Development of Affordable Bioelectronic Devices Based on Soluble and Membrane Proteins
80th ACS Colloids and Surface Science Symposium
University of Colorado at Boulder June 20, 2006
Brian L. Hassler, Aaron J. Greiner, Sachin Jadhav, Neeraj Kohli, Robert M. Worden, Robert Y. Ofoli, Ilsoon Lee
Department of Chemical Engineering and Materials ScienceMichigan State UniversityEast Lansing, MI 48823
Outline• Motivation• Interface chemistry for both soluble and
membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions
Motivation• Rapid detection• Multi-analyte identification• High throughput screening for the
pharmaceutical industry• Identification of pathogens• Affordable fabrication
Interface for dehydrogenase enzymes
• Mediator integration Linear approach
• Electron mediator Pyrroloquinoline quinone (PQQ)
ENZ
MED
ne-
ne-
GOLD
ENZ MED
ne-
ne-
GOLD
• Mediator integration Linear approach Branched approach
• Electron mediators Neutral red Nile blue A Toluidine blue O
ENZ MED
ne-
ne-
GOLDZayats et al., Journal of the American Chemical
Society, 124, 14724-15735 (2002)
Reaction Mechanism
Hassler et. al, Biosensors and Bioelectronics, 77, 4726-4733 (2006)
Mobile lipid
Reservoir lipid
Spacer molecule
Membrane protein
Interface for membrane proteins
Gold electrode
Raguse et. al, Langmuir, 14, 648 (1998)
Outline• Motivation• Interface chemistry for both soluble and
membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions
Chronoamperometry• Technique:
Induce 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
Cyclic voltammetry• Technique:
Conduct potential sweep Measure current density
• Parameters obtained: Peak current
• Electrode area (A)• Scan rate (v)
• Concentration (CA)
Sensitivity Maximum turnover (TRmax) Potential
Cu
rre
nt
Time
Po
ten
tia
l
E1
E2
E1
Constant potential amperometry• Technique:
Set constant potential Vary analyte concentration
• Parameters obtained: Sensitivity (slope)
Time
Cu
rre
nt
Concentration
Cu
rre
nt
Impedance spectroscopy• Technique:
Apply sinusoidal AC voltage (Vac) on top of a constant DC voltage (Vdc):
Measure resistance
• Parameters obtained: Membrane capacitance (CM)
Membrane resistance (RM)
Vapplied = Vdc + Vac sin ωt
Model equivalent circuit
RM: Resistance of the membrane containing the ion channels
CM: Capacitance of membrane
RS: Resistance of the solution
CDL: Double layer capacitance
RS
CM
RM
CDL
Outline• Motivation• Interface chemistry for both soluble and
membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions
Experimental protocol• Secondary alcohol dehydrogenase (2 ADH)• Bacteria: Thermoanaerobacter ethanolicus
Thermostable Cofactor dependent
• Reaction mechanism
2-Propanol+NADP+ Acetone +NADPH
MEDOX+NADPH MEDRED+NADP+
MEDRED MEDOX
2 ADH
Chronoamperometry results• Cofactor: NADP+
• Equation:et etI = k Qexp(-k t)
ket= 4.8×102 s-1
= 2.1×10-11 mol cm-2
-2.50E-05
2.50E-05
7.50E-05
1.25E-04
1.75E-04
0 0.01 0.02 0.03 0.04 0.05
Time (s)
Cu
rren
t (A
)
Zayats et al., Journal of the American Chemical Society, 124, 14724-15735 (2002)
Cyclic voltammetry results• Concentration range: 5 – 25 mM• Sensitivity: 3.8 A mM-1 cm-2
• TRmax=37 s-1
-30
-20
-10
0
10
20
30
-2000200400
Voltage (mV)
Cu
rren
t (
A)
0
5
10
15
20
25
0 10 20 30Concentration (mM)
Cu
rren
t (
A)
Amperometric detection• Potential: -200 mV• Concentration range: 1-6 mM• Sensitivity: 2.81 A mM-1 cm-2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0 20 40Time (s)
Cu
rre
nt
(A
)
0
1
2
3
4
0 2 4 6 8
Concentration (mM)
Cu
rren
t ( A
)
Impedance spectroscopy• Membrane capacitance: 1.17 µF cm-2
• Membrane resistance: 0.68 M cm2
• Resistance with valinomycin: 0.19 M cm2
4.5
4.7
4.9
5.1
5.3
5.5
5.7
5.9
-2 -1 0 1
Log(freq) (Hz)
Lo
g Z
(
)
After addition of valinomycin
Before addition of valinomycin
Outline• Motivation• Interface chemistry for both soluble and
membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions
Motivation for use of microfluidics• Precise control over channel geometry• Precise control over flow conditions• Small sample volumes• Ease of fabrication using PDMS
Integration with microfluidics• Soft lithography• Channel dimensions: (300µm x 35µm)
Si
PDMS
Glass
PDMS
Layout of microfluidics system
Working Electrodes
Auxiliary Electrode
Inlet Outlet
Torque-Actuated Valves
Inlet/Outlet Ports
Microfluidic Channels
Torque-actuated valves
Glass
PDMS
Urethane
Whitesides et al., Analytical Chemistry, 77, 4726-4733 (2005)
Zayats model
Torque-actuated valves
Torque-actuated valves
Outline• Motivation• Interface chemistry for both soluble and
membrane proteins• Electrochemical characterization• Experimental results• Integration with microfluidics• Conclusions
Conclusions• Developed self-assembling biosensor interfaces
Dehydrogenases Ionophores
• Characterized interfaces electrochemically Chronoamperometry Cyclic voltammetry Constant potential amperometry Impedance spectroscopy
• Fabricated electrode arrays with microfluidics Photolithography Soft lithography Torque-actuated valves
Acknowledgments• Yue Huang:
Electrical Engineering (MSU)
• Dr. J. Gregory Zeikus: Biochemistry and Molecular Biology (MSU)
• Ted Amundsen Chemical Engineering (MSU)
Thank you
Questions?