1 / 66

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.

tabib
Télécharger la présentation

High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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)

More Related