Integrated microfluidic system for non-invasive electrophysiological measurements on Xenopus oocytes

We developed a new non-invasive integrated microsystem for electrophysiological measurements on Xenopus laevis oocytes. The Xenopus oocyte is a well-known expression system for various kinds of ion channels that are of potential interest for drug screening. In the traditional "Two Electrode Voltage Clamp" (TEVC) method, delicate micromanipulation is required to impale an oocyte with two microelectrodes. In our system, a non-invasive electrical access to the cytoplasm is provided by permeabilizing the cell membrane with an ionophore (e.g. nystatin). Unlike for the classical patch-clamp or "macropatch" techniques, this method does not require removal of the fibrous vitelline membrane. Cell handling is significantly simplified, resulting in more robust recordings with increased throughput. Moreover, because only part of the oocyte surface is exposed to reagents, the required volume of reagent solutions could be reduced by an order of magnitude compared to the TEVC method. In a second part of this work, a rapid fluidic exchange system was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimeter-size cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The fabrication process for this disposable microchip, based on PDMS micromolding, is cost-efficient and simple. Moreover, an innovative integration of agar gel-based reference electrodes was developed. A conductive liquid junction is injected by capillary force filling of suitable microchannels, overcoming the problems encountered with the integration of Ag/AgCl thin film or wire reference electrodes. We tested this new microdevice by performing electrophysiological measurements on oocytes expressing the human Epithelial Sodium Channel (hENaC). Transmembrane currents could be recorded with a large signal-to-noise ratio. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of hENaC. Our results show that the response time of this ion channel to a specific reactant is about one order of magnitude faster than estimated with the traditional TEVC technique. We conclude that this new microdevice has high potential for improved electrophysiological investigations with oocytes in the field of pharmacology and toxicology.

Rapid Prototyping of Microfluidic Devices

Meng Shen

With the growing importance of miniaturized energy applications and the development of micro Total Analysis Systems (μTAS), we have realized microfluidic devices, namely, magnetic micropumps, microfluidic fuel cells and membrane-based protein preconcentrators, all having high application potential in future. The choice of rapid prototyping microfabrication technologies and the selection of affordable materials are important aspects, when thinking of commercialization. Thus, we have employed powder blasting, polymer molding and assembly technologies during devices fabrication throughout the thesis. The first type of microfluidic device that we present is a poly(methyl methacrylate) (PMMA) ball-valve micropump with two different designs of the electromagnetic actuator, as optimized by the finite element method. The integration of a permanent magnet in a flexible polydimethylsiloxane (PDMS) membrane, which is clamped into PMMA structure, is proposed for providing a large stroke of the pumping membrane, making the micropump bubble-tolerant and self-priming Focusing on low power consumption for μTAS integration, another type of magnetic micropump with active valves is realized. It consists of a microfluidic chamber structure in glass that is assembled with a PDMS sheet, which comprises two valving membranes and a central actuation membrane, having each an integrated permanent magnet that is peristaltically actuated by a rotating arc-shaped permanent magnets assembly. A lumped circuit model is developed to predict and describe the frequency-dependent flow rate behavior for this type of pump. Powder blasting and PDMS molding rapid prototyping technologies are employed for realization of these two types of micropumps. Fuel cells with fluid delivery and removal options, having chemical reaction sites and electrode structures that can be realized in a microfluidic format, have high potential for applications. Therefore, microfluidic direct methanol fuel cells with embedded ion- permselective medium are studied and such type of fuel cell is realized by integrating a narrow Nafion strip in a molded elastomeric structure. A mechanical clamping assembly technology enables leakage-free operation and stable performance. The characterization reveals its output power density, using H2O2-based oxidant, is among the high-performance direct methanol fuel cells in microscale. Re-using the technology of the fuel cell chip, with its particular ion-permselective Nafion membrane and assembly method, we also have developed a protein preconcentrator with high purification performance. Our device can preconcentrate negatively charged biomolecules located at the anodic compartment side of the Nafion strip within only a few minutes with a high preconcentration factor. Moreover, a complex microfluidic finite element model is proposed to study and understand the physics of the preconcentration effect. Finally, we conclude the thesis with an outlook on future developments based on our work of the project and on the assembly technologies for microfluidic device integration.

