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Membranes on nanopores for multiplexed single-transporter analyses

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Abstract

The study of membrane proteins as prime drug targets has led to intensified efforts to characterize their structure and function. With regards to the structural analysis of membrane proteins, there have been considerable technological innovations in cryo-EM and X-ray crystallography, but advancements in the elucidation of membrane protein function, especially on a single-molecule level, have been struggling to bridge from basic science to high-throughput applications. There is a need for advanced biosensor platforms allowing membrane protein-mediated transport and potential suppressor libraries to be characterized. Membrane proteins facilitating the translocation of non-electrogenic substrates particularly suffer from a lack of such techniques to date. Here, we summarize recent developments in the field of membrane protein analysis, with a special focus on micro- and nanostructured platforms for purpose of high-throughput screening using fluorescent read-out systems. Additionally, their use as novel biosensor platforms to elucidate non-electrogenic substrate translocation is described. This overview contains 82 references.

Micro- and nanostructured surfaces provide highly parallel sensor systems for the study of transmembrane processes. Deploying multiple fluorescence signals as readout, multiplexing can be achieved and even non-electrogenic substrate translocation detected in real-time, paving the way for high-content studies.

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References

  1. Yıldırım MA, Goh K-I, Cusick ME, Barabási A-L, Vidal M (2007) Drug—target network. Nat Biotechnol 25(10):1119

    Article  Google Scholar 

  2. Arinaminpathy Y, Khurana E, Engelman DM, Gerstein MB (2009) Computational analysis of membrane proteins: the largest class of drug targets. Drug Discov Today 14(23):1130–1135

    Article  CAS  Google Scholar 

  3. Rawlins MD (2004) Cutting the cost of drug development? Nat Rev Drug Discov 3(4):360–364

    Article  CAS  Google Scholar 

  4. Nathan C (2004) Antibiotics at the crossroads. Nature 431(7011):899–902

    Article  CAS  Google Scholar 

  5. Morgan S, Grootendorst P, Lexchin J, Cunningham C, Greyson D (2011) The cost of drug development: a systematic review. Health Policy 100(1):4–17

    Article  Google Scholar 

  6. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth F (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch EJP 391(2):85–100

    Article  CAS  Google Scholar 

  7. Osaki T, Suzuki H, Le Pioufle B, Takeuchi S (2009) Multichannel simultaneous measurements of single-molecule translocation in α-hemolysin nanopore array. Anal Chem 81(24):9866–9870

    Article  CAS  Google Scholar 

  8. Carrillo L, Cucu B, Bandmann V, Homann U, Hertel B, Hillmer S, Thiel G, Bertl A (2015) High‐resolution membrane capacitance measurements for studying endocytosis and exocytosis in yeast. Traffic. doi:10.1111/tra.12275

    Google Scholar 

  9. Fertig N, Blick RH, Behrends JC (2002) Whole cell patch clamp recording performed on a planar glass chip. Biophys J 82(6):3056–3062. doi:10.1016/S0006-3495(02)75646-4

    Article  CAS  Google Scholar 

  10. Stoelzle S, Obergrussberger A, Brüggemann A, Haarmann C, George M, Kettenhofen R, Fertig N (2011) State-of-the-art automated patch clamp devices: heat activation, action potentials, and high throughput in ion channel screening. Front Pharmacol 2

  11. Schroeder K, Neagle B, Trezise DJ, Worley J (2003) IonWorks (TM) HT: a new high-throughput electrophysiology measurement platform. J Biomol Screen 8(1):50–64. doi:10.1177/1087057102239667

    Article  CAS  Google Scholar 

  12. Dunlop J, Bowlby M, Peri R, Vasilyev D, Arias R (2008) High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 7(4):358–368

    Article  CAS  Google Scholar 

  13. Uehlein N, Otto B, Eilingsfeld A, Itel F, Meier W, Kaldenhoff R (2012) Gas-tight triblock-copolymer membranes are converted to CO2 permeable by insertion of plant aquaporins. Sci Rep 2

  14. Horn C, Steinem C (2005) Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes. Biophys J 89(2):1046–1054

    Article  CAS  Google Scholar 

  15. Weiskopf D, Schmitt EK, Klühr MH, Dertinger SK, Steinem C (2007) Micro-BLMs on highly ordered porous silicon substrates: rupture process and lateral mobility. Langmuir 23(18):9134–9139

