High-rate cofactor regeneration at nanostructured interfaces for bioelectrocatalysis

1

High-rate cofactor regeneration at nanostructured interfaces for

bioelectrocatalysisHanzi 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

Cathode

Anode

Power supply

e-

Dual Chamber Catalysis

Why electrode: Cofactor electrochemical regeneration

3

Dehydrogenase-based electrochemical conversion

• Dihydroxyaceton(DHA): Sunless tanning cream; Precursor to pharmaceuticals• Mannitol: Natural sugar alcohol sweetener; Additive to food and pharmaceuticals

GlycerolGlyDH

FructoseMtDH

Mannitol DHA

NAD+ NADH

NADH NAD+

4

• Thermodynamically, NADH oxidation should be observed at low potential.

Cofactor electroregeneration

ProductEnzyme

NADHNAD+

Substrate

-0.49 V/Ag|AgCl at pH 6

2electrodeNADH NAD e H

CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.

NAD+

5

Cofactor electroregeneration

• Cyclic voltammograms in 0.5 mM NADH at glassy carbon electrode, 50 mV/s, 0.1 M PBS, pH 6

60

50

40

30

20

10

0

-10

Curre

nt de

nsity

(µA/

cm2 )

1.21.00.80.60.40.20.0-0.2

Potential (V) vs. Ag/AgClGlassy carbon

Electrode

NADH NAD+

E0’ = -0.49 V/Ag|AgCl at pH 6

• Direct NADH oxidation requires high overpotential; Reaction rate is low.

Typical planar electrode:Glassy carbon electrode ( 3 mm diameter)

CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.

6

High-performance cofactor regeneration

NADH NAD+

Electrode

• Achieve high-rate kinetics for NADH oxidation by electrode modification

• Analyze the conversions in NADH oxidation using modified electrode as working electrode

7

Bioelectrocatalysis involving cofactor regeneration

Substrat

e

NAD+

NADHAnode

catalyst red

catalyst ox

Product

Enzym

e• Evaluate bioelectrocatalysis

based on NADH electrocatalysis

• Model glycerol oxidation and fructose reduction coupled with cofactor regeneration

8

Electropolymeried azine electrodes modified with carbon nanotubes for NADH oxidation

May 13th, 2011

Part 1

9

Electrode modification

High-surface area material to

increase active site density

NADH NAD+

Glassy carbon

Electrode

NADH NAD+

Glassy carbon

Electrode

High surface area material

NADH NAD+

Glassy carbon

Electrode

Catalystox Catalystred

Electrocatalyst

to decrease activation energy

NADH NAD+

Glassy carbon

Electrode

Catalyst ox Catalystred

High-surface area material

1. Gorton, L.; Dominguez, E. J Biotechnol 2002, 82, 371.2. Zhao, X.; Lu, X.; Tze, W. T. Y.; Wang, P. Biosensors and Bioelectronics 2010, 25, 2343. 3. Villarrubia, C. W. N.; Rincon, R. A.; Atanassov, P.; Radhakrishnan, V.; Davis, V. ECS Meeting Abstracts 2010, 1001, 443.

10

• 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. Glassy carbon

Electrode

Drop Casting CNT ink

Carboxylated CNT (Nanocyl)

http://www.nanocyl.com/

Modify electrode with CNT

SEM image of CNT on electrode surface

1. Li, H.; Wen, H.; Calabrese Barton, S. In Electroanalysis, 2011.2. Wen, H.; Nallathambi, V.; Chakraborty, D.; Calabrese Barton, S. Microchim. Acta, 1.

111. Barton, S. C.; Sun, Y.; Chandra, B.; White, S.; Hone, J. Electrochemical and Solid-State Letters 2007, 10, B96.

2. Kinoshita, K.; Carbon: Electrochemical and Physicochemical Properties; 1st ed.; Wiley-Interscience, 1988.

CNT-GC: High-surface area material

Capacitance (mF/cm2) in 1 M sulfuric acid

Active surface area / Geometric surface area(Assuming 25 µF/cm2)

1600

1400

1200

1000

800

600

400

200

00.80.60.40.20.0

CNT loading (mg/cm2)

40

30

20

10

01.00.80.60.40.20.0

CNT Loading (mg/cm2)

12

Toluidine Blue O

Methylene Green

Glassy carbon

Electrode

Poly(azine) ox

Poly(azine) red

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

Coat electrocatalyst: Electropolymerization

1. Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.

