1. 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, 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. Interface for dehydrogenase enzymes Mediator integration  Linear approach Electron mediator  Pyrroloquinoline quinone (PQQ) ENZ MED ne - GOLD ENZMED ne - GOLD Mediator integration  Linear approach  Branched approach Electron mediators  Neutral red  Nile blue A  Toluidine blue O ENZMED ne - GOLD 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. Mobile lipid Reservoir lipid Spacer molecule Membrane protein Interface for membrane proteins 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 (k et )  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 (C A )  Sensitivity  Maximum turnover (TR max )

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

11. Impedance spectroscopy Technique:  Apply sinusoidal AC voltage (V ac ) on top of a constant DC voltage (V dc ):  Measure resistance Parameters obtained:  Membrane capacitance (C M )  Membrane resistance (R M ) V applied = V dc + V ac sin ωt

12. Model equivalent circuit R M : Resistance of the membrane containing the ion channels C M : Capacitance of membrane R S : Resistance of the solution C DL: Double layer capacitance RSRS C M R M C DL

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-Propanol+NADP + Acetone +NADPH MED OX +NADPH MED RED +NADP + MED RED MED OX 2 ADH

15. Chronoamperometry results Cofactor: NADP + Equation : k et =4.8×10 2 s -1 == 2.1×10 -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 A mM -1 cm -2 TR max =37 s -1

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

18. Impedance spectroscopy Membrane capacitance: 1.17 µF cm -2 Membrane resistance: 0.68 M cm 2 Resistance with valinomycin: 0.19 M cm 2 After addition of valinomycin Before 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. Integration with microfluidics Soft lithography Channel dimensions: (300µm x 35µm) Si PDMS Glass PDMS

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

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

24. Zayats model

25. Torque-actuated valves

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

28. 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?

31. FTIR of Cysteine

32. FTIR of TBO

33. FTIR of NAD

34. 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, 1991Katz and Willner, Langmuir, 1997

35. Cyclic Voltammetry Assumptions  Nernstian behavior  Single species  No other reaction occurs Governing Equations Turnover ratio Nicholson and Shain, Analytical Chemisitry, 1964

36. Lipids used