DNA Sizing with pg/µL Sensitivity for Cell-Free DNA Analysis with the µLAS Technology.

The µLAS technology enables in-line DNA concentration and separation in a microchannel. Here, we describe its operation to analyze the size profile of cell-free DNA (cfDNA) extracted from blood plasma. Operated on commercial systems for capillary electrophoresis, we provide the size distribution of healthy individuals or patients using an input of 10 µL.

Size Fractionation of Milliliter DNA Samples in Minutes Controlled by an Electric Field of ∼10 V.

DNA size fractionation is an essential tool in molecular biology and is used to isolate targets in a mixture characterized by a broad molecular-weight distribution. Microfluidics was thought to provide the opportunity to create devices capable of enhancing and speeding up the classical fractionation processes. However, this conjecture met limited success due to the low mass or volume throughput of these technologies. We describe the µLAF (µ-laboratory for DNA fractionation) technology for DNA size selection based on the stacking of molecules on films of ∼100 µm in thickness with 105 cm-2 pores ∼2 µm in diameter. Size selection is achieved by controlling the regime of electrohydrodynamic migration through the temporal modulation of an electric field. This technology allows the processing of milliliter-scale samples containing a DNA mass of several hundreds of ng within ∼10 min and the selection of DNA in virtually any size window spanning 200 to 1000 bp. We demonstrate that one operation suffices to fractionate sheared genomic DNA in up to six fractions with collection efficiencies of ∼20-40% and enrichment factors of ∼1.5-3-fold. These performances compare favorably in terms of speed and versatility to those of the current standards.

Hybridization-based DNA biosensing with a limit of detection of 4 fM in 30 s using an electrohydrodynamic concentration module fabricated by grayscale lithography.

Speeding up and enhancing the performances of nucleic acid biosensing technologies have remained drivers for innovation. Here, we optimize a fluorimetry-based technology for DNA detection based on the concentration of linear targets paired with probes. The concentration module consists of a microfluidic channel with the shape of a funnel in which we monitor a viscoelastic flow and a counter-electrophoretic force. We report that the technology performs better with a target longer than 100 nucleotides (nt) and a probe shorter than 30 nt. We also prove that the control of the funnel geometry in 2.5D using grayscale lithography enhances sensitivity by 100-fold in comparison to chips obtained by conventional photolithography. With these optimized settings, we demonstrate a limit of detection of 4 fM in 30 s and a detection range of more than five decades. This technology hence provides an excellent balance between sensitivity and time to result.

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.

Diazonium Salt-Based Surface-Enhanced Raman Spectroscopy Nanosensor: Detection and Quantitation of Aromatic Hydrocarbons in Water Samples.

Here, we present a surface-enhanced Raman spectroscopy (SERS) nanosensor for environmental pollutants detection. This study was conducted on three polycyclic aromatic hydrocarbons (PAHs): benzo[a]pyrene (BaP), fluoranthene (FL), and naphthalene (NAP). SERS substrates were chemically functionalized using 4-dodecyl benzenediazonium-tetrafluoroborate and SERS analyses were conducted to detect the pollutants alone and in mixtures. Compounds were first measured in water-methanol (9:1 volume ratio) samples. Investigation on solutions containing concentrations ranging from 10-6 g L-1 to 10-3 g L-1 provided data to plot calibration curves and to determine the performance of the sensor. The calculated limit of detection (LOD) was 0.026 mg L-1 (10-7 mol L-1) for BaP, 0.064 mg L-1 (3.2 × 10-7 mol L-1) for FL, and 3.94 mg L-1 (3.1 × 10-5 mol L-1) for NAP, respectively. The correlation between the calculated LOD values and the octanol-water partition coefficient (Kow) of the investigated PAHs suggests that the developed nanosensor is particularly suitable for detecting highly non-polar PAH compounds. Measurements conducted on a mixture of the three analytes (i) demonstrated the ability of the developed technology to detect and identify the three analytes in the mixture; (ii) provided the exact quantitation of pollutants in a mixture. Moreover, we optimized the surface regeneration step for the nanosensor.

Investigation of aromatic hydrocarbon inclusion into cyclodextrins by Raman spectroscopy and thermal analysis.

Among various cavitand molecules, cyclodextrins are extensively studied due to their ability to form host-guest complexes with small hydrophobic molecules. Aiming to explore cyclodextrin implementation on the scopes related to the environmental pollution monitoring or remediation, extensive studies for understanding the cyclodextrin-based host-guest complex formation with selected targeted substances are conducted. In this context, two polycyclic aromatic hydrocarbons, naphthalene and fluoranthene as well as toluene as a member of volatile organic compounds, were studied regarding their ability to encapsulate into cyclodextrin cavities. Synthesised complexes were examined by thermogravimetric analysis combined with Raman spectroscopy. The obtained results demonstrated that the size between targeted molecules and the cyclodextrin cavities strongly correlates with its ability to engage in complexation. Thus, this latter parameter plays an important role in the inclusion complex formation as well as in the strength of the interaction between the molecules.

Soft UV nanoimprint lithography-designed highly sensitive substrates for SERS detection.

We report on the use of soft UV nanoimprint lithography (UV-NIL) for the development of reproducible, millimeter-sized, and sensitive substrates for SERS detection. The used geometry for plasmonic nanostructures is the cylinder. Gold nanocylinders (GNCs) showed to be very sensitive and specific sensing surfaces. Indeed, we demonstrated that less than 4 ×10(6) avidin molecules were detected and contributed to the surface-enhanced Raman scattering (SERS) signal. Thus, the soft UV-NIL technique allows to obtain quickly very sensitive substrates for SERS biosensing on surfaces of 1 mm (2).