2. Zeng, J.; Wei, W.; Wu, L.; Liu, X.; Liu, K.; Li, Y. Journal of Electroanalytical Chemistry 2006, 595, 152.

13

NADH NAD+

Glassy carbon

Electrode

Poly(azine) ox

Poly(azine) red

CNT

, oxNADH ads Pi k

,NADH adsPox

ox redNADH P NAD P H

max

exp ( ) /1 exp ( ) /

NADH

S NADH

V U bCi iK C V U b

1. Kar, P.; Barton, S. C. ECS Meeting Abstracts 2010, 1001, 405.2. Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Schmidt, H. L. Electroanalysis 1999, 11, 553.

NADH electrocatalysis

14

NADH electrocatalysis

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.

a&c: PTBO ; b&d: PMG 1: Bare GC; 2: 0.21 mg/cm2 CNT-GC; 3: 0.85 mg/cm2 CNT-GC

max

exp ( ) /1 exp ( ) /

NADH

S NADH

V U bCi iK C V U b

Electrodes imax (mA/cm2)

PTBO-0.21 mg/cm2 CNT-

GC 4.2 ± 0.8

PTBO-0.85 mg/cm2 CNT-

GC 8.4 ± 1.9

PMG-0.21 mg/cm2 CNT-GC 15 ± 3.2

PMG-0.85 mg/cm2 CNT-GC 26 ± 4.1

NADH electrocatalysis

15

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

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.

Batch reactor to study the conversion

NADH

NAD

+

Carbon Paper CNT-

PMG

PMG-CNT acts as electrocatalyst for NADH oxidation

• 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.

Electrocatalysis:

Decay

ox redNADH Catalyst NAD Catalyst H

max exp ( ) /1 exp ( ) /

NADHelectro

S NADH

V U bj A Cj ArnFV nFV K C V U b

decayNADH NADHr kC

k=(1.0± 0.1 ) ×10-3 min-1

Conversions in NADH bulk oxidation

19

NADH consumption:

NADH electro decayr r r

NADH concentration profile can be simulated.

20

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

1.0

0.8

0.6

0.4

0.2

0.0

NA

DH

con

cent

ratio

n / m

M

140120100806040200

Reaction time / min

1.0

0.8

0.6

0.4

0.2

0.0

NA

D+ concentration / m

M

NADH concentration Expected NAD+

a

0 , ,_ , t measured decayedNADH NADH t NADH tExpected NAD tC C C C

Conversions in NADH bulk oxidation

21

www.bioassaysys.com

Initially: LDH, Lactate, Diaphorase, MTTox

Lactate

NAD+

NADH

Pyruvate Diaphorase

MTTox

MTTred

LDH

Very fast Relatively slow

565, ([ ] [ ])tA NADH NAD in the solution

Enzyme cycling assay for detecting bioactive NAD+

• During electraocatalysis and after electrocatalysis, enzyme assay was employed for bulk solution

0 , ,_ , t measured decayedNADH NADH t NADH tExpected NAD tC C C C

,_ , ( _ ), measuredNADH tActive NAD t Active NAD NADH tC C C

22

Applied potential Yield of NAD+ at end (%)500 mV 88 ± 2.3150 mV 82 ± 3.6

1.0

0.8

0.6

0.4

0.2

0.0

Enz

ymat

ical

ly a

ctiv

e N

AD

+ / m

M

1.00.80.60.40.20.0

Expected NAD+ / mM

500 mV / Ag|AgCl 150 mV / Ag|AgCl

Bioactive NAD+

23

Part 3

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

24

Aryl amine

N6 –linked-NAD+/NADH by Vieille Lab

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

NAD+

25

Typical RDE Set-up 40 µl - Electrolyte Set-up

900 rpm, 30 °C, At least 10 ml solution, Purged Ar

5 µmoles NADH is needed for 0.5 mM solution

40 µl, Room temperature0.02 µmoles NADH is needed for 0.5 mM

solution

Electrochemical activity of N6-linked NADH

electrode

ω

electrolyte

electrode

electrolyte

26

Polarization curvesSteady-state data from chronoamperometry , pH 6 PBS, Standard NADH solution: 0.5 mM

• The lower activity may due too Limited mass transporto O2 present

Electrochemical activity of N6-linked NADH

• Can be fixed byo Compare RDE data in 0

rpm and in air o (Experiment in N2 or Ar)