    Article  CAS  Google Scholar 

  16. Kalsi S, Powl AM, Wallace BA, Morgan H, de Planque MR (2014) Shaped apertures in photoresist films enhance the lifetime and mechanical stability of suspended lipid bilayers. Biophys J 106(8):1650–1659

    Article  CAS  Google Scholar 

  17. Funakoshi K, Suzuki H, Takeuchi S (2006) Lipid bilayer formation by contacting monolayers in a microfluidic device for membrane protein analysis. Anal Chem 78(24):8169–8174

    Article  CAS  Google Scholar 

  18. Holden MA, Needham D, Bayley H (2007) Functional bionetworks from nanoliter water droplets. J Am Chem Soc 129(27):8650–8655

    Article  CAS  Google Scholar 

  19. Bayley H, Cronin B, Heron A, Holden MA, Hwang WL, Syeda R, Thompson J, Wallace M (2008) Droplet interface bilayers. Mol Biosyst 4(12):1191–1208

    Article  CAS  Google Scholar 

  20. Czekalska MA, Kaminski TS, Jakiela S, Sapra KT, Bayley H, Garstecki P (2015) A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings. Lab Chip 15(2):541–548

    Article  CAS  Google Scholar 

  21. Castell OK, Berridge J, Wallace MI (2012) Quantification of membrane protein inhibition by optical ion flux in a droplet interface bilayer array. Angew Chem Int Ed 51(13):3134–3138

    Article  CAS  Google Scholar 

  22. Nisisako T, Portonovo SA, Schmidt JJ (2013) Microfluidic passive permeability assay using nanoliter droplet interface lipid bilayers. Analyst 138(22):6793–6800. doi:10.1039/C3AN01314F

    Article  CAS  Google Scholar 

  23. Poulos JL, Jeon T-J, Damoiseaux R, Gillespie EJ, Bradley KA, Schmidt JJ (2009) Ion channel and toxin measurement using a high throughput lipid membrane platform. Biosensors Bioelectron 24(6):1806–1810. doi:10.1016/j.bios.2008.08.041

    Article  CAS  Google Scholar 

  24. Jeon T-J, Malmstadt N, Schmidt JJ (2006) Hydrogel-encapsulated lipid membranes. J Am Chem Soc 128(1):42–43. doi:10.1021/ja056901v

    Article  CAS  Google Scholar 

  25. X-f K, Cheley S, Rice-Ficht AC, Bayley H (2007) A storable encapsulated bilayer chip containing a single protein nanopore. J Am Chem Soc 129(15):4701–4705. doi:10.1021/ja068654g

    Article  Google Scholar 

  26. Tamm LK, McConnell HM (1985) Supported phospholipid bilayers. Biophys J 47(1):105–113

    Article  CAS  Google Scholar 

  27. Sackmann E (1996) Supported membranes: scientific and practical applications. Science 271(5245):43–48

    Article  CAS  Google Scholar 

  28. Cremer PS, Boxer SG (1999) Formation and spreading of lipid bilayers on planar glass supports. J Phys Chem B 103(13):2554–2559

    Article  CAS  Google Scholar 

  29. Castellana ET, Cremer PS (2006) Solid supported lipid bilayers: from biophysical studies to sensor design. Surf Sci Rep 61(10):429–444

    Article  CAS  Google Scholar 

  30. Tanaka M, Sackmann E (2006) Supported membranes as biofunctional interfaces and smart biosensor platforms. Phys Status Solidi A Appl Res 203(14):3452–3462

    Article  CAS  Google Scholar 

  31. Janshoff A, Steinem C (2006) Transport across artificial membranes—an analytical perspective. Anal Bioanal Chem 385(3):433–451

    Article  CAS  Google Scholar 

  32. Reimhult E, Kumar K (2008) Membrane biosensor platforms using nano- and microporous supports. Trends Biotechnol 26(2):82–89. doi:10.1016/j.tibtech.2007.11.004

    Article  CAS  Google Scholar 

  33. Suzuki H, Takeuchi S (2008) Microtechnologies for membrane protein studies. Anal Bioanal Chem 391(8):2695–2702

    Article  CAS  Google Scholar 

  34. He L, Robertson JW, Li J, Kärcher I, Schiller SM, Knoll W, Naumann R (2005) Tethered bilayer lipid membranes based on monolayers of thiolipids mixed with a complementary dilution molecule. 1. Incorporation of channel peptides. Langmuir 21(25):11666–11672

