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A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules. Brian Hassler, Megan Dennis, Maris Laivenieks * , Robert Y. Ofoli, J. Gregory Zeikus * , and R. Mark Worden Chemical Engineering and Material Science * Biochemistry and Molecular Biology Michigan State University - PowerPoint PPT Presentation
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Center for Nanostructured Biomimetic Interfaces
A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules
Brian Hassler, Megan Dennis, Maris Laivenieks*, Robert Y. Ofoli, J. Gregory Zeikus*, and R. Mark Worden
Chemical Engineering and Material Science*Biochemistry and Molecular Biology
Michigan State UniversityEast Lansing, Michigan
Presented at 2004 Annual AIChE ConferenceNovember 7 - 12, 2004, Austin, TX
Center for Nanostructured Biomimetic Interfaces
Presentation Outline
Background Dehydrogenase enzyme Bioelectronic interface
Project goals Site directed enzyme mutagenesis Characterization of bioelectronic interface
Cyclic voltammetry Chronoamperometry
Conclusions
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Background
Dehydrogenase enzymes Catalyze electron transfer reactions
• Activity easily measured electrochemically• Bioelectronic applications
Often require cofactor (e.g., NAD(P)+) Challenge: regenerating cofactor after reaction
S
P
NAD(P)+
NAD(P)HDehydrogenase
Enzyme Reaction
cofactorcofactorenzymeenzyme
MEDox
MEDred
Cofactor Regeneration
mediatormediator
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Background on Enzyme
Model enzyme secondary alcohol dehydrogenase (sADH) Thermoanaerobacter ethanolicus
Thermal stability Activity range: 7°C – 95°C
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Background on Enzyme
Cofactor specificity: NADP+
Amino acids affecting NADP+ affinity binding 198, 199, 200, 203, 218
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Electron mediator required Shuttles electrons between electrode and
cofactor Prevents cofactor degradation
Linear structure Mediator requirements
• Two unique functional groups– Bind to electrode– Bind to cofactor
• Few suitable mediators
Background on Cofactor Regeneration
Med ElecCofEnz
2 e- 2 e-
(Zayats, et al., J. Am. Chem. Soc. 2002, 124, 14724-14735)
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Research Goals
Enhance enzyme activity with NAD+
Retain thermal stability Generate a unique electron transfer scaffold
Using a hetro-trifunctional linking molecule Suitable for wider range of electron mediators
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Enzyme Mutagenesis
5’ primer
Mutant primer
3’ primer
Wild type template Wild type template
endMutant 5’- Mutant 3’ end-
PCR amplification 1
PCR amplification 2
Complete mutant
5’ primer
3’ primer
5’ primer
3’ primer
Mutant primer
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Clone adhB Gene Into pCR 2.1 Vector
Insert mutant gene into lacZ gene
Cells with plasmid will have ampicillin & kanamycin resistance
Transformed cell containing the PCR product will grow white on X-gal
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Enzymatic Activities of Wild Type, Mutant Strains
NADP+
NAD+
EnzymeSpecific Activity
(units/mg)Ratio
Wild-Type 46.5 1.00Y218 F Mutant 36.5 0.78
EnzymeSpecific Activity
(units/mg)Ratio
Wild-Type 23.4 1.00Y218 F Mutant 28.7 1.23
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Linear structure Mediator requirements
• Two unique functional groups• Few suitable mediators
Branched structure Mediator requirements
• Single functional group• Many suitable mediators
Cofactor Regeneration by Electrode
Med ElecCofEnz
2 e- 2 e-
Med
Elec
CofEnz
2 e-
2 e-
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gold electrode
NAD+
cysteine
TBO
Enzyme Interface Assembly
Cysteine: branched, trifunctional linker Thiol group: self assembles on gold Carboxyl group: binds to electron mediator Amine group: binds to
phenylboronic acid
Mediators used Toluidine Blue O (TBO) Nile Blue A Neutral Red
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Characterization Tools
Cyclic Voltammetry Calibration plots Turnover ratio Effects of increased temperatures
Chronoamperometry Electrode kinetics
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Cyclic Voltammetry Y218F-mutant sADH
Cyclic voltammetry
Substrate: Isopropanol, in phosphate buffer, pH=7.4
• High voltage: 400mV• Low voltage: -200 mV• Scan rate: 100 mV/s• Electrode area: 1 cm2
Calibration plot:• Slope: 1 A/mM
• Isat= 42A
Turnover ratio: 65 s-1
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Cyclic Voltammetry Wild-Type sADH
Cyclic voltammetry
Substrate: Isopropanol, in phosphate buffer, pH=7.4• High voltage: 400mV• Low voltage: -200 mV• Scan rate: 100 mV/s• Electrode area: 1 cm2
Calibration plot:• Slope: 1.67 A/mM
• Isat= 80A
Turnover ratio: 450 s-1
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Chronoamperometry
Procedure Step change in potential
• Initial Potential (E1): -200 mV
• Final Potential (E2): 400 mV
Plot current vs. time
Characterization Equation
• Measurable variables
• ket= Electron transfer constant
• Q= Charge associated with oxidation/reduction
I=ket’Q’exp(-ket
’t)+ket”Q”exp(-kett)
(Forster, R. J. Langmuir 1995, 11, 2247-2255)
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Chronoamperometry Y218F-mutant sADH-NAD+
Forster equation
Best fit ket values ket
’= 7.0x104 s-1
ket”= 5.5x103 s-1
Surface coverage=Q/nFA
’= 9.56x10-13 mol cm-2
”= 7.55x10-12 mol cm-2
I=ket’Q’exp(-ket
’t)+ket”Q”exp(-kett)
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Chronoamperometry Wild Type-sADH
Forster equation
Best fit ket values ket= 7.0x104 s-1
Surface coverage
= 2.34x10-12 mol cm-2
I=ket’Q’exp(-ket
’t)+ket”Q”exp(-kett)I=ket’Q’exp(-kett)
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Determination of Thermostability
Temperatures Measured 25 °C (I= 9 A) 35 °C (I= 15 A) 45 °C (I= 21 A) 50 °C (I= 25 A) 60 °C (I= 38 A) 65 °C (I= 8 A) 0
0.5
1
1.5
2
2.5
3
3.5
4
0.0029 0.003 0.0031 0.0032 0.0033 0.0034
(1/Temperature) (1/K)
Ln
Cu
rren
t
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Conclusions
Mutant sADH developed Increased activity with NAD+
Novel electron transfer scaffold developed Trifunctional linking molecule Wider range of mediators
Bioelectronic interface with sADH developed Electrode kinetics measured Calibration curves developed Stable up to 60 °C
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Acknowledgements
Funding Michigan Technology Tri-Corridor Department of Education GAANN Fellowship
Undergraduate students involved John Baldrey Timothy Howes
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