Black lipid membranes (BLMs) provide a biomimetic model system for studying cellular membrane processes, and are important tools in drug screening and biosensing applications. BLMs offer advantages over liposomes and solid-supported lipid bilayers in applications where access to both leaflets of the bilayer is critical. Reliable and repeatable formation of BLMs presents a major challenge, especially in systems that require interrogation of the membrane via optical microscopy. BLMs for optical interrogation are often formed by the manual painting method, which is tedious and has a high failure rate because it involves manual manipulation of nanoscale liquid films for membrane self-assembly. Here, we describe a fully automated technique for the formation of BLMs within the imaging plane of an inverted fluorescence microscope. The technique utilizes hydrostatic pressure manipulations within a simple microfluidic device, which are feedback controlled via confocal fluorescence monitoring of the BLM formation process. An algorithm for monitoring and precision control of BLM formation is devised and optimized to yield an 80% success rate for the formation of BLMs, with formation times on the order of 78 min. Membranes formed via the automated procedure are confirmed to be fluid and biomimetic via spontaneous insertion of α-hemolysin pores with characteristic conductance of ca. 1 nS.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Cooper MA. Optical biosensors in drug discovery. Nat Rev Drug Discov. 2002;1:515–28.
Castellana ET, Cremer PS. Solid supported lipid bilayers: from biophysical studies to sensor design. Surf Sci Rep. 2006;61:429–44.
Mirzabekov TA, Silberstein AY, Kagan BL.  Use of planar lipid bilayer membranes for rapid screening of membrane active compounds. Ion Channels Part C Elsevier. 1999;294:661–74.
Mueller P, Rudin DO, Tien HT, Wescott WC. Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature. 1962;194:979–80.
Mueller P, Rudin DO. Induced excitability in reconstituted cell membrane structure. J Theor Biol. 1963;4:268–80.
Tien HT, Carbone S, Dawidowicz EA. Formation of “black” lipid membranes by oxidation products of cholesterol. Nature. 1966;212:718–9.
Yang T, Jung S-Y, Mao H, Cremer PS. Fabrication of phospholipid bilayer-coated microchannels for on-chip immunoassays. Anal Chem. 2001;73:165–9.
Stoddart A, Dykstra ML, Brown BK, Song W, Pierce SK, Brodsky FM. Lipid rafts unite signaling cascades with clathrin to regulate BCR internalization. Immunity. 2002;17:451–62.
Qi SY, Groves JT, Chakraborty AK. Synaptic pattern formation during cellular recognition. Proc Natl Acad Sci U S A. 2001;98:6548–53.
Kasahara K, Sanai Y. Functional roles of glycosphingolipids in signal transduction via lipid rafts. Glycoconj J. 2000;17:153–62.
Tanaka M, Sackmann E. Polymer-supported membranes as models of the cell surface. Nature. 2005;437:656–63.
Wiegand G, Arribas-Layton N, Hillebrandt H, Sackmann E, Wagner P. Electrical properties of supported lipid bilayer membranes. J Phys Chem B. 2002;106:4245–54.
Hirano-Iwata A, Aoto K, Oshima A, Taira T, Yamaguchi R-T, Kimura Y, et al. Free-standing lipid bilayers in silicon chips-membrane stabilization based on microfabricated apertures with a nanometer-scale smoothness. Langmuir. 2010;26:1949–52.
Oshima A, Hirano-Iwata A, Mozumi H, Ishinari Y, Kimura Y, Niwano M. Reconstitution of human ether-a-go-go-related gene channels in microfabricated silicon chips. Anal Chem. 2013;85:4363–9.
Bright LK, Baker CA, Agasid MT, Ma L, Aspinwall CA. Decreased aperture surface energy enhances electrical, mechanical, and temporal stability of suspended lipid membranes. ACS Appl Mater Interfaces. 2013;5:11918–26.
White RJ, Zhang B, Daniel S, Tang JM, Ervin EN, Cremer PS, et al. Ionic conductivity of the aqueous layer separating a lipid bilayer membrane and a glass support. Langmuir. 2006;22:10777–83.
Baker CA, Bright LK, Aspinwall CA. Photolithographic fabrication of microapertures with well-defined, three-dimensional geometries for suspended lipid membrane studies. Anal Chem. 2013;85:9078–86.
Bright LK, Baker CA, Bränström R, Saavedra SS, Aspinwall CA. Methacrylate polymer scaffolding enhances the stability of suspended lipid bilayers for ion channel recordings and biosensor development. ACS Biomater Sci Eng. 2015;1:955–63.
Montal M, Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci. 1972;69:3561–6.
Pantoja R, Sigg D, Blunck R, Bezanilla F, Heath JR. Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip. Biophys J. 2001;81:2389–94.
Ryu H, Choi S, Park J, Yoo Y-E, Yoon JS, Seo YH, et al. Automated lipid membrane formation using a polydimethylsiloxane film for ion channel measurements. Anal Chem. 2014;86:8910–5.
Thapliyal T, Poulos JL, Schmidt JJ. Automated lipid bilayer and ion channel measurement platform. Biosens Bioelectron. 2011;26:2651–4.
Czekalska M, Kaminski T, Horka M, Jakiela S, Garstecki P. An automated microfluidic system for the generation of droplet interface bilayer networks. Micromachines. 2017;8:93.
Baker CA, Bulloch R, Roper MG. Comparison of separation performance of laser-ablated and wet-etched microfluidic devices. Anal Bioanal Chem. 2011;399:1473–9.
Kawano R, Schibel AEP, Cauley C, White HS. Controlling the translocation of single-stranded DNA through alpha-hemolysin ion channels using viscosity. Langmuir. 2009;25:1233–7.
Conflict of interest
The authors declare that there are no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry with guest editors Erin Baker, Kerstin Leopold, Francesco Ricci, and Wei Wang.
About this article
Cite this article
Dugger, M.E., Baker, C.A. Automated formation of black lipid membranes within a microfluidic device via confocal fluorescence feedback-controlled hydrostatic pressure manipulations. Anal Bioanal Chem 411, 4605–4614 (2019). https://doi.org/10.1007/s00216-018-1550-4
- Black lipid membrane
- Suspended bilayer
- Confocal fluorescence