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Biomimetic Interfaces Based on Membrane Proteins for Bioelectronic Applications

Biomimetic Interfaces Based on Membrane Proteins for Bioelectronic Applications. Sachin R. Jadhav 1 , R. Michael Garavito 2 and R. Mark Worden 1 1 Department of Chemical Engineering and Materials Science, 2 Department of Biochemistry, Michigan State University, East Lansing, MI.

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Biomimetic Interfaces Based on Membrane Proteins for Bioelectronic Applications

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  1. 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

  2. Outline • Biomimetic interfaces • Tethered bilayer lipid membrane (tBLM) • Electrochemical impedance spectroscopy (EIS) • Methodology for tBLM fabrication • Functional characterization of tBLM • Conclusion AIChE Annual Meeting 2006

  3. Biological cell membrane • Phospholipid molecules self-assemble forming BLM • Embedded membrane proteins contribute activity www.ee.bilkent.edu.tr AIChE Annual Meeting 2006

  4. 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 AIChE Annual Meeting 2006

  5. 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 AIChE Annual Meeting 2006

  6. Components of tBLM Ion channel Raguse et al. Langmuir 14, 648 (1998) AIChE Annual Meeting 2006

  7. Characteristics of an ideal tBLM It should be- • highly insulating • fluid • having an ion reservoir • stable • easy to fabricate AIChE Annual Meeting 2006

  8. Electrochemical impedance spectroscopy (EIS) • Potential of working electrode • Fixed dc potential with superimposed ac signal • V = Vdc + Vacsinωt • Impedance (Z) is calculated and plotted • Bode plot: Z vs ω • Resistance and capacitance of interface determined from data using circuit model AIChE Annual Meeting 2006

  9. Bode plot Z ~1/ωCdl Z~ Rm Z ~ Cm Z~ Rs Naumann et al. J Electroanal Chem 550, 241 (2003) AIChE Annual Meeting 2006

  10. Bode plot after ion channel addition Bilayer Bilayer containing ion channel at different ion concentrations Naumann et al. J Electroanal Chem 550, 241 (2003) AIChE Annual Meeting 2006

  11. 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) AIChE Annual Meeting 2006

  12. + + + + Ionophore + + + + + + Tethering Lipid Liposome Ion channel Methodology for tBLM fabrication Gold Slide AIChE Annual Meeting 2006

  13. Lipids used Tether Lipid-1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) AIChE Annual Meeting 2006

  14. TEM characterization of liposome Average Particle size analysis using dynamic light scattering- 48 nm AIChE Annual Meeting 2006

  15. EIS of tBLM AIChE Annual Meeting 2006

  16. Cyclic voltammetry ____ Blank gold ____ Tether lipid monolayer ____ DOPC bilayer AIChE Annual Meeting 2006

  17. Cyclic voltammetry ____ Tether lipid monolayer ____ DOPC bilayer AIChE Annual Meeting 2006

  18. tBLM with ionophore valinomycin Electrochemical Characteristics Cm= 1.1 µF/cm2 Rm= 850 Kcm2 Rm after 5 µM valinomycin addition= 192 Kcm2 AIChE Annual Meeting 2006

  19. 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 AIChE Annual Meeting 2006

  20. 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 AIChE Annual Meeting 2006

  21. tBLM in barium chloride AIChE Annual Meeting 2006

  22. TEM characterization of microsome Average Particle size analysis using dynamic light scattering- 89 nm AIChE Annual Meeting 2006

  23. tBLM using microsomes AIChE Annual Meeting 2006

  24. 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 AIChE Annual Meeting 2006

  25. 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 AIChE Annual Meeting 2006

  26. Acknowledgement • Michigan Technology Tri-Corridor program through Michigan Economic Development Corporation (MEDC) AIChE Annual Meeting 2006

  27. Thank You AIChE Annual Meeting 2006

  28. 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 AIChE Annual Meeting 2006

  29. 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 AIChE Annual Meeting 2006

  30. 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). AIChE Annual Meeting 2006

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