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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

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

dehydrogenase based electrochemical conversion
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+

slide4

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.

slide5

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.

slide6

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
slide7

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
electropolymeried azine electrodes modified with carbon nanotubes for nadh oxidation

Part 1

Electropolymeried azine electrodes modified with carbon nanotubes for NADH oxidation

May 13th, 2011

slide9

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.

slide10

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.

slide11

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.

slide12

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.

slide13

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.

slide14

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.

slide16

Part 2

  • Analysis of the bulk rate of cofactor electroregeneration
cnt modified carbon paper toray
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

nadh oxidation using pmg cnt toray
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.

conversions in nadh bulk oxidation
Conversions in NADH bulk oxidation

Electrocatalysis:

NADH consumption:

Decay

k=(1.0± 0.1 ) ×10-3 min-1

NADH concentration profile can be simulated.

conversions in nadh bulk oxidation1
Conversions in NADH bulk oxidation

NADH concentration was measured using UV-Vis spectra during NADH bulk oxidation

enzyme cycling assay for detecting bioactive nad
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

slide23

Part 3

  • Immobilization of enzymes and cofactors on poly(azine)-CNT modified electrodes to achieve high-performance bioelectrocatalysis
slide24

N6 –linked-NAD+/NADH by Vieille Lab

NAD+

Aryl amine

Lindberg, M.; Larsson, P.-O.; Mosbach, K. European Journal of Biochemistry 1973, 40, 187

typical rde set up

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
polarization curves

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
biosensor based on electronic interface
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
biosensor towards malate concentration
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

back up plan for cofactor enzyme immobilization
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.

linear model
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 :

non dimensionalization
Non-dimensionalization

Damkohler numbers

Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.

porous model
Porous model

Mass balance:

Boundary conditions:

slide34

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.

slide35

Simulation results

Linear model:

Porous model:

  • DaNAD+ = 16
  • Daglycerol= 0.0013;
  • DaaNAD+ = 406;
slide36

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.
biosensor towards malate concentration1
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

slide41

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.

slide42

Acknowledgements

  • Collaborators
    • Dr. Mark Worden
    • Dr. Claire Vieille
    • Justin Beauchamp
  • The National Science Foundation
  • (Award CBET-0756703)
slide43

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

slide44

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

slide45

Modified electrodes

High-surface area electrodes for NADH electrocatalysis

why mannitol
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
overall objective
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
slide48

Nicotinamide

Dinucleotide

Adenine

slide49

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

for the reduction of u in polarization curves
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)

cnt gc
CNT-GC
  • High-surface area of CNT-GC: Good utilization of CNT
  • 115 m2/g (capacitive surface area) vs. 80-140 m2/g (BET)
    • Carboxylated multiwall carbon nanotubes (CNT) instead of untreated CNT were used: hydrophilic property of COOH-CNT make it possible to utilize the good properties of CNT for electrochemical experiments
    • Dimethylformamid (DMF) is used as solvent to form CNT-ink:
      • Organic solvent, disperse CNT well; Can evaporate; Miscible in water

Wen, H.; Nallathambi, V.; Chakraborty, D.; Barton, S. C. ECS Meeting Abstracts 2010, 1002, 366.

characterization of ptbo and pmg films
Characterization of PTBO and PMG films
  • CV in pH 6 0.1 M PBS, 50 mV/s 30 oC

Poly(azine) ox

Poly(azine) red

Glassy carbon Electrode

CNT

process of catalytic reaction of nadh
Process of catalytic reaction of NADH

Thecatalysis efficiency varies with polymers. Even though the mechanisms are not well developed, it is reported that the differences of azine chemical structures affect the electrocatalytic activity toward NADH oxidation. For instance, the additional electron acceptor groups in the aromatic ring always lead to higher electrocatalytic activity, while the additional proton donor groups cause lower electrocatalytic activity. [13, 36] Methylene green has an additional –NO2 group and toluidine blue has an additional -CH3 group. Thus PMG-modified electrodes tend to show higher activity especially at high positive potential region and high NADH concentration.

