Real-time droplet monitoring for digital Polymerase Chain Reaction in microfluidic chip

• Autor(a) principal: Mota, Ana Catarina Candeias
• Data de Publicação: 2020
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/117487
• topic: dPCR; Microfluidic; Droplet generator; Droplet-based microfluidic; label-free; real-time droplet monitoring; Domínio/Área Científica::Engenharia e Tecnologia::Nanotecnologia

Current cancer diagnosis techniques are often dependent on the collection of tumour tissue, involving invasive processes for the patient. Circulating Tumour DNA (ctDNA) emerges as an alternative resource for cancer detection and monitoring, that can be har vested from simple blood samples. Digital Polymerase Chain Reaction (dPCR) is a fast and sensitive technique for DNA amplification, suitable for low DNA concentrations such as ctDNA. Advances in microfluidics allow the partition of PCR samples into droplets based in water-in-oil emulsions, so that PCR amplification occurs within each droplet. In this way, the PCR reaction is a well controlled process with a low probability of contami nation and allowing a high throughput analysis. The aimed of this work was to develop droplet-based microfluidic device for application to dPCR technique coupled with real-time droplet monitoring. This work focused on the design and fabrication of a microfluidic device capable of producing a large number of uniform droplets with volumes in the nanoliter range and constant frequency. For this, a polydimethylsiloxane (PDMS) droplet generator device was developed, through photo and soft-lithography techniques, and tested with several oil/water flow rates ratios. Then, the droplets generated were characterized in terms of droplet size, velocity and frequency through the implementation of a powerful open-source software for real-time analysis. Several tests on different devices were carried out to evaluate the device reproducibility. Finally, the droplet generator was incorporated with a serpentine design, allowing the PCR cycles to occur in continuous flow. The results revealed that was possible to generate droplets with radius between 22-99 µm and a coefficient of variation bellow 10%. The correspondents volumes ranged between 90 pL-4.18 nL. Moreover, the velocities obtained situated between 0.05 mm/s-7.62 mm/s with droplet generating frequency of 2-50 Hz. Regarding to the droplet monitoring, the results of the workflows developed revealed similarity with the results obtained trough a widely used software for this purposes, with the advantage of allowing real-time analysis for a larger sample of results.