Continuous-flow separation of cells in a lab-on-a-chip using "liquid electrodes" and multiple-frequency dielectrophoresis

This thesis reports on the integration of continuous-flow cell separation method for lab-on-a-chip applications. Cell separation methods are widely used in biology to prepare samples prior to analysis. There is a need for a highly sensitive separation method that is capable to quickly isolate a particular cell type in a single manipulation step. We attempt to provide such a separation method that discriminates between cell types according to their dielectric properties such as the membrane permittivity and the cytoplasm conductivity. The dielectric properties are intrinsic to each cell type and thus prevent the need of a specific cell labeling as discriminating factor. To guaranty a high throughput, the cell separation method we propose is performed in a continuous flow. The continuous-flow cell separation method presented in this thesis makes use of electrical forces to achieve the separation of different cell types. Dielectrophoresis is a phenomenon that describes the electrical force exerted on a dielectric particle such as a biological cell in presence of a non-uniform AC electric field. The combination of several dielectrophoretic forces at multiple frequencies produces a distinct dielectric response for each cell type. The method is integrated into a microfluidic platform of 20 mm long by 15 mm wide. The microfabrication of the device consists of two successive steps of photolithography to define the metal electrodes in platinum and the microfluidic network in SU-8 photoresist. In the so-called separation chamber, an array of "liquid electrodes" is localized along the 20 µm deep central channel. The technological development of "liquid electrodes" allow us to produce horizontal dielectrophoretic force and the array of these electrodes opposes two fields of such forces. This opposition of two force fields defines an equilibrium position towards which the cells that flow through the central channel are focused. There is a relationship between the equilibrium position and the dielectric properties of the cells which allows a flow-through dielectric characterization of the cells by the cell position readout. Using this microfluidic platform that integrate a method of continuous-flow cell separation based on multiple-frequency dielectrophoresis, we succeeded in purifying fractions of viable and nonviable yeast cells that were initially mixed. Due to its sensitivity, the method also allowed to increase the infection rate of a cell culture up to 50% of parasitemia percentage, which facilitates the study of the parasite cycle. The method was finally applied to the biological issue of cell synchronization. By isolating cells that are at a particular phase within their cell cycle, our method prevents the use of metabolic agents in order to arrest a cell culture by disrupting the cell physiology. The synchronization method we proposed and based on multiplefrequency dielectrophoresis is to our best knowledge the most powerful one in terms of synchrony level reported so far.

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