Transcript Document

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
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
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
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
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
P
enzyme
S
MEDred
NAD(P)+
Dehydrogenase
Enzyme
Reaction
cofactor
MEDox
NAD(P)H
Center for Nanostructured Biomimetic Interfaces
Cofactor
Regeneration
IEEE Sensors Conf., Oct. 2004
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Bioelectronic Interface
• Enzyme, cofactor, mediator bound to electrode
2 e• Linear structure
– ref: Willner and Katz
Enz
2 e-
Med
Cof
Elec
– Mediator requires two unique binding sites
• few mediators have two unique binding sites
• limits range of suitable mediators
• New branched structure
Enz
– Mediator needs only one unique binding site
• expands range of suitable mediators
Center for Nanostructured Biomimetic Interfaces
Cof
2
Elec
eMed
2 e-
IEEE Sensors Conf., Oct. 2004
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
cofactor
– Thiol group: self assembles on gold
– Carboxyl group: binds to mediator
– Amine group: binds to
phenylboronic acid
NAD+
• phenylboronic acid spontaneously
binds to cofactor
• Mediators used
– Toluidine Blue O (TBO)
– Nile Blue A
– Neutral Red
Center for Nanostructured Biomimetic Interfaces
TBO
mediator
cysteine
gold electrode linker
IEEE Sensors Conf., Oct. 2004
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Branched Trifunctional Linker
cofactor
NAD+
TBO
mediator
gold electrode
Center for Nanostructured Biomimetic Interfaces
cysteine
linker
IEEE Sensors Conf., Oct. 2004
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Ion Channel Sensor
• Membrane proteins
– found embedded in lipid bilayer
– require bilayer for activity
Protein channel
• Biomimetic sensor interface
Lipid
bilayer
– synthetic bilayer on electrode
– protein embedded in bilayer
• Example: ion-gated channel
protein
Aqueous
layer
– Gramicidin D
• from Bacillus brevis
– Ion selectivity
Electrode
Spacer
molecules
• monovalent cations
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004
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
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Prototype Integrated 3-Electrode System
• Conventional electrochemistry
Ag
KCl
AE
Ag/AgCl
Reference Electrode
RE
WE
Conventional
Top View
• Integrated 3-electrode system
– CMOS compatible
– presented at Sensors 2003
PR
AE
WE
RE
Nafion
Ag/AgCl
Ag
Ti/Au
SiO2
Si
Cross Section View
Integrated Electrode
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004
Introduction
Biosensor Interfaces
Results
Integrated System
Conclusion
Integrated 3-Electrode System: Test Results
• Fabricated macro-scale prototype integrated EC system
Working Electrode
O-ring: Sample Container
Auxilary Electrode
Reference Electrode
conventional
instrument
silicon-based three electrode system (left) with
and (right) without a test sample
• Test setup and results
integrated
3-electrode system
_
Vapp
+
A
PR
AE
WE
 Cyclic voltammetry setup
 ferricyanide electrochemical cell
 applied voltage: -350mV ~ +350mV
 output: current (A range)
RE
 temp: room temperature
Center for Nanostructured Biomimetic Interfaces
effect of ferricyanide
concentration: cyclic
voltammograms
obtained using the
integrated threeelectrode system
IEEE Sensors Conf., Oct. 2004
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
Center for Nanostructured Biomimetic Interfaces
Comparison of cyclic voltammograms for
(a) bare gold substrate, (b) thiol modified
substrate, and (c) lipid modified substrate.
IEEE Sensors Conf., Oct. 2004
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 A/mM
– electrode area: 1.21cm2
test results for
enzyme biosensor
sADH
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004
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
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004
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
Counter Reference
Vsrc
Vref
Working
– three-electrode electrochemical system
Phi1
phi2
phi2
Cint
Cf
Phi1 Cs
Current from
working elec
Phi1
Vout
phi2
Sample To A/D
and
Hold
• 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
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
Integrated 3-Electrode System: Process Steps
Four mask design
Si/SiO 2
1.
2.
3.
4.
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)
Mask 1: Deposited and Lift-off Ti/Au
Mask 2: Deposited and Lift-off Ag
Mask 3: AgCl Reference Electrode
Mask 4: Passivation Layer Opening
prototype integrated electrode process flow
Center for Nanostructured Biomimetic Interfaces
IEEE Sensors Conf., Oct. 2004