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Biomimetic Interfaces for a Multifunctional B iosensor Array Microsystem

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

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  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  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

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