Impedance Spectroscopy for Single Cell Analysis and Dispensing

Flow cytometry is an essential tool in biology and medicine with applications in immunodeficiency diagnostic, immunosurveillence, organ transplantation in addition to its basic research usage. Before the widespread use of the Fluorescent-Activated Cell Sorting (FACS), the first flow cytometer was invented by Wallace Coulter and was embodied in a remarquably simple device. The Coulter counter principle consists in passing particles in an aperture concurrently with an electric current to measure the related impedance variations. In the fifties, the Coulter counter has established as a gold standard for cell counting and sizing. More recently, thanks to advances in miniaturization, micromachined impedance spectroscopy flow cytometers extended their analysis capabilities to the dielectric properties of cells. This opened the way to label-free and non-invasive methods for cell population differentiations such as leukocytes clustering. However, these flow cytometers were not designed for cell retrieval neither provided the ability to work with a limited number of scarce cells. First, this thesis extends the capability of impedance based flow cytometers to perform single cell isolation on disposable devices. Today, many biological methods are based on single cell isolation. In cell lines development, the gold standard procedure involves serial dilution. However, this approach is time-consuming as it needs to be repeated over several weeks to ensure clonality. In this thesis, a tool enabling single cell isolation in one step and based on impedance spectroscopy is developed. The modeling, designing and testing of a disposable pipette tip integrating a cell sensor based on the Coulter principle is reported. Coupled with an instrumented pipette, this disposable sensing tip enables single cell dispensing. Furthermore, this system allows recording the impedance trace to be used as proof of single cell isolation. Second, this thesis translates the concept of disposable, sterile and low-cost single-cell dispensing device on a standard planar microfabrication technology. Using a planar microfabrication technology enables a better control of the fluidic behavior and permits the integration of more complex features on the dispensing device. However, a disposable device requires a large-scale and cost-effective production method. A planar fabrication method based on the industrially standardized printed circuit board (PCB) manufacturing process is assessed to produce different topologies of flow cytometers with emphasis on the disposable aspect of the devices required for cell culture. Third, this thesis is dedicated to further exploring the cell parameters that can be analyzed by impedance spectroscopy. Ligand-gated ion channels are cellular membrane proteins reacting very specifically and rapidly to the binding of a ligand molecule and modulate the membrane permeability. This cellular mechanism was proposed as the transducing elements for highly sensitive and specific chemical biosensors. However, this implies the engineering challenges of a long-term, automated and integrated cellular electrophysiology monitoring. Finally, in this thesis, the monitoring of ligand-gated ion channel permeability is investigated using impedance spectroscopy as a real-time, non-invasive and label free analytical technique.