Integrated genetic analysis systems

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Brennan, Desmond
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University College Cork
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The overall objective of this thesis is to integrate a number of micro/nanotechnologies into integrated cartridge type systems to implement such biochemical protocols. Instrumentation and systems were developed to interface such cartridge systems: (i) implementing microfluidic handling, (ii) executing thermal control during biochemical protocols and (iii) detection of biomolecules associated with inherited or infectious disease. This system implements biochemical protocols for DNA extraction, amplification and detection. A digital microfluidic chip (ElectroWetting on Dielectric) manipulated droplets of sample and reagent implementing sample preparation protocols. The cartridge system also integrated a planar magnetic microcoil device to generate local magnetic field gradients, manipulating magnetic beads. For hybridisation detection a fluorescence microarray, screening for mutations associated with CFTR gene is printed on a waveguide surface and integrated within the cartridge. A second cartridge system was developed to implement amplification and detection screening for DNA associated with disease-causing pathogens e.g. Escherichia coli. This system incorporates (i) elastomeric pinch valves isolating liquids during biochemical protocols and (ii) a silver nanoparticle microarray for fluorescent signal enhancement, using localized surface plasmon resonance. The microfluidic structures facilitated the sample and reagent to be loaded and moved between chambers with external heaters implementing thermal steps for nucleic acid amplification and detection. In a technique allowing probe DNA to be immobilised within a microfluidic system using (3D) hydrogel structures a prepolymer solution containing probe DNA was formulated and introduced into the microfluidic channel. Photo-polymerisation was undertaken forming 3D hydrogel structures attached to the microfluidic channel surface. The prepolymer material, poly-ethyleneglycol (PEG), was used to form hydrogel structures containing probe DNA. This hydrogel formulation process was fast compared to conventional biomolecule immobilization techniques and was also biocompatible with the immobilised biomolecules, as verified by on-chip hybridisation assays. This process allowed control over hydrogel height growth at the micron scale.
Molecular diagnostics , Microfluidics , PCR , Hydrogel
Brennan, D. 2014. Integrated genetic analysis systems. PhD Thesis, University College Cork.