Date

2018

Author

Çağlayan, Zeynep

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Circulating tumor cells (CTCs) are cancerous cells detached from a primary tumor site and enter the bloodstream, causing the development of new tumors in a secondary site. Therefore, their detection in blood is critical to assess the metastatic progression and to guide the line of the therapy. However, the rarity of CTCs in the bloodstream and the lack of suitable detection tool hinders their use as a biomarker in malignancies. Recent advances in microfluidic technologies enabled development of point-of-care (POC) medical diagnostic tools, which offers low cost, rapid, and sensitive analysis of variety of clinical disorders, especially in resource-limited settings. Integration of microfluidic systems with on-chip mechanical and electronic parts have been enabled by Micro-Electro-Mechanical Systems (MEMS) technology, allowing low-cost fabrication of fully integrated microfluidic detection tools. Dielectrophoresis (DEP) is a technique used for separating particles with different sizes and/or dielectric properties. Fabrication of microelectrodes thanks to MEMS technology, allows DEP to be applied in biomedical applications such as manipulation, separation and enrichment of targeted cells from untargeted ones without any labeling. Among numerous applications, rare cell detection from blood occupies an important place in diagnostics of fatal diseases such as cancer. Because of the difficulties in detecting only a few rare cells inside of billions of blood cells in 1 ml blood, rare cell enrichment from blood becomes essential. Starting from this point of view, enrichment of CTCs from blood by using DEP is decided as the main objective of the thesis. One of the most critical issue related to successful design of a DEP-based enrichment device is that DEP spectra of the targeted particles should accurately be known. In this content, DEP spectrum analysis method was developed. This study presents an approach for analyzing the dielectrophoretic (DEP) spectra of biological cells without ascertaining their membrane and cytoplasmic properties. For the proof of DEP spectrum investigation of the biological cells with the proposed analysis method, MEMS-based DEP spectrum device was designed. In this design, reciprocal V-shaped planar electrodes were utilized to generate non-uniform electric field in the chamber that holds the cell solution. Fabrication flow for this design was developed and fabrication of these devices was performed. Testing of the proof of concept DEP spectrum devices was carried out with polymoprhonuclear leukocytes and mononuclear leukocytes obtained from 2 different healthy donors and MCF7 cells (human breast adenocarcinoma cell line) for 15 different frequencies in the range from 100 kHz to 50 MHz at 10 Vpp. The results reveal that at 1 MHz, a significant velocity difference occurs between MCF7 cells (33.99 μm/s) and leukocytes, mononuclear leukocytes (7.9 μm/s) and polymorphonuclear leukocytes (13.82 μm/s), which can be utilized as the working frequency for DEP-based enrichment of MCF7 cancer cells from WBCs as a future work. Considering the problems associated with the experimental setup, testing and post-processing of the proof of concept devices, the proposed DEP spectrum study was improved. The design of the device used in the improved DEP spectrum study was the same as the proof of concept device, except the gap between the electrodes was increased from 20 µm to 30 µm in order to generate more proper electric field gradient distribution on the surface. Testing of the improved analysis was carried out with leukocytes obtained from single donor and MCF7 cells at 10 Vpp for 9 different frequencies in the range from 500 kHz to 10 MHz, by considering the results presented for the proof of concept analysis. The results reveal that there are significant velocity differences occur between MCF7 cells and leukocytes at 500 kHz, 850 kHz and 1 MHz with the ratios 3.58, 3.37 and 3.12, respectively. With the improved DEP spectrum study, the examination method was automated and approximately 130 MCF7 cells were examined for each frequency value. By considering the results obtained from DEP spectrum analysis, DEP-based enrichment microfluidic device was designed. In this design, rectangular and evenly spaced planar electrodes rotated with a certain degree relative to the main flow (13º) were utilized at the bottom of parylene microchannel with 1000 µm in wide. The proposed structure performs the separation of the cells focused on the microchannel wall (with the help of hydrodynamic focusing principle) by using the positive dielectrophoresis (pDEP) method in the active DEP area. Fabrication flow for this design was developed and fabrication of these devices was performed. Testing of the fabricated devices was started with the selection of experimental parameters to examine hydrodynamic focusing and expected cell movements. Later, CTC enrichment experiments of the DEP-based enrichment devices were conducted with MCF7 and leukocyte mixture. Recovery rate result for MCF7 cells were calculated as 83.3% at 10 Vpp. Cell enrichment factor for these rare cells were calculated as 3, which means the desired rare cell ratio was increased to 3 fold at the output relative to the input.

Subject Keywords

Leucocytes., Breast, Dielectrophoresis., Cancer cells, Microelectromechanical systems.

URI

http://etd.lib.metu.edu.tr/upload/12623008/index.pdf
https://hdl.handle.net/11511/27963

Collections

Graduate School of Natural and Applied Sciences, Thesis