Optimization Study of Polyethyleneimine Surface Coating for Microtip DNA Purification
Cairns, Andrew Alexander
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DNA purification is a process to separate and extract DNA from various samples. As such, it is an initial step in molecular diagnostics, forensics, pharmacogenomics, and many other genetic analysis based disciplines. Current commercial solutions employ microfilters, centrifugation, or magnetic beads to extract DNA. Recently, silicon microtips have shown equivalent yield to the aforementioned methods, while using fewer reagents and shorter processing time. The working principle of the microtip system relies on dielectrophoretic force and capillary action to concentrate and bind DNA to the microtip surface. There remain two challenges associated with the microtip method: <bold>(1)</bold> increase efficacy of the process in the presence of a high concentration of inhibitors, such as in blood and <bold>(2)</bold> improve stability of the microtip surface chemistry during storage. In this thesis, we investigate a polymer film of Polyethyleneimine (PEI) as a potential surface layer to address these challenges. To evaluate the performance of PEI, a coating is applied to the microtips, and their purification performance is tested in a variety of conditions. The operation parameters including curing temperature, capture time, elution time, and elution temperature are optimized. Subsequently the PEI tips are tested after storage; results are consistent for up to 1 month. To address the need for analysis of blood samples, PEI tips are tested with a modified procedure including "washing" steps. Using PEI tips, the overall results with blood are comparable to other commercial kits. By optimizing the conditions of the PEI layer, the challenges associated with microtip based DNA purification systems have been addressed to enhance the DNA purity without compromising yield.
- Mechanical engineering