodynamic flow in viscoelastic liquids, in which we show the occurrence of transverse forces oriented toward the walls. These forces increase with DNA molecular weight (MW) and hence induce a progressive reduction in DNA migration speed that triggers size separation in microfluidic channels as well as in capillaries. The separation of MW markers in the range 1-50 kbp is achieved in 15 minutes, thus outperforming gel electrophoresis that takes ∼3 hours for this sample. Furthermore, the use of a funnel, where electric and flow fields are modulated spatially, enables us to adjust the transverse forces so as to stall the motion of DNA molecules at a position where they accumulate at factors of up to 1000 per minute. In this configuration, we establish that the operations of DNA enrichment and separation can be carried out simultaneously for the bands of a DNA MW marker between 0.2-1.5 kbp diluted at 0.02 ng µL(-1) in 30 s. Altogether, our technology, which can readily be integrated as an in-line module in Lab-on-Chips, offers unique opportunities for sample preparation and analysis of minute genomic samples.

Metrology of confined flows using wide field nanoparticle velocimetry.

The manipulation of fluids in micro/nanofabricated systems opens new avenues to engineer the transport of matter at the molecular level. Yet the number of methods for the in situ characterization of fluid flows in shallow channels is limited. Here we establish a simple method called nanoparticle velocimetry distribution analysis (NVDA) that relies on wide field microscopy to measure the flow rate and channel height based on the fitting of particle velocity distributions along and across the flow direction. NVDA is validated by simulations, showing errors in velocity and height determination of less than 1% and 8% respectively, as well as with experiments, in which we monitor the behavior of 200 nm nanoparticles conveyed in channels of ~1.8 µm in height. We then show the relevance of this assay for the characterization of flows in bulging channels, and prove its suitability to characterize the concentration of particles across the channel height in the context of visco-elastic focusing. Our method for rapid and quantitative flow characterization has therefore a broad spectrum of applications in micro/nanofluidics, and a strong potential for the optimization of Lab-on-Chips modules in which engineering of confined transport is necessary.