Development of Cell Lysis Techniques in Lab on a chip
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The recent breakthroughs in genomics and molecular diagnostics will not be reflected in health-care systems unless the biogenetic or other nucleic acid-based tests are transferred from the laboratory to clinical market. Developments in microfabrication techniques brought lab-on-a-chip (LOC) into being the best candidate for conducting sample preparation for such clinical devices, or point-of-care testing set-ups. Sample preparation procedure consists of several stages including cell transportation, separation, cell lysis and nucleic acid purification and detection. LOC, as a subset of Microelectromechanical systems (MEMS), refers to a tiny, compact, portable, automated and easy-to-use microchip capable of performing the sample-preparation stages together. Complexity in micro-fabrications and inconsistency of the stages oppose integration of them into one chip. Among the variety of mechanisms utilized in LOC for cell lysis, electrical methods have the highest potential to be integrated with other microchip-based mechanisms. There are, however, major limitations in electrical cell lysis methods: the difficulty and high-cost fabrication of microfluidic chips and the high voltage requirements for cell lysis. Addressing these limitations, the focus of this thesis is on realization of cell lysis microchips suitable for LOC applications. We have developed a new methodology of fabricating microfluidic chips with electrical functionality. Traditional lithography of microchannel with electrode, needed for making electro-microfluidic chips, is considerably complicated. We have combined several easy-to-implement techniques to realize electro-microchannel with laser-ablated polyimide. The current techniques for etching polyimide are by excimer lasers in bulky set-ups and with involvement of toxic gas. We present a method of ablating microfluidic channels in polyimide using a 30W CO2 laser. Although this technique has poorer resolution, this approach is more cost effective, safer and easier to handle. We have verified the performance of the fabricated electro-microfluidic chips on electroporation of mammalian cells. Electrical cell lysis mechanisms need an operational voltage that is relatively high compared to other cell manipulation techniques, especially for lysing bacteria. Microelectro-devices have dealt with this limitation mostly by reducing the inter-distance of electrodes. The technique has been realized in tiny flow-through microchips with built-in electrodes in a distance of a few micrometers which is in the scale of cell size. In addition to the low throughput of such devices, high probability of blocking cells in such tiny channels is a serious challenge. We have developed a cell lysis device featured with aligned carbon nanotube (CNT) to reduce the high voltage requirement and to improve the throughput. The vertically aligned CNT on an electrode inside a MEMS device provides highly strengthened electric field near the tip. The concept of strengthened electric field by means of CNT has been applied in field electron emission but not in cell lysis. The results show that the incorporation of CNT in lysing bacteria reduces the required operational voltage and improves throughput. This achievement is a significant progress toward integration of cell lysis in a low-voltage, high-throughput LOC. We further developed the proposed fabrication methodology of micro-electro-fluidic chips, described earlier, to perform electroporation of single mammalian cell. We have advanced the method of embedding CNT in microchannel so that on-chip fluorescent microscopy is also feasible. The results verify the enhancement of electroporation by incorporating CNT into electrical cell lysis. In addition, a novel methodology of making CNT-embedded microfluidic devices has been presented. The embedding methodology is an opening toward fabrication of a CNT-featured LOC for other applications.
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Mehdi Shahini (2013). Development of Cell Lysis Techniques in Lab on a chip. UWSpace. http://hdl.handle.net/10012/7976