Microfluidic Formation of Double-Stacked Planar Bilayer Lipid Membranes by Controlling the Water-Oil Interface

This study reports double-stacked planar bilayer lipid membranes (pBLMs) formed using a droplet contact method (DCM) for microfluidic formation with five-layered microchannels that have four micro guide pillars. pBLMs are valuable for analyzing membrane proteins and modeling cell membranes. Furthermore, multiple-pBLM systems have broadened the field of application such as electronic components, light-sensors, and batteries because of electrical characteristics of pBLMs and membrane proteins. Although multiple-stacked pBLMs have potential, the formation of multiple-pBLMs on a micrometer scale still faces challenges. In this study, we applied a DCM strategy to pBLM formation using microfluidic techniques and attempted to form double-stacked pBLMs in micro-meter scale. First, microchannels with micro pillars were designed via hydrodynamic simulations to form a five-layered flow with aqueous and lipid/oil solutions. Then, pBLMs were successfully formed by controlling the pumping pressure of the solutions and allowing contact between the two lipid monolayers. Finally, pore-forming proteins were reconstituted in the pBLMs, and ion current signals of nanopores were obtained as confirmed by electrical measurements, indicating that double-stacked pBLMs were successfully formed. The strategy for the double-stacked pBLM formation can be applied to highly integrated nanopore-based systems.

[1]  Takehiko Kitamori,et al.  Three-layer flow membrane system on a microchip for investigation of molecular transport. , 2002, Analytical chemistry.

[2]  D. DeVoe,et al.  Visualizing the growth and dynamics of liquid-ordered domains during lipid bilayer folding in a microfluidic chip. , 2012, Small.

[3]  R. Latorre,et al.  Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Howorka,et al.  Self-assembled DNA nanopores that span lipid bilayers. , 2013, Nano letters.

[5]  T. Ganz,et al.  Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[6]  A. van den Berg,et al.  High yield, reproducible and quasi-automated bilayer formation in a microfluidic format. , 2013, Small.

[7]  Stephen A. Sarles,et al.  Hydrodynamic trapping for rapid assembly and in situ electrical characterization of droplet interface bilayer arrays. , 2016, Lab on a chip.

[8]  H. Bayley,et al.  Stochastic Sensing with Protein Pores , 2000 .

[9]  T. G. Martin,et al.  Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures , 2012, Science.

[10]  Masahiro Takinoue,et al.  Nanopore Logic Operation with DNA to RNA Transcription in a Droplet System. , 2017, ACS synthetic biology.

[11]  William L. Hwang,et al.  Droplet networks with incorporated protein diodes show collective properties. , 2009, Nature nanotechnology.

[12]  L. P. Hromada,et al.  Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip. , 2008, Lab on a chip.

[13]  H. Bayley,et al.  Functional aqueous droplet networks. , 2017, Molecular bioSystems.

[14]  H Morgan,et al.  Microfluidic array platform for simultaneous lipid bilayer membrane formation. , 2009, Biosensors & bioelectronics.

[15]  Norihisa Miki,et al.  Automated Parallel Recordings of Topologically Identified Single Ion Channels , 2013, Scientific Reports.

[16]  H. Bayley,et al.  Continuous base identification for single-molecule nanopore DNA sequencing. , 2009, Nature nanotechnology.

[17]  Stephen A. Sarles,et al.  Adsorption Kinetics Dictate Monolayer Self-Assembly for Both Lipid-In and Lipid-Out Approaches to Droplet Interface Bilayer Formation. , 2015, Langmuir : the ACS journal of surfaces and colloids.

[18]  K. Jensen,et al.  Multiphase microfluidics: from flow characteristics to chemical and materials synthesis. , 2006, Lab on a chip.

[19]  Hiroaki Suzuki,et al.  Planar lipid bilayer reconstitution with a micro-fluidic system. , 2004, Lab on a chip.

[20]  Ryuji Kawano,et al.  Amplification and Quantification of an Antisense Oligonucleotide from Target microRNA Using Programmable DNA and a Biological Nanopore. , 2017, Analytical chemistry.

[21]  P. Garstecki,et al.  An Automated Microfluidic System for the Generation of Droplet Interface Bilayer Networks , 2017, Micromachines.

[22]  D. DeVoe,et al.  Rapid Microfluidic Perfusion Enabling Kinetic Studies of Lipid Ion Channels in a Bilayer Lipid Membrane Chip , 2011, Annals of Biomedical Engineering.

[23]  William L. Hwang,et al.  Droplet interface bilayers. , 2008, Molecular bioSystems.

[24]  William L. Hwang,et al.  Electrical behavior of droplet interface bilayer networks: experimental analysis and modeling. , 2007, Journal of the American Chemical Society.

[25]  R. French,et al.  Single sodium channels from rat brain incorporated into planar lipid bilayer membranes , 1983, Nature.

[26]  Shoji Takeuchi,et al.  Multichannel simultaneous measurements of single-molecule translocation in alpha-hemolysin nanopore array. , 2009, Analytical chemistry.

[27]  Michael A Nash,et al.  Automated formation of lipid-bilayer membranes in a microfluidic device. , 2006, Nano letters.

[28]  Shoji Takeuchi,et al.  Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis. , 2006, Analytical chemistry.

[29]  S. Hladky,et al.  Discreteness of Conductance Change in Bimolecular Lipid Membranes in the Presence of Certain Antibiotics , 1970, Nature.

[30]  Michele Zagnoni,et al.  Miniaturised technologies for the development of artificial lipid bilayer systems. , 2012, Lab on a chip.

[31]  Norihisa Miki,et al.  Logic Gate Operation by DNA Translocation through Biological Nanopores , 2016, PloS one.

[32]  Z. Siwy,et al.  Nanopore analytics: sensing of single molecules. , 2009, Chemical Society reviews.

[33]  Roland Kieffer,et al.  Stable Free-Standing Lipid Bilayer Membranes in Norland Optical Adhesive 81 Microchannels. , 2016, Analytical chemistry.

[34]  Norihisa Miki,et al.  A Portable Lipid Bilayer System for Environmental Sensing with a Transmembrane Protein , 2014, PloS one.

[35]  K. Tsumoto,et al.  Analysis of Pore Formation and Protein Translocation Using Large Biological Nanopores. , 2017, Analytical chemistry.

[36]  David Needham,et al.  Functional bionetworks from nanoliter water droplets. , 2007, Journal of the American Chemical Society.

[37]  Stacked biofuel cells separated by artificial lipid bilayers , 2015, 2015 Transducers - 2015 18th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS).

[38]  M. Simon,et al.  Diphtheria toxin forms transmembrane channels in planar lipid bilayers. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Shoji Takeuchi,et al.  Microfluidic lipid membrane formation on microchamber arrays. , 2011, Lab on a chip.

[40]  R. Kawano,et al.  Channel Current Analysis for Pore-forming Properties of an Antimicrobial Peptide, Magainin 1, Using the Droplet Contact Method , 2016, Analytical sciences : the international journal of the Japan Society for Analytical Chemistry.

[41]  M. Niederweis,et al.  Nanopore DNA sequencing with MspA , 2010, Proceedings of the National Academy of Sciences.