Biomimetic Interfaces Based on Membrane Proteins for Bioelectronic Applications

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


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

www.ee.bilkent.edu.tr


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


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


Components of tBLM

Ion channel

Raguse et al. Langmuir 14, 648 (1998)


Characteristics of an ideal tBLM

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


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


Naumann et al. J Electroanal Chem 550, 241 (2003)

Z ~ Cm

Z~ Rs

Z~ Rm

Z ~1/ωCdl

Bode plot


Bilayer

Naumann et al. J Electroanal Chem 550, 241 (2003)

Bilayer containing ion channel at different ion concentrations

Bode plot after ion channel addition


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)


Tethering Lipid

Methodology for tBLM fabrication

Gold Slide

Ionophore

Liposome

++

++

+

++

++

+

Ion channel


Lipids used

Tether Lipid-1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol

1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC)


TEM characterization of liposome

Average Particle size analysis using dynamic light scattering- 48 nm


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

)


DOPC bilayer


Cyclic voltammetry

____ Blank gold____ Tether lipid monolayer____ DOPC bilayer


Cyclic voltammetry

____ Tether lipid monolayer____ DOPC bilayer


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


tBLM with gramicidin ion channel


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


tBLM in ammonium chloride


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


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


TEM characterization of microsome

Average Particle size analysis using dynamic light scattering- 89 nm


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)


Bilayer usingmicrosome


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


Cm= 0. 98 µF/cm2

Rm= 1.09 Mcm2

Rm after 1 µM gramicidin addition= 320 Kcm2


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


Acknowledgement

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


Thank You


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


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


Surface confined membrane models

sBLM

Polymer cushioned BLM

BLM on gold usingSAM of alkanethiols

Freely suspended BLMRichter et al. Langmuir 22, 3497 (2006).

Biomimetic Interfaces Based on Membrane Proteins for Bioelectronic Applications

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