A Digital Microfluidics Platform for Loop-Mediated Isothermal Amplification of DNA

• Autor(a) principal: Coelho, Beatriz Jorge
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/99431
• topic: Digital Microfluidics; Loop-mediated Isothermal Amplification; c-Myc; lab-on-chip; point-of-care diagnostics; Domínio/Área Científica::Engenharia e Tecnologia::Nanotecnologia

Digital Microfluidics (DMF) is an innovative technology for liquid manipulation at microliter- to picoliter-scale, with tremendous potential of application in biosensing. DMF allows maneuvering single droplets over an electrode array, by means of electrowetting-on-dielectric (EWOD), that allows changing the contact angle of a droplet over a dielectric. Each droplet is thus considered a microreactor, with an unparalleled potential to perform chemical and biological reactions. Several aspects inherent to DMF platforms, such as multiplex assay capability and integration capability, make them promising for lab-on-chip and point-of-care (PoC) applications, e.g. DNA amplification assays or disease detection. DNA detection strategies for PoC have been profiting from recent development of isothermal amplification schemes, of which Loop-mediated Isothermal Amplification (LAMP) is a major methodology, allowing a 109-fold amplification efficiency in one hour. Here, I demonstrate for the first time the effective coupling of DMF and LAMP, resulting in a DMF device capable of performing LAMP reactions. This novel DMF platform has been developed and characterised, which allows successful amplification of a c-Myc gene fragment by LAMP. Precise temperature control is achieved by using a transparent heating element, connected to a looping feedback control system. This platform is able to amplify just 0.5 ng/μL of the target DNA, in only 45 minutes, for a device temperature of 65 °C and a reaction volume of 1.62 μL, one of the lowest volumes ever reported. Moreover, the electrophoretic analysis indicates that the amplification efficiency of the on-chip LAMP is considerably higher than that from the bench-top reaction.