< Terug naar vorige pagina

Publicatie

Advancements in compartmentalized microfluidic platforms for microwell-assisted digital bioassays

Boek - Dissertatie

Currently, the in-vitro-diagnostics field (IVD) is focusing at strengthening the combination of those tests conceived to ensure automation, simplicity, portability and affordability (also known as point-of-care (POC) tests with those ensuring highly sensitive and specific analytical performances. In this context, microfluidics, the science describing and controlling the fluid behaviour at the microscale, and microwell-assisted digital bioassays, biosensing platforms which allow single-target molecule resolution, have proved to be between the most remarkable technological drivers. Hence, the integration of microwell-assisted digital bioassays in microfluidics would result in a revolutionary biosensor platform which could potentially fulfill many of the abovementioned requirements at once. However, the integration of microwell-assisted digital bioassays in microfluidics is still far from full development in terms of bioassay versatility and device engineering. As a matter of fact, albeit microwell-assisted digital bioassays have been largely performed for the sensitive detection of proteins, cells and enzymes, their applicability for oligonucleotides detection is still an unxplored territory. Moreover, several engineering challenges remain: (i) the generation of smart fabrication microwell arrays for reliable digital bioassays, (ii) the compatible implementation of these microwell arrays with microfluidics, (iii) the high fabrication throughput and functionality of the microfluidic devices. In this dissertation, advancements in compartmentalized microfluidic platforms for the generation of microwell-assisted digital bioassays have been carried out, focusing on the troubleshooting of the above-mentioned challenges. The first part of the dissertation focuses on the development of new microwell-assisted digital bioassays for DNA and single-nucleotide polymorphisms (SNPs). A limit-of detection in the attomolar range for DNA strands of different lengths was obtained and discrimination of DNA strands containing a single SNP (mutant strands) from DNA strands without SNP (wildtype strands) was demonstrated using ligation steps. In the second part of the dissertation alternative materials and methods to improve digital microfluidics (DMF) and DMF-compatible microwell array fabrication is presented. Hereto, the embossing of Telfon on glass substrates using a Si-micropillar mould was used to shape wells able to host a single beads per well while obtaining beads seeding and reagents sealing efficiency close to 100 %, thus ensuring the suitability of the these arrays for digital bioassays. Following, new DMF bottom plates were re-designed and fabricated using a laser-patterned FR4 board as alternative arrays of electrodes. OSTE+ polymer was used as alternative dielectric layer allowing user-friendly deposition and low-actuation voltages. The OSTE+ layer was dielectrically characterized and implemented on the new FR4 substrates allowing lower voltage actuation in perfect accordance with theoretical estimation. Finally, the third part of the dissertation focused on the development of plastic-based microwell-arrays and their implementation in plastic-based self-powered continuous microfluidic systems. Here, again microwells able to host one single bead were fabricated through reactive-ion-etching approaches. Surface wettability was modified in order to obtain 100 % reagent sealing. Beads seeding and reagents sealing efficiencies in Poly(methyl methacrylate) microwells were characterized on the self-powered microfluidic platform and confirmed a plastic modification which allowed an 100 % reagent sealing. A preliminary experiment for oligonucleotides detection was also performed presenting for the first time the potential of this new approach. To conclude, in this dissertation, new bioassays and technologies were established to progress the implementation of microwell-assisted digital bioassays in microfluidics beyond the state-of-the-art, aiming at the fulfillment of the emerging diagnostics requirements while keeping the ultimate implementation successful.
Jaar van publicatie:2020
Toegankelijkheid:Open