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Development of Affordable Bioelectronic Devices Based on Soluble and Membrane Proteins

Development of Affordable Bioelectronic Devices Based on Soluble and Membrane Proteins. 80 th ACS Colloids and Surface Science Symposium University of Colorado at Boulder June 20, 2006 Brian L. Hassler, Aaron J. Greiner, Sachin Jadhav, Neeraj Kohli,

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Development of Affordable Bioelectronic Devices Based on Soluble and Membrane Proteins

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  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

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

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

  4. ENZ ne- MED ne- GOLD ne- ne- ENZ ENZ MED MED ne- ne- GOLD GOLD 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)

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

  6. Interface for membrane proteins Mobile lipid Reservoir lipid Spacer molecule Membrane protein Gold electrode Raguse et. al, Langmuir, 14, 648 (1998)

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

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

  9. 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)

  10. Constant potential amperometry • Technique: • Set constant potential • Vary analyte concentration • Parameters obtained: • Sensitivity (slope)

  11. 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

  12. C M CDL RS R M 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

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

  14. Experimental protocol • Secondary alcohol dehydrogenase (2 ADH) • Bacteria: Thermoanaerobacter ethanolicus • Thermostable • Cofactor dependent • Reaction mechanism 2 ADH 2-Propanol+NADP+ Acetone +NADPH MEDOX+NADPH MEDRED+NADP+ MEDRED MEDOX

  15. Chronoamperometry results • Cofactor: NADP+ • Equation: 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 • TRmax=37 s-1

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

  18. 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

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

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

  21. PDMS Si PDMS Glass Integration with microfluidics • Soft lithography • Channel dimensions: (300µm x 35µm)

  22. Auxiliary Electrode Inlet/Outlet Ports Microfluidic Channels Torque-Actuated Valves Outlet Inlet Working Electrodes Layout of microfluidics system

  23. Torque-actuated valves Urethane PDMS Glass Whitesides et al., Analytical Chemistry, 77, 4726-4733 (2005)

  24. Zayats model

  25. Torque-actuated valves

  26. Torque-actuated valves

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

  28. 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

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

  30. Thank you Questions?

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