Automated formation of black lipid membranes within a microfluidic device via confocal fluorescence feedback-controlled hydrostatic pressure manipulations


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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Cooper MA. Optical biosensors in drug discovery. Nat Rev Drug Discov. 2002;1:515–28.

    Article  CAS  PubMed  Google Scholar 

  2. 2.

    Castellana ET, Cremer PS. Solid supported lipid bilayers: from biophysical studies to sensor design. Surf Sci Rep. 2006;61:429–44.

    Article  CAS  Google Scholar 

  3. 3.

    Mirzabekov TA, Silberstein AY, Kagan BL. [35] Use of planar lipid bilayer membranes for rapid screening of membrane active compounds. Ion Channels Part C Elsevier. 1999;294:661–74.

    Article  CAS  Google Scholar 

  4. 4.

    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.

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Mueller P, Rudin DO. Induced excitability in reconstituted cell membrane structure. J Theor Biol. 1963;4:268–80.

    Article  CAS  PubMed  Google Scholar 

  6. 6.

    Tien HT, Carbone S, Dawidowicz EA. Formation of “black” lipid membranes by oxidation products of cholesterol. Nature. 1966;212:718–9.

    Article  CAS  Google Scholar 

  7. 7.

    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.

    Article  CAS  PubMed  Google Scholar 

  8. 8.

    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.

    Article  CAS  PubMed  Google Scholar 

  9. 9.

    Qi SY, Groves JT, Chakraborty AK. Synaptic pattern formation during cellular recognition. Proc Natl Acad Sci U S A. 2001;98:6548–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Kasahara K, Sanai Y. Functional roles of glycosphingolipids in signal transduction via lipid rafts. Glycoconj J. 2000;17:153–62.

    Article  CAS  PubMed  Google Scholar 

  11. 11.

    Tanaka M, Sackmann E. Polymer-supported membranes as models of the cell surface. Nature. 2005;437:656–63.

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    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.

    Article  CAS  Google Scholar 

  13. 13.

    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.

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    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.

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    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.

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    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.

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    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.

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    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.

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Thapliyal T, Poulos JL, Schmidt JJ. Automated lipid bilayer and ion channel measurement platform. Biosens Bioelectron. 2011;26:2651–4.

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    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.

    Article  PubMed Central  Google Scholar 

  24. 24.

    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.

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    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.

Download references

Author information



Corresponding author

Correspondence to Christopher A. Baker.

Ethics declarations

Conflict of interest

The authors declare that there are no conflicts of interest.

Additional information

Publisher’s note

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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).

Download citation


  • Black lipid membrane
  • Suspended bilayer
  • Automation
  • Confocal fluorescence
  • Microfluidic