biomimetic interfaces based on membrane proteins for bioelectronic applications
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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
AIChE Annual Meeting 2006 11/13/2006
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 11/13/2006
Biological cell membrane Phospholipid molecules self-assemble forming BLM Embedded membrane proteins contribute activity
www.ee.bilkent.edu.tr
AIChE Annual Meeting 2006 11/13/2006
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 11/13/2006
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 11/13/2006
Components of tBLM
Ion channel
Raguse et al. Langmuir 14, 648 (1998)
AIChE Annual Meeting 2006 11/13/2006
Characteristics of an ideal tBLM
It should be- highly insulating fluid having an ion reservoir stable easy to fabricate
AIChE Annual Meeting 2006 11/13/2006
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 11/13/2006
Naumann et al. J Electroanal Chem 550, 241 (2003)
Z ~ Cm
Z~ Rs
Z~ Rm
Z ~1/ωCdl
Bode plot
AIChE Annual Meeting 2006 11/13/2006
Bilayer
Naumann et al. J Electroanal Chem 550, 241 (2003)
Bilayer containing ion channel at different ion concentrations
Bode plot after ion channel addition
AIChE Annual Meeting 2006 11/13/2006
Rs
CM
RM
Cdl
Rm: Resistance of the bilayer containing the ion channels
Cm: Capacitance of bilayer
Rs: Resistance of the solution
Cdl : Capacitance of double layer
Equivalent circuit for impedance data
Raguse et al. Langmuir 14, 648 (1998)
AIChE Annual Meeting 2006 11/13/2006
Tethering Lipid
Methodology for tBLM fabrication
Gold Slide
Ionophore
Liposome
++
++
+
++
++
+
Ion channel
AIChE Annual Meeting 2006 11/13/2006
Lipids used
Tether Lipid-1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol
1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC)
AIChE Annual Meeting 2006 11/13/2006
TEM characterization of liposome
Average Particle size analysis using dynamic light scattering- 48 nm
AIChE Annual Meeting 2006 11/13/2006
EIS of tBLM
2
3
4
5
6
7
-2 -1 0 1 2 3 4
Log Frequency (Hz)
Lo
g Z
(O
hm
)
Tether lipidmonolayer
DOPC bilayer
10
20
30
40
50
60
70
80
90
-2 -1 0 1 2 3 4
Log Frequency (Hz)
- Pha
se a
ngle
(Deg
rees
)
Tether lipidmonolayer
DOPC bilayer
AIChE Annual Meeting 2006 11/13/2006
Cyclic voltammetry
____ Blank gold____ Tether lipid monolayer____ DOPC bilayer
AIChE Annual Meeting 2006 11/13/2006
Cyclic voltammetry
____ Tether lipid monolayer____ DOPC bilayer
AIChE Annual Meeting 2006 11/13/2006
tBLM with ionophore valinomycin
Electrochemical Characteristics
Cm= 1.1 µF/cm2
Rm= 850 Kcm2
Rm after 5 µM valinomycin addition= 192 Kcm2
4.5
5
5.5
6
6.5
7
-2 -1.5 -1 -0.5 0 0.5 1
Log Frequency (Hz)
Log
Z (
Ohm
s)
DOPC Bilayer
Bilayer aftervalinomycin addition
AIChE Annual Meeting 2006 11/13/2006
tBLM with gramicidin ion channel
Electrochemical Characteristics
Cm= 0.78 µF/cm2
Rm= 1.61 Mcm2
Rm after 1 µM gramicidin addition= 100 Kcm2
4.5
5
5.5
6
6.5
7
7.5
-2 -1.5 -1 -0.5 0 0.5 1 1.5
Log Frequency (Hz)
Lo
g Z
(O
hm
s)
DOPC bilayer
Bilayer aftergramicidin addition
AIChE Annual Meeting 2006 11/13/2006
tBLM in ammonium chloride
Electrochemical Characteristics
Cm= 0.7 µF/cm2
Rm= 1.8 Mcm2
Rm after 1 µM gramicidin addition= 1.54 Mcm2
4.5
5
5.5
6
6.5
7
7.5
-2 -1.5 -1 -0.5 0 0.5 1Log Frequency (Hz)
Log
Z (
Ohm
s)
DOPC Bilayer
Bilayer after gramicidinaddition
AIChE Annual Meeting 2006 11/13/2006
tBLM in barium chloride
5
5.5
6
6.5
7
7.5
-2 -1.5 -1 -0.5 0 0.5 1
Log Frequency (Hz)
Lo
g Z
(O
hm
)DOPC bilayer
Bilayer aftergramicidin addition
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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
22.5
33.5
44.5
55.5
66.5
7
-2 -1 0 1 2 3 4 5
Log Frequency (Hz)
Lo
g Z
(Oh
m)
Tether lipidmonolayer
Bilayer usingmicrosome
AIChE Annual Meeting 2006 11/13/2006
tBLM using microsome with gramicidin
5
5.5
6
6.5
7
-2 -1.5 -1 -0.5 0 0.5 1
Log Frequency (Hz)
Log
Z (O
hm)
Bilayer usingmicrosomeBilayer aftergramicidin
Electrochemical Characteristics
Cm= 0. 98 µF/cm2
Rm= 1.09 Mcm2
Rm after 1 µM gramicidin addition= 320 Kcm2
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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
AIChE Annual Meeting 2006 11/13/2006
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 11/13/2006
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 11/13/2006
Surface confined membrane models
sBLM
Polymer cushioned BLM
BLM on gold usingSAM of alkanethiols
Freely suspended BLMRichter et al. Langmuir 22, 3497 (2006).