Applications of novel three-dimensional well electrodes in toxicological studies

• Main Author: Hubner, Yvonne
• Published: University of Surrey 2010
• Subjects: 615.9
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.538036

Dielectrophoresis (DEP), the induced motion of cells in a non-uniform electric field, has increasingly been used for cell analysis since its discovery in 1951 by Herbert Pohl. By studying the motion of cells when exposed to electric fields of different frequencies, the electrical properties of ceils can be derived. It is a non-invasive and marker-free method, using low cost and mass-producible technology to rapidly determine drug-cell interactions. Despite its many advantages, uptake from industry has been low and it has largely remained in mostly in academic laboratories. This thesis sets out to describe a new method of performing DEP experiments based on laminate manufacturing methods. These use a 3D "well" structure, similar in size and pitch to conventional microtiter well plates, but offer electrodes along the inner surface to allow easy measurement of cell properties across the whole population. The dielectrophoretic force can then be monitored using a microscope, but the technology used could potentially be integrated into a conventional well-plate reader setup. In this thesis, these electrodes are benchmarked against standard DEP assays using a variety of different cell types, ranging in size and form from oblate red blood cells to spherical yeast. Jurkat and K562 cells. Both impedance measurements and light intensity change are measured to obtain dielectrophoretic spectra. The effects of ion channel blockers on the electrophysical properties of Jurkat cells are determined. In later experiments, changes in the conductivity and permittivity of the membrane and cytoplasm of red blood cells are detected after the treatment with membrane-altering drugs such as valinomycin and saponin. Following on from those experiments, the dielectrophoretic spectra of multiple populations within one well are measured simultaneously. When mixing known concentrations of viable and non-viable yeast cells experiments show that there are characteristic changes in the DEP spectra which indicate the ratio of the mixture and the dielectric properties of both populations. Using the same mathematical analysis, changes in the dielectric properties of K562 cells are examined after incubation with the cytotoxic drug doxorubicin. Those results are compared to viability tests using trypan blue and flow cytometry. Dielectrophoresis was found to enable a much more rapid identification of cytotoxicity. Finally, well electrodes are used to examine the dielectric properties of Jurkat cells after incubation with anti-Fas antibody and staurosporine. Again, DEP allowed the much more rapid identification of the induction of cell death by apoptosis as compared with flow cytometry.