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Title: An integrated chip-based device for droplet-flow polymerase chain reaction
Author: Sim, Steven Poh Chuen
ISNI:       0000 0004 6495 8779
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2016
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The polymerase chain reaction (PCR) is an important in-vitro technique in molecular biology for amplifying trace quantities of deoxyribonucleic acid (DNA). PCR is carried out by mixing the DNA molecules to be amplified with primers, polymerase enzymes and deoxynucleotide triphosphates (dNTPs) in a suitable buffer solution. A conventional thermal-cycler is then used to cycle the PCR mixture between multiple temperatures for denaturation, annealing and extension. Bench-top thermal cyclers have large thermal masses and use large sample volumes, leading to overly long cycling times, excessive energy and material consumption, inhomogeneity in the reaction environment, and an inability to handle large numbers of small volume aliquots. Microfluidic technologies overcome many of the limitations of bench-top thermal cyclers, providing a more controlled approach to PCR. Droplet-flow is one of the most promising microfluidic methods for carrying out PCR. The droplet-flow approach uses small water-in-oil droplets for compartmentalisation of the PCR reaction mixture, with each droplet behaving like an individual reaction chamber. By flowing the droplets over different temperatures for denaturation, annealing and extension, rapid thermal cycling can be achieved, greatly reducing the reaction time relative to bench-top thermal cyclers. The use of an oil phase to encapsulate the aqueous PCR mixture as droplets also prevents unwanted surface interactions and flow dispersion that can adversely affect the PCR yield. Here we describe an integrated microfluidic device for carrying out droplet-flow PCR. Instead of using multiple temperature zones to thermally-cycle the flowing droplets, the device used an on-chip radial temperature gradient. The droplets passed through microchannels arranged in a spoke-like geometry, causing them to pass backwards and forwards along the radial temperature gradient and so undergo the repeated thermal cycling required for PCR. The device reported here builds on an earlier plastic microfluidic PCR device (by Schaerli et al.) in which the radial temperature gradient was generated using a bulky external heater and a thermoelectric cooler, together with heat sinks and fans. In the silicon- and glass-based device reported here, integrated heaters, temperature sensors and air gaps (for passive cooling) were used to generate the temperature gradient, leading to significant miniaturisation of the device. The dimensions of the complete device assembly were 6.0 cm x 5.0 cm x 2.0 cm compared to 25.0 cm x 25.0 cm x 25.0 cm for the device by Schaerli et al. Despite the small size of the device, the achievable temperature gradient on the chip was sizeable. For instance, when the central heater was set to 92.0 °C, the temperature at the periphery was ~60.0 °C, corresponding to a temperature difference of ~32.0 °C – easily sufficient for PCR applications. Using chemical modification, the hydrophilic walls of the microchannel were rendered hydrophobic. An on-chip T-junction or flow-focusing junction was subsequently used to merge the oil and aqueous streams to generate the PCR-containing water-in-oil droplets. A PCR recipe was optimised on a bench-top thermal cycle. With this recipe, droplet-flow PCR was conducted on the PCR device by flowing the generated droplets up and down the radial temperature gradient to induce thermal cycling. Gel electrophoresis analysis of the collected droplets from the device showed the presence of the PCR product, confirming the ability of the integrated device to conduct droplet-flow PCR. By varying the central temperature of the PCR device and the flow rate of the droplets, the yield of the PCR product could be tuned. By serially diluting the concentration of the DNA molecules, it was found that the PCR device was able to amplify concentrations as low as 0.01 pM to a level detectable by gel electrophoresis. When coupled to a laser-induced fluorescence detection system, the emission from the PCR mixture in the water-in-oil droplets could be successfully detected for each PCR cycle. The increase in the fluorescence over successive PCR cycles once again verified the feasibility of carrying out droplet-flow PCR on the integrated device.
Supervisor: deMello, John ; deMello, Andrew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral