1. Michigan State University
Biomimetic Interfaces for a Multifunctional Biosensor Array Microsystem
Brian Hassler, R. Mark Worden, Andrew Mason+, Peter Kim+, Neeraj Kohli, J. Gregory Zeikus*, Maris Laivenieks*, and Robert Ofoli
Chemical Engineering and Material Science
+Electrical and Computer Engineering
*Biochemistry and Molecular Biology
Michigan State University
East Lansing, Michigan/USA
Presented at 3rd IEEE Conference on Sensors
Vienna, Austria, October 24-27, 2004
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

2. Integrated Biosensor Arrays
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Integrated Biosensor Arrays
Concept
biosensor array on a CMOS chip
readout/control circuitry
multiple nanostructured biosensor interfaces attached to the array
Advantages
extend range of measurable analytes
increase sensitivity
continuous, real-time multi-analyte measurements
easy to use: single chip, compact size
Challenges
post-CMOS integration
high performance readout circuitry
new interfaces for protein-based biosensors
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

3. Motivation Multiparameter biosensors valuable in many applications
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Motivation
Multiparameter biosensors valuable in many applications
healthcare, biomedical research, environmental monitoring, etc.
Proteins make excellent biochemical recognition elements
great diversity of molecules recognized
high specificity and sensitivity
diverse mechanisms of interaction with target molecules
Nanostructured biomimetic interfaces
pseudo-natural environments for proteins  maximize activity
nanometer dimensions  possibility of single-molecule detection
 fast response
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

4. Develop a versatile biosensor platform
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Project Goal
Develop a versatile biosensor platform
supports diverse sensing mechanisms
enzymatic reactions (generate/consume electrons)
dehydrogenase enzyme
membrane-bound protein reactions
ion channel protein (selectively transport certain ions)
can be implemented in an array on a microelectronics chip
electrically measurable outputs
electrochemical
impedance spectroscopy
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

5. Enzyme Biosensor Interfaces
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Enzyme Biosensor Interfaces
Dehydrogenase enzymes
one of few enzymes that directly transfer electrons
ideal for biosensors, easily measured (amperometry)
electrons transferred via cofactor molecule (e.g., NADH)
Challenge: regenerating cofactor after electron transfer
mediator: electron transfer without cofactor degradation S P
NAD(P)+
NAD(P)H
MEDox
MEDred
Dehydrogenase Enzyme Reaction
Cofactor Regeneration
enzyme
cofactor
S=substrate, P=product
transfer of e- directly to electrode required high electrical potentials that degrades cofactor. mediator allows e- transfer without degrading cofactor
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

6. Bioelectronic Interface
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Bioelectronic Interface
Enzyme, cofactor, mediator bound to electrode
Linear structure
ref: Willner and Katz
Mediator requires two unique binding sites
few mediators have two unique binding sites
limits range of suitable mediators
New branched structure
Mediator needs only one unique binding site
expands range of suitable mediators
Med
Elec
Cof
Enz
2 e-
Med
Elec
Cof
Enz
2 e-
transfer of e- directly to electrode required high electrical potentials that degrades cofactor. mediator allows e- transfer without degrading cofactor
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

7. Enzyme Interface Assembly
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Enzyme Interface Assembly
Secondary alcohol dehydrogenase (sADH)
from Thermoanaerobacter ethanolicus
Activity range: 15°C – 95°C
Cofactor: NADP+
Cysteine: branched, trifunctional linker
Thiol group: self assembles on gold
Carboxyl group: binds to mediator
Amine group: binds to
phenylboronic acid
phenylboronic acid spontaneously
binds to cofactor
Mediators used
Toluidine Blue O (TBO)
Nile Blue A
Neutral Red
cofactor
gold electrode
NAD+
cysteine
TBO
mediator
linker
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

8. Branched Trifunctional Linker
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Branched Trifunctional Linker
cofactor
gold electrode
NAD+
cysteine
TBO
mediator
linker
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

9. Ion Channel Sensor Membrane proteins Biomimetic sensor interface
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Ion Channel Sensor
Membrane proteins
found embedded in lipid bilayer
require bilayer for activity
Biomimetic sensor interface
synthetic bilayer on electrode
protein embedded in bilayer
Example: ion-gated channel protein
Gramicidin D
from Bacillus brevis
Ion selectivity
monovalent cations
Protein channel
Lipid bilayer
Aqueous layer
Spacer molecules
Electrode
bacillus brevis is a microorganism
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

