Plateforme microfluidique en polymere integrant une microelectrode ionique selective pour la detection de la mort cellulaire.

摘要

In order to study the reaction of cells exposed to various environments and agents, it is helpful to integrate microwells for cellular culture in a "lab-on-a-chip". For the characterization of the time evolution of cellular functions, microsensors are integrated into a microfluidic platform. During this M.Sc.A project, the objective has been to design a microfluidic platform made of a polymer that integrates a cellular culture microwell, able to detect cell death.;With the detection of K+ ions, it is possible to study cell death, because extracellular potassium effluxes are early indicators of such cell death. Ion-selective electrodes are well suited for this project, since they convert the activity of free ions into an electric potential. Ion-selective electrodes do not require any tracer, they have a great selectivity and a rapid response time, thereby allowing measurements in real-time.;The microfluidic platform which integrates an ion-selective microelectrode and a cellular microwell is composed of two parts. The first, made of PDMS, consists of a microchannel connecting two reservoirs. It is microfabricated by moulding the PDMS on a thick layer of photoresist that has been microstructured by photolithography. Rectangular microchannel dimensions are of 50mum width and 65mum height and each cylindrical reservoir has a 250nL volume. The second part of the platform is a glass substrate on which a platinum microstrip is fabricated by a lift-off process. The assembly of the two parts is achieved by centering the microchannel of PDMS on the platinum microstrip.;The last step of fabrication is the introduction of the K+-selective solution into the first reservoir. The solution covers the platinum microstrip by completely filling the microchannel, and it reaches the second reservoir by capillarity forces. This solution, called ion-selective membrane, becomes solid after the evaporation of its solvent. The second reservoir, used as a cell microwell, is thus in contact with the ion-selective membrane on a surface of 50mum x 65mum. Because of the toxicity of the membrane, this design has the advantage of minimizing the surface of contact with the cell medium, while ensuring a good stability of measurements, thanks to anchoring of the membrane inside the microchannel and relatively to the large membrane reservoir.;Polydimethylsiloxane (PDMS) is the polymer chosen to create the microfluidic platform because of its transparency at visible wavelengths, allowing the cellular observation by inverse microscopy. Moreover, its great permeability to gases allows the easy integration of cellular cultures. Rapidity and low cost of PDMS microfabrication by the technique of rapid prototyping allows one to create disposable platform, which represents an important characteristic for biomedical applications.;The functionality of the K+-selective electrode within the platform is characterized by measurement of its sensitivity. The platform is placed in a fluidic manifold which circulates a series of solutions of varying potassium concentrations ([K+]) in the reservoir designed for the cell culture. A reference electrode is included in the manifold, and it bathes in the same solutions. Variations in potential differences between the ion-selective electrode and the reference electrode are recorded via the platinum microstrip. These potential difference variations versus K+ concentration are described by the Nernst equation. A variation of 59 mV is typically observed per decade of [K+], noted pK+, for ion concentrations of 10-5 M [K+] 0,1 M.;The concept of integration of an ion-selective electrode into a PDMS platform was initially verified by using an electrode of macroscopic dimensions. A quasi-Nemstian response was obtained and it remained stable for more than 4 days. However, its sensitivity decreased and its survival appeared to be less than 8 days. This degradation is probably caused by incompatibility between the PDMS and solvent contained in the ion-selective membrane. Indeed, cyclohexanone contained in the membrane damages the PDMS by dissolving and deforming it, which results in deterioration of the adhesion between the PDMS and glass and weakens the contact between the membrane and the platinum.;In order to raise the lifetime of the ion-selective electrode, it is necessary to protect the PDMS from cyclohexanone by a diffusion barrier placed between the membrane and the PDMS. This barrier must have a good chemical resistance, a large electrical resistance, it must be transparent and not interfere with the ion-selective electrode. Diffusion barriers created by the deposition by sol-gel chemistry and by UV/O3 treatment proved inefficient, but a better option was deposition of a thin SiO2 layer (150nm) by plasma-enhanced chemical vapour deposition (PECVD). The SiO 2 is well-suited for diffusion barrier because of its chemically inert character, its robustness and its dielectric properties. However, handling of the sample deforms the PDMS and creates a mechanical stress in the rigid SiO2 layer, which in turn generates cracks. In order to avoid these, it is necessary to reduce the difference in thickness between the PDMS and SiO2, in accordance to the Stoney equation. However, for proof-of-concept performed with a macroscopic electrode, it is difficult to work with a device of total thickness less than a millimeter. The choice thus turns to a flexible diffusion barrier, like Parylene. Thanks to a layer of 5mum deposited at room temperature, the ion-selective electrode preserves a sensitivity of 57 mV/pK+, which remains stable for more than one week.;An ion-selective electrode with micrometric dimensions (50mum x 65mum) shows a quasi-Nernstian response of 55 mV/pK+. With a preliminary cellular essay, it was possible to detect extracellular potassium effluxes with an ion-selective microelectrode. This test comprises inducing a hypoosmotic shock to the cells (mouse embryo fibroblasts), to make them activate their mechanism of Regulatory Volume Decrease (RVD), during which extracellular potassium effluxes are emitted. By maintaining these hypoosmotic conditions, the RVD is not able to restore equilibrium and cells suffer necrosis. The success of the cellular essays enables us to conclude that it is possible to detect cellular death with an ion-selective microelectrode integrated into a microfuidic platform made of PDMS.