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Biomimetic Interfaces for a Multifunctional B iosensor 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

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biomimetic interfaces for a multifunctional b iosensor array microsystem

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

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

motivation

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

project goal

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

enzyme biosensor interfaces

Introduction

Biosensor Interfaces

Results

Integrated System

Conclusion

NAD(P)+

MEDred

S

enzyme

cofactor

Dehydrogenase Enzyme Reaction

MEDox

P

NAD(P)H

Cofactor Regeneration

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

Center for Nanostructured Biomimetic Interfaces

bioelectronic interface

Introduction

Biosensor Interfaces

Results

Integrated System

Conclusion

Med

Med

Enz

Enz

Cof

Cof

Elec

Elec

2 e-

2 e-

2 e-

2 e-

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

Center for Nanostructured Biomimetic Interfaces

enzyme interface assembly

Introduction

Biosensor Interfaces

Results

Integrated System

Conclusion

NAD+

TBO

cysteine

gold electrode

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

mediator

linker

Center for Nanostructured Biomimetic Interfaces

branched trifunctional linker

Introduction

Biosensor Interfaces

Results

Integrated System

Conclusion

NAD+

TBO

cysteine

gold electrode

Branched Trifunctional Linker

cofactor

mediator

linker

Center for Nanostructured Biomimetic Interfaces

ion channel sensor

Introduction

Biosensor Interfaces

Results

Integrated System

Conclusion

Protein channel

Lipid bilayer

Aqueous layer

Spacer molecules

Electrode

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

Center for Nanostructured Biomimetic Interfaces

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

Center for Nanostructured Biomimetic Interfaces

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

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

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

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

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

Center for Nanostructured Biomimetic Interfaces

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

conclusions

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

integrated 3 electrode system process steps
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