A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules

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


Presentation Outline

Background Dehydrogenase enzyme Bioelectronic interface

Project goals Site directed enzyme mutagenesis Characterization of bioelectronic interface

Cyclic voltammetry Chronoamperometry

Conclusions


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


Background on Enzyme

Model enzyme secondary alcohol dehydrogenase (sADH) Thermoanaerobacter ethanolicus

Thermal stability Activity range: 7°C – 95°C


Background on Enzyme

Cofactor specificity: NADP+

Amino acids affecting NADP+ affinity binding 198, 199, 200, 203, 218


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)


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


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


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


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


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-


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


Characterization Tools

Cyclic Voltammetry Calibration plots Turnover ratio Effects of increased temperatures

Chronoamperometry Electrode kinetics


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


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


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)


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


’t)+ket”Q”exp(-kett)


Chronoamperometry Wild Type-sADH

Forster equation

Best fit ket values ket= 7.0x104 s-1

Surface coverage

= 2.34x10-12 mol cm-2


’t)+ket”Q”exp(-kett)I=ket’Q’exp(-kett)


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


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


Acknowledgements

Funding Michigan Technology Tri-Corridor Department of Education GAANN Fellowship

Undergraduate students involved John Baldrey Timothy Howes


Thank you

A Bioelectronic Sensor Interface Based on Trifunctional Linking Molecules

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