Biomimetic Interfaces Based on Membrane Proteins for Bioelectronic Applications

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Biomimetic Interfaces Based on Membrane Proteins
for Bioelectronic Applications

• Sachin R. Jadhav1, R. Michael Garavito2 and
• R. Mark Worden1
• 1Department of Chemical Engineering and
• Materials Science,
• 2Department of Biochemistry,
• Michigan State University, East Lansing, MI

AIChE Annual Meeting 2006 San Francisco
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Outline

• Biomimetic interfaces
• Tethered bilayer lipid membrane (tBLM)
• Electrochemical impedance spectroscopy (EIS)
• Methodology for tBLM fabrication
• Functional characterization of tBLM
• Conclusion

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Biological cell membrane

• Phospholipid molecules self-assemble forming BLM
• Embedded membrane proteins contribute activity

www.ee.bilkent.edu.tr
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Biomimetic interfaces

• Biomimetic interfaces are capable of reproducing
  the biological functions of cell membrane in
  vitro
• Applications
• Biophysical studies on cell membrane
• Design of biosensors for membrane proteins
• High-throughput drug screening
• These interfaces can be characterized using
  electrochemical and optical techniques

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Tethered bilayer lipid membrane (tBLM)

• tBLM decouples the bilayer membrane from an
  electrode surface
• The space between the surface and the BLM acts as
  ion reservoir and accommodates transmembrane
  proteins
• Overcomes limitations of unsupported and
  supported BLMs

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Components of tBLM
Ion channel
Raguse et al. Langmuir 14, 648 (1998)
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Characteristics of an ideal tBLM

• It should be-
• highly insulating
• fluid
• having an ion reservoir
• stable
• easy to fabricate

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Electrochemical impedance spectroscopy (EIS)

• Potential of working electrode
• Fixed dc potential with superimposed ac signal
• V Vdc Vacsin?t
• Impedance (Z) is calculated and plotted
• Bode plot Z vs ?
• Resistance and capacitance of interface
  determined from data using circuit model

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Bode plot
Z 1/?Cdl
Z Cm
Z Rm
Z Rs
Naumann et al. J Electroanal Chem 550, 241 (2003)
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Bode plot after ion channel addition
Bilayer
Bilayer containing ion channel at different ion
concentrations
Naumann et al. J Electroanal Chem 550, 241 (2003)
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Equivalent circuit for impedance data
Rm Resistance of the bilayer containing the ion
channels Cm Capacitance of bilayer Rs
Resistance of the solution Cdl Capacitance of
double layer
Raguse et al. Langmuir 14, 648 (1998)
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Methodology for tBLM fabrication
Gold Slide
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Lipids used

• Tether Lipid-1,2-Dipalmitoyl-sn-Glycero-3-Phosphot
  hioethanol
• 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC)

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TEM characterization of liposome
Average Particle size analysis using dynamic
light scattering- 48 nm
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EIS of tBLM
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Cyclic voltammetry
____ Blank gold ____ Tether lipid
monolayer ____ DOPC bilayer
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Cyclic voltammetry
____ Tether lipid monolayer ____ DOPC
bilayer
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tBLM with ionophore valinomycin
Electrochemical Characteristics Cm 1.1
µF/cm2 Rm 850 K?cm2 Rm after 5 µM valinomycin
addition 192 K?cm2
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tBLM with gramicidin ion channel
Electrochemical Characteristics Cm 0.78
µF/cm2 Rm 1.61 M?cm2 Rm after 1 µM gramicidin
addition 100 K?cm2
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tBLM in ammonium chloride
Electrochemical Characteristics Cm 0.7
µF/cm2 Rm 1.8 M?cm2 Rm after 1 µM gramicidin
addition 1.54 M?cm2
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tBLM in barium chloride
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TEM characterization of microsome
Average Particle size analysis using dynamic
light scattering- 89 nm
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tBLM using microsomes
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tBLM using microsome with gramicidin
Electrochemical Characteristics Cm 0. 98
µF/cm2 Rm 1.09 M?cm2 Rm after 1 µM gramicidin
addition 320 K?cm2
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Conclusion

• Biomimetic interfaces based on tBLM were
  fabricated
• Liposome
• Microsome
• Cyclic voltammetry was used to show tBLM
  formation on a gold electrode
• Impedance spectroscopy was used to characterize
  biomimetic interfaces
• Potassium transport by valinomycin
• Ion selectivity passage by gramicidin

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Acknowledgement

• Michigan Technology Tri-Corridor program through
  Michigan Economic Development Corporation (MEDC)

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• Thank You

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Bilayer lipid membranes

• Unsupported BLM can be formed by painting lipid
  solution over a small aperture (1 mm)
• Advantages
• Easy to fabricate
• Can carry out ion channel assays
• Limitations
• Fragility of BLM
• Stable only for couple of hours

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Supported bilayer lipid membrane (sBLM)

• BLM is deposited over hydrophilic substrates-
  glass, silica, mica, gold
• For gold substrates, self assembled monolayer
  (SAM) of alkanethiols is formed
• Advantages
• Stable and robust interfaces
• Limitations
• Lack of ion reservoir
• Steric hindrances for transmembrane proteins

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Surface confined membrane models
BLM on gold using SAM of alkanethiols
sBLM
Freely suspended BLM
Polymer cushioned BLM
Richter et al. Langmuir 22, 3497 (2006).