8

6

4

2

0

Cur

rent

den

sity

/ µA

cm

-2

0.50.40.30.20.10.0-0.1

E / V | Ag/AgCl

RDE New set-up

27

Biosensor based on electronic interfaceReference electrode

Malate

NAD+

NADH

Anode

catalyst red

catalyst ox

Oxaloacetate

MDH

Kinetics:

electrodeNADH NAD

MDHMalate NAD Oxaloacetate NADH

Step 1 relectro

Step 2 renzyme

• Evaluate the whole process by monitoring the responding current

28

Biosensor towards malate concentration

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

100

80

60

40

20

0

Cur

rent

den

sity

/ µA

cm

-2

300250200150100500

Malate Concentration / mM

#1 Electrode #2 Electrode

• Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH (immobilization method by Worden Lab)

29

Back-up plan for cofactor/enzyme immobilization

Zhou, H.; Zhang, Z.; Yu, P.; Su, L.; Ohsaka, T.; Mao, L. Langmuir 2010, 26, 6028.

• Cofactor is non-covalently attached to CNT via π-π stacking interaction

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

Model glycerol oxidation and fructose reduction coupled with cofactor regeneration

Part 4

31

Linear model

Mass balance involving kinetics and diffusion within film :

2

2Nh enzyme

NAD ND r

t x

2

2Gly enzyme

Glycerol GlyD r

t x

Steady-state within film :

2

2Nh enzyme

d ND r

dx

2

2Gly enzyme

d GlyD r

dx

Boundary conditions:

0

d Glydx

0d NAD

dx

max 0

0

[ ] exp(( ) / ) 1[ ] 1 exp(( ) / )

x

Nh x

d NAD j Nh V U bdx K Nh V U b nF

0x

x l [ ] [ ]Glycerol Glycerol

Glycero

l

NAD+

NADHAnode

catalyst red

catalyst ox

Dihydroxyacetone

(DHA)

GlyDH

X=0 X=l

32

Non-dimensionalization

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

2'

2 NADa enzyme

d N D rdw

2'

2 Glycerola enzymed G D rdw

0dGdw

0dNdw

0x

x w

1G

'

NADaa electro

dN D rdw

2[ ][ ]Glycerol

catfa

Glycerol

k E LD

D Glycerol

max

0

exp(( ) / ) 1[ ] 1 exp(( ) / )NAD

aaNAD

j L V U bDD NADH V U b nF

0

2[ ][ ]NAD

catfa

NAD

k E LD

D NADH

Damkohler numbers

33

Porous model

Boundary conditions:

Mass balance: 2

' '2 NAD NAD

a enzyme aa elelctrod N D r D rdw

2'

2 Glycerola enzymed G D rdw

0dGdw

0dNdw

0x

x l

1G

0dNdw

34

Parameters

Parametera Value SourceNADH concentration, [NADH]0 10 mM SetGlycerol bulk concentration, [Glycerol]∞ 1 M SetReactor volume, Vol 10 cm3 SetElectrode geometric surface area, cm2 1 cm2 SetEnzyme concentration, [Enzyme] 1 mM SetEquilibrium constant for enzyme reaction, Keq 4×10-4 Vieille labTurnover number of glycerol oxidation, kf 9.1 s-1 Vieille labTurnover number of DHA reduction, kr 9.1 s-1 Vieille labMichaelis-menten constant for NAD+, KmA 12 µM Vieille labMichaelis-menten constant for glycerol, KmB 440 mM Vieille labMichaelis-menten constant for NADH, KmQ 14 µM Vieille labMichaelis-menten constant for DHA, KmP 13 mM Vieille labDissociation constant of NAD+, Kia 1.09 mM 2

Dissociation constant of glycerol, Kib 1.5×104 mM 2,3

Dissociation constant for NADH, Kiq 25 µM 2

Dissociation constant for DHA, Kip 11 mM 3

Film thickness, L 10 µm SetDiffusion coefficient for NADH/NAD+, DNh/DN 3.3×10-8 cm2 s-1 1

Diffusion coefficient for glycerol/DHAb, DGly 4.0×10-6 cm2 s-1 1

a: parameter values regarding NADH electrocatalytic reaction have been shown in Project 1b: assumed to be the same as methanol

1. Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.2. Nishise, H.; Nagao, A.; Tani, Y.; Yamada, H. Agricultural and Biological Chemistry 1984, 48,

1603.3. Gartner, G.; Kopperschlager, G. J. Gen. Microbiol. 1984, 130, 3225.

35

enzymeR A r dl

Simulation results

Linear model: Porous model:

'enzymeR A r dw 4porous

linear

RR

DaNAD+ = 16

Daglycerol = 0.0013;DaaNAD

+ = 406;

36

• 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

• Nondimensional Damkohler numbers can provide useful approach to simulate, predict and evaluate performance of bioreactor.

