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High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis. Hanzi Li Comprehensive oral presentation Advisor: Dr. Scott Calabrese Barton Department of Chemical Engineering and Materials Science Michigan state University. Nov , 2011. Introduction and background.

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High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

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  1. High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis Hanzi Li Comprehensive oral presentation Advisor: Dr. Scott Calabrese Barton Department of Chemical Engineering and Materials Science Michigan state University Nov , 2011

  2. Introduction and background

  3. Dehydrogenase-based electrochemical conversion • Dihydroxyaceton(DHA): Sunless tanning cream; Precursor to pharmaceuticals • Mannitol: Natural sugar alcohol sweetener; Additive to food and pharmaceuticals • Why electrode: Cofactor electrochemical regeneration Dual Chamber Catalysis e- NAD+ NADH Anode GlyDH Power supply Glycerol DHA MtDH Cathode Fructose Mannitol NADH NAD+

  4. Cofactor electroregeneration • Thermodynamically, NADH oxidation should be observed at low potential. Enzyme NAD+ Product -0.49 V/Ag|AgClat pH 6 NADH NAD+ Substrate CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.

  5. Cofactor electroregeneration • Direct NADH oxidation requires high overpotential; Reaction rate is low. Glassy carbon Electrode Typical planar electrode: Glassy carbon electrode ( 3 mm diameter) NADH NAD+ E0’ = -0.49 V/Ag|AgClat pH 6 • Cyclic voltammograms in 0.5 mM NADH at glassy carbon electrode, 50 mV/s, 0.1 M PBS, pH 6 CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.

  6. High-performance cofactor regeneration • Achieve high-rate kinetics for NADH oxidation by electrode modification NADH NAD+ Electrode • Analyze the conversions in NADH oxidation using modified electrode as working electrode

  7. Bioelectrocatalysis involving cofactor regeneration NAD+ NADH • Evaluate bioelectrocatalysis based on NADH electrocatalysis catalyst red catalyst ox Substrate Anode Enzyme Product • Model glycerol oxidation and fructose reduction coupled with cofactor regeneration

  8. Part 1 Electropolymeried azine electrodes modified with carbon nanotubes for NADH oxidation May 13th, 2011

  9. Electrode modification NADH • High-surface area material to increase active site density NAD+ NADH NAD+ Glassy carbon Electrode NADH NAD+ Catalyst ox NADH NAD+ Catalystox • Electrocatalyst • to decrease activation energy High surface area material High-surface area material Glassy carbon Electrode Glassy carbon Electrode Glassy carbon Electrode Catalystred Catalystred Gorton, L.; Dominguez, E. J Biotechnol 2002, 82, 371. Zhao, X.; Lu, X.; Tze, W. T. Y.; Wang, P. Biosensors and Bioelectronics 2010, 25, 2343. Villarrubia, C. W. N.; Rincon, R. A.; Atanassov, P.; Radhakrishnan, V.; Davis, V. ECS Meeting Abstracts 2010, 1001, 443.

  10. Modify electrode with CNT • CNT-GC: CNT were coated on glassy carbon electrode surface (3 mm diameter RDE) by drop-casting 5 µl CNT ink on the surface of GC electrode and drying in vacuum. Drop Casting CNT ink SEM image of CNT on electrode surface Carboxylated CNT (Nanocyl) Glassy carbon Electrode http://www.nanocyl.com/ Li, H.; Wen, H.; Calabrese Barton, S. In Electroanalysis, 2011. Wen, H.; Nallathambi, V.; Chakraborty, D.; Calabrese Barton, S. Microchim. Acta, 1.

  11. CNT-GC: High-surface area material Active surface area / Geometric surface area (Assuming 25 µF/cm2) Capacitance (mF/cm2) in 1 M sulfuric acid Barton, S. C.; Sun, Y.; Chandra, B.; White, S.; Hone, J. Electrochemical and Solid-State Letters 2007, 10, B96. Kinoshita, K.; Carbon: Electrochemical and Physicochemical Properties; 1st ed.; Wiley-Interscience, 1988.

  12. Coat electrocatalyst: Electropolymerization Toluidine Blue O Methylene Green Poly(azine) ox Poly(azine) red Glassy carbon Electrode CNT Cyclic voltammograms of PTBO (Right: Top) and PMG (Right: Bottom) electropolymerization on 0.85 mg cm2- CNT-coated GC, 20 cycles, 50 mV/s, 0.4 mM TBO, 0.01 M borate buffer pH 9.1, 0.1M NaNO3, 30 ºC Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553. Zeng, J.; Wei, W.; Wu, L.; Liu, X.; Liu, K.; Li, Y. Journal of Electroanalytical Chemistry 2006, 595, 152.

