Optical and Electrical Characterization of Lipid Bilayers on Various Substrates

Shepard GroupBioIGERT RPUAshley Nagel, Columbia UniversitySijun Yang, Cornell University

What is a lipid bilayer?How is it formed?How are they characterized?What substrates are used?What additional experiments are done?What are the project’s future directions &

eventual applications?

Contents

What is a lipid bilayer?A lipid consists of a hydrophilic

phosphate group head, and a

hydrophobic fatty acid tail.

Lipid Bilayers

Lipid Bilayers

Hydrophobic tails stick together to form liposomes, micelles, and bilayers (energetically favorable)

Lipid bilayers are what compose our cell membranes. Bilayers can be formed artificially as well.

Lipid Bilayers

Supported Lipid Bilayers Supported lipid bilayers are formed by collapsing liposomes onto a

hydrophilic substrate

Process is spontaneous

Lipid Bilayers

Transmembrane ProteinsOne of many functions of proteins is to transport molecules

and ions across the cell membrane

ATPase and Gramicidin are used in this project

ATP is used by ATPase in active transport of ions against concentration gradient

Techniques

Techniques

Why Different Substrates?Glass is standard, but non-conductive. Bottom electrode is

necessary to allow for electrical measurements.

Lipids Used: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)

1% Fluorescent lipids used for imaging experiments

Fluorescein isothiocyanate (FITC)

488nm wavelength excitation, 530nm emission

Lipids provided by Avanti Polar Lipids, Inc.

Techniques

ProcedureCleaning

Reconstitution

Extruding

Formation

Rinsing

Techniques

Optical Characterizations:Fluorescence Recovery After Photobleaching (FRAP)

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Un-smoothed, un-normalized

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Smoothed, Normalized

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Position Position

Position

Techniques

Optical Characterizations:Line Profiles (saline-washed glass)

Good Bilayer RecoveryDiffusion is GaussianSlope of normalized line profile decreases

Bad Bilayer RecoveryBleaching resembles diffusion recoverySlope of line profile remains constant

Techniques

Optical Characterizations:Line Profiles: Good vs Bad

Axelrod & Soumpasis Method:

Kapitza Method:

Expected Diffusion Coefficients for Glass: 0.1 to 10 um^2/sec

Techniques

Optical Characterizations:Diffusion Coefficient

Diffusion (Axelrod) coefficients:18.3419.4720.5321.1619.522.3918.14

Avg: 19.93

Diffusion (Kapitza) coefficients:28.8624.326.8

29.8125.7332.5128.32

Avg: 28.05

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Time (s) Time (s)

TechniquesOptical Characterizations:Matlab Plotted Intensity Profiles (saline-washed glass)

– Uses the line profiles to measure rate of diffusion in both X and Y directions through use of Fast Fourier Transform

– Gave much smaller but inconsistent values

– Diffusion coefficient seemed too dependent on time

D0.550.200.360.241.108.70

Time (s)60120180240300360

Techniques

Optical Characterizations:Diffusion Coefficient - Matlab code provided by Professor

Lance Kam

log(Freq/Hz)

log(Freq/Hz)

Electrical Characterizations: Cyclic Voltammetry (CV)

– Potential sweep gives qualitative information about redox reactions

Electrical Impedance Spectroscopy (EIS)– Bode Plot: Phase angle and Magnitude

– Data fitted circuit models of bilayer and substrate

log(

imp

edan

ce/O

hms)

Ph

ase

Ang

le

90°

0°

Techniques

Well-formed Bilayer Badly-formed Bilayer

Techniques

Electrical Characterizations:EIS: Good vs Bad

Substrates

Indium-Tin Oxide (ITO)Transparent conductor coated on glass

Shown in literature to support bilayers

Substrates

Indium-Tin Oxide (ITO)Optical Characterization

– Line profile shows improper bilayer recovery

FRAP Line Profile

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Position

Substrates

Indium-Tin Oxide (ITO)Electrical Characterization

– Supports FRAP evidence of unstable bilayer formation

Substrates

PEDOT-PSS Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate)

Transparent polymer

Conductor

Applied by spin coating

Substrates

PEDOT-PSSOptical Characterization

– Line profile suggests improper bilayer formation

FRAP Line Profile

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ty

Position

Substrates

PEDOT-PSSElectrical Characterization

– Supports FRAP evidence of unstable bilayer formation

– Data does not conform to expectations

AFM image of bilayer over patterned substrate

SiO2

Electrode/Silicon

Substrates

Nano-Fabricated Patterned Substrate

Substrates

Nano-Fabricated Patterned SubstrateOptical Characterization

– Line profile shows clear diffusive recovery

FRAP Line Profile

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Position

SubstratesNano-Fabricated Patterned SubstrateElectrical Characterization

– EIS supports promising bilayer formation

Substrates

Tethered Bilayer (Hydrophobic Substrate)Constructed by adsorbing a functional group to the surface

of an otherwise non-hydrophilic substrate

Substrates

Tethered Bilayer: GoldGold slide treatments allow for the formation of a Self-

Assembled Monolayer (SAM)

Changes the surface chemistry of the substrate to allow for self-assembly of lipid bilayers– Cysteamine(2-aminoethanethiol)

– Silane((3-mercaptopropyl)trimethoxysilane)

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Gold: Thiol SolutionOptical Characterization

