Optical and Electrical Characterization of Lipid Bilayers on Various Substrates
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1 Optical and Electrical Characterization of Lipid Bilayers on Various Substrates Shepard Group BioIGERT RPU Ashley Nagel, Columbia University Sijun Yang, Cornell University
2 Contents 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?
3 Lipid Bilayers What is a lipid bilayer? A lipid consists of a hydrophilic phosphate group head, and a hydrophobic fatty acid tail.
4 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.
5 Lipid Bilayers Supported Lipid Bilayers Supported lipid bilayers are formed by collapsing liposomes onto a hydrophilic substrate Process is spontaneous
6 Lipid Bilayers Transmembrane Proteins One 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
7 Techniques
8 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.
9 Techniques Procedure Cleaning Reconstitution Extruding Formation Rinsing
10 Techniques Optical Characterizations: Fluorescence Recovery After Photobleaching (FRAP)
11 Techniques Optical Characterizations: Line Profiles (saline-washed glass) Exponentially smoothed, un-normalized Intensity Position Un-smoothed, un-normalized Smoothed, Normalized Intensity Intensity Position Position
12 Techniques Optical Characterizations: Line Profiles: Good vs Bad Good Bilayer Recovery Diffusion is Gaussian Slope of normalized line profile decreases Bad Bilayer Recovery Bleaching resembles diffusion recovery Slope of line profile remains constant
13 Techniques Optical Characterizations: Diffusion Coefficient Axelrod & Soumpasis Method: Kapitza Method: Expected Diffusion Coefficients for Glass: 0.1 to 10 um^2/sec
14 Techniques Optical Characterizations: Intensity Intensity Difference Matlab Plotted Intensity Profiles (saline-washed glass) Time (s) Diffusion (Axelrod) coefficients: Avg: Time (s) Diffusion (Kapitza) coefficients: Avg: 28.05
15 Techniques Optical Characterizations: Diffusion Coefficient - Matlab code provided by Professor Lance Kam 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 Time (s) D
16 Techniques Electrical Characterizations: Cyclic Voltammetry (CV) Potential sweep gives qualitative information about redox reactions Electrical Impedance Spectroscopy (EIS) Bode Plot: Phase angle and Magnitude log(freq/hz) Phase Angle log(impedance/ohms) Data fitted circuit models of bilayer and substrate 90 0 log(freq/hz)
17 Techniques Electrical Characterizations: EIS: Good vs Bad Well-formed Bilayer Badly-formed Bilayer
18 Substrates Indium-Tin Oxide (ITO) Transparent conductor coated on glass Shown in literature to support bilayers
19 Substrates Indium-Tin Oxide (ITO) Optical Characterization Intensity Line profile shows improper bilayer recovery Position FRAP Line Profile
20 Substrates Indium-Tin Oxide (ITO) Electrical Characterization Supports FRAP evidence of unstable bilayer formation
21 Substrates PEDOT-PSS Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) Transparent polymer Conductor Applied by spin coating
22 Substrates PEDOT-PSS Optical Characterization Intensity Line profile suggests improper bilayer formation Position FRAP Line Profile
23 Substrates PEDOT-PSS Electrical Characterization Supports FRAP evidence of unstable bilayer formation Data does not conform to expectations
24 Substrates Nano-Fabricated Patterned Substrate SiO2 Electrode/Silicon AFM image of bilayer over patterned substrate
25 Substrates Nano-Fabricated Patterned Substrate Optical Characterization Intensity Line profile shows clear diffusive recovery Position FRAP Line Profile
26 Substrates Nano-Fabricated Patterned Substrate Electrical Characterization EIS supports promising bilayer formation
27 Substrates Tethered Bilayer (Hydrophobic Substrate) Constructed by adsorbing a functional group to the surface of an otherwise non-hydrophilic substrate
28 Substrates Tethered Bilayer: Gold Gold slide treatments allow for the formation of a SelfAssembled Monolayer (SAM) Changes the surface chemistry of the substrate to allow for self-assembly of lipid bilayers Cysteamine(2-aminoethanethiol) Silane((3-mercaptopropyl)trimethoxysilane)
29 Substrates Gold: Thiol Solution Optical Characterization Line profile shows inconclusive recovery Some bilayers are stable: maybe promising Intensity Position FRAP Line Profile
30 Substrates Gold: Thiol Solution Electrical Characterization Results are preliminary
31 Substrates Gold: Stamping with Cysteamine, Silane or Ethanol Control Soak PDMS stamp in SAM solution Dry and lightly press onto gold slide
32 Substrates Gold: Stamping with Cysteamine, Silane or Ethanol Control Bilayer forms in stamped regions
33 Vertical Bilayer 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
34 Vertical Bilayer Vertical Bilayers: Electrical Characterization Blue: Teflon Brown: Teflon with holes Red: Bilayer over hole
35 Future Steps Further characterization of substrates with actual membrane protein Consistent diffusion coefficient analysis Electrical settings optimization Perfection of tethering/lipid bilayer formation procedures AFM images of bilayers with proteins and of the bilayer on all the substrates Eventual biobattery/ biosensor using lipid bilayer research and transmembrane proteins
36 Bye, lab!
37 Acknowledgments 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!
38 References Alvarez, 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: 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 apoptosis, Electrochemistry Communications, p for studying Janshoff, Andreas. Claudia Steinem., 2006, Transport across artificial membranes an analytical perspective., Anal Bioanal Chem (2006) 385: Kaufmann, S., Kumar, K., and Reimhult, E., 2011, Preparation and Dynamic Patterning of Supported Lipid Membranes Mimicking Cell Membranes, Methods in ch. 28. Molecular Biology, v. 751, Vladimir Atanasov., 2005, Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces., Biophysical Journal,Vol.89,September 2005, p 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 Srinivasan, M.P, T. V. Ratto, P. Stroeve, and M. L. Longo., 2001, Patterned Supported Bilayers on Self-Assembled Monolayers: Confinement of Adjacent Mobile Bilayers., Langmuir, 17, Steinem, Claudia, Andreas Janshoff, Wolf-Peter Ulrich, Manfred Sieber, Hans-Joachim Galla., 1996, Impedance analysis of supported lipid bilayer membranes: a scrutiny of preparation techniques., Biochimica et Biophysica Acta 1279 (1996) different Tien, H.T.,1984, Cyclic Voltammetry of Bilayer Lipid Membranes., J. Phys. Chem. 1984,88, 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 Vladimir Atanasov., 2005, Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces., Biophysical Journal,Vol.89,September 2005, p Images q=pdms+stamping&um=1&hl=en&tbm=isch&tbnid=xhn_ypzmubcwom:&imgrefurl= =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
39 Questions?
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