Transcript Development of Affordable Bioelectronic Interfaces Using

Development of Affordable
Bioelectronic Interfaces Using
Medically Relevant Soluble
Enzymes
2006 AIChE Annual Meeting
San Francisco, CA
Brian L. Hassler1, Maris Laivenieks2, Claire Vieille2, J.
Gregory Zeikus2, and Robert M. Worden1
1-Department
of Chemical Engineering and Materials Science
2-Department of Biochemistry and Molecular Biology
Michigan State University, East Lansing, Michigan
Presentation Outline
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Motivation
Dehydrogenase enzymes
Formation of bioelectronic interfaces
Characterization techniques
Results
Summary
Motivation
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Rapid detection
Identification of multiple analytes
High throughput screening
Affordable fabrication
Dehydrogenase Enzymes
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Catalyze electron transfer reactions
Cofactor dependence: NAD(P)+
Challenge: cofactor recycling
Substrate
enzyme
Substrate
Product
NAD(P)H NAD(P)HMEDox
Product
Dehydrogenase Dehydrogenase
Cofactor
Enzyme
Reaction
cofactor
enzyme
NAD(P)+
Enzyme
Reaction
cofactor mediator
+
NAD(P)MED
red
Regeneration
Enzyme Interface Assembly
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Cysteine: branched, trifunctional linker
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Thiol group: self assembles on gold
Carboxyl group: binds to electron mediator
Amine group: binds to cofactor
OH
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Mediator used
N
O
OH
O
NH2
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Toluidine Blue O (TBO)
O
O
P
O
H3C
N
O
O
P
HO
N
O
H3C
N
O
S
N
O
O
NH
O
HN
O
HS
N
O
CH3
O
NH2
B
O
N
Reaction Mechanism
TBO
CBA
EDC+/NHS*
EDC/NHS
Cysteine
Gold
Gold
Gold
NAD(P)+
Gold
Gold
Protein
Gold
Gold
*N-Hydroxysulfosuccinimide
+N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
Hassler et al., Biosensors and Bioelectronics, 21(11), 2146-2154 (2006)
Presentation Outline
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Motivation
Sensing mechanisms
Formation of bioelectronic interfaces
Characterization techniques
Results
Summary
Chronoamperometry
Technique:
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Step change in potential
Measure current vs. time
E2
Potential
Current
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E1
Parameters obtained:
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Time
Time
Electron transfer coefficients (ket)
Charge (Q)
*
*
'
'
'
"
'
"
Surface
coverage
I =k
exp(-k etet()
t)
ketetQ
Qexp(-k
t)+k et Qexp(-k et t)
Q

nFA
Zayats et al.,
Katz,
Journal
E. and
of the
I. Willner,
American
Langmuir,
Chemical
13(13),
Society,
3364-3373
124, 14724-15735
(1997)
(2002)
Cyclic Voltammetry
Technique:
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E1
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Sensitivity (slope)
Maximum turnover (TRmax)
TRmax
sat
I cat
 Io

FnA
E1
Potential
Time
Parameters obtained:
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Current
Conduct potential sweep
Measure current
Potential
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E2
Current
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Concentration
Constant Potential Amperometry
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Set constant potential
Vary analyte concentration
Current
Technique:
Parameters obtained:
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Time
Sensitivity
Current
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Concentration
Presentation Outline
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Motivation
Sensing mechanisms
Formation of bioelectronic interfaces
Characterization techniques
Results
Summary
The Current System
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Protein array
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4 working electrodes
Diameter: 3 mm
Counter electrode
Electrode formation:
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Reservoir in PDMS*
Molecular self-assembly
Different enzymes
* Polydimethylsiloxane (PDMS)
Sorbitol Dehydrogenase (SDH)
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Organism: Pseudomonas sp. KS-E1806
Cofactor dependence: NAD+
Temperature stability: 30-50C
Sorbitol
Fructose
enzyme
Dehydrogenase
Enzyme
Reaction
cofactor
mediator
NAD+
MEDred
NADH
MEDox
Cofactor
Regeneration
Chronoamperometric Response
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Substrate: Sorbitol
Concentration: 5 mM
Kinetic parameters:
k’=
 k”= 87 s-1
Surface coverage:
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690 s-1
’=
”= 8.010-12 mol cm-2
8.710-12 mol
cm-2
Current (mA)
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120
100
80
60
40
20
0
0
0.01
0.02
0.03
Time (s)
0.04
0.05
Cyclic Voltammetric Response
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Concentration range: 3-21 mM
Sensitivity: 3.4 mA mM-1 cm-2
TRmax=38 s-1
5
0
300
100
-5-100
-10
-15
Voltage (mV)
-300
Current (mA)
10
Current (mA)
15
14
12
10
8
6
4
2
0
0
10
20
Concentration (mM)
30
Amperometric Response
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Potential: -200 mV
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Concentration range: 1-6 mM
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Sensitivity: 2.8 mA mM-1 cm-2
10
Current (mA)
Current (mA)
5
4
3
2
1
8
6
4
2
0
0
0
20
40
Time (s)
60
80
0
2
4
6
Concentration (mM)
8
Other Enzymes Used
Mannitol dehydrogenase
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Organism: Lactobacillus reuteri
Reaction: Fructose
Mannitol
Cofactor specificity: NAD+
Thermal stability: 50C-90C
Other Enzymes Used
Secondary alcohol dehydrogenase
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Organism: Thermoanaerobacter ethanolicus
Reaction: 2-Propanol
Acetone
Cofactor specificity: NADP+
Thermal stability: 30C-100C
Chronoamperometric Results
Enzyme
Electron Transfer Coefficient
Substrate
-1
SDH
MDH
2ADH
Sorbitol
Mannitol
2-Propanol
k'et(s )
6843.2
5059.3
69013
* Chronoamperometric
-1
k"et(s )
870.3
452.1
NA
Surface Coverage
'(10-12 mol cm-2)
8.70.4
7.20.3
161.3
"(10-12 mol cm-2)
8.00.9
6.00.1
NA
measurements were made at a
concentration of 5 mM of the substrate.
Cyclic Voltammetry Results
Enzyme
Substrate
SDH
MDH
2ADH
Sorbitol
Mannitol
2-Propanol
Saturation Current
(Isat-mA)
11.60.3
9.90.1
7.10.4
Sensitivity
(mA mM-1 cm-2)
3.40.4
8.40.5
2.50.2
Concentration Range Turnover Rate
Low (mM) High (mM)
(s-1)
3
21
38.11.2
1
11
20.10.3
28.50.4
3
21
Conclusions
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Developed self-assembling biosensor array
Multiple analyte detection
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Sorbitol
Mannitol
2-Propanol
Characterized interfaces electrochemically
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Chronoamperometry
Cyclic voltammetry
Constant potential amperometry
Acknowledgments
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Ted Amundsen (CHEMS-MSU)
Yue Huang (EECS-MSU)
Kikkoman Corporation
Funding sources
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Michigan Technology Tri-Corridor (MTTC)
IRGP programs at MSU
Department of Education GAANN Fellowship
Thank you
Questions?