– Line profile shows inconclusive recovery

– Some bilayers are stable: maybe promising

FRAP Line Profile

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Substrates

Gold: Thiol SolutionElectrical Characterization

– Results are preliminary

Substrates

Gold: Stamping with Cysteamine, Silane or Ethanol Control

Soak PDMS stamp in SAM solution

Dry and lightly press onto gold slide

Substrates

Gold: Stamping with Cysteamine, Silane or Ethanol Control

Bilayer forms in stamped regions

Forming a Vertical Bilayer: Separate two saline solution chambers

with thin Teflon

Poke hole through Teflon

Fill chambers with lipids

Change solution level of chambers on both side such that lipids “zip” across the hole in Teflon, forming a lipid bilayer

Disadvantage: bilayers last about 6hrs at longest

Vertical Bilayer

Vertical Bilayers: Electrical Characterization

Blue: TeflonBrown: Teflon with holesRed: Bilayer over hole

Vertical Bilayer

Further characterization of substrates with actual membrane protein

Consistent diffusion coefficient analysisElectrical settings optimizationPerfection of tethering/lipid bilayer formation

proceduresAFM images of bilayers with proteins and of the

bilayer on all the substrates

Eventual biobattery/ biosensor using lipid bilayer research and transmembrane proteins

Future Steps

Bye, lab!

Thank You!

To our mentors:

Siddharth Ramakrishnan, PhD

Jared Roseman

And to:

Professor Ken Shepard

Professor Lance Kam

Ria Miranda

And to Viewers like you!

Acknowledgments

ReferencesAlvarez, PE, C. A. Gervasi, A. E. Vallejo., 2007, Impedance Analysis of Ion Transport Through Supported Lipid Membranes Doped with Ionophores: A New Kinetic Approach., Journal Biol Phys 33:421–431

Erbe, A., 2007, Calculation of the diffusion coefficient from FRAP (FPR) data.

Feng, J., Ci, Y. X., Zhang, C. Y., Ottova, A. L., Tien, H. T., 1999, Photoelectric measurements of self-assembled and supported planar lipid bilayers: a new technique for studying apoptosis, Electrochemistry Communications, p. 145 - 147.

Janshoff, Andreas . Claudia Steinem., 2006, Transport across artificial membranes–an analytical perspective., Anal Bioanal Chem (2006) 385: 433–451.

Kaufmann, S., Kumar, K., and Reimhult, E., 2011, Preparation and Dynamic Patterning of Supported Lipid Membranes Mimicking Cell Membranes, Methods in Molecular Biology, v. 751, ch. 28.

Vladimir Atanasov., 2005, Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces., Biophysical Journal,Vol.89,September 2005, p.1780–1788.

Ro¨mer, Wilfred, Claudia Steinem.,2004, Impedance Analysis and Single-Channel Recordings on Nano-Black Lipid Membranes Based on Porous Alumina., Biophysical Journal Volume 86 February 2004 955–965.

Srinivasan, M.P, T. V. Ratto, P. Stroeve, and M. L. Longo., 2001, Patterned Supported Bilayers on Self-Assembled Monolayers: Confinement ofAdjacent Mobile Bilayers., Langmuir, 17, 7951-7954.

Steinem, Claudia, Andreas Janshoff, Wolf-Peter Ulrich, Manfred Sieber, Hans-Joachim Galla., 1996, Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques., Biochimica et Biophysica Acta 1279 (1996) 169-180.

Tien, H.T.,1984, Cyclic Voltammetry of Bilayer Lipid Membranes., J. Phys. Chem. 1984,88, 3172-3174.

Tien H,T., L. Ottovaa,1998, Supported planar lipid bilayers (s-BLMs) as electrochemical biosensors., Electrochimica Acta, Vol. 43, No. 23, pp. 3587±3610.

Urisu, T., Rahman, M., Uno, H., Tero, R., and Nonogaki, Y., 2005, Formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes, Nanomedicine, p. 317 - 322.

Vladimir Atanasov., 2005, Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces., Biophysical Journal,Vol.89,September 2005, p.1780–1788.

http://www.embl.de/eamnet/frap/html/overview.html

Images

http://www.quora.com/What-keeps-a-phospholipid-bilayer-of-one-cell-from-fusing-with-the-bilayer-of-another-cellhttp://www.avantilipids.com/index.php?option=com_content&view=article&id=1786&Itemid=460&catnumber=770375http://bio1151b.nicerweb.com/Locked/media/ch07/bilayer.htmlhttp://upload.wikimedia.org/wikipedia/commons/thumb/c/c6/Phospholipids_aqueous_solution_structures.svg/300px-Phospholipids_aqueous_solution_structures.svg.pnghttp://popups.ulg.ac.be/Base/docannexe/image/6568/img-11.jpghttp://esciencenews.com/files/images/20080723320550.jpghttp://rxrecall.com/wp-content/uploads/2011/05/proton_pump.jpghttp://www.avantilipids.com/index.php?option=com_content&view=article&id=532&Itemid=296http://www.cf.gu.se/digitalAssets/725/725440_FRAP_recovery.gifhttp://www.sigmaaldrich.com/prodimages/i/ito_slides.jpghttp://www.hindawi.com/journals/ijp/2009/650509.fig.001.jpghttp://www.google.com/imgres?q=pdms+stamping&um=1&hl=en&tbm=isch&tbnid=xhn_ypZmuBcWoM:&imgrefurl=http://gmwgroup.harvard.edu/research/soft_lithography_and_nanofabrication/&docid=ZeUDkFWDhXS3gM&w=500&h=384&ei=0eMyToOLJs3pgQeGpdGUDQ&zoom=1&iact=hc&vpx=178&vpy=424&dur=127&hovh=196&hovw=255&tx=122&ty=94&page=1&tbnh=136&tbnw=178&start=0&ndsp=28&ved=1t:429,r:14,s:0&biw=1639&bih=800

Questions?