Development of Affordable Bioelectronic Devices Based on Soluble and Membrane Proteins

80th ACS Colloids and Surface Science Symposium

University of Colorado at Boulder June 20, 2006

Brian L. Hassler, Aaron J. Greiner, Sachin Jadhav, Neeraj Kohli, Robert M. Worden, Robert Y. Ofoli, Ilsoon Lee

Department of Chemical Engineering and Materials ScienceMichigan State UniversityEast Lansing, MI 48823

Outline• Motivation• Interface chemistry for both soluble and

membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions

Motivation• Rapid detection• Multi-analyte identification• High throughput screening for the

pharmaceutical industry• Identification of pathogens• Affordable fabrication

Interface for dehydrogenase enzymes

• Mediator integration Linear approach

• Electron mediator Pyrroloquinoline quinone (PQQ)

ENZ

MED

ne-

ne-

GOLD

ENZ MED

ne-

ne-

GOLD

• Mediator integration Linear approach Branched approach

• Electron mediators Neutral red Nile blue A Toluidine blue O

ENZ MED

ne-

ne-

GOLDZayats et al., Journal of the American Chemical

Society, 124, 14724-15735 (2002)

Reaction Mechanism

Hassler et. al, Biosensors and Bioelectronics, 77, 4726-4733 (2006)

Mobile lipid

Reservoir lipid

Spacer molecule

Membrane protein

Interface for membrane proteins

Gold electrode

Raguse et. al, Langmuir, 14, 648 (1998)

Outline• Motivation• Interface chemistry for both soluble and

membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions

Chronoamperometry• Technique:

Induce step change in potential Measure current vs. time

• Parameters obtained: Electron transfer coefficients (ket) Charge (Q) Surface coverage ()

Time

Po

ten

tia

l

E1

E2

Time

Cu

rre

nt

Cyclic voltammetry• Technique:

Conduct potential sweep Measure current density

• Parameters obtained: Peak current

• Electrode area (A)• Scan rate (v)

• Concentration (CA)

Sensitivity Maximum turnover (TRmax) Potential

Cu

rre

nt

Time

Po

ten

tia

l

E1

E2

E1

Constant potential amperometry• Technique:

Set constant potential Vary analyte concentration

• Parameters obtained: Sensitivity (slope)

Time

Cu

rre

nt

Concentration

Cu

rre

nt

Impedance spectroscopy• Technique:

Apply sinusoidal AC voltage (Vac) on top of a constant DC voltage (Vdc):

Measure resistance

• Parameters obtained: Membrane capacitance (CM)

Membrane resistance (RM)

Vapplied = Vdc + Vac sin ωt

Model equivalent circuit

RM: Resistance of the membrane containing the ion channels

CM: Capacitance of membrane

RS: Resistance of the solution

CDL: Double layer capacitance

RS

CM

RM

CDL

Outline• Motivation• Interface chemistry for both soluble and

membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions

Experimental protocol• Secondary alcohol dehydrogenase (2 ADH)• Bacteria: Thermoanaerobacter ethanolicus

Thermostable Cofactor dependent

• Reaction mechanism

2-Propanol+NADP+ Acetone +NADPH

MEDOX+NADPH MEDRED+NADP+

MEDRED MEDOX

2 ADH

Chronoamperometry results• Cofactor: NADP+

• Equation:et etI = k Qexp(-k t)

ket= 4.8×102 s-1

= 2.1×10-11 mol cm-2

-2.50E-05

2.50E-05

7.50E-05

1.25E-04

1.75E-04

0 0.01 0.02 0.03 0.04 0.05

Time (s)

Cu

rren

t (A

)

Zayats et al., Journal of the American Chemical Society, 124, 14724-15735 (2002)

Cyclic voltammetry results• Concentration range: 5 – 25 mM• Sensitivity: 3.8 A mM-1 cm-2

• TRmax=37 s-1

-30

-20

-10

0

10

20

30

-2000200400

Voltage (mV)

Cu

rren

t (

A)

0

5

10

15

20

25

0 10 20 30Concentration (mM)

Cu

rren

t (

A)

Amperometric detection• Potential: -200 mV• Concentration range: 1-6 mM• Sensitivity: 2.81 A mM-1 cm-2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 20 40Time (s)

Cu

rre

nt

(A

)

0

1

2

3

4

0 2 4 6 8

Concentration (mM)

Cu

rren

t ( A

)

Impedance spectroscopy• Membrane capacitance: 1.17 µF cm-2

• Membrane resistance: 0.68 M cm2

• Resistance with valinomycin: 0.19 M cm2

4.5

4.7

4.9

5.1

5.3

5.5

5.7

5.9

-2 -1 0 1

Log(freq) (Hz)

Lo

g Z

(

)

After addition of valinomycin

Before addition of valinomycin

Outline• Motivation• Interface chemistry for both soluble and

membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions

Motivation for use of microfluidics• Precise control over channel geometry• Precise control over flow conditions• Small sample volumes• Ease of fabrication using PDMS

Integration with microfluidics• Soft lithography• Channel dimensions: (300µm x 35µm)

Si

PDMS

Glass

PDMS

Layout of microfluidics system

Working Electrodes

Auxiliary Electrode

Inlet Outlet

Torque-Actuated Valves

Inlet/Outlet Ports

Microfluidic Channels

Torque-actuated valves

Glass

PDMS

Urethane

Whitesides et al., Analytical Chemistry, 77, 4726-4733 (2005)

Zayats model

Torque-actuated valves

Torque-actuated valves

Outline• Motivation• Interface chemistry for both soluble and

membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions

Conclusions• Developed self-assembling biosensor interfaces

Dehydrogenases Ionophores

• Characterized interfaces electrochemically Chronoamperometry Cyclic voltammetry Constant potential amperometry Impedance spectroscopy

• Fabricated electrode arrays with microfluidics Photolithography Soft lithography Torque-actuated valves

Acknowledgments• Yue Huang:

Electrical Engineering (MSU)

• Dr. J. Gregory Zeikus: Biochemistry and Molecular Biology (MSU)

• Ted Amundsen Chemical Engineering (MSU)

Thank you

Questions?