Summary

37

Thank you.

38

Supplemental information

39

Biosensor towards malate concentration

80

60

40

20Cur

rent

den

sity

/ µA

cm

-2

2000150010005000

Time / s

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

100

80

60

40

20

0

Cur

rent

den

sity

/ µA

cm

-2

300250200150100500

Malate Concentration / mM

#1 Electrode #2 Electrode

• Start with free diffusing cofactor, MDH and malate, will be extended to immobilized cofactor/MDH (immobilization method by Worden Lab)

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

• Collaborators

• Dr. Mark Worden

• Dr. Claire Vieille

• Justin Beauchamp

Acknowledgements

• The National Science Foundation

(Award CBET-0756703)

43

www.bioassaysys.com

Lactate

NAD+

NADH

Pyruvate Diaphorase

MTTox

MTTred

LDH

Initially: LDH, Lactate, Diaphorase, MTTox

Very fast

Principle of LDH-MTT Assay

1. When NAD+ presents in the sample, it is converted to NADH in LDH and lactate.

2. MTTox uses NADH to oxidize into MTTred. The NADH is thus converted back to NAD+.

3. The enzyme cycle starts over.

Relatively slow

Once the cycle starts, NADH concentration in the assay is not changing = [NAD]+[NADH] in the sample

44

www.bioassaysys.com

Lactate

NAD+

NADH

Pyruvate Diaphorase

MTTox

MTTred

LDH

Initially: LDH, Lactate, Diaphorase, MTTox

565,0 15min 565, 15min 565, 0min 15min 0( ) ([ ] [ ] )t t red t red tA A A MTT MTT

Kinetics assay using LDH-MTT Assay Kit

565, 0 15min [ ]tA NADH

565 [ ]redA MTT

15min 0([ ] [ ] ) 15min [ ] 15minred t red tMTT MTT R k NADH • Linear kinetics within 15 mins

565, 0 15min ([ ] [ ])tA NADH NAD in the sample

0.8

0.6

0.4

0.2

0.0

A56

5

1086420

Pyridine necleotide/ µM

BioAssay using NAD only NADH only NAD NADH:NAD=1:1

45

High-surface area electrodes for NADH electrocatalysis

Modified electrodes

Data source Approach Applied potential E

vs. RHE (mV)

imax

(µA/cm2)

imax

(µA)

(Villarrubia, Rincon et al.

2010)

PMG -“Bucky

paper”

913 ------------ 600

(Yang and Liu 2009) PBCB-SWCNT-GC 685 ------------ 1.2

(Doaga, McCormac et al.

2009)

p-DAB-MB-SWCNT-

GC

663 8.49 0.6

(Zhu, Zhai et al. 2007) Meldola blue-CNT-

GC

505 1.6 0.4

(Huang, Jiang et al.

2007)

Thionine-CNT-

Nafion/GC

537 28.3 2

(Zeng, Wei et al. 2006) TBO-MWNT-GC 655 ------------ 45

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

• 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

Adenine

Nicotinamide

Dinucleotide

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 overpotentialData source Approach Applied

potential E vs. RHE (mV)

Vmax(uA/cm2)

Vmax(uA)

(Palmore, Bertschy et al. 1998)

Free diffusing DI+ BV-GC ---------- --------- -----------

(Dilgin, Gorton et al. 2007) PTBO-GC: photoelectrocatalytic 755 25.4 5(Radoi, Compagnone et al.

