1. Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes Brian L. Hassler 1, Maris Laivenieks 2, Claire Vieille 2, J. Gregory Zeikus 2, and Robert M. Worden 1 1 -Department of Chemical Engineering and Materials Science 2 -Department of Biochemistry and Molecular Biology Michigan State University, East Lansing, Michigan 2006 AIChE Annual Meeting San Francisco, CA

2. Presentation Outline  Motivation  Dehydrogenase enzymes  Formation of bioelectronic interfaces  Characterization techniques  Results  Summary

3. Motivation  Rapid detection  Identification of multiple analytes  High throughput screening  Affordable fabrication

4. Dehydrogenase Enzymes  Catalyze electron transfer reactions  Cofactor dependence: NAD(P) +  Challenge: cofactor recycling Substrate Product NAD(P) + NAD(P)H Dehydrogenase Enzyme Reactioncofactorenzyme Substrate Product NAD(P) + NAD(P)H Dehydrogenase Enzyme Reactioncofactorenzyme MED ox MED red Cofactor Regeneration mediator

5. Enzyme Interface Assembly  Cysteine: branched, trifunctional linker Thiol group: self assembles on gold Carboxyl group: binds to electron mediator Amine group: binds to cofactor  Mediator used Toluidine Blue O (TBO)

6. Reaction Mechanism Hassler et al., Biosensors and Bioelectronics, 21(11), 2146-2154 (2006) Cysteine TBO EDC + /NHS * CBA EDC/NHS Gold NAD(P) + Protein Gold * N-Hydroxysulfosuccinimide + N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide

7. Presentation Outline  Motivation  Sensing mechanisms  Formation of bioelectronic interfaces  Characterization techniques  Results  Summary

8. Chronoamperometry  Technique: Step change in potential Measure current vs. time  Parameters obtained: Electron transfer coefficients (k et ) Charge (Q) Surface coverage (  ) * * Zayats et al., Journal of the American Chemical Society, 124, 14724-15735 (2002)Katz, E. and I. Willner, Langmuir, 13(13), 3364-3373 (1997)

9. Cyclic Voltammetry  Technique: Conduct potential sweep Measure current  Parameters obtained: Sensitivity (slope) Maximum turnover (TR max )

10. Constant Potential Amperometry  Technique: Set constant potential Vary analyte concentration  Parameters obtained: Sensitivity

11. Presentation Outline  Motivation  Sensing mechanisms  Formation of bioelectronic interfaces  Characterization techniques  Results  Summary

12. The Current System  Protein array 4 working electrodes Diameter: 3 mm Counter electrode  Electrode formation: Reservoir in PDMS * Molecular self-assembly Different enzymes * Polydimethylsiloxane (PDMS)

13. Sorbitol Dehydrogenase (SDH)  Organism: Pseudomonas sp. KS-E1806  Cofactor dependence: NAD +  Temperature stability: 30-50  C Sorbitol Fructose NAD + NADH Dehydrogenase Enzyme Reactioncofactorenzyme MED ox MED red Cofactor Regeneration mediator

14. Chronoamperometric Response  Substrate: Sorbitol  Concentration: 5 mM  Kinetic parameters: k’= 690 s -1 k”= 87 s -1  Surface coverage:  ’= 8.7  10 -12 mol cm -2  ”= 8.0  10 -12 mol cm -2

15. Cyclic Voltammetric Response  Concentration range: 3-21 mM  Sensitivity: 3.4  A mM -1 cm -2  TR max =38 s -1

16. Amperometric Response  Potential: -200 mV  Concentration range: 1-6 mM  Sensitivity: 2.8  A mM -1 cm -2

17. Other Enzymes Used Mannitol dehydrogenase Organism: Lactobacillus reuteri Reaction: Fructose Mannitol Cofactor specificity: NAD + Thermal stability: 50  C-90  C

18. Other Enzymes Used Secondary alcohol dehydrogenase Organism: Thermoanaerobacter ethanolicus Reaction: 2-Propanol Acetone Cofactor specificity: NADP + Thermal stability: 30  C-100  C

19. Chronoamperometric Results * Chronoamperometric measurements were made at a concentration of 5 mM of the substrate.

20. Cyclic Voltammetry Results

21. Conclusions  Developed self-assembling biosensor array  Multiple analyte detection Sorbitol Mannitol 2-Propanol  Characterized interfaces electrochemically Chronoamperometry Cyclic voltammetry Constant potential amperometry

22. Acknowledgments-  Ted Amundsen (CHEMS-MSU)  Yue Huang (EECS-MSU)  Kikkoman Corporation  Funding sources Michigan Technology Tri-Corridor (MTTC) IRGP programs at MSU Department of Education GAANN Fellowship

23. Thank you Questions?

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

25. Secondary Alcohol Dehydrogenase (2  ADH)  Organism: Thermoanaerobacter ethanolicus  Cofactor dependence: NADP +  Temperature stability: 30-100  C 2-propanol Acetone NADP + NADPH Dehydrogenase Enzyme Reactioncofactorenzyme MED ox MED red Cofactor Regeneration mediator

26. Mannitol Dehydrogenase  Organism: Lactrobacillus reuteri  Cofactor dependence: NAD +  Temperature stability: 50-80  C Mannitol Fructose NAD + NADH Dehydrogenase Enzyme Reactioncofactorenzyme MED ox MED red Cofactor Regeneration mediator

27. Chronoamperometry response with MDH  Electron transfer coefficients k’ et = 5.0×10 2 s -1 k” et = 4.5×10 1 s -1  Surface coverage  ’= 7.2×10 -12 mol cm -2  ”= 6.0×10 -12 mol cm -2

28. Chronoamperometry response with 2  ADH  Electron transfer coefficients k et = 6.9×10 2 s -1  Surface coverage  ’= 1.6×10 -11 mol cm -2