    Article  CAS  Google Scholar 

  35. Purrucker O, Hillebrandt H, Adlkofer K, Tanaka M (2001) Deposition of highly resistive lipid bilayer on silicon–silicon dioxide electrode and incorporation of gramicidin studied by ac impedance spectroscopy. Electrochim Acta 47(5):791–798

    Article  CAS  Google Scholar 

  36. Rhoten MC, Burgess JD, Hawkridge FM (2002) The reaction of cytochrome c from different species with cytochrome c oxidase immobilized in an electrode supported lipid bilayer membrane. J Electroanal Chem 534(2):143–150

    Article  CAS  Google Scholar 

  37. Bin X, Zawisza I, Goddard JD, Lipkowski J (2005) Electrochemical and PM-IRRAS studies of the effect of the static electric field on the structure of the DMPC bilayer supported at a Au (111) electrode surface. Langmuir 21(1):330–347

    Article  CAS  Google Scholar 

  38. Devadoss A, Palencsár MS, Jiang D, Honkonen ML, Burgess JD (2005) Enzyme modification of platinum microelectrodes for detection of cholesterol in vesicle lipid bilayer membranes. Anal Chem 77(22):7393–7398

    Article  CAS  Google Scholar 

  39. Bazzone A, Costa WS, Braner M, Călinescu O, Hatahet L, Fendler K (2013) Introduction to solid supported membrane based electrophysiology. J Vis Exp (75)

  40. Mager T, Braner M, Kubsch B, Hatahet L, Alkoby D, Rimon A, Padan E, Fendler K (2013) Differential effects of mutations on the transport properties of the Na+/H+ antiporter NhaA from Escherichia coli. J Biol Chem 288(34):24666–24675

    Article  CAS  Google Scholar 

  41. Wagner ML, Tamm LK (2000) Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker. Biophys J 79(3):1400–1414

    Article  CAS  Google Scholar 

  42. Naumann CA, Prucker O, Lehmann T, Rühe J, Knoll W, Frank CW (2002) The polymer-supported phospholipid bilayer: tethering as a new approach to substrate-membrane stabilization. Biomacromolecules 3(1):27–35

    Article  CAS  Google Scholar 

  43. Naumann R, Schiller SM, Giess F, Grohe B, Hartman KB, Kärcher I, Köper I, Lübben J, Vasilev K, Knoll W (2003) Tethered lipid bilayers on ultraflat gold surfaces. Langmuir 19(13):5435–5443

    Article  CAS  Google Scholar 

  44. Kuhner M, Tampe R, Sackmann E (1994) Lipid mono- and bilayer supported on polymer films: composite polymer-lipid films on solid substrates. Biophys J 67(1):217–226. doi:10.1016/S0006-3495(94)80472-2

    Article  CAS  Google Scholar 

  45. Hillebrandt H, Wiegand G, Tanaka M, Sackmann E (1999) High electric resistance polymer/lipid composite films on indium-tin-oxide electrodes. Langmuir 15(24):8451–8459

    Article  CAS  Google Scholar 

  46. Tanaka M, Sackmann E (2005) Polymer-supported membranes as models of the cell surface. Nature 437(7059):656–663

    Article  CAS  Google Scholar 

  47. Purrucker O, Gönnenwein S, Förtig A, Jordan R, Rusp M, Bärmann M, Moroder L, Sackmann E, Tanaka M (2007) Polymer-tethered membranes as quantitative models for the study of integrin-mediated cell adhesion. Soft Matter 3(3):333–336

    Article  CAS  Google Scholar 

  48. Stamou D, Duschl C, Delamarche E, Vogel H (2003) Self‐assembled microarrays of attoliter molecular vessels. Angew Chem Int Ed 115(45):5738–5741

    Article  Google Scholar 

  49. Christensen SM, Stamou D (2007) Surface-based lipid vesicle reactor systems: fabrication and applications. Soft Matter 3(7):828–836

    Article  CAS  Google Scholar 

  50. Christensen SM, Stamou DG (2010) Sensing-applications of surface-based single vesicle arrays. Sensors 10(12):11352–11368

    Article  CAS  Google Scholar 

  51. Bolinger P-Y, Stamou D, Vogel H (2004) Integrated nanoreactor systems: triggering the release and mixing of compounds inside single vesicles. J Am Chem Soc 126(28):8594–8595

