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Design and Fabrication of Centrifugal Microfluidic Disc for Cell Lysis and DNA Extraction

Jalalian, Pourya | 2023

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 55903 (06)
  4. University: Sharif University of Technology
  5. Department: Chemical and Petroleum Engineering
  6. Advisor(s): Saadatmand, Maryam; Garshasbi, Masoud
  7. Abstract:
  8. Centrifugal microfluidic systems due to their unique properties, including the ease of sample transfer, high controllability, the need for samples and reagents in a small volume scale, the possibility of embedding a complete laboratory and modeling a variety of processes on a disk, they have great potential to be used in the field of point of care diagnosis. Such devices can perform several different processes simultaneously and continuously by using inertial and pneumatic forces as well as a complex network of microchannels. The purpose of this research is to design and build a centrifugal microfluidic disc for lysing amniotic stem cells, followed by DNA extraction, in order to detect fetal genetic abnormalities. The implementation of the process in the proposed disc relies on the internal forces of centrifugal systems and magnetic force, which does not require external equipment. The rapture of cells was done by chemical reaction and extraction of DNAs was done using magnetic nanoparticles coated with silica as a movable adsorbent substrate, which fixed magnets embedded in the upper and lower part of the disk were used to guide them and control the sequence of the process. Also, with the aim of investigating and optimizing the effective design parameters in cell rupture, the simulation of the mixing unit and the siphon-inertia valve was carried out with Comsol software. The results of the simulation show that the simultaneous emptying of the mixing chambers and the increase of the mixing efficiency happen in the design with symmetrical output. Also, the results of the inertial valve simulation confirm the success of the valve in complete discharge. After fabricating the disk, the results of the hydrodynamic test with colored solutions were presented to check the proposed design in the implementation of the process. The results indicate the success of the disk in controlling the process sequence, and the duration of the entire extraction process by the disk was estimated to be 16 minutes and the manual method was 39 minutes. Also, the amount of the initial sample required for extraction by disk was 150 microliters and 1500 microliters for the manual method. In the next step, the extraction process was carried out, and with the aim of checking the efficiency of the disk and comparing the purity of the extraction with the manual method in the laboratory, optical density test, gene amplification technique with PCR, RT-PCR and gel electrophoresis were performed. The average purity and concentration of extraction in manual method were 0.71 and 1.714 μg/ml, respectively, and 1.73 and 14.86 μg/ml in disc extraction, which indicated the superiority of microfluidic disc extraction compared to manual method. The results of the gel electrophoresis section showed that the process was carried out without contamination and due to the formation of single bands in a weight balance, exclusive gene amplification was successful. However, in the results of the RT-PCR test, it was observed that the process of gene amplification for the DNAs extracted by the disc was done with 2 cycles of delay, which is justified due to the injection of the input sample in smaller amounts, and is not a defect in the process. Extraction with the presented system, in addition to reducing costs, will bring ease in carrying out the process and perform separation in the least possible time and with high purity, therefore it has a significant advantage compared to laboratory methods
  9. Keywords:
  10. Lab-on-a-CD (LOCD) ; DNA Extraction ; Stem Cell ; COMSOL Software ; Anomaly Detection ; Fetal Genetic Abnormalities Detection ; Cell Disruption ; Amniotic Stem Cells

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