Photocured Microfluidic Device-based Integrated Platform For Nucleic Acid Extraction And Detection

Nucleic acids detection is essential in many fields such as biomedical research and disease diagnosis.With the rapid development of microfluidics,digital PCR(d PCR)has grown into a powerful technique,which is capable of absolute quantification of nucleic acids with ultrahigh sensitivity and precision.Droplet d PCR(dd PCR),in particular,has been widely explored in recent years owing to the ease of chip fabrication and the flexibility of droplets generation.Nevertheless,there remain several practical issues in current dd PCR technique,including the possible droplets fusion/fission during PCR thermocycling,droplets movement-caused incapability of real-time fluorescence monitoring,and droplet loss-related wasting of precious samples/reagents,as well as difficulties in precisely metering their volume.Additionally,the nucleic acid extraction,as an indispensable step for dd PCR,is usually performed off-chip,leaving the whole process at low integration/automation levels,and high risk of aerosol contamination.It is therefore very necessary to develop a fully integrated platform for successive nucleic acid extraction and detection.Toward this end,this thesis is mainly focused on UV photocuring method,trying to progressively solved the practical issues such as droplets stability,loss-free droplets trapping,and the integration of channel-based microfluidics(CMF)with digital microfluidics(DMF).On these bases,a fully integrated microfluidic platform that involves both DMF-based nucleic acid extraction and dd PCR-based nucleic acid detection has been developed.The main contributions include:1)A photoimmobilized planar droplet array for dd PCR has been developed.First,a photocurable mixture has been proposed as the continuous oil phase,which enables not only the generation of uniform water-in-oil droplets of various sizes,but also the rapid immobilization of droplets into stable planar array,thereby,effectively preventing the droplets form movement,fusion or fission.By further developing a multi-layer chip design,the severe evaporation issue that commonly exist in polydimethylsiloxane(PDMS)-based devices has also been successfully overcome.These features could make the dd PCR experiments more robust,and show a promising potential for real-time fluorescence monitoring.2)A photofabricated micropillar array for loss-free droplets trapping has been developed.First,a honeycomb-configuration micropillar array has been optimally designed,which can make the trapping density up to 160-250 droplets/mm~2.Moreover,there is no droplet loss through outlet,thereby,effectively avoiding the waste of precious samples/reagents,and facilitating the precise metering of sample volume.A facile photofabrication technique of microfluidic chips has also been developed,which allows the optimal material selection for dd PCR application,and thus,thoroughly avoid the sample evaporation and surface modification,thereby making the chip fabrication more convenient.Additionally,by taking advantage of the photocurable oil,the trapped droplet array can be further immobilized into more stable format,and thus become more promising in real-time fluorescence dd PCR.3)An integrated channel-to-digital microfluidic chip for successive nucleic acid extraction and detection has been developed.For the first time,a novel photocured composite hydrophobic dielectric layer with both high dielectric constant and strong hydrophobic property has been proposed.Meanwhile,a photocured layer with channel structures has been fabricated by using a hydrophobic photopolymer.These enables the easy formation of a fully integrated channel-to-digital microfluidic chip,and makes the whole fabrication process very simple,without the requirement for tedious surface hydrophobization treatment.Moreover,it also facilitates the incorporation of indium tin oxide(ITO)film,thereby,forming the necessary two-plate configuration and realizing all basic DMF operations,such as droplets generation,splitting,movement and fusion.On these bases,an integrated platform,involving both DMF-based nucleic acid extraction and dd PCR detection,has been developed after optimally designing the microfluidic channel and electrode structures,which is expected to play important roles in many areas such as point-of-care test(POCT),and clinical diagnosis.