Development of a Microfluidic Lipid Bilayer Membrane System Integrated with an On-Site Nanoinjector and a Nanoporous Support

Abstract

Ion channels are of great interest as subjects of basic biophysical studies, and recognized as a major class for drug targets. For over four decades, planar bilayer method has been extensively used in investigating the properties of ion channels. The method has also provided stable bilayer lipid membranes (BLMs) by narrowing the aperture to submicro- and nanoscale. Within the broad-ranging development of microfluidics, there has been an increasing interest in manipulating and analyzing channel proteins in a miniaturized BLM system, owing to the advantages, such as high level of integration, portability, rapid and controllable solution perfusion, etc. This dissertation focuses on the development of a microchip-based nano-BLM system coupled with the technique of direct solution injection on the membrane site. This on-site injection technique can reduce the amount of sample by 103 fold (from μL to nL). Stable BLMs are formed on a multilayer-stack polycarbonate (MSPC) membrane with micro- or nanopores, which offers a facile and low-cost approach to the common, microfabrication-based preparation of nanopores. The MSPC membrane is then integrated into the poly(dimethylsiloxane) (PDMS) microchip by an epoxy bonding technique that we have developed to provide strong bonding between PDMS and MSPC even under organic solvents. To integrate commercial filter membranes into a microfluidic device, three incorporation methods, namely, PDMS gluing, 3-amino-propyltriethoxysilane (APTES) conjugation and epoxy bonding method, have been investigated. Fluorescence-dye-assisted leakage and peel test have been adopted to evaluate the bonding resistance to aqueous and organic solvent. By comparison, epoxy bonding method is proved to provide a reliable and robust sealing to withstand a tress that is greater than the bulk strength of PDMS and PC, even in a harsh and long-time organic environment. A facile and cost-effective process has been developed to fabricate nanoporous BLM support from polycarbonate (PC) membranes. Among commercially available filter membranes, PC membrane has become one of the most suitable porous supports for the pore-spanning BLM formation due to its circular aperture of the greater regularity and high resistivity. Because of its high pore population, PC-supported BLMs are very susceptible to hydrodynamic pressure. For this reason, such BLMs have not been formed in a microfluidic system where hydraulic fluid is involved. To address this issue, multiple PC membranes are thermally bonded into a multilayer stack as a nanoporous BLM support. The cross-sectional views of an MSPC membrane demonstrate that the stacking efficiently blocks most of pores in the bottom-most layer. To our knowledge, the bonding of such ultrathin thermoplastic layers (~10 μm) has not been reported before. A microchip-based BLM system coupled with the technique of direct solution injection on the membrane site has been developed. The microchip has a special structural component: a guiding channel for an injection micropipette. The 350-μm-thickness PDMS wall withstands micropipette insertion for at least 20 times. This on-site injection technique can reduce the amount of sample by 103 fold (from μL to nL), compared with the sample introduction in conventional microchips where solutions are delivered through microchannels. Incorporating gramicidin ion channels into BLMs has further confirmed the formation of single BLMs, which is based on the observation of current signals with 20 pS conductance that is typical to single channel opening. Through the bilayer capacitance measurement, about 15% of the through pores across the MSPC membrane are estimated to be covered with BLMs. This microfluidic platform for ion channel study would facilitate extension to multistation systems with different kinds of ion channels because the on-site injection technique can greatly simplify microfluidics control and thus microfluidic network design.