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?