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Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes

Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes. 2006 AIChE Annual Meeting San Francisco, CA. Brian L. Hassler 1 , Maris Laivenieks 2 , Claire Vieille 2 , J. Gregory Zeikus 2 , and Robert M. Worden 1.

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Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes

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  1. Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes 2006 AIChE Annual Meeting San Francisco, CA Brian L. Hassler1, Maris Laivenieks2, Claire Vieille2, J. Gregory Zeikus2, and Robert M. Worden1 1-Department of Chemical Engineering and Materials Science 2-Department of Biochemistry and Molecular Biology Michigan State University, East Lansing, Michigan

  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. enzyme cofactor mediator MEDred MEDox NAD(P)H Dehydrogenase Enzyme Reaction Cofactor Regeneration NAD(P)+ NAD(P)+ enzyme cofactor Substrate Substrate NAD(P)H Dehydrogenase Enzyme Reaction Product Product Dehydrogenase Enzymes • Catalyze electron transfer reactions • Cofactor dependence: NAD(P)+ • Challenge: cofactor recycling

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

  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 (ket) • 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 (TRmax)

  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. enzyme cofactor mediator MEDred MEDox NADH Dehydrogenase Enzyme Reaction Cofactor Regeneration NAD+ Sorbitol Fructose Sorbitol Dehydrogenase (SDH) • Organism: Pseudomonas sp. KS-E1806 • Cofactor dependence: NAD+ • Temperature stability: 30-50C

  14. Chronoamperometric Response • Substrate: Sorbitol • Concentration: 5 mM • Kinetic parameters: • k’= 690s-1 • k”= 87s-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 mA mM-1 cm-2 • TRmax=38 s-1

  16. Amperometric Response • Potential: -200 mV • Concentration range: 1-6 mM • Sensitivity: 2.8 mA 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?

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