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A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules

A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules. Brian Hassler, Megan Dennis, Maris Laivenieks * , Robert Y. Ofoli, J. Gregory Zeikus * , and R. Mark Worden Chemical Engineering and Material Science * Biochemistry and Molecular Biology Michigan State University

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A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules

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  1. A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules Brian Hassler, Megan Dennis, Maris Laivenieks*, Robert Y. Ofoli, J. Gregory Zeikus*, and R. Mark Worden Chemical Engineering and Material Science *Biochemistry and Molecular Biology Michigan State University East Lansing, Michigan Presented at 2004 Annual AIChE Conference November 7 - 12, 2004, Austin, TX Center for Nanostructured Biomimetic Interfaces

  2. Presentation Outline • Background • Dehydrogenase enzyme • Bioelectronic interface • Project goals • Site directed enzyme mutagenesis • Characterization of bioelectronic interface • Cyclic voltammetry • Chronoamperometry • Conclusions Center for Nanostructured Biomimetic Interfaces

  3. NAD(P)+ S enzyme cofactor mediator MEDred NAD(P)H MEDox P Dehydrogenase Enzyme Reaction Cofactor Regeneration Background • Dehydrogenase enzymes • Catalyze electron transfer reactions • Activity easily measured electrochemically • Bioelectronic applications • Often require cofactor (e.g., NAD(P)+) • Challenge: regenerating cofactor after reaction Center for Nanostructured Biomimetic Interfaces

  4. Background on Enzyme • Model enzyme • secondary alcohol dehydrogenase (sADH) • Thermoanaerobacter ethanolicus • Thermal stability • Activity range: 7°C – 95°C Center for Nanostructured Biomimetic Interfaces

  5. Background on Enzyme • Cofactor specificity: NADP+ • Amino acids affecting NADP+ affinity binding • 198, 199, 200, 203, 218 Center for Nanostructured Biomimetic Interfaces

  6. Med Enz Cof Elec 2 e- 2 e- Background on Cofactor Regeneration • Electron mediator required • Shuttles electrons between electrode and cofactor • Prevents cofactor degradation • Linear structure • Mediator requirements • Two unique functional groups • Bind to electrode • Bind to cofactor • Few suitable mediators (Zayats, et al., J. Am. Chem. Soc. 2002, 124, 14724-14735) Center for Nanostructured Biomimetic Interfaces

  7. Research Goals • Enhance enzyme activity withNAD+ • Retain thermal stability • Generate a unique electron transfer scaffold • Using a hetro-trifunctional linking molecule • Suitable for wider range of electron mediators Center for Nanostructured Biomimetic Interfaces

  8. Mutant 3’ - end Enzyme Mutagenesis PCR amplification 1 5’ primer 5’ primer Wild type template Wild type template 3’ primer 3’ primer 5’ primer Mutant primer - end Mutant 5’ Mutant primer 3’ primer PCR amplification 2 Complete mutant Center for Nanostructured Biomimetic Interfaces

  9. Clone adhB Gene Into pCR 2.1 Vector Insert mutant gene into lacZ gene Transformed cell containing the PCR product will grow white on X-gal Cells with plasmid will have ampicillin & kanamycin resistance Center for Nanostructured Biomimetic Interfaces

  10. Enzymatic Activities of Wild Type, Mutant Strains • NADP+ • NAD+ Center for Nanostructured Biomimetic Interfaces

  11. Med Enz Cof Elec Med Enz Cof 2 e- 2 e- Elec 2 e- 2 e- Cofactor Regeneration by Electrode • Linear structure • Mediator requirements • Two unique functional groups • Few suitable mediators • Branched structure • Mediator requirements • Single functional group • Many suitable mediators Center for Nanostructured Biomimetic Interfaces

  12. NAD+ TBO cysteine gold electrode Enzyme Interface Assembly • Cysteine: branched, trifunctional linker • Thiol group: self assembles on gold • Carboxyl group: binds to electron mediator • Amine group: binds to phenylboronic acid • Mediators used • Toluidine Blue O (TBO) • Nile Blue A • Neutral Red Center for Nanostructured Biomimetic Interfaces

  13. Characterization Tools • Cyclic Voltammetry • Calibration plots • Turnover ratio • Effects of increased temperatures • Chronoamperometry • Electrode kinetics Center for Nanostructured Biomimetic Interfaces

  14. Cyclic Voltammetry Y218F-mutant sADH • Cyclic voltammetry • Substrate: Isopropanol, in phosphate buffer, pH=7.4 • High voltage: 400mV • Low voltage: -200 mV • Scan rate: 100 mV/s • Electrode area: 1 cm2 • Calibration plot: • Slope: 1 mA/mM • Isat= 42mA • Turnover ratio: • 65 s-1 Center for Nanostructured Biomimetic Interfaces

  15. Cyclic Voltammetry Wild-Type sADH • Cyclic voltammetry • Substrate: Isopropanol, in phosphate buffer, pH=7.4 • High voltage: 400mV • Low voltage: -200 mV • Scan rate: 100 mV/s • Electrode area: 1 cm2 • Calibration plot: • Slope: 1.67 mA/mM • Isat= 80mA • Turnover ratio: • 450 s-1 Center for Nanostructured Biomimetic Interfaces

  16. Chronoamperometry • Procedure • Step change in potential • Initial Potential (E1): -200 mV • Final Potential (E2): 400 mV • Plot current vs. time • Characterization • Equation • Measurable variables • ket= Electron transfer constant • Q= Charge associated with oxidation/reduction I=ket’Q’exp(-ket’t)+ket”Q”exp(-kett) (Forster, R. J. Langmuir1995, 11, 2247-2255) Center for Nanostructured Biomimetic Interfaces

  17. Chronoamperometry Y218F-mutant sADH-NAD+ • Forster equation • Best fit ket values • ket’= 7.0x104 s-1 • ket”= 5.5x103 s-1 • Surface coverage =Q/nFA • ’= 9.56x10-13 mol cm-2 • ”= 7.55x10-12 mol cm-2 I=ket’Q’exp(-ket’t)+ket”Q”exp(-kett) Center for Nanostructured Biomimetic Interfaces

  18. Chronoamperometry Wild Type-sADH • Forster equation • Best fit ket values • ket= 7.0x104 s-1 • Surface coverage • = 2.34x10-12 mol cm-2 I=ket’Q’exp(-kett) I=ket’Q’exp(-ket’t)+ket”Q”exp(-kett) Center for Nanostructured Biomimetic Interfaces

  19. Determination of Thermostability • Temperatures Measured • 25 °C (I= 9 mA) • 35 °C (I= 15 mA) • 45 °C (I= 21 mA) • 50 °C (I= 25 mA) • 60 °C (I= 38 mA) • 65 °C (I= 8 mA) Center for Nanostructured Biomimetic Interfaces

  20. Conclusions • Mutant sADH developed • Increased activity with NAD+ • Novel electron transfer scaffold developed • Trifunctional linking molecule • Wider range of mediators • Bioelectronic interface with sADH developed • Electrode kinetics measured • Calibration curves developed • Stable up to 60 °C Center for Nanostructured Biomimetic Interfaces

  21. Acknowledgements • Funding • Michigan Technology Tri-Corridor • Department of Education GAANN Fellowship • Undergraduate students involved • John Baldrey • Timothy Howes Center for Nanostructured Biomimetic Interfaces

  22. Thank you Center for Nanostructured Biomimetic Interfaces

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