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
1 , 2011
Introduction and background
2
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
eNAD+
GlyDH
Glycerol
Power
supply
NADH
DHA
MtDH
Fructose
NADH
Anode
Mannitol
NAD+
Cathode
3
Cofactor electroregeneration
• Thermodynamically, NADH oxidation should be observed at low potential.
Enzyme
Substrate
NAD+
Product
NADH
NAD+


N A D H   
 N A D  2e  H
electrode

-0.49 V/Ag|AgCl at pH 6
CRC Handbook of Chemistry and Physics, 91st ed.; Haynes, W. M., Ed.; 2010.
4
Cofactor electroregeneration
• Direct NADH oxidation requires high overpotential; Reaction rate is low.
E0’ = -0.49 V/Ag|AgCl at pH 6
NADH
NAD+
50
2
Typical planar electrode:
Glassy carbon electrode ( 3 mm diameter)
Current density (µA/cm )
60
40
30
20
10
0
-10
Glassy carbon
Electrode
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Potential (V) vs. Ag/AgCl
• 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.
5
High-performance cofactor regeneration
NADH
NAD+
• Achieve high-rate kinetics for NADH oxidation
by electrode modification
Electrode
• Analyze the conversions in NADH oxidation
using modified electrode as working
electrode
6
Bioelectrocatalysis involving cofactor regeneration
catalyst ox
Substrate
NADH
Anode
Enzyme
catalyst red
NAD+
Product
• Evaluate bioelectrocatalysis based
on NADH electrocatalysis
• Model glycerol oxidation and
fructose reduction coupled with
cofactor regeneration
7
Part 1
Electropolymeried azine electrodes modified
with carbon nanotubes for NADH oxidation
th
8
Electrode modification
NADH
NAD+
 High-surface area
material to
increase active
site density
NADH
NAD+
NADH
High surface
area material
NAD+
Glassy carbon
Electrode
Glassy carbon
Electrode
Catalyst ox
 Electrocatalyst
NADH
to decrease
activation energy
Catalystox
NAD+
Catalystred
Catalystred
High-surface
area material
Glassy carbon
Electrode
Glassy carbon
Electrode
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.
9
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.
Carboxylated CNT
(Nanocyl)
Drop Casting
CNT ink
Glassy carbon
Electrode
SEM image of CNT on electrode
surface
http://www.nanocyl.com/
1. Li, H.; Wen, H.; Calabrese Barton, S. In Electroanalysis, 2011.
2. Wen, H.; Nallathambi, V.; Chakraborty, D.; Calabrese Barton, S. Microchim. Acta, 1.
10
CNT-GC: High-surface area material
Active surface area / Geometric surface area
(Assuming 25 µF/cm2)
(mF/cm2)
Capacitance
in 1 M sulfuric acid
1600
40
1400
1200
30
1000
800
20
600
400
10
200
0
0.0
0.2
0.4
0.6
0.8
2
CNT Loading (mg/cm )
1.0
0
0.0
0.2
0.4
0.6
0.8
2
CNT loading (mg/cm )
1. 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.
11
Coat electrocatalyst: Electropolymerization
Toluidine Blue O
Poly(azine) oxPoly(azine) red
Methylene Green
CNT
Glassy carbon
Electrode
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
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.
12
NADH electrocatalysis
NAD+
NADH


N AD H  Pox  N AD  Pred H
i  k  N AD H , ads Pox
Poly(azine) Poly(azine)
red
ox
CNT
Glassy carbon
Electrode
 N A D H , ads
P
ox

  exp  (V  U ) / b  
C NADH
i  im ax 

 
K

C
1

exp
(
V

U
)
/
b


NADH  
 S

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.
13
NADH electrocatalysis
 a&c: PTBO ; b&d: PMG
 1: Bare GC; 2: 0.21 mg/cm2 CNT-GC; 3: 0.85 mg/cm2 CNT-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.
14
NADH electrocatalysis

  exp  (V  U ) / b  
C NADH
i  im ax 

 
K

C
1

exp
(
V

U
)
/
b


NADH  
 S

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
15
Part 2
Analysis of the bulk rate of cofactor
electroregeneration
16
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
17
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) CNTToray was used for further modification and working electrode.
Batch reactor to study the conversion
PMG-CNT acts as electrocatalyst for NADH oxidation
Carbon
Paper
NADH
CNT-PMG
NAD+
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.
18
Conversions in NADH bulk oxidation
NADH consumption:
rN A D H   relectro  rdecay
Electrocatalysis:


N AD H  C atalyst ox  N AD  C atalyst red H
relectro 
j A
nF V

  exp  (V  U ) / b  
j m ax  A 
C NADH



nF V  K S  C N A D H   1  exp  (V  U ) / b  
Decay
rN A D H d eca y  kC N A D H
k=(1.0± 0.1 ) ×10-3 min-1
NADH concentration profile can be simulated.
19
Conversions in NADH bulk oxidation
1.0
1.0
0.8
0.6
0.6
+
0.8
NAD concentration / mM
NADH concentration / mM
a
NADH concentration
0.4
Expected NAD
+
0.2
0.4
0.2
0.0
0.0
0
20
40
60
80
100
120
140
Reaction time / min
NADH concentration was measured using UV-Vis spectra during NADH bulk oxidation
C Expected _ N AD  ,t  C N AD H t  0  C N AD H m easured , t  C N AD H decayed , t
20
Enzyme cycling assay for detecting bioactive NAD+
• During electraocatalysis and after electrocatalysis, enzyme assay was
employed for bulk solution
Initially: LDH,
Lactate,
Diaphorase, Pyruvate
MTTox
MTTox
NAD+
Diaphorase
LDH
Lactate
MTTred
NADH
www.bioassaysys.com
Very fast
 A5 6 5 ,
Relatively slow

t
 ([ N A D H ]  [ N A D ])
in the solution
21
Bioactive NAD+
C Active _ N AD  ,t  C ( Active _ N AD   N AD H ),t  C N AD H m easured , t
C Expected _ N AD  ,t  C N AD H t  0  C N AD H m easured , t  C N AD H decayed , t
0.8
+
Enzymatically active NAD / mM
1.0
0.6
0.4
0.2
0.0
0.0
500 mV / Ag|AgCl
150 mV / Ag|AgCl
0.2
0.4
0.6
0.8
1.0
+
Expected NAD / mM
Applied potential
Yield of NAD+ at end (%)
500 mV
88 ± 2.3
150 mV
82 ± 3.6
22
Part 3
Immobilization of enzymes and cofactors on
poly(azine)-CNT modified electrodes to achieve
high-performance bioelectrocatalysis
23
N6 –linked-NAD+/NADH by Vieille Lab
NAD+
Aryl amine
Lindberg, M.; Larsson, P.-O.; Mosbach, K. European Journal of Biochemistry 1973, 40, 187
24
Electrochemical activity of N6-linked NADH
Typical RDE Set-up
40 µl - Electrolyte Set-up
ω
electrolyte
electrode
electrode
electrolyte
40 µl, Room temperature
0.02 µmoles NADH is needed for 0.5 mM solution
900 rpm, 30 °C, At least 10 ml solution, Purged Ar
5 µmoles NADH is needed for 0.5 mM solution
25
Electrochemical activity of N6-linked NADH
Polarization curves
Steady-state data from chronoamperometry , pH 6 PBS, Standard NADH solution: 0.5 mM
RDE
New set-up
Current density / µA cm
-2
8
6
4
2
0
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
E / V | Ag/AgCl
•
The lower activity may due to
o
Limited mass transport
o
O2 present
•
Can be fixed by
o Compare RDE data in 0 rpm
and in air
o (Experiment in N2 or Ar)
26
Biosensor based on electronic interface
catalyst ox
Malate
NADH
Anode
MDH
catalyst red
NAD+
Oxaloacetate
Reference
electrode
Kinetics:
Step 1 relectro
Step 2 renzyme
N A D H   
 NAD
electrode


M alate  N A D   O xaloacetate  N A D H
MDH
• Evaluate the whole process by monitoring the responding current
27
Biosensor towards malate concentration
100
Current density / µA cm
-2
80
60
#1 Electrode
#2 Electrode
40
20
0
0
50
100
150
200
250
300
Malate Concentration / mM
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
• Start with free diffusing cofactor, MDH and malate, will be
extended to immobilized cofactor/MDH
(immobilization method by Worden Lab)
28
Back-up plan for cofactor/enzyme immobilization
• Cofactor is non-covalently
attached to CNT via π-π
stacking 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 .
29
Part 4
Model glycerol oxidation and fructose
reduction coupled with cofactor regeneration
30
Linear model
NADH
Anode
catalyst ox
catalyst red
Glycerol
GlyDH
NAD+
Dihydroxyacetone
(DHA)
X=0
X=l
Mass balance involving kinetics and
diffusion within film :

