On chip cellular dielectric spectroscopy : monitoring of glucose effect on erythrocytes membrane and analysis of the physiological role of anthrax toxin receptor in endothelial cells

In the recent years, the development of automated systems for cellular monitoring has been highly promoted in basic biological research as well as in industries and medical diagnostic. Several methods for transduction of cellular signals have been investigated and it has been demonstrated that impedance-based techniques have great potential for real-time, non invasive cell-based assays for both high throughput screening and research laboratory environments. This thesis presents the fabrication and design of a microelectrodes-based dielectric sensor for cellular analysis, and its application to monitor in-vitro physiological functions of different cell types. A dual array of planar interdigitated microelectrodes has been patterned on the bottom of two microchambers used for cells culture. The system allows real-time dynamic cellular monitoring with high temporal resolution and high sensitivity. The device enables multifrequency characterization (frequency between 100Hz and 30MHz), so that the integral cellular response is obtained and several independent effects can be monitored in parallel at different frequencies. Analytical models are used to evaluate the evolution of various cell parameters, such as cell membrane capacitance and cytoplasm conductivity, and to obtain information about cell morphology and adhesion. To interface the microelectrode array to the external instrumentation, a printed circuit board has been developed, which includes the circuitry controlling the switch between reference/sensing chamber and calibration/measurement mode. In addition, the device is controlled by a computer interface, to automatize the acquisition of the impedance spectra and select long or short term response monitoring. Two innovative biological applications are proposed for the presented device. The effect of D-glucose on biconcave erythrocytes has been investigated. The variation of glucose concentration in plasma is an important biological process and it has been extensively studied because of its implication in diseases like diabetes. Measurements have been performed on erythrocyte suspensions exposed to several D-glucose concentrations. The results have shown that glucose induces variations in the dielectric properties of erythrocytes and affects mainly the plasma membrane capacitance. A better understanding of this mechanism is fundamental for a future development of impedance glucose sensors based on the evaluation of the dielectric properties of the red blood cells. Furthermore, a functional assay of the anthrax toxin receptor on endothelial cells has been performed using the developed device. This transmembrane protein is encoded by the Capillary Morphogenesis Gene 2 (CMG2), which was identified as a gene overexpressed in endothelial cells during capillary morphogenesis. This protein is known as an anthrax toxin receptor and it has been discovered that its mutations lead to a rare human disease (Hyaline Fibromatosis Symdrome). On the contrary, i