2007)Bulk screen-printed electrodes

modified with Prussian blue (PB)445 3.54 0.25

(Zhang, Smith et al. 2004) MWCNT-Chitosan-GC 1037 85 6(Liu, Zhang et al. 2010) Magnetic chitosan microspheres -

Polythionine-GC705 141 10

(Zhao, Lu et al. 2010) Single-carbon fiber microelectrode with CNT

1331 ------------- 1.7

(Villarrubia, Rincon et al. 2010)

PMG SWCNTs-based “Bucky paper”

913 (pH 7 solution)

------------ 600

(Yang and Liu 2009) PBCB-SWCNT-GC 685 ------------ 1.2(Zeng, Wei et al. 2006) TBO/MWNTs adduct-GC 655 ------------ 45

(Doaga, McCormac et al. 2009)

p-DAB-MB/SWCNTs/GC 663 8.49 0.6

(Zhu, Zhai et al. 2007) Meldola’s blue adsorbed-CNT-GC 505 1.6 0.4(Huang, Jiang et al. 2007) Thionie incorporated by

DMF/CNTs-Nafion/GC537 28.3 2

(Kim, Kim et al. 2010) Iron oxide/carbon black-GC 643 16 4

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Curre

nt de

nsity

(mA/

cm2 )

0.40.30.20.10.0-0.1-0.2

0.035

0.030

0.025

0.020

0.015

0.010

0.005

0.000

PTBO-0.85 mg/cm2 CNT-GC PTBO-Bare GC

Proposed reason: Impact of Mass-transport

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

Controlled by electron-transfer rate (controlled by applied potential)

Controlled by mass-transport(not controlled by applied potential)

Mixed Control (By both applied potential and mass-

transport)

51

• 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

CNT-GC

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

52

Characterization of PTBO and PMG films

• CV in pH 6 0.1 M PBS, 50 mV/s 30 oC

-4

-2

0

2

4

Curre

nt de

nsity

(mA/

cm2 )

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6E (V) vs. Ag/AgCl

PTBO/bare GC PTBO/ 0.21 mg/cm2 CNT-GC PTBO/ 0.85 mg/cm2 CNT-GC

-4

-2

0

2

4

Curre

nt de

nsity

(mA/

cm2 )

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6E (V) vs. Ag/AgCl

PMG/bare GC PMG/ 0.21 mg/cm2 CNT-GC PMG/ 0.85 mg/cm2 CNT-GC

Glassy carbon

Electrode

Poly(azine) ox

Poly(azine) red

CNT

53

• DMF: Dimethylformamid (CH3)2NC(O)H

54

Process of catalytic reaction of NADH

Or 2NADH NAD H e

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.

The catalysis 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.

55

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 Glassy

carbon

Electrod

e

Drop Casting CNT

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

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

1.0

0.5

0.0

-0.5

-1.0

Curre

nt (m

A/cm

2 )

0.400.380.360.340.320.30E (V) vs. Ag/AgCl

30 mV/s 50 mV/s 60 mV/s 80 mV/s 100 mV/s

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Char

ging C

urre

nt (m

A/cm

2 )

120100806040200Scan rate (mV/s)

Charging current Linear fit: slope is 12.5 mF/cm2

56

Proposed structure of Poly (MB)

S

N

N NCH3

CH3

CH3

H3C

NH3C

S

N

NCH3

CH3

H N

S NCH3

CH3N

H3C

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

S

N

N NCH3

CH3

CH3

H3C

NO2

Methylene Green

57

Imax after MT correction

25

20

15

10

5

0

i max (

mA/cm

2 )

1.00.80.60.40.20.0Loading of CNT (mg/cm2)

PMG-CNT-GC PTBO-CNT-GC

58Solid lines : kinetic controlDotted lines : partially MT

limited

Effect of Mass transport

Mass balance:( ) S

dS S

k Ck C Cs

K C

0 ( )x dCi nFD nFk C Csx

Electrochemical

experiments:

Obtain i’max and K’s for pure kinetic control

MT Correction

59

Bioreactor based on electronic interface

Glycerol

NAD+

NADHAnode

catalyst red

catalyst ox

Dihydroxyaceto

ne(DHA)

GlyDH

Power supply

Reference

electrode

electrodeNADH NAD GlyDHGlycerol NAD DHA NADH H Step 1

r1

Step 2 r2

Kinetics:

1[ ][ ]electro

Nh

k SS NADHr inF K NADH nFV

[ ][ ][ ][ ] [ ][ ] [ ]

catenzyme

GlycerolNAD

k Enzyme Glycerol NADrK Glycerol Glycerol NAD K NAD

100

80

60

40

20

0

Glyc

erol

conc

entra

tion (

mM)

1086420Time (hr)