1. Qi-Jin, C. and D. Shao-Jun, Journal of Molecular Catalysis A: Chemical, 1996. 105(3): p. 193-201.

2. Cooney, M.J., et al., Energy & Environmental Science, 2008. 1(3): p. 320-337.

Or

cnt modified gc electrode capacitance measurement
CNT-modified GC electrode: Capacitance measurement
  • Carbon nanotube is coated on glassy carbon electrode surface (3 mm diameter RDE) by drop-casting:
  • Each CNT layer: 5ug 1mg/ml CNT-DMF suspension

Example of capacitance measurement: 0.50 mg/cm2 CNT loaded on GC

Drop Casting CNT

Glassy carbon Electrode

Capacitance data were obtained by cyclic voltammetry in the 0.3 to 0.4 V vs. Ag/AgCl at 0.01 M borate buffer pH 9.1, 0.1 M NaNO3, 30 oC

proposed structure of poly mb
Proposed structure of Poly (MB)

Karyakin, A.A., et al., 1999. 11(8): p. 553-557.

slide58

Effect of Mass transport

MT Correction

Mass balance:

Electrochemical experiments:

Obtain i’maxand K’s for pure kinetic control

Solid lines : kinetic control

Dotted lines : partially MT limited

bioreactor based on electronic interface
Bioreactor based on electronic interface

Power supply

NADH

NAD+

Reference

electrode

catalyst red

catalyst ox

Glycerol

Anode

GlyDH

  • Step 2 r2
  • Step 1 r1

Dihydroxyacetone

(DHA)

Kinetics:

concentration profile for substrate conversion
Concentration profile for substrate conversion

For the whole batch reactor:

  • Initial values: t=0, [NADH] = [NADH]0 ;
  • [NAD+]=0;
  • [Glycerol] = [Glycerol]0
  • [NADH]0=20 mM;
  • [Glycerol]0 =100 mM;
  • V = 20 cm3;
  • S = 1 cm2;
  • E = 10 µM;
  • KNADH =7.0 mM;
  • kcat= 9.1s-1;
  • Kglycerol= 11 mM
  • KNAD+ = 25 µM;
  • 3.02 hrs
  • Key parameters:
    • Sk1/nFV ( µM/s ) 2. [Enzyme] (µM or mM)
fabrication of pmg cnt toray
Fabrication of PMG-CNT-Toray

CNT-Toray: Spray-coat (air-brushing) CNT ink on Toray paper surface and dry in vacuum.

Toray paper: 3.5 cm × 3.5 cm; 100 µm thickness

CNT ink: 20 mg CNT dispersed in 10 ml DMF

Exposed surface area of Toray to CNT ink: 2.5 cm × 2.5 cm

Resulted loading: 1.1 mg ± 0.11 CNT/ cm2

Bare Toray: 0.16 m2/cm3

For 0.9 cm2 bare Toray, active surface area: 14.4 cm2

Capacitance: 608 uF/cm2

nondimensionalization
Nondimensionalization
  • DEQs
  • Boundary conditions:
  • t=0, x=1, y=0, f=1
  • Important parameters:
  • Variables:
  • Parameters:
  • Equilibrium constant, representing key operation conditions
  • Time constants for step 1, step 2 and their ratio
current work enzyme kinetics of mtdh
Current work: Enzyme kinetics of MtDH
  • Ordered bi bi kinetics

NADH

Fructose

Mannitol

NAD

k1

k2

k3

k4

k-1

k-2

k-3

k-4

A: NADH

B: Fructose

P: Mannitol

Q: NAD

1. Segel, Irwin H. (1993). New York: Wiley

enzyme kinetics of mtdh
Enzyme kinetics of MtDH
  • Definition of parameters

A: NADH

B: Fructose

P: Mannitol

Q: NAD

  • The values of 10 parameters I extracted based on experimental data

Vf and Vr: U/mg; All Km’s and Ki’s: mM; Keq: dimensionless 2. For 60 oC, pH 6.1

1. Segel, Irwin H. (1993). New York: Wiley 2. SeungHoon, S., N. Ahluwalia, et al. (2008). "Applied Microbiology and Biotechnology: 81 (3) 485-495 81(3): 485-495.

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