2
  N AD 
 N 
 D Nh
 renzym e
2
t
x
  G lycerol 
t
 D G ly

2
 G ly 
x
2
Steady-state within film :
D Nh
D G ly
d
2
N 
dx
d
2
2
  renzym e
 G ly 
dx
2
 renzym e
 renzym e
Boundary conditions:
x0
d  G ly 
0
dx

d  N AD 
j m ax [ N h ] x  0
exp((V  U ) / b )
1


dx
K N h  [ N h ] x  0 1  exp((V  U ) / b ) nF
x l
[ G lycerol ]  [ G lycerol ]

d  N AD 
0
dx
31
Non-dimensionalization
x0
2
d N
dw
2
  Da
N AD

r
'
enzym e
dG
0
dw
dN
dw
 D aa
 relectro
'
NAD

2
d G
dw
2
 D a G lycerol  renzym e
'
xw
dN
0
dw
G 1
Damkohler numbers
Da
N AD

k catf [ E ] L

D N AD  [ N AD H ] 0
D a G lycerol 
D aa
NAD
2

k catf [ E ] L
2
D G lycerol [ G lycerol ] 

j m ax L

exp((V  U ) / b )
1
D N A D  [ N A D H ] 0 1  exp((V  U ) / b ) nF
Kar, P.; Wen, H.; Li, H.; Minteer, S. D.; Barton, S. C. J. Electrochem. Soc. 2011, 158, B580.
32
Porous model
Boundary conditions:
Mass balance:
x0
2
d N
dw
2
  Da
 renzyme  D aa
'
NAD

 r elelctro
'
NAD

dG
0
dw
dN
0
dw
2
d G
dw
2
 D aGlycerol  renzyme
'
x l
dN
0
dw
G 1
33
Parameters
Parametera
NADH concentration, [NADH]0
Glycerol bulk concentration, [Glycerol]∞
Reactor volume, Vol
Electrode geometric surface area, cm2
Enzyme concentration, [Enzyme]
Equilibrium constant for enzyme reaction, Keq
Turnover number of glycerol oxidation, kf
Turnover number of DHA reduction, kr
Michaelis-menten constant for NAD+, KmA
Michaelis-menten constant for glycerol, KmB
Michaelis-menten constant for NADH, KmQ
Michaelis-menten constant for DHA, KmP
Dissociation constant of NAD+, Kia
Dissociation constant of glycerol, Kib
Dissociation constant for NADH, Kiq
Dissociation constant for DHA, Kip
Film thickness, L
Diffusion coefficient for NADH/NAD+, DNh/DN
Value
10 mM
1M
10 cm3
1 cm2
1 mM
4×10-4
9.1 s-1
9.1 s-1
12 µM
440 mM
14 µM
13 mM
1.09 mM
1.5×104 mM
25 µM
11 mM
10 µm
3.3×10-8 cm2 s-1
Source
Set
Set
Set
Set
Set
Vieille lab
Vieille lab
Vieille lab
Vieille lab
Vieille lab
Vieille lab
Vieille lab
Diffusion coefficient for glycerol/DHAb, DGly
4.0×10-6 cm2 s-1
1
2
2,3
2
3
Set
1
a: parameter values regarding NADH electrocatalytic reaction have been shown in Project 1
b: 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.
34
Simulation results
DaNAD+ = 16
Daglycerol = 0.0013;
DaaNAD+ = 406;
Linear model:
Porous model:
R  A   renzym e dl
R  A   renzym e dw
'
R porous
4
R linear
35
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
• Nondimensional Damkohler numbers can provide useful approach
to simulate, predict and evaluate performance of bioreactor.
36
Thank you.
37
Supplemental information
38
Biosensor towards malate concentration
• Start with free diffusing cofactor, MDH and malate, will be
extended to immobilized cofactor/MDH
(immobilization method by Worden Lab)
60
40
20
100
500
1000
1500
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
2000
80
-2
0
Current density / µA cm
Current density / µA cm
-2
80
60
#1 Electrode
#2 Electrode
40
20
0
0
50
100
150
200
250
300
Malate Concentration / mM
39
Cystein
40
•
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.
41
Acknowledgements
• Collaborators
• Dr. Mark Worden
• Dr. Claire Vieille
• Justin Beauchamp
• The National Science Foundation
(Award CBET-0756703)
42
Principle of LDH-MTT Assay
Initially: LDH,
Lactate,
Diaphorase,
MTTox
Pyruvate
MTTox
NAD+
Diaphorase
LDH
Lactate
NADH
MTTred
www.bioassaysys.com
Very fast
Relatively slow
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.
Once the cycle starts, NADH concentration in the assay is not
changing = [NAD]+[NADH] in the sample
43
Kinetics assay using LDH-MTT Assay Kit
www.bioassaysys.com
Initially: LDH,
Lactate,
Diaphorase,
MTTox
MTTox
NAD+
Diaphorase
LDH
Lactate
MTTred
NADH
 A565 ,0 15 m in  ( A565 , t 15 m in  A565 , t  0 m in )  ([ M T Tred ]t 15 m in  [ M T T red ]t  0 )
A565  [ M TTred ]
•
Pyruvate
Linear kinetics
within 15 mins
([ M TTred ]t 15 min  [ M TTred ]t  0 )  R  15 min  k [ N AD H ]  15 min
0.8
BioAssay using NAD
only NADH
only NAD
NADH:NAD=1:1
 A565 , t  0 15 m in  [ N A D H ]