    Article  CAS  Google Scholar 

  52. Christensen SM, Bolinger P-Y, Hatzakis NS, Mortensen MW, Stamou D (2012) Mixing subattolitre volumes in a quantitative and highly parallel manner with soft matter nanofluidics. Nat Nanotechnol 7(1):51–55

    Article  CAS  Google Scholar 

  53. Christensen AL, Lohr C, Christensen SM, Stamou D (2013) Single vesicle biochips for ultra-miniaturized nanoscale fluidics and single molecule bioscience. Lab Chip 13(18):3613–3625

    Article  CAS  Google Scholar 

  54. Dekker C (2007) Solid-state nanopores. Nat Nanotechnol 2(4):209–215

    Article  CAS  Google Scholar 

  55. Howorka S, Siwy Z (2009) Nanopore analytics: sensing of single molecules. Chem Soc Rev 38(8):2360–2384

    Article  CAS  Google Scholar 

  56. Basit H, Gaul V, Maher S, Forster RJ, Keyes TE (2015) Aqueous-filled polymer microcavity arrays: versatile & stable lipid bilayer platforms offering high lateral mobility to incorporated membrane proteins. Analyst 140(9):3012–3018

    Article  CAS  Google Scholar 

  57. Simon A, Girard-Egrot A, Sauter F, Pudda C, D’Hahan NP, Blum L, Chatelain F, Fuchs A (2007) Formation and stability of a suspended biomimetic lipid bilayer on silicon submicrometer-sized pores. J Colloid Interface Sci 308(2):337–343

    Article  CAS  Google Scholar 

  58. Han X, Studer A, Sehr H, Geissbühler I, Di Berardino M, Winkler FK, Tiefenauer LX (2007) Nanopore arrays for stable and functional free‐standing lipid bilayers. Adv Mater 19(24):4466–4470

    Article  CAS  Google Scholar 

  59. Schmidt C, Mayer M, Vogel H (2000) A chip‐based biosensor for the functional analysis of single ion channels. Angew Chem Int Ed 112(17):3267–3270

    Article  Google Scholar 

  60. Buchholz K, Tinazli A, Kleefen A, Dorfner D, Pedone D, Rant U, Tampé R, Abstreiter G, Tornow M (2008) Silicon-on-insulator based nanopore cavity arrays for lipid membrane investigation. Nanotechnology 19(44):445305

    Article  CAS  Google Scholar 

  61. Kleefen A, Pedone D, Grunwald C, Wei R, Firnkes M, Abstreiter G, Rant U, Tampé R (2010) Multiplexed parallel single transport recordings on nanopore arrays. Nano Lett 10(12):5080–5087

    Article  CAS  Google Scholar 

  62. Wu H-L, Chen P-Y, Chi C-L, Tsao H-K, Sheng Y-J (2013) Vesicle deposition on hydrophilic solid surfaces. Soft Matter 9(6):1908–1919

    Article  CAS  Google Scholar 

  63. Kumar K, Isa L, Egner A, Schmidt R, Textor M, Reimhult E (2011) Formation of nanopore-spanning lipid bilayers through liposome fusion. Langmuir 27(17):10920–10928. doi:10.1021/la2019132

    Article  CAS  Google Scholar 

  64. Urban M, Kleefen A, Mukherjee N, Seelheim P, Windschiegl B, Vor der Brüggen M, Koçer A, Tampé R (2014) Highly parallel transport recordings on a membrane-on-nanopore chip at single molecule resolution. Nano Lett 14(3):1674–1680

    Article  CAS  Google Scholar 

  65. Kaufeld T, Steinem C, Schmidt CF (2015) Microporous device for local electric recordings on model lipid bilayers. J Phys D Appl Phys 48(2):025401

    Article  Google Scholar 

  66. Kusters I, Van Oijen AM, Driessen AJ (2014) Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion. ACS Nano 8(4):3380–3392

    Article  CAS  Google Scholar 

  67. Frese D, Steltenkamp S, Schmitz S, Steinem C (2013) In situ generation of electrochemical gradients across pore-spanning membranes. RSC Adv

  68. Danelon C, Perez J-B, Santschi C, Brugger J, Vogel H (2006) Cell membranes suspended across nanoaperture arrays. Langmuir 22(1):22–25

