Date

2014

Author

Laçin, Özgen Sümer

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In medical research, cell counting is of a great importance for the indication of the health status of the patients, diagnosis of the illness, and the detection of the progress of a disease. In clinics, cell counting and sorting is carried out by Coulter Counter devices, in which the electrical potential or resistance change is measured when the cells flow through the defined aperture. Coulter Counter devices perform rapid and accurate analysis of the biological particles in terms of their size and dielectric properties. However, traditional Coulter Counters are bulky and expensive requiring large sample volume. Therefore, miniaturizing this device, utilizing micro-electromechanical systems (MEMS) technology, simply reduces the cost and provides rapid analysis and ease of use. In this study, two different MEMS-based Coulter Counter designs, employing single and double channels, were developed. The novel double channel Coulter Counter design allows the detection of the particles simultaneously by forming sub-channels inside a main channel, increasing the throughput and obviating the need for hydrodynamic focusing of the particles. Two different electrode geometries, coplanar surface Au electrodes and 3D Cu-electroplated electrodes, were employed in the same microchannel, to compare the sensitivities and allow double counting in a single channel. Before fabricating each of these designs, simulations regarding the surface velocity field, electrical field change, and electrical resistance change were accomplished in COMSOL Multiphysics 4.3b. It was observed that designed channels do not face a turbulent flow or clogging problem at the detection points, where channels narrow down. Flow is stable and accurate. Electric field (E-field) simulations proved that when there is no particle in the sensing zone, E-field has the highest value; while when the particle starts to enter the sensing zone, this value decreases gradually and reaches to its minimum when the particle is at the center of the sensing zone or aperture. The existence of a particle in the sensing zone removes the amount of liquid in proportion to the volume of the particle; hence, decreases the area of the aperture. This leads to an increase in the electrical resistance. After getting satisfactory results from the simulations, both of the designs were fabricated. A four-mask fabrication protocol, including the electrode lithography, Cu-electroplating and channel lithography, was developed for the fabrication. Channels were formed of Parylene C, a biocompatible polymer, possessing excellent properties of the chemical resistance for biological analysis, and have long shelf life. Focusing of the particles is achieved by adjusting the channel dimensions relative to the particle diameter; hence, the additional inlet requirement for hydrodynamic focusing is eliminated. For our case, we aimed to sense polystyrene microbeads (10 µm) and K562 leukemia cancer cells (13-22 µm); therefore, a channel having a diameter of approximately 30µm is adequate for sensing and counting. The excitation voltage (5 Vpp AC) and the frequency (55 kHz) were adjusted to prevent Electrical Double Layer (EDL) and electrode degeneration. A custom-made electrical detection circuit was built to filter out undesired frequencies and detect the passage of the particles. All results were recorded with a Data Acquisition Board (National Instruments 6337) and illustrated with LabVIEW SignalExpress software. An algorithm for counting the number of negative peaks was developed on MATLAB R2013b. A flow rate of 1µl/min was introduced to the main channel and this rate increased up to 7.5µl/min in single channel design and 4.5µl/min in double channel design. 10 µm polystyrene microbeads and 17 µm K562 cancer cells were counted separately and in mixture, with different flow rates and frequencies. It was proven that both of the designs are capable of counting the particles larger than 10µm with a flow rate of maximum 75 µl/min. In conclusion, two different Coulter Counters were developed. Satisfactory results were obtained from both simulations and experiments. Different flow rates and frequencies were employed to investigate flow rate profile and electrical double layer effect on the measurements. Both of the devices sensed and counted the microparticles introduced in a conductive medium. With further improvements, granular analysis of the cell, in addition to the size based detection, and reaching faster detection rate are the ultimate goals of this study.

Subject Keywords

Cytometry., Coulter principle., Particle size determination., Microelectromechanical systems.

URI

http://etd.lib.metu.edu.tr/upload/12617565/index.pdf
https://hdl.handle.net/11511/23744

Collections

Graduate School of Natural and Applied Sciences, Thesis