  13. NADH electrocatalysis NADH NAD+ Poly(azine) ox Poly(azine) red Glassy carbon Electrode CNT Kar, P.; Barton, S. C. ECS Meeting Abstracts 2010, 1001, 405. Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.

  14. NADH electrocatalysis • a&c: PTBO ; b&d: PMG • 1: Bare GC; 2: 0.21 mg/cm2 CNT-GC; 3: 0.85 mg/cm2CNT-GC NADH concentration study of PTBO-CNT-GC (a) and PMG-CNT-GC (b) at 50 mV/Ag|AgCl; Polarization curves of PTBO-CNT-GC (c) and PMG-CNT-GC (d) in 0.5 mM NADH. 0.1 M phosphate buffer pH 6.0, 900 rpm, 30 ºC. Markers: Experimental data; Solid line: Fitting using mass-transport corrected model; Dash line: Simulation for mass-transport corrected curves.

  15. NADH electrocatalysis

  16. Part 2 • Analysis of the bulk rate of cofactor electroregeneration

  17. CNT modified carbon paper (Toray) Active surface area / Geometric surface area (Assuming 25 µF/cm2) Capacitance was obtained in 0.01 M borate buffer pH 9.1, 0.1 M NaNO3, 30 ºC

  18. NADH Oxidation Using PMG-CNT-Toray • CNT-Toray: CNT were coated on carbon paper surface (2.5×2.5 cm2) by air-brushing 2 mg ml-1 CNT ink on the surface and drying in vacuum. • 1.2×0.8 cm2 (Exposed surface area 1.0×0.8 cm2 , CNT loading 0.9 ± 0.1 mg/cm2) CNT-Toray was used for further modification and working electrode. NADH NAD+ Batch reactor to study the conversion CNT-PMG PMG-CNT acts as electrocatalyst for NADH oxidation Carbon Paper NADH oxidation was performed with initial NADH concentration 0.94 mM in 20 ml pH 6 phosphate buffer, constant applied potential 0.5 V/ Ag|AgCl, 1200 rpm magnetically stirred, 30 ºC.

  19. Conversions in NADH bulk oxidation Electrocatalysis: NADH consumption: Decay k=(1.0± 0.1 ) ×10-3 min-1 NADH concentration profile can be simulated.

  20. Conversions in NADH bulk oxidation NADH concentration was measured using UV-Vis spectra during NADH bulk oxidation

  21. Enzyme cycling assay for detecting bioactive NAD+ • During electraocatalysis and after electrocatalysis, enzyme assay was employed for bulk solution NAD+ NADH MTTox Pyruvate Diaphorase Initially: LDH, Lactate, Diaphorase, MTTox LDH Lactate MTTred Very fast Relatively slow www.bioassaysys.com in the solution

  22. Bioactive NAD+

  23. Part 3 • Immobilization of enzymes and cofactors on poly(azine)-CNT modified electrodes to achieve high-performance bioelectrocatalysis

  24. N6 –linked-NAD+/NADH by Vieille Lab NAD+ Aryl amine Lindberg, M.; Larsson, P.-O.; Mosbach, K. European Journal of Biochemistry 1973, 40, 187

  25. Electrochemical activity of N6-linked NADH Typical RDE Set-up 40 µl - Electrolyte Set-up ω electrolyte electrode electrode 40 µl, Room temperature 0.02 µmoles NADH is needed for 0.5 mM solution electrolyte • 900 rpm,30 °C, At least 10 ml solution, Purged Ar • 5 µmoles NADH is needed for 0.5 mM solution

  26. Electrochemical activity of N6-linked NADH Polarization curves Steady-state data from chronoamperometry , pH 6 PBS, Standard NADH solution: 0.5 mM • Can be fixed by • Compare RDE data in 0 rpm and in air • (Experiment in N2 or Ar) • The lower activity may due to • Limited mass transport • O2 present

  27. Biosensor based on electronic interface NADH NAD+ Reference electrode catalyst red catalyst ox Anode Malate MDH Kinetics: Oxaloacetate • Step 1 relectro • Step 2 renzyme • Evaluate the whole process by monitoring the responding current

  28. Biosensor towards malate concentration • Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH • (immobilization method by Worden Lab) PMG-CNT-GC, chronoamperometry , E=0.4 V vs. Ag|AgCl, 900 rpm, pH 6 PBS, 30 oC, MDH 0.83 µM, initially NAD+ 10 mM

  29. Back-up plan for cofactor/enzyme immobilization • Cofactor is non-covalently attached to CNT via π-πstacking interaction Zhou, H.; Zhang, Z.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. Langmuir 2010, 26, 6028. CVs obtained at the MWCNT-modified Pt electrodes in 0.1 PBS buffet before (blue curve) and after (black curve) at the electrodes were first immersed into the aqueous solution of 10 mM NAD+ for 1 h and then thoroughly rinsed with distilled water. Scant rate: 50 mV/s. Inset: structure of NAD+ cofactor.

