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

Presentation Outline Motivation Dehydrogenase enzymes Formation of bioelectronic interfaces Characterization techniques Results Summary

Motivation Rapid detection Identification of multiple analytes High throughput screening Affordable fabrication

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

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

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

Presentation Outline Motivation Sensing mechanisms Formation of bioelectronic interfaces Characterization techniques Results Summary

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)

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

Constant Potential Amperometry Technique:

Set constant potential Vary analyte concentration

Parameters obtained: Sensitivity

Time

Cu

rre

nt

Concentration

Cu

rren

t

Presentation Outline Motivation Sensing mechanisms Formation of bioelectronic interfaces Characterization techniques Results Summary

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)

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

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)

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)

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)

Other Enzymes UsedMannitol dehydrogenase

Organism: Lactobacillus reuteri Reaction: Fructose Mannitol Cofactor specificity: NAD+

Thermal stability: 50C-90C

Other Enzymes UsedSecondary alcohol dehydrogenase

Organism: Thermoanaerobacter ethanolicus Reaction: 2-Propanol Acetone Cofactor specificity: NADP+

Thermal stability: 30C-100C

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

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

Conclusions Developed self-assembling biosensor array Multiple analyte detection

Sorbitol Mannitol 2-Propanol

Characterized interfaces electrochemically Chronoamperometry Cyclic voltammetry Constant potential amperometry

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

Thank you

Questions?