Dielectric spectroscopy in a microfabricated flow cytometer

In this thesis, a new microfabricated flow cytometer for single cell discrimination based on dielectric spectroscopy is presented. Integrated tools for rapid in-flow cell handling are also demonstrated. Signal amplification and analysis as well as the chip fabrication process are described. Finally, measurements on beads and cells are presented and discussed. Due to differences in their complex dielectric properties and dimensions, specific information from cell sub-structures such as the cell membrane, cytoplasm or nucleus can be obtained at distinct frequencies. The dielectric spectroscopy approach is particularly suited to operate at the micrometer scale which permits the analysis of single cell dielectric properties. Interrogating the cell simultaneously at different frequencies of interest can thus bring a wealth of information which is presently available only with slower or more expensive instruments. Furthermore, it is a labelfree technique, which offers the unique advantage that cells are unaltered by the measurement process. An original clean room fabrication technique was developed which uses photosensitive polyimide precursor to pattern the microchannel geometry on glass substrates. Two symmetrical wafers are aligned and bonded to form the final chip. This technique is particularly advantageous in that platinum microelectrodes can be patterned on opposing sides of the channel to produce precise micrometer size electric fields within the liquid flowing in the capillary. Dielectrophoretic commutable barriers are used to direct particles in-flow. In particular, cell focusing and sorting techniques are presented. An original differential amplification scheme based on two measurement detection volumes is used to reduce the measurement noise and fluctuation. In order to provide practical fluidic and electric interconnection, a modular setup is described which allows fast connection and replacement of the chip. The setup is mounted on a printed circuit board which includes high-gain differential signal amplification electronics with an effective bandwidth of 10 kHz to 50 MHz. Analytic and finite element models as well as electrical simulations were used to understand and validate the system sensitivity to various cell parameters including cell size, membrane capacitance and cytoplasm resistance. The influence of the cell position in an inhomogeneous electric field on the measured signal is of particular interest. It is shown that the ratio of signals measured at two different frequencies could be used to reduce this artifact. The presented instrument can easily discriminate sub-micrometer differences in calibrated polystyrene particle diameters. Normal and fixed red blood cells as well as ghost cells are used to determine the system sensitivity to different dielectric properties. Discrimination of white blood cell lymphocytes and granulocytes subpopulation was achieved and the results are compared with standard light scatter techniques used in optical flow cytometry. Compared to commercially available instruments, the proposed approach offers significant advantages in terms of costs and dimensions and could be used in point-ofcare diagnostics. In the future, other functionalities supporting applications such as controlled single-cell electroporation, drug testing, and cell culturing can be integrated with the final aim of building a complete lab-on-a-chip.