  30. Part 4 Model glycerol oxidation and fructose reduction coupled with cofactor regeneration

  31. Linear model NADH NAD+ X=0 X=l Mass balance involving kinetics and diffusion within film : catalyst red Glycerol catalyst ox Anode GlyDH Boundary conditions: Dihydroxyacetone (DHA) Steady-state within film :

  32. Non-dimensionalization Damkohler numbers Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.

  33. Porous model Mass balance: Boundary conditions:

  34. Parameters a: parameter values regarding NADH electrocatalytic reaction have been shown in Project 1 b: assumed to be the same as methanol Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580. Nishise, H.; Nagao, A.; Tani, Y.; Yamada, H. Agricultural and Biological Chemistry 1984, 48, 1603. Gartner, G.; Kopperschlager, G. J. Gen. Microbiol. 1984, 130, 3225.

  35. Simulation results Linear model: Porous model: • DaNAD+ = 16 • Daglycerol= 0.0013; • DaaNAD+ = 406;

  36. Summary • Fabricated poly(azine)-CNT-GC demonstrates high-rate for NADH electrocatalysis. • NADH bulk oxidation shows 80% conversion of 1 mM NADH in 1 hr. Bioactive NAD+ was verified. • Calibration curve for immobilized cofactor evaluation and dehydrogenase-based biosensor are proposed • NondimensionalDamkohler numbers can provide useful approach to simulate, predict and evaluate performance of bioreactor.

  37. Thank you.

  38. Supplemental information

  39. Biosensor towards malate concentration • Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH • (immobilization method by Worden Lab) PMG-CNT-GC, chronoamperometry , E=0.4 V vs. Ag|AgCl, 900 rpm, pH 6 PBS, 30 oC, MDH 0.83 µM, initially NAD+ 10 mM

  40. Cystein

  41. The decay of NADH in 0.1 M phosphate buffer pH 6.0, magnetic stirred speed 1200 rpm, 30 ºC. a. At varies NADH initial concentrations, NADH decay was monitored using UV-Vis spectra at 340 nm; b. The slopes in a. varying with NADH initial concentration.

  42. Acknowledgements • Collaborators • Dr. Mark Worden • Dr. Claire Vieille • Justin Beauchamp • The National Science Foundation • (Award CBET-0756703)

  43. Principle of LDH-MTT Assay NAD+ NADH Initially: LDH, Lactate, Diaphorase, MTTox MTTox Pyruvate Diaphorase LDH www.bioassaysys.com Lactate MTTred Very fast Relatively slow When NAD+ presents in the sample, it is converted to NADH in LDH and lactate. MTTox uses NADH to oxidize into MTTred. The NADH is thus converted back to NAD+. The enzyme cycle starts over. Once the cycle starts, NADH concentration in the assay is not changing = [NAD]+[NADH] in the sample

  44. Kinetics assay using LDH-MTT Assay Kit www.bioassaysys.com NAD+ NADH Initially: LDH, Lactate, Diaphorase, MTTox MTTox Pyruvate Diaphorase LDH Lactate MTTred • Linear kinetics within 15 mins in the sample

  45. Modified electrodes High-surface area electrodes for NADH electrocatalysis

  46. Why Mannitol? • Mannitol is a natural sugar alcohol sweetener. • Mannitol is especially useful as an additive to food and pharmaceuticals • It has low caloric and cariogenic properties • It is not metabolized by the body • It has a cool sweet taste • Currently mannitol is produced by hydrogenating a 1:1 fructose/glucose syrup • Very high temperatures, pressure and a Raney nickel catalyst • Needs highly purified substrates • Energy intensive • Costly purification • Low yield (15%) • Enzymatic catalysis reducing fructose to mannitol • Potential applications to other dehydrogenases

  47. Overall Objective • Glucose  fructose using a thermostable glucose isomerase • Triple mutant of Thermotoga neapolitana xylose isomerase (TNXI 1F1) • Optimized for high activity at 60°C, and high activity at pH 6.0 while maintaining glucose activity • Fructose  mannitol • NADH regeneration from cathodic current pulls reaction towards mannitol production

  48. Nicotinamide Dinucleotide Adenine

  49. Literature review about NADH electrocatalytic oxidation: The reported steady-state current densities for NADH oxidation were far less than 1 mA cm-2 under low overpotential

  50. For the reduction of U in polarization curves Take one PTBO-0.85 mg/cm2 CNT-GC and PTBO-GC as an example: Polarization curve: 0.5 mM NADH , 900 rpm, pH 6 PBS, 30 oC Proposed reason: Impact of Mass-transport Controlled by mass-transport (not controlled by applied potential) Mixed Control (By both applied potential and mass-transport) Controlled by electron-transfer rate (controlled by applied potential)

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