Dielectrophoretic cytometry for measurement of live cell dielectric signatures on population level

Flow cytometry in combination with staining and antibody labelling presents one of the most valuable tools in current biotechnology providing information about cell population heterogeneity, cell size and volume, as well as expression of certain surface and intracellular molecules. The increased cost and the fundamental difficulty of these methods, however, are attributed to the requirement of the surface marker molecules. Attractive alternatives to flow cytometry are label-free methods, such as micro-filtration, dielectric spectroscopy, and electro-kinetic methods. Out of these methods, dielectrophoresis (DEP) was selected as the most promising approach. This thesis focuses on improvements of current DEP-based technologies, and development and establishment of a new method to address the issues of dielectrophoretic (DEP) cytometry enabling label-free non-invasive probabilistic measurement of cell dielectric (DE) signatures on population level, as well as enabling identification of reliable biomarkers for cell changes.First, improvements of DEP cytometry based on DEP-induced lateral cell translation through fabrication are explored. A benchmark microfluidic live cell sorting system is presented, and the effect of microchannel misalignment above electrode topologies on live cell DEP is discussed in detail. Simplified model of budding S. cerevisiae cell is presented and validated experimentally in fabricated microfluidic devices. A novel fabrication process enabling rapid prototyping of microfluidic devices with well-aligned integrated electrodes is presented and the process flow is described. Identical devices were produced with standard PDMS soft lithography processes. The presented fabrication process significantly improved the alignment of the microstructures. According to the benchmark study, the standard PDMS procedure fell well outside the range required for reasonable cell sorting efficiency. The fabrication time and costs of the proposed methodology were found to be roughly the same.Second, a method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a DEP-induced vertical translation of live cells in conjunction with PIV in order to measure probabilistic distribution of live cell DE signatures on an entire cell population. The method was integrated in a microfluidic device with integrated electrodes. Cells passing through the microchannel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the DE signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using particle image velocimetry (PIV). Experimental data quantify the DE signature of a S. cerevisiae population and Human immortalised myelogenous leukaemia cell line K562. First, the effect of over-expression of certain membrane protein was studied in S. cerevisiae cells. Measured distribution of DEP forces was compared to cell population expressing a cytoplasmic protein at the same rate. Second, 2DEP cytometry was applied to K562 cell line. Effects of stress response triggered by various inducers on the DE signature of the cell population were analysed.Finally, statistical data analysis defined adjusted kernel density estimation to overcome the finite nature of the measured data. In combination with Wasserstein pseudometrics from sampled data, the Wasserstein distance spectra, denoted as Wasserstein signatures, were quantified and linked to certain cell changes. These signatures may be used as reliable biomarkers for cell changes.In conclusion, 2DEP cytometry showed it is sensitive enough to identify certain changes in cell states. The novel 2DEP cytometry device is therefore a promising alternative to conventional flow cytometry