A novel approach for chip-based digital LAMP towards the quantification of prostate cancer biomarkers

• Autor(a) principal: Oliveira, Ana Beatriz Brito de
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/88336
• topic: Lab-on-a-chip; Microfluidics device; isothermal DNA amplification; digital LAMP; c-Myc; prostate cancer; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Nucleic acids amplification-based methods can profit from the features offered by Lab-on-a-chip technologies, in particular those that aimed for molecular diagnosis purposes. Currently, isothermal amplification approaches, more precisely LAMP, have become promising alternatives to the current gold standard technology (PCR). Regardless the amplification mechanism, accurate target quantification is still challenging. To this end, the development of digital amplification methods has helped to circumvent this limitation. This thesis focused on the development of a chip-based digital LAMP system towards the quantification of prostate cancer biomarkers. For this, LAMP was integrated with droplet-based digital amplification concept. LAMP positive amplification was achieved after 60 minutes, leading to a 2-fold increase in fluorescence when compared to the negative amplification controls, in a vortex-based droplet generation approach. However, aspects inherent to this method prevented a quantitative assessment of LAMP amplification. In order to overcome these limitations, a novel microfluidics chip-based device was developed and implemented towards dLAMP quantification of c-Myc gene. The T-junction type droplet generator chip achieved droplets of 1.5 nL with a coefficient of variation bellow 3%, in line with the standard for this technique. This system showed a sharp response to template concentration, observable by the raise in the fraction of positive droplets. Additionally, the target quantification proven to be precise (R2 =0.99) for 4 orders of magnitude of copies/µL (5 copies/µL - 5x105 copies/µL) after Poisson’s modulation. Aiming for the implementation of this chip-based dLAMP system into the detections of prostate cancer-associated biomarkers, amplification reactions of SChLAP1 and PCA3 genes were developed and further optimized for real-time fluorescence monitoring. As a result, it was possible to develop a quantitative method for cDNA amplification, that presented higher amplification efficiencies and a reduction on the overall reaction time, when compared to the gold standard RT-PCR. Furthermore, the proposed strategy is compatible with the integration into the chip-based microfluidics device, hence easily extended to the monitorization of gene expression levels.