Integrated Nanoscale Ion-Channel Sensor

1. Integrated Nanoscale Ion-Channel Sensor

2. AgCl Electrode Oxide SU-8 Resist Si Project Goals Goal 1:Embed channels in an integrated device that maintains stable potential across them and allows recording of stable, artifact free current through them. Goal 2: Find simulants that bind and transiently block conduction of ions through OmpF.** we shall work with DARPA and other groups within the MOLDICE network to incorporate ion channels that show desired properties Lipid Bilayer with Ion Channels Important building blocks of a fully integrated biosensor with on-chip sensing and signal processing

3. Technical Approach For the fabrication … Challenges we are facing • silicon substrates are used • layers are structured by • conventional optical lithography • the aperture that supports the • bilayers is constructed using • deep silicon dry etching • relation between the size of the • lipid bilayer and its stability and • the signal-to-noise ratio of the • ion channel response • ultimate limit for the size scaling • of the sensor • optimal surface treatment for • bilayer attachment • stability of the integrated • reversible Ag/AgCl electrodes • manufacturability of the sensor • usability issues (reusability, • cleaning, automation) Experiments involve … • impedance analysis of bilayers • current-voltage measurements • of bilayers and porin channels • studying the influence of surface modification layers on bilayer Gigaseal formation

4. Summary sheet Milestones Accomplishments • maintain stable potential • (± 1 mV for 1 hour) across a • single channel of OmpF porin • recording of stable, artifact- • free current voltage curves • (± 100 pA for 1 hour) from a • single channel of OmpF porin • using external electrodes • recording stable current • voltage curves using inte- • grated Ag/AgCl electrodes • design and process flow-chart • for a silicon bilayer support chip • working proof-of-concept in form • of a silicon chip as a direct Teflon • membrane replacement • Gigaseal formation proven • channel insertion succeeded • PTFE layers deposited by plasma • CVD facilitate bilayer formation • planar AgCl electrodes exhibit • desired properties

5. Summary sheet Demonstration of Results Technology Transition • measure sealing resistance • on samples with different • geometries and surface • properties • measure Nernst potential of • Ag/AgCl electrodes • measure DC potential across • porin • measure current through porin • construct a silicon-based sensor • template (reusable if possible) • along with a fixture to allow easy • bilayer formation and protein • insertion • development of a procedure to • reproducibly create bilayers with • Gigaseals • work with DARPA and other • groups within the MOLDICE net- • work to incorporate ion channels • that show desired properties

6. Microfabrication Details(ASU)

7. 150 mm 150 mm 825 Resist, 1 mm thickness SU-8 Resist Si Si Si Substrate AZ 4330 Resist, 2.6 mm thickness AgCl Electrode, up to 1 mm thickness Thermally Grown Oxide, d = 500 nm AgCl SU-8 Resist Si Si Si 250 mm 1 mm Hydrophobic Layer 300 mm 150 mm 50 mm SU-8 Resist Photoresist Si AgCl Si SU-8 Resist Si Bilayer Process Flow Resist for Initial Hole Etching Small Hole Etching AgCl Electrode Large Hole Etching Thermal Oxidation Surface Modification Layer Resist for Small Hole Etching SU-8 Resist (Epoxy) Lipid Bilayer Attachment

8. Process optimization 250 mm • deep silicon etch process that • is optimized on high etch rate • (4.7 mm/min), good selectivity • (220:1) and a concave bottom • profile • etch process that exhibits vertical • sidewalls and a low aspect ratio • dependent etch rate of 3.7 mm/min • with planar bottom profiles below • 100 mm ridge width

9. Process optimization 250 mm • switch to double-side polished • 100 mm (4”) wafer with 380 mm • thickness allows the fabrication • of multiple samples per run with • identical geometry • front and backside have a smooth • surface and the etching does not • roughen the lower surface • optimized backside alignment re- • sults in good centering of the hole

10. Sample comparison 250 mm • conventional hole preparation • using electrical discharge to • create an aperture in a PTFE • sheet of 25 mm thickness • using deep silicon dry etching • and back side alignment photo- • lithography a small hole (150 mm) • was created inside a recess

11. PTFE Surface Modification • the stability of the lipid bilayer • is related to the contact angle • between the bilayer and the • supporting substrate • water contact angle measure- • ments can be used to determine • the substrate’s surface energy Torus g Bilayer Substrate • coating the oxide surface with a • Teflon film changes its properties • from hydrophilic to hydrophobic • (small to large contact angle) • using Plasma CVD is a novel • method that provides an easy • way to deposit thick PTFE layers

12. Lipid Bilayer Experiments(Rush)

13. Hole diameter = 150 mm PTFE coated surface Lipid Bilayer Experiments • Experiment showing the opening of a single • OmpF porin channel. The vertical lines • through the red current trace are an artifact • from stirring of the bath to facilitate the • insertion of porin into the bilayer membrane. • Plot showing the different levels of OmpF • porin (Trimer). Level 1 is not shown. All the • traces in the above plot are from the same • OmpF porin bilayer experiment using the • silicon wafer coated with PTFE (Teflon).