EPFL2010

MEMS based nanopatterning

Jürgen Brugger, Christian Santschi, Nao Takano, Marc Antonius Friedrich van den Boogaart

Sub-micron lithography processes are continuously optimized for semiconductor products where the enormous market potential justifies investment in advanced methods and extremely sophisticated equipment. Due to the extreme process costs and due to the limited process versatility in terms of substrates, processing and materials, these high-end methods can not be applied for the engineering of multi-material and multifunctional nano/micro-electro-mechanical systems (NEMS/MEMS), such as polymer-based electronic and sensor devices, 3D microfluidics, and bio-analytical systems. Often standard lithography methods can not be applied because the surfaces to be structured are either mechanically unstable, such as for cantilevers and membranes, and/or chemically functionalised for bio-sensor applications. To overcome the cost and process issues, a series of alternative surface patterning methods are currently being developed. The do not rely on photoresist technology and thin-film etching, and they are potentially low-cost. Well-known methods are e.g. scanning probe lithography, DipPen or NADIS lithography, or replication using soft-lithography and nanoimprint lithography. Another emerging method is based on the direct local deposition of thin film patterns through the apertures in miniature shadow-masks or nanostencils. E-beam or focused ion beam prepared stencils demonstrated sub-100 nm scale shadow nanofabrication on a small surface area. Recently we have made further progress in scaling up the nanostencil method to full-wafer scale, and by using it for various deposition methods on various sample surfaces. The presentation will first give an overview of the state-of-the-art of emerging nanopatterning methods that are based on micro-mechanical (MEMS) tools such as scanning probes and thin membranes. In particular, details on a combined DUV-MEMS process will be presented on the fabrication and application of nanostencils on a 100-mm size full-wafer.

2004

Molybdenum disulphide nanoporous membranes as nanofluidic platforms - large-area engineering and study

Michal Daniel Macha

The work demonstrated in this thesis represent the path towards developing the large-scale fabrication of two-dimensional nanoporous membrane devices and use thereof as platforms to study nanoscale physics of water and ion flow through confined channels. Methods developed here are used to fabricate molybdenum disulfide (MoS2) membrane devices and investigate the fundamental physics of nanopore systems and thin films in aqueous solutions. In particular, the second chapter presents the accomplishments in the large-area MoS2 synthesis via chemical vapor deposition. Process modifications such as the use of spin-coated precursors/growth promoters and addition of active gases such as H2O vapor or moderate amount of O2 enable to scale up the growth reaction into the synthesis of a continuous monolayer films on 2-, 3- and 4-inch substrates. Third chapter shows the application of focused ion beam irradiation for engineering, imaging and studying optically active defects in MoS2 and hexagonal boron nitride films. On top of that, the ion irradiation protocol was used for establishing the tunable and large-throughout nanopore drilling process on suspended MoS2 membranes. With the high-resolution, scanning electron transmission microscopy, nanopores were investigated with an analysis script and classified based on the pore geometries and edge composition. Coupled with molecular dynamics simulations the membranes populated with ~1nm nanopores were assessed in the context of emerging ion- and water- permeation properties. The last, fourth chapter presents the application of nanoporous membranes discussed in previous chapters in the nanofluidics study application. A particular emphasis is put on the application of hydrostatic pressure in nanopore experiments as an additional measurement probe. Experimental results shown in this chapter uncover the non-linear signals originating from nanobubbles pinning, improper wetting and membrane adhesion issues. With the assistance of hydrostatic pressure, the nanoporous MoS2 suspended membranes are then further investigated to explore the ion transport properties and nanopores behaviour under applied strain in the context of artificial mechanosensing as well as osmotic power generation in salinity gradients.The ion irradiated, atomically thin films proved to be a highly attractive membrane materials and demonstrated a great promise in broad nanofluidic applications. Insights acquired during the thesis study were used to advance the state-of-art knowledge in field of the synthesis and application of 2D membranes in the nanofluidics research.

EPFL2022