Development of Affordable Bioelectronic Devices Based on Soluble and Membrane Proteins

1
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
  Science
• Michigan State University
• East Lansing, MI 48823

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Outline

• Motivation
• Interface chemistry for both soluble and membrane
  proteins
• Electrochemical characterization
• Experimental results
• Integration with microfluidics
• Conclusions

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Motivation

• Rapid detection
• Multi-analyte identification
• High throughput screening for the
• pharmaceutical industry
• Identification of pathogens
• Affordable fabrication

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Interface for dehydrogenase enzymes

• Mediator integration
• Linear approach
• Electron mediator
• Pyrroloquinoline quinone (PQQ)

• Mediator integration
• Linear approach
• Branched approach
• Electron mediators
• Neutral red
• Nile blue A
• Toluidine blue O

Zayats et al., Journal of the American Chemical
Society, 124, 14724-15735 (2002)
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Reaction Mechanism
Hassler et. al, Biosensors and Bioelectronics,
77, 4726-4733 (2006)
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Interface for membrane proteins
Mobile lipid
Reservoir lipid
Spacer molecule
Membrane protein
Gold electrode
Raguse et. al, Langmuir, 14, 648 (1998)
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Outline

• Motivation
• Interface chemistry for both soluble and membrane
  proteins
• Electrochemical characterization
• Experimental results
• Integration with microfluidics
• Conclusions

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Chronoamperometry

• Technique
• Induce step change in potential
• Measure current vs. time
• Parameters obtained
• Electron transfer coefficients (ket)
• Charge (Q)
• Surface coverage (?)

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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)

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Constant potential amperometry

• Technique
• Set constant potential
• Vary analyte concentration
• Parameters obtained
• Sensitivity (slope)

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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 ?t
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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
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Outline

• Motivation
• Interface chemistry for both soluble and membrane
  proteins
• Electrochemical characterization
• Experimental results
• Integration with microfluidics
• Conclusions

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Experimental protocol

• Secondary alcohol dehydrogenase (2? ADH)
• Bacteria Thermoanaerobacter ethanolicus
• Thermostable
• Cofactor dependent
• Reaction mechanism

2? ADH
2-PropanolNADP Acetone NADPH
MEDOXNADPH MEDREDNADP
MEDRED MEDOX
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Chronoamperometry results

• Cofactor NADP
• Equation

ket 4.8102 s-1
? 2.110-11 mol cm-2
Zayats et al., Journal of the American Chemical
Society, 124, 14724-15735 (2002)
16
Cyclic voltammetry results

• Concentration range 5 25 mM
• Sensitivity 3.8 mA mM-1 cm-2
• TRmax37 s-1

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Amperometric detection

• Potential -200 mV
• Concentration range 1-6 mM
• Sensitivity 2.81 mA mM-1 cm-2

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Impedance spectroscopy

• Membrane capacitance 1.17 µF cm-2
• Membrane resistance 0.68 M? cm2
• Resistance with valinomycin 0.19 M? cm2

Before addition of valinomycin
After addition of valinomycin
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Outline

• Motivation
• Interface chemistry for both soluble and membrane
  proteins
• Electrochemical characterization
• Experimental results
• Integration with microfluidics
• Conclusions

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Motivation for use of microfluidics

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

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Integration with microfluidics

• Soft lithography
• Channel dimensions (300µm x 35µm)

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Layout of microfluidics system
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Torque-actuated valves
Urethane
PDMS
Glass
Whitesides et al., Analytical Chemistry, 77,
4726-4733 (2005)
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Zayats model
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Torque-actuated valves
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Torque-actuated valves
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Outline

• Motivation
• Interface chemistry for both soluble and membrane
  proteins
• Electrochemical characterization
• Experimental results
• Integration with microfluidics
• Conclusions

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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

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Acknowledgments

• Yue Huang
• Electrical Engineering (MSU)
• Dr. J. Gregory Zeikus
• Biochemistry and Molecular Biology (MSU)
• Ted Amundsen
• Chemical Engineering (MSU)

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Thank you

• Questions?

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FTIR of Cysteine
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FTIR of TBO
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FTIR of NAD
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Chronoamperometry

• Governing equations
• Cottrell equation
• Chidsey model
• Katz model
• Pertinent information
• Electron transfer coefficients
• Charge
• Surface coverage

Delahay, et al., J. Am. Chem., 1952
Chidsey, Science, 1991
Katz and Willner, Langmuir, 1997
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Cyclic Voltammetry

• Assumptions
• Nernstian behavior
• Single species
• No other reaction occurs
• Governing Equations
• Turnover ratio

Nicholson and Shain, Analytical Chemisitry, 1964
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Lipids used