14. Lipid Bilayer Experiments • physiological behavior of OmpF • response is indistinguishable • from channels in Teflon • supported membranes • reproducibility of measurements • and voltage dependence • indicates that switching is not an • artifact but real channel activity

15. Ag/AgCl Electrodes(ASU)

16. Integrated AgCl Electrodes AgCl ring on oxide AgCl ring on SU-8 (chloridized) Schematic view of the electrode layout 3 mm • silver is evaporated on both • sides of the wafer (> 500 nm) • layer patterning by photo- • lithography and etching • chloridization in 5% NaOCl • for 30 sec • measurements are performed • using 0.1M or 0.5M KCl • reference solutions

17. Integrated AgCl Electrodes AgCl layer, chloridized in 5% NaOCl 10 8 0.5M (trans) and 0.6M (cis) 0 KCl Test solutions 6 -20 4 2 Potential difference (mV) -40 Potential difference (mV) 0 -2 -60 -4 -6 -80 Simulation -8 Measurement -10 -100 0 1 2 3 4 5 0 1 2 3 4 5 Time (h) AgCl Electrode Potential, Single substrate 0.1M KCl Reference solution KCl Molarity difference (M) • minimal difference between the • expected and measured Nernst • potential variation with KCl • concentration • no notable difference between • electrodes on oxide and epoxy • good potential stability of • the microstructured electrodes

18. AgCl Electrode AgCl layer after 5 h measurement AgCl layer before measurement • difference between expected • and measured potential due to • partially chloridized surface • longterm failure mechanism: • AgCl gets dissolved in the KCl • electrolyte

19. Making a Calcium Channel(Rush)

20. Theory, Simulation, Experiment show Crowded Charge  Selectivity Make a Calcium Channel by Site-directed Mutagenesis George Robillard, Henk Mediema, Wim Meijberg BioMaDe Corporation, Groningen, Netherlands

21. D113 D113 E117 E117 Site-directed R132 E132 mutagenesis R82 A82 R42 E42 Wild type WT EAE mutant Strategy Use site-directed mutagenesis to put in extraglutamates and create anEEEElocusin the selectivity filter of OmpF George Robillard, Henk Mediema, Wim Meijberg BioMaDe Corporation, Groningen, Netherlands

22. Ca2+ over Cl- selectivity (PCa/PCl) recorded in 1 : 0.1 M CaCl2 IV-Plot Zero-current potential or reversal potential = measure of ion selectivity Henk Mediema Wim Meijberg

23. Make a Calcium Channel by constructing the right Charge, Volume, Dielectric Selectivity arises from Electrostatics and Crowding of Charge Precise Arrangement of Atoms is not involved

24. Conclusions Accomplishments Future work under Phase I • a silicon bilayer support chip has • been constructed and successful • Gigaseal formation has been • demonstrated • channel insertion succeeded • first milestones have been • achieved • integration of the reversible • electrodes demonstrated • PTFE layers deposited by plasma • CVD exhibit excellent properties • measure single channels in an integrated device • study the relation between the size of the lipid bilayer and the signal-to-noise ratio • find optimal surface treatment for bilayer attachment • find simulants that bind and transiently block conduction of ions through ompF • work with DARPA and other groups MOLDICE groups to incorporate ion channels that show desired properties

25. Si Bilayer 2) Project Goals 1) Project Details Title: Integrated Nanoscale Ion Channel Sensor Start Date: December 15th 2003 End Date: December 31st 2004 (Phase I) Partners: Marco Saraniti (IIT) Bob Eisenberg (Rush) Steve Goodnick (ASU) Trevor Thornton (ASU) • embed channels in a membrane device that maintains stable potential across them and allows recording of stable, artifact free current through them. • Simulants will be found that bind and transiently block conduction of ions through ompF. Plus: Dr. J. Tang (Rush), Dr. M. Goryll (ASU), Dr. G. Laws (ASU), Mr. S