EWOD Digital Microfluidic Device for Single Cells Sample Preparation and Gene Expression Analysis - Arnaud RIVAL
EWOD Digital Microfluidic Device for Single Cells Sample Preparation and Gene Expression Analysis
Arnaud RIVAL, M.S.
Cyril Delattre, Yves Fouillet, Claude Chabrol, Gaël Castellan, Fréderic Bottausci, Nadine David, Christine Peponet, Françoise Vinet, Dorothée Jary
Single cells sample preparation and analysis become today of major interest in biology laboratories, including studies on small and precious samples such as biopsies and stem cells, or even single cell for gene expression analysis. Despite new microsystems and technological developments, single cells manipulation, mRNA capture and purification, and sensitive qRT-PCR (quantitative Reverse Transcription – Polymerase Chain Reaction) are still critical points. Moreover, most of the time the devices proposed do not allow a complete on-chip protocol.
At LETI, we developed a fully integrated and automated EWOD (ElectroWetting On Dielectric) Lab On Chip for biology and healthcare. EWOD digital microfluidics offers very well reduced, controlled, and reproducible reaction chambers, droplets, for lab on chip applications. The microfluidic device is a silicon chip coated with a SiOC hydrophobic layer instead of the commonly used Teflon®. A pattern of metallic electrodes is used to make the droplets move on precise positions when applying a voltage across it. The whole process is made with classical cleanrooms machines at 200mm wafer scale. A specific experimental setup enables direct and reliable electrical connections thanks to spring loaded pins instead of usual PCB with wire connections. EWOD chip is manually placed on a heat regulation module (Peltier module and regulation fans) for temperature cycles required in biological experiments.
The chip is designed to perform the whole sample preparation and analysis for gene expression directly on a single chip, in volumes ranging from 1µL down to a few tens of nanoliters. It includes cells loading and selection, cells lysis, capture of mRNA by functionalized magnetic beads (moved to a microcontroller permanent magnet), elution of the mRNA, and the expressed genes quantification by qRT-PCR. So far, each step of the sample preparation protocol for gene expression like single cells manipulation and mRNA capture and purification by magnetic beads has been validated on chip and gives better yield on reduced samples than tube extractions. Moreover, qRT-PCR performed on mRNA down to a single cell scale has been successfully performed in 64nL droplets and gives similar results compared to qRT-PCR in 10 µL tubes. Current results are encouraging for future experiments, i.e. a complete gene expression study, starting from a few single cells, up to the quantification by qRT-PCR, is now possible on EWOD lab on chip.
This EWOD microsystem is adapted for single cells experiments since it enhances sensitivity by reducing volume scale during mRNA purification steps, and thus, it becomes a promising tool for molecular biology on small and precious samples.