    Article  CAS  Google Scholar 

  69. Hennesthal C, Drexler J, Steinem C (2002) Membrane‐suspended nanocompartments based on ordered pores in alumina. Chemphyschem 3(10):885–889

    Article  CAS  Google Scholar 

  70. Drexler J, Steinem C (2003) Pore-suspending lipid bilayers on porous alumina investigated by electrical impedance spectroscopy. J Phys Chem B 107(40):11245–11254

    Article  CAS  Google Scholar 

  71. Römer W, Lam YH, Fischer D, Watts A, Fischer WB, Göring P, Wehrspohn RB, Gösele U, Steinem C (2004) Channel activity of a viral transmembrane peptide in micro-BLMs: Vpu1-32 from HIV-1. J Am Chem Soc 126(49):16267–16274

    Article  Google Scholar 

  72. Römer W, Steinem C (2004) Impedance analysis and single-channel recordings on nano-black lipid membranes based on porous alumina. Biophys J 86(2):955–965

    Article  Google Scholar 

  73. Schmitt EK, Vrouenraets M, Steinem C (2006) Channel activity of OmpF monitored in nano-BLMs. Biophys J 91(6):2163–2171

    Article  CAS  Google Scholar 

  74. Steltenkamp S, Müller MM, Deserno M, Hennesthal C, Steinem C, Janshoff A (2006) Mechanical properties of pore-spanning lipid bilayers probed by atomic force microscopy. Biophys J 91(1):217–226

    Article  CAS  Google Scholar 

  75. Hennesthal C, Steinem C (2000) Pore-spanning lipid bilayers visualized by scanning force microscopy. J Am Chem Soc 122(33):8085–8086

    Article  CAS  Google Scholar 

  76. Stutzmann M, Garrido JA, Eickhoff M, Brandt MS (2006) Direct biofunctionalization of semiconductors: a survey. Phys Status Solidi A Appl Res 203(14):3424–3437

    Article  CAS  Google Scholar 

  77. Korman CE, Megens M, Ajo-Franklin CM, Horsley DA (2013) Nanopore-spanning lipid bilayers on silicon nitride membranes that seal and selectively transport ions. Langmuir 29(14):4421–4425. doi:10.1021/la305064j

    Article  CAS  Google Scholar 

  78. Lazzara TD, Carnarius C, Kocun M, Janshoff A, Steinem C (2011) Separating attoliter-sized compartments using fluid pore-spanning lipid bilayers. ACS Nano 5(9):6935–6944. doi:10.1021/nn201266e

    Article  CAS  Google Scholar 

  79. Watanabe R, Soga N, Yamanaka T, Noji H (2014) High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition. Sci Rep 4

  80. Sumitomo K, McAllister A, Tamba Y, Kashimura Y, Tanaka A, Shinozaki Y, Torimitsu K (2012) Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate. Biosensors Bioelectron 31(1):445–450. doi:10.1016/j.bios.2011.11.010

    Article  CAS  Google Scholar 

  81. Watanabe R, Soga N, Fujita D, Tabata KV, Yamauchi L, Kim SH, Asanuma D, Kamiya M, Urano Y, Suga H (2014) Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity. Nat Commun 5

  82. Kolesinska B, Podwysocka DJ, Rueping MA, Seebach D, Kamena F, Walde P, Sauer M, Windschiegl B, Meyer‐Ács M, Vor der Brüggen M (2013) Permeation through phospholipid bilayers, skin‐cell penetration, plasma stability, and cd spectra of α‐and β‐oligoproline derivatives. Chem Biodivers 10(1):1–38

    Article  CAS  Google Scholar 

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Acknowledgments

The work was supported by the German Research Foundation Grant (CRC 807 – Transport and Communication across Biological Membranes; EXC 115 – Macromolecular Complexes), the German-Israeli Project Cooperation (DIP), and the Federal Ministry of Economics and Technology (ZIM R&D Project).

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  1. Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany

    Michael Urban & Robert Tampé

  2. Cluster of Excellence - Macromolecular Complexes, Goethe University Frankfurt, Frankfurt, Germany

    Robert Tampé

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  1. Michael Urban
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Correspondence to Robert Tampé.

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Urban, M., Tampé, R. Membranes on nanopores for multiplexed single-transporter analyses. Microchim Acta 183, 965–971 (2016). https://doi.org/10.1007/s00604-015-1676-4

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