10. Ion Channel Interface Assembly
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Ion Channel Interface Assembly
PEG spacer molecule
Thiol end group binds to gold
Lipid end group binds to bilayer
Provides space between BLM and electrode
room for proteins to extend beyond BLM
space for ions traveling through protein
Bilayer deposited from liposomes
Dioleoylphosphatidylcholine (DOPC) lipid
Gramicidin embedded in liposomes
Deposited on PEG spacer molecule
“peg” strand of polymer, chain of molecules
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

11. Prototype Integrated 3-Electrode System
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Prototype Integrated 3-Electrode System
Conventional electrochemistry
Integrated 3-electrode system
CMOS compatible
presented at Sensors 2003 Ag
KCl AE RE
Ag/AgCl
Reference Electrode WE
Conventional
Top View
Nafion
Ag/AgCl Ag PR
Ti/Au AE WE RE
SiO2 Si
Cross Section View
Integrated Electrode
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

12. Integrated 3-Electrode System: Test Results
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Integrated 3-Electrode System: Test Results
Fabricated macro-scale prototype integrated EC system
Test setup and results
conventional
instrument
silicon-based three electrode system (left) with
and (right) without a test sample
integrated
3-electrode system _
Vapp +
Cyclic voltammetry setup
ferricyanide electrochemical cell
applied voltage: -350mV ~ +350mV
output: current (A range)
temp: room temperature A
effect of ferricyanide concentration: cyclic voltammograms obtained using the integrated three-electrode system PR AE WE RE
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

13. Results with a Tethered Lipid Bilayer
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Results with a Tethered Lipid Bilayer
Biosensor interface formation
lipid bilayer on gold electrode
Test results
minimal current leakage
good insulation between electrode and sample
temperature stability
tested at 4°C, 25°C and 40°C
Comparison of cyclic voltammograms for (a) bare gold substrate, (b) thiol modified substrate, and (c) lipid modified substrate.
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

14. Test Results: Enzyme Interface Assembly
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Test Results: Enzyme Interface Assembly
Using Cyclic Voltammetry
Isopropanol detected
Concentration varied
5 to 35mM
Linear calibration plot
slope: 1.7 mA/mM
electrode area: 1.21cm2
test results for
enzyme biosensor
sADH
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

15. Test Results: Ion Channel Interface Assembly
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Test Results: Ion Channel Interface Assembly
Using Cyclic Voltammetry
Thallium detected by sensor and passed to electrode
Monovalent cations passed by gramicidin
Ferricyanide not detected at electrode
Anions not passed by gramicidin
test results for
ion channel membrane protein biosensor
Gramicidin D
peak for ferricyanide would be off scale to the left, mV. just not shown on this plot. would have large current even at range shown in plot.
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

16. Electrochemical Electrode Array on a CMOS Chip
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Electrochemical Electrode Array on a CMOS Chip
CMOS chip with potentiostat
low-noise current measurement (~1pA)
cyclic voltammetry
Post-CMOS electrode array
three-electrode electrochemical system
Benefits
integrate sensors & circuitry
lower noise = higher resolution
microfabrication
high density biosensor arrays
utilize versatile biosensor interfaces
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

17. Conclusions Protein-based biosensor interfaces developed
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Conclusions
Protein-based biosensor interfaces developed
versatile, suitable for broad classes of proteins
dehydrogenase enzymes
channel proteins
suitable for electrical measurements
Biosensor interfaces bound to gold electrodes on a silicon substrate
sensor operation verified, analytes measured
Future Work
combine sensors with on-chip readout circuitry
form fully integrated biosensor array microsystem
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004

18. Integrated 3-Electrode System: Process Steps
Four mask design
Mask#1 – Patterned three electrodes (200Å Ti/ 1500Å Au)
Mask#2 – Reference electrode (1500Å Ag)
Mask #3 – To formed Ag/AgCl with Nafion coated layer
Mask #4 – Passivation opening (PR or SiO2)
prototype integrated electrode process flow
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004