In situ formed magnetic ionic liquids: DNA extraction performance and fluorescence-compatibility in bioanalytical applications

Bowers, Ashley
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Extracting genomic DNA from complex biological sample matrices is often the first step in numerous molecular biology procedures such as polymerase chain reaction (PCR), cloning, and gene therapy. Obtaining high yields and pure DNA presents a significant sample preparation challenge in nucleic acid analysis. Current methodologies such as the phenol-chloroform extraction use toxic organic solvents and commercially available kits are often very expensive and have limited reusability. Magnetic ionic liquids (MILs) have gained popularity as inexpensive, environmentally benign and tunable extraction solvents. MILs are a subclass of ionic liquids containing a paramagnetic component in the cation or anion, allowing them to be manipulated using an external magnetic field. This thesis describes the use of a new class of MILs featuring metal-containing cations for DNA extraction and their compatibility with fluorescence-based detection methods. Two studies were conducted to address this goal. The first study focused on the DNA extraction efficiency of a new class of MILs using in situ dispersive liquid-liquid microextraction (DLLME) versus conventional DLLME to assess the extraction of DNA sequences of varied sizes. Extraction efficiencies were obtained using indirect detection using anion-exchange high performance liquid chromatography with diode array detection and fluorescence spectroscopy. However, to minimize steps in the sample preparation process, it is useful to directly analyze the DNA within the enriched MIL microdroplet therefore, in a second study the fluorescence quenching effects of the MIL were evaluated. These studies provide an insight into how the paramagnetic metal (Ni, Co, Mn) and ligand used in the design of the MIL can be tailored in order to achieve highly efficient DNA extraction and the subsequent influence of the MIL on the fluorescence signal in downstream analysis.

DNA, fluorescence spectroscopy, high performance liquid chromatography, in situ dispersive liquid-liquid microextraction, magnetic ionic liquids, SYBR Green I