 A5 6 5 , t  0 1 5 m in  ([ N A D H ]  [ N A D ])
in the sample
A565
0.6
0.4
0.2
0.0
0
2
4
6
8
10
Pyridine necleotide/ µM
44
Modified electrodes
High-surface area electrodes for NADH electrocatalysis
Data source
Applied potential E vs.
imax
imax
RHE (mV)
(µA/cm2)
(µA)
PMG -“Bucky paper”
913
------------
600
(Yang and Liu 2009)
PBCB-SWCNT-GC
685
------------
1.2
(Doaga, McCormac et al.
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-
537
28.3
2
655
------------
45
(Villarrubia, Rincon et al.
Approach
2010)
2009)
Nafion/GC
(Zeng, Wei et al. 2006)
TBO-MWNT-GC
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
Nicotinamide
Dinucleotide
Adenine
48
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
Vmax
Vmax
(Palmore, Bertschy et al.
1998)
(Dilgin, Gorton et al. 2007)
(Radoi, Compagnone et al.
2007)
(Zhang, Smith et al. 2004)
(Liu, Zhang et al. 2010)
(Zhao, Lu et al. 2010)
(Villarrubia, Rincon et al.
2010)
(Yang and Liu 2009)
(Zeng, Wei et al. 2006)
(Doaga, McCormac et al.
2009)
(Zhu, Zhai et al. 2007)
(Huang, Jiang et al. 2007)
(Kim, Kim et al. 2010)
Free diffusing DI+ BV-GC
PTBO-GC: photoelectrocatalytic
Bulk screen-printed electrodes
modified with Prussian blue (PB)
MWCNT-Chitosan-GC
Magnetic chitosan microspheres Polythionine-GC
Single-carbon fiber microelectrode
with CNT
PMG SWCNTs-based “Bucky
paper”
PBCB-SWCNT-GC
TBO/MWNTs adduct-GC
p-DAB-MB/SWCNTs/GC
Meldola’s blue adsorbed-CNT-GC
Thionie incorporated by
DMF/CNTs-Nafion/GC
Iron oxide/carbon black-GC
potential E vs.
RHE (mV)
----------
(uA/cm2)
(uA)
---------
-----------
755
445
25.4
3.54
5
0.25
1037
705
85
141
6
10
1331
-------------
1.7
913 (pH 7
solution)
685
655
663
------------
600
----------------------8.49
1.2
45
0.6
505
537
1.6
28.3
0.4
2
643
16
4
49
For the reduction of U in polarization curves
Take one PTBO-0.85 mg/cm2 CNT-GC and PTBO-GC as an example:
Proposed reason: Impact of Mass-transport
Polarization curve: 0.5 mM NADH , 900 rpm, pH 6 PBS, 30 oC
Controlled
0.035 by mass-transport
(not controlled by applied potential)
0.7
2
Current density (mA/cm )
0.6
2
PTBO-0.85 mg/cm CNT-GC
PTBO-Bare GC
0.030
0.5
0.025
0.4
0.020
0.3
0.015
Mixed Control
(By both applied potential and mass-transport)
0.2
0.010
0.1
0.005
0.0
-0.2
0.000
-0.1
0.0
0.1
0.2
0.3
0.4
Controlled by electron-transfer rate
(controlled by applied potential)
50
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
51
Wen, H.; Nallathambi, V.; Chakraborty, D.; Barton, S. C. ECS Meeting Abstracts 2010, 1002, 366.
Characterization of PTBO and PMG films
Poly(azine) oxPoly(azine) red
•
CV in pH 6 0.1 M PBS, 50 mV/s 30 oC
CNT
Glassy carbon
Electrode
4
Current density (mA/cm )
2
2
2
Current density (mA/cm )
4
0
-2
PTBO/bare GC
2
PTBO/ 0.21 mg/cm CNT-GC
2
PTBO/ 0.85 mg/cm CNT-GC
-4
-0.8
-0.6
-0.4
-0.2
0.0
E (V) vs. Ag/AgCl
0.2
0.4
2
0
-2
PMG/bare GC
2
PMG/ 0.21 mg/cm CNT-GC
2
PMG/ 0.85 mg/cm CNT-GC
-4
0.6
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
E (V) vs. Ag/AgCl
52
0.6
• DMF: Dimethylformamid (CH3)2NC(O)H
53
Process of catalytic reaction of NADH
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.
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.

