micro-RNA 21 detection with a limit of 2 pM in 1 min using a size-accordable concentration module operated by electrohydrodynamic actuation.

We present a fluorimetry-based technology for micro-RNA-21 (miR-21) sensing based on the concentration of miR-molecular beacon (MB) complexes and flushing of unbound MB. This concentration module consists of a microfluidic channel with the shape of a funnel operated with electrohydrodynamic actuation. We report a limit of detection of 2 pM in less than 1 min for miR-21 alone, and then demonstrate that miR-21 levels, measured in fine needle biopsy samples, from patients with pancreatic cancer correlate with the reference technique of reverse-transcription polymerase chain reaction (RT-PCR). Altogether, this technology has promising clinical performances for the follow-up of patients with cancer.

Characterization and minimization of band broadening in DNA electrohydrodynamic migration for enhanced size separation.

The combination of hydrodynamic actuation with an opposing electrophoretic force in viscoelastic liquids enables the separation, concentration, and purification of DNA. Obtaining good analytical performances despite the use of hydrodynamic flow fields, which dramatically enhance band broadening due to Taylor dispersion, constitutes a paradox that remains to be clarified. Here, we study the mechanism of band broadening in electrohydrodynamic migration with an automated microfluidic platform that allows us to track the migration of a 600 bp band in the pressure-electric field parameter space. We demonstrate that diffusion in the electrohydrodynamic regime is controlled predominantly by the electric field and marginally by the hydrodynamic flow velocity. We explain this response with an analytical model of diffusion based on Taylor dispersion arguments. Furthermore, we demonstrate that the electric field can be modulated over time to monitor and minimize the breadth of a DNA band, and suggest guidelines to enhance the resolution of DNA separation experiments. Altogether, our report is a leap towards to the development of high-performance analytical technologies based on electrohydrodynamic actuation.

Single-step electrohydrodynamic separation of 1-150 kbp in less than 5 min using homogeneous glass/adhesive/glass microchips.

Electrohydrodynamic migration, which is based on hydrodynamic actuation with an opposing electrophoretic force, enables the separation of DNA molecules of 3-100 kbp in glass capillary within 1 h. Here, we wish to enhance these performances using microchip technologies. This study starts with the fabrication of microchips with uniform surfaces, as motivated by our observation that band splitting occurs in microchannels made out of heterogeneous materials such as glass and silicon. The resulting glass-adhesive-glass microchips feature the highest reported bonding strength of 11 MPa for such materials (115 kgf/cm2), a high lateral resolution of critical dimension 5 µm, and minimal auto-fluorescence. These devices enable us to report the separation of 13 DNA bands in the size range of 1-150 kbp in one experiment of 5 min, i.e. 13 times faster than with capillary. In turn, we observe that bands split during electrohydrodynamic migration in heterogeneous glass-silicon but not in homogeneous glass-adhesive-glass microchips. We suggest that this effect arises from differential Electro-Osmotic Flow (EOF) in between the upper and lower walls of heterogeneous channels, and provide evidence that this phenomenon of differential EOF causes band broadening in electrophoresis during microchip electrophoresis. We finally prove that our electrohydrodynamic separation compares very favorably to microchip technologies in terms of resolution length and features the broadest analytical range reported so far.

µLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes.

We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.