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Design and Fabrication of an Integrated Microfluidic System for Cell Lysis, DNA Purification and Amplification in the Continuation of the Cell Isolation Stage for Biological Diagnosis Purposes

Rahbary Moghadam, Ali | 2024

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 57675 (08)
  4. University: Sharif University of Technology
  5. Department: Mechanical Engineering
  6. Advisor(s): Shamloo, Amir; Kazemzadeh Hanani, Siamak
  7. Abstract:
  8. Analytical biological research has gained significant attention due to the critical importance of processes such as cell separation, cell lysis, DNA purification, and amplification for disease diagnosis and genetic studies. These processes are essential in biological assays for molecular pathogen detection, immunological assessments, and studies on the effects of drugs on diseases. The objective of this research is to design and develop microfluidic systems in three main areas: cell separation, DNA extraction, and amplification for the identification of BRCA1 mutations. The cell separation platform is solely designed for simulation on a microfluidic chip, while the extraction and amplification platforms are developed on a centrifugal microfluidic system, which has also been constructed and experimentally tested. In addition to the microfluidic platforms designed for biological assays, a driver-thermocycler device was also fully developed in this project, which provides rotational movement and heating at the required stages for the centrifugal systems. For the extraction process on the disk, which used a column-based protocol, results were tested with one, two, and three silica filters. The platform with three filters achieved a 260/280 absorbance ratio of 1.773, closely matching the standard kit with an absorbance ratio of 1.889 (yielding 93% efficiency). Moreover, for the amplification section, a biological design based on cDNA amplification was developed, where specific primers and a master mix were designed and used. After extracting RNA (with an absorbance ratio of 1.836) from mutated samples off-chip, cDNA was synthesized from these samples (with an absorbance ratio of 1.741). The master mix and primers were used, and amplification and detection were successfully achieved on the centrifugal system with an acceptable percentage. Gene amplification was also successfully performed using the micro-PCR (polymerase chain reaction) device, confirming the accuracy of the primer design. In the final stage, the genetic amplification platform on the centrifugal disk was successfully tested. In the design of the cell separation system, microchannels with specific geometries, such as circular expansion-contraction and parabolic shapes, were tested. In simulations, the circular geometry achieved a separation purity of 78.9% and a recovery rate of 90%, whereas the parabolic geometry performed better, reaching 100% tumor cell separation purity and 75% recovery. These results indicate that geometric configurations can help optimize the separation process. Overall, the results of this research show that optimally designed microfluidic systems can serve as efficient and cost-effective tools in bioanalytical assays and, with further optimization, can enhance the accuracy and efficiency of diagnostic processes
  9. Keywords:
  10. Inertial Microfluidic Device ; Cancer Cells ; Polymerase Chain Reaction (PCR) ; Cell Separation ; Gene Amplification ; DNA Extraction ; Cell Lysis

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