60

Concentration profile for substrate conversion

Initial values: t=0, [NADH] = [NADH]0 ; [NAD+]=0; [Glycerol] =

[Glycerol]0

[ ] [ ]electro enzyme

d NAD d NADH r rdt dt

[ ]enzyme

d Glycerol rdt

[NADH]0=20 mM; [Glycerol]0 =100

mM;V = 20 cm3;S = 1 cm2;E = 10 µM;

KNADH =7.0 mM;kcat = 9.1 s-1;

Kglycerol = 11 mMKNAD

+ = 25 µM;

3.02 hrs

• Key parameters:1. Sk1/nFV ( µM/s ) 2. [Enzyme] (µM or mM)

For the whole batch reactor:

61

Fabrication of PMG-CNT-Toray

1. 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 thicknessCNT 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

62

63

How Sk1/nFV or/and [Enzyme] impact Time constant?

Sk1/nFV ( µM/s )

Sk1/V ( A/cm3 ) E (µM)

10 - 1000 0.002 – 0.2 1-100

Zoom in5

4

3

2

1

0

Time (

hr)

6 7 8 9100

2 3 4 5 6 7 8 91000

Sk1/nFV (uM/s)

[E] = 0.1 uM [E] = 1 uM [E] = 10 uM [E] = 100 uM

30

25

20

15

10

5

0

Time (

hr)

102 3 4 5 6 7 8 9

1002 3 4 5 6 7 8 9

1000

Sk1/nFV (uM/s)

[E] = 0.1 uM [E] = 1 uM [E] = 10 uM [E] = 100 uM

64

Nondimensionalization

1 2N F Nh

dx dy x yfrho K rho Kd d x y f yf

2F Nh

df yfM rho Md y f yf

DEQs

Boundary conditions:

t=0, x=1, y=0, f=1

Important parameters

:

Time constants for step 1,

step 2

and their ratio

0

[ ][ ]NADxNAD

0

[ ][ ]NADHyNAD

0

[ ][ ]FructosefFructose

t

Parameters:

Variables:

1

[ ]cat

Enzyme VK k nFSk

01

2 0

[ ][ ]

NADM KFructose

01

1

[ ]V NAD nFS k

02

[ ][ ]cat

Fructosek Enzyme

Equilibriu

m constant, representing key operation condition

s

0[ ]NAD

N

KNAD

0[ ]NADH

NhKNAD

0[ ]Fructose

FK

Fructose

65

Current work: Enzyme kinetics of MtDH

p

p

k

kE NADH F E M NAD

• Ordered bi bi

kineticsNADH Fructose Mannitol NAD

k1 k-1 k2 k-2 k3 k-3 k4 k-4

[ ][ ]([ ][ ] )

[ ] [ ][ ] [ ] [ ][ ]

[ ][ ] [ ][ ] [ ][ ][ ][ ][ ] [ ][ ][ ]

f req

f mQ f mPr ia mB r mB r mA r

eq eq

f mQ f fr mA r

eq ia eq iq ip ib eq

P QV V A BK

v V K P V K QV K K V K A V K B V A B

K K

V K A P V P Q V B P QV K B Q V A B PK K K K K K K

A: NAD

HB:

FructoseP:

MannitolQ:

NAD

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

66

Enzyme kinetics of MtDH

• Definition of parameters1

1ia

kKk

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

A: NAD

HB:

FructoseP:

MannitolQ:

NAD

4

4iq

kKk

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

KmA KmB KmP KmQ Kia Kiq Vf Vr Keq

[P]/Kip=0Kib

0.0371 39.89 8.06 0.0181 0.033 0.222 19.38 0.445 59.2 4.7e4Kip1. Vf and Vr: U/mg; All Km’s and Ki’s: mM; Keq: dimensionless 2. For 60 oC,

pH 6.1

1 2

4 1 2 1 1 2( )P

mQP P P

k k kKk k k k k k k k k

1 2 3 2 3

3 1 2 1 1 2

( )( )

P PmP

P P P

k k k k k k kKk k k k k k k k k

4 3

1 4 3 4 4 3( )P

mAP P P

k k kKk k k k k k k k k

4 2 3 3 2

2 4 3 4 4 3

( )( )

P PmB

P P P

k k k k k k kKk k k k k k k k k

2f iq mP f ip mQ

eqr ia mB r ib mA

V K K V K KK

V K K V K K