NADH  NAD  H

 2e

Or
54
CNT-modified GC electrode: Capacitance measurement
•
Carbon nanotube is coated on glassy carbon
electrode surface (3 mm diameter RDE) by dropcasting:
Drop Casting
CNT
Each CNT layer: 5ug 1mg/ml CNT-DMF suspension
Example of capacitance measurement: 0.50 mg/cm2 CNT loaded on GC
1.4
2
Current (mA/cm )
2
Charging Current (mA/cm )
1.0
0.5
0.0
-0.5
30 mV/s
50 mV/s
60 mV/s
80 mV/s
100 mV/s
-1.0
0.30
0.32
0.34
0.36
0.38
1.2
1.0
0.8
0.6
0.4
Glass
y
carbo
n
Elect
rode
Charging current
0.2
Linear fit: slope is 12.5 mF/cm
0.0
0.40
E (V) vs. Ag/AgCl
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
0
20
40
60
80
100
2
120
Scan rate (mV/s)
55
Proposed structure of Poly (MB)
N
H 3C
N
S
CH3
N
CH3
CH3
NO2 CH3
H 3C
M e th y le n e G re e n
N
S
H
N
N
CH3
N
H 3C
N
S
N
H 3C
N
CH3
S
N
CH3
CH3
N
CH3
CH3
Karyakin, A.A., et al., 1999. 11(8): p. 553-557.
56
Imax after MT correction
25
2
imax (mA/cm )
20
PMG-CNT-GC
PTBO-CNT-GC
15
10
5
0
0.0
0.2
0.4
0.6
0.8
1.0
2
Loading of CNT (mg/cm )
57
Effect of Mass transport
MT Correction
Mass balance:
 k  CS 
k d (C  C s )  

 K S  CS 
Electrochemical experiments:
i  nF D
C
x
x0
 nF k d ( C  C s )
Obtain i’max and K’s for pure kinetic control
Solid lines : kinetic
58
Bioreactor based on electronic interface
Power
supply
Anode
GlyDH
NAD+
catalyst red
Referen
ce
electro
de
Glycerol
NADH
catalyst ox
Dihydroxyaceton
e
(DHA)
Kinetics:
Step 1 r1 N A D H
  
 NAD
electrode
Step 2 r2


G lycerol  N AD    D H A  N AD H  H
GlyDH


relectro  i 
S
nF

[ NADH ]
K Nh  [ N A D H ]

k1 S
nF V
renzym e 
k cat [ E nzym e ][ G lycerol ][ N A D ]


K N A D  [ G lycerol ]  [ G lycerol ][ N A D ]  K G lycerol [ N A D ]
59
Concentration profile for substrate conversion
For the whole batch reactor:

d [ N AD ]

dt
d [ N AD H ]
dt
d [ G lycerol ]
dt
  relectro  renzym e
Initial values: t=0, [NADH] = [NADH]0 ;
[NAD+]=0;
[Glycerol] = [Glycerol]0
  renzym e
[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 mM
KNAD+ = 25 µM;
Glycerol concentration (mM)
100
80
60
40
20
0
0
2
4
6
8
10
Time (hr)
1.
3.02
hr
•
Key parameters:
Sk1/nFV ( µM/s ) 2. [Enzyme] (µM or mM)
60
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 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
61
62
How Sk1/nFV or/and [Enzyme] impact Time constant?
30
[E] = 0.1 uM
[E] = 1 uM
[E] = 10 uM
[E] = 100 uM
25
Zoom in
5
15
10
3
2
1
5
06
0
10
[E] = 0.1 uM
[E] = 1 uM
[E] = 10 uM
[E] = 100 uM
4
Time (hr)
Time (hr)
20
2
3
4
5 6 7 89
100
2
3
4
7 89
5 6 7 89
1000
100
2
3
4
5 6 7 89
1000
Sk1/nFV (uM/s)
Sk1/nFV (uM/s)
Sk1/nFV ( µM/s )
Sk1/V ( A/cm3 )
E (µM)
10 - 1000
0.002 – 0.2
1-100
63
Nondimensionalization
Variables:

[ NAD ]
x

[ N A D ]0
 Nh 
[ NADH ]
y

[ N A D ]0
N 
[ F ructose ]
f 
[ F ructose ] 0
 
Important
parameters:
Parameter
s:
F 
t
K  k cat nF 
K NADH

1 
K N AD 
V  [ N A D ] 0  nF
S  k1

[ N A D ]0
2 
K F ructose
[ F ructose ] 0
M 

dx
d
df
d

dy
d
 rho1  K  rho 2 
  M  rho 2   M 
Sk 1

[ N A D ]0
[ F ructose ] 0
k cat [ E nzym e ]
1
2

 K
DEQs

[ E nzym e ]V
x
N  x
K
yf
 F y   N h f  yf
yf
 F y   N h f  yf
[ N AD ] 0
[ Fructose ]0
Equili
brium
const
Time
ant,
const
repre
ants
sentin
for
g key
step
opera
Boundary conditions:
t=0, x=1, y=0, f=1
64
Current work: Enzyme kinetics of MtDH
• Ordered
bi bi
kinetics
NADH
k1
k-1
Fructose
Mannitol
k2
k3
k-2
NAD
k-3
k4
k-4
k
p

 
E  N AD H  F 
E  M  N AD

k
p
A:
NADH
B:
Fructo
V f V r ([ A ][ B ] 
v
V r K ia K m B  V r K m B [ A ]  V r K m A [ B ] 

V f K m Q [ A ][ P ]
K eq K ia

V f [ P ][ Q ]
K eq

[ P ][ Q ]
)
K eq
V f K mQ [ P ]

V f K m P [Q ]
K eq
V r K m A [ B ][ Q ]
K iq
1. Segel, Irwin H. (1993). New York: Wiley

K eq
V r [ A ][ B ][ P ]
K ip

 V r [ A ][ B ]
V f [ B ][ P ][ Q ]
K ib K eq
65
Enzyme kinetics of MtDH
• Definition of
parameters
A:
NADH
B:
Fructo
se
V K K
V  K K
K 


V K K
V

 K K
P:
• The values of 10 parameters
Manni I
extracted based on
tol
experimental data
K ia 
k 1
K mA 
K mB 
K iq 
k1
k4k3k P
k4
k 4
K mQ 
k1 ( k 4 k 3  k 4 k  P  k 4 k P  k 3 k P )
k 4 ( k 2 k 3  k 3 k P  k 2 k  P )
K mP 
k2 (k4 k3  k 4 k  P  k 4k P  k3k P )
k 1k 2 k  P
k 4 ( k 1k 2  k 1k P  k 1k  P  k 2 k  P )
k 1 ( k 2 k 3  k 2 k  P  k 3 k P )
k 3 ( k 1k 2  k 1k P  k 1k  P  k 2 k  P )
2
f
iq
mP
f
ip
mQ
r
ia
mB
r
ib
mA
eq
KmA
KmB
KmP
KmQ
Kia
Kiq
Vf
Vr
Keq
0.0371
39.89
8.06
0.0181
0.033
0.222
19.38
0.445
59.2
[P]/Kip=0
Kib
4.7e4Kip
1. 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. Seung Hoon, S., N. Ahluwalia, et al. (2008). "Applied Microbiology and Biotechnology: 81 (3) 485-495 81(3): 485-495.
66