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Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes
Brian L. Hassler1, Maris Laivenieks2, Claire Vieille2, J. Gregory Zeikus2, and Robert M. Worden1
1-Department of Chemical Engineering and Materials Science2-Department of Biochemistry and Molecular Biology
Michigan State University, East Lansing, Michigan
2006 AIChE Annual MeetingSan Francisco, CA
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Presentation Outline Motivation Dehydrogenase enzymes Formation of bioelectronic interfaces Characterization techniques Results Summary
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Motivation Rapid detection Identification of multiple analytes High throughput screening Affordable fabrication
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Dehydrogenase Enzymes Catalyze electron transfer reactions Cofactor dependence: NAD(P)+
Challenge: cofactor recycling
Substrate
Product
NAD(P)+
NAD(P)HDehydrogenase
Enzyme Reaction
cofactorcofactorenzymeenzymeSubstrate
Product
NAD(P)+
NAD(P)HDehydrogenase
Enzyme Reaction
cofactorcofactorenzymeenzyme
MEDox
MEDred
Cofactor Regeneration
mediatormediator
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Enzyme Interface Assembly Cysteine: branched, trifunctional linker
Thiol group: self assembles on gold Carboxyl group: binds to electron mediator Amine group: binds to cofactor
Mediator used Toluidine Blue O (TBO)
HS
O
CH3
N
S
N
H3C
H3C
NH
HN
O
O
O B
P
O
O
O
O P
O
O
HO
O
N
N
NN
NH2
O
OH
OHN
O
NH2
O
O
O
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Reaction Mechanism
Hassler et al., Biosensors and Bioelectronics, 21(11), 2146-2154 (2006)
Cysteine TBO
EDC+/NHS*
CBA
EDC/NHSGold Gold Gold Gold
NAD(P)+ Protein
Gold Gold Gold
*N-Hydroxysulfosuccinimide +N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
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Presentation Outline Motivation Sensing mechanisms Formation of bioelectronic interfaces Characterization techniques Results Summary
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Chronoamperometry Technique:
Step change in potential Measure current vs. time
Parameters obtained: Electron transfer coefficients (ket) Charge (Q) Surface coverage ()
Time
Po
ten
tia
l
E1
E2
Time
Cu
rre
nt
Q
nFA
' ' " "
et et et et
' 'I = k Qexp(-k t)+k Qexp(-k t)*
et etI = k Q exp(-k t)
*
Zayats et al., Journal of the American Chemical Society, 124, 14724-15735 (2002)Katz, E. and I. Willner, Langmuir, 13(13), 3364-3373 (1997)
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Cyclic Voltammetry Technique:
Conduct potential sweep Measure current
Parameters obtained: Sensitivity (slope) Maximum turnover (TRmax)
max
satcat oI I
TRFn A
Time
Po
ten
tia
l
E1
E2
E1
Potential
Cu
rre
nt
ConcentrationC
urr
ent
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Constant Potential Amperometry Technique:
Set constant potential Vary analyte concentration
Parameters obtained: Sensitivity
Time
Cu
rre
nt
Concentration
Cu
rren
t
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Presentation Outline Motivation Sensing mechanisms Formation of bioelectronic interfaces Characterization techniques Results Summary
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The Current System Protein array
4 working electrodes Diameter: 3 mm Counter electrode
Electrode formation: Reservoir in PDMS*
Molecular self-assembly Different enzymes
* Polydimethylsiloxane (PDMS)
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Sorbitol Dehydrogenase (SDH) Organism: Pseudomonas sp. KS-E1806 Cofactor dependence: NAD+
Temperature stability: 30-50C
Sorbitol
Fructose
NAD+
NADHDehydrogenase
Enzyme Reaction
cofactorcofactorenzymeenzyme
MEDox
MEDred
Cofactor Regeneration
mediatormediator
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Chronoamperometric Response Substrate: Sorbitol Concentration: 5 mM Kinetic parameters:
k’= 690 s-1
k”= 87 s-1
Surface coverage: ’= 8.710-12 mol cm-2
”= 8.010-12 mol cm-2
0
20
40
60
80
100
120
0 0.01 0.02 0.03 0.04 0.05
Time (s)
Cu
rren
t (m
A)
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Cyclic Voltammetric Response Concentration range: 3-21 mM Sensitivity: 3.4 mA mM-1 cm-2
TRmax=38 s-1
-15
-10
-5
0
5
10
15
-300-100100300
Voltage (mV)
Cu
rren
t (m
A)
0
2
4
6
8
10
12
14
0 10 20 30
Concentration (mM)
Cu
rren
t (m
A)
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Amperometric Response Potential: -200 mV Concentration range: 1-6 mM Sensitivity: 2.8 mA mM-1 cm-2
0
1
2
3
4
5
0 20 40 60 80
Time (s)
Cu
rren
t (m
A)
0
2
4
6
8
10
0 2 4 6 8
Concentration (mM)
Cu
rren
t (m
A)
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Other Enzymes UsedMannitol dehydrogenase
Organism: Lactobacillus reuteri Reaction: Fructose Mannitol Cofactor specificity: NAD+
Thermal stability: 50C-90C
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Other Enzymes UsedSecondary alcohol dehydrogenase
Organism: Thermoanaerobacter ethanolicus Reaction: 2-Propanol Acetone Cofactor specificity: NADP+
Thermal stability: 30C-100C
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Chronoamperometric Results
* Chronoamperometric measurements were made at a concentration of 5 mM of the substrate.
Enzyme Substrate
k'et(s-1) k"et(s
-1) '(10-12 mol cm-2) "(10-12 mol cm-2)SDH Sorbitol 6843.2 870.3 8.70.4 8.00.9MDH Mannitol 5059.3 452.1 7.20.3 6.00.1
2 ADH 2-Propanol 69013 NA 161.3 NA
Electron Transfer Coefficient Surface Coverage
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Cyclic Voltammetry Results
Enzyme Substrate Saturation Current Sensitivity Turnover Rate
(Isat-mA) (mA mM-1 cm-2) Low (mM) High (mM) (s-1)SDH Sorbitol 11.60.3 3.40.4 3 21 38.11.2MDH Mannitol 9.90.1 8.40.5 1 11 20.10.32 ADH 2-Propanol 7.10.4 2.50.2 3 21 28.50.4
Concentration Range
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Conclusions Developed self-assembling biosensor array Multiple analyte detection
Sorbitol Mannitol 2-Propanol
Characterized interfaces electrochemically Chronoamperometry Cyclic voltammetry Constant potential amperometry
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Acknowledgments- Ted Amundsen (CHEMS-MSU) Yue Huang (EECS-MSU) Kikkoman Corporation Funding sources
Michigan Technology Tri-Corridor (MTTC) IRGP programs at MSU Department of Education GAANN Fellowship
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Thank you
Questions?