Assessment of changes in the dielectric properties of multidrug resistant cancer cells by electrorotation technique /

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2014
Bahrieh, Garsha
With recent advances in microfabrication technologies different methodologies are employed to study the dielectric properties of the biological systems. These include electrorotation (ER), dielectrophoresis, microelectrical impedance spectroscopy, and impedance flow cytometry. Among them, ER is utilized as a high accuracy approach in determining the membrane and interior dielectric properties of single cells. In addition, in cancer therapy, cancer cells are developing resistance toward the chemotherapeutic drugs and their biophysical properties are showing variations. Therefore, study of changes in the physical and electrical properties of resistant cells is important to make differentiation between drug sensitive and resistant cells. This thesis presents the design, fabrication, and implementation of ER devices with 3D electrodes for the dielectric characterization of cells. In addition, as an experimental application of the extracted dielectric properties of the cells (by ER), the DEP method is utilized. 1st generation of the ER chips with pyramidal planar (2D) electrodes was utilized for the proof of concept and study of the feasibility of dielectric characterization of cells using ER technique. First analyses were carried out using yeast cells and imatinib resistant human leukemia cells (K562-IMA). 3D FEM analyses and experimental results showed that the use of 2D (planar) structures can lead to errors in the measurement. In 2D structures, because of the negligible height of the electrodes (~300nm) compared to the biological cells (~10-25 µm), the variation of the rotational torque (ROT-T) is significant along the z-axis. In addition, due to high current density passing through the 2D electrodes, the thermal heating of the electrodes was significant. This can result in variations in the medium temperature due to the thermal resistive heating of the electrodes. Therefore, variations in the conductivity of the medium were unavoidable. It can alter the ER characterization results, because of the high sensitivity of the ER method to the conductivity of the medium. 3D electrode structures can provide a high sustainability of the ROT-T along the z-axis, by encompassing the whole cell body and eliminating the fringing electric field effect on the cells rotation. In these electrodes, due to increased volume of the electrodes, the current density passing through the electrodes drops significantly. Therefore, the thermal heating reduces by more than an order of magnitude, which guarantees consistency of the medium conductivity during the ER experiments. The second generation of the ER devices with 3D pyramidal electrodes were designed and fabricated to overcome the problems with the planar ER devices. These devices were fabricated with 3D electrodes on az glass substrate. However, there were a number of problems which were faced in the experimental analyses of these devices. Firstly, due to the glass substrate, assessment of the rotation of the cells was difficult. Moreover, small reservoir size and hydrophobicity of the parylene had made entrance of the cells into the electrode area difficult. In addition, due the complexity of the fabrication process and limited number of ER chips on each wafer, the 2nd generation of the ER chips was unsuitable for the practical uses. The third generation of the ER devices, with 3D electrodes was designed to overcome the problems in the previous generations. The fabrication process of these devices needs a single mask. In addition, to study the electrode geometry effects in the ER method, ER devices with six different electrode geometries were designed and fabricated. In order to make a comparison between the electrical characteristics of the each electrode type, experimental and numerical investigations were carried out. For this purpose, 3D rotational torque and effective electric field distributions were characterized using experimental and FEM analyses (COMSOL). The ER devices with polynomial electrodes were chosen to have the most practical compromise between the uniformity and sustainability of the generated rotational torque. Using polynomial quadrupole electrodes, 9 different cell populations, including K562 human leukemia cells and MCF7 breast cancer cells and their multidrug resistance (MDR) counterparts were characterized. For this purpose, the rotational behavior of the cells was studied in four different medium conductivities (1-8 mS/m) in the frequency range of 1-100 kHz. Subsequent to determination of peak rotation frequencies in all media, an automated MATLAB program was used to calculate the dielectric properties of the cells. It was observed that, sensitive and MDR cancer cells show significant variation in their dielectric properties, which can be used to identify and separate MDR cells from sensitive ones through DEP-based methods. In addition, to investigate the feasibility of separation of the cancerous cells and their multidrug resistance (MDR) derivation from the human leukocyte cell mixture, DEP chips were designed and fabricated. These chips have five active electrodes and total of 12 potentially floating electrodes, which had 3D structures, located in the parylene microchannel side walls. 3D COMSOL simulations were utilized in the design and studying the separation performance of the DEP chips. In addition, FEM analyses were implemented in the optimization of fluidic flow rates and applied voltages. Using 3D simulations, the separation efficiency of the designed DEP devices was calculated as 100%. The initial experimental analysis of fabricated DEP devices with K562-IMA cells showed a successful separation of the cell under both n-DEP and p-DEP forces to the intended channels. In conclusion, three generations of the ER devices with more than eight different electrode geometries were designed and fabricated. 3D FEM simulations and experimental analyses were utilized in the characterization of the ER devices. The polynomial electrodes were chosen to have the most practical compromise between the rotational torque uniformity and magnitude. Using the polynomial electrodes, different cell populations, including K562 leukemia cancer cells and its imatinib (0.2, 0.3, and 0.5 µM) and doxorubicin (0.1, 0.3, and 0.5 µM) resistant counterparts and MCF7 and its 1 µM doxorubicin resistant derivation were characterized. As an application for extracted dielectric properties of the cells, DEP chips with novel potentially floating electrode configuration was designed and fabricated. Results show that sensitive and resistant cancer cells can be separated from each other via DEP-based methods, and ER is a powerful and sensitive dielectric characterization technique for the biological cells.

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Citation Formats
G. Bahrieh, “Assessment of changes in the dielectric properties of multidrug resistant cancer cells by electrorotation technique /,” M.S. - Master of Science, Middle East Technical University, 2014.