A magnetic bead-based DNA extraction and purification device has been designed to be used for extraction of the target DNA molecules from whole blood sample. Mixing and separation steps are performed using functionalised superparamagnetic beads suspended in the cell lysis buffer in a circular chamber that is sandwiched between two electromagnets. Non-uniform nature of the magnetic field causes temporal and spatial distribution of the beads within the chamber. This process efficiently mixes the lysis buffer and whole blood in order to extract DNA from target cells. Functionalized surface of the magnetic beads then attract the exposed DNA molecules. Finally, DNA-attached magnetic beads are attracted to the bottom of the chamber by activating the bottom electrode. DNA molecules are extracted from the magnetic beads by washing and re-suspension processes. The numerical simulation approach has been adopted in order to design the magnetic field source. The performance of the magnetic field source has been investigated against different physical and geometrical parameters and optimised dimensions are obtained with two different magnetic field sources; integrated internal source and external source. A new magnetic field pattern has been introduced in order to efficiently control the bulk of magnetic beads inside the mixing chamber by dynamic shifting of magnetic field regions from the centre of the coils to the outer edge of the coils and vice versa. A Matlab code has been developed to simulate beads trajectories inside the designed extraction chip in order to investigate the efficiency of the magnetic mixing. A preliminary target molecule capturing simulation has also been performed using the simulated bead trajectories to evaluate the DNA-capturing efficiency of the designed extraction chip. The performance of the designed extraction chip has been tested by conducting a series of biological experiments. Different magnetic bead-based extraction kits have been used in a series of preliminary experiments in order to extract a more automation friendly extraction protocol. The efficiency of the designed device has been evaluated using the spiked bacterial DNA and non-pathogenic bacterial cell cultures (B. subtilis, Gram positive bacteria and E. coli, Gram negative bacteria) into the blood sample. Excellent DNA yields and recovery rates are obtained with the designed extraction chip through a simple and fast extraction protocol.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:521000 |
| Date | January 2010 |
| Creators | Azimi, Sayyed Mohamad |
| Contributors | Balachandran, W. ; Slijepcevic, P. |
| Publisher | Brunel University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://bura.brunel.ac.uk/handle/2438/4520 |
Page generated in 0.0019 seconds