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

Abstract

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.

Trapping DNA with a high throughput microfluidic device

“…The mechanism described in the previous paragraph is different from the focusing mechanism described in Refs. [6,7], despite the similar geometries and outcomes. In those studies the shear rate and concentration of neutral polymer in the buffer solution were an order of magnitude higher than in our work, leading to significant viscoelastic migration.…”

“…Methods of preconcentration include isotachophoresis [3] and field-amplified stacking [4], which utilize gradients in the electrophoretic velocities of the DNA. More recently proposed approaches include rheotaxis [5] and methods [6] that have been attributed to elastic migration of DNA molecules during flow and electrophoresis [7]. trapped for considerable lengths of time within the device, while simultaneously allowing spherical particles, having a similar size and electrophoretic mobility as the DNA, to pass through.…”

High‐Performance Nucleic Acid Sensors for Liquid Biopsy Applications

“…[59] Angewandte Chemie The speed and sensitivity of this method shows potential for use in patient samples.…”

High‐Performance Nucleic Acid Sensors for Liquid Biopsy Applications

“…A final example of the application of microfluidics and DEP in cfDNA analysis is a commercial capillary electrophoresis instrument that uses electro‐hydrodynamic actuation to perform capture and concentration analysis of dsDNA with a sensitivity of 10 fg μL −1 in an operating time of 20 min. The speed and sensitivity of this method shows potential for use in patient samples …”