Novel polymers and electrophoretic methods for biomolecule purification and separation

Interest in microfluidic devices has grown significantly in recent years due to the potential of this analytical platform to provide rapid, inexpensive analyses in a closed system. The capability to integrate multiple processes into a single device to create a sample-in-answer-out 'lab-on-a-chip' has been the end-goal of this field of research. The focal point for many years was device fabrication and translating benchtop methods to microfluidic devices, but did not concentrate on whether the methods were compatible and could be integrated with other processes needed for a complete analysis. Therefore, integration has been limited by the lack of materials designed specifically for microfluidic devices with the ability to integrate with other processes a primary consideration. The aim of this research was to develop polymer materials and electrophoretic methods to overcome these limitations. The first aim demonstrated the ability of an acid-labile surfactant (ALS) that is mass spectrometry compatible to replace SDS for microchip protein separations. ALS was demonstrated to provide suitable separation efficiency with poly-N-hydroxyethylacrylamide providing the highest separation efficiency of five common wall coatings tested. Next, two methods of nucleic acid purification were investigated as this is a critical component of any genetic analysis required PCR amplification. Transient isotachophoresis, which is based on a discontinuous buffer system, was shown to be able to concentrate and separate DNA from serum proteins for PCR. A polymer capture matrix, which uses an oligonucleotide covalently bound to a polymer, was developed for selectively purifying target DNA or RNA. This method overcomes the major limitations of the widely used solid-phase extraction method such as limited sample capacity and the use of PCR inhibiting reagents. Extraction of 37.5 copies of RNA per microliter from a 10% serum solution is demonstrated. Finally, the physical properties and DNA sieving capability of thermoreponsive alkoxyalkylacryalmide polymers were investigated. It is shown that separation efficiencies higher than that of LPA can be achieved if the polymer hydrophobicity is controlled. This research provides a foundation for overcoming a number of the limitations facing the current purification and separation methods that can facilitate the development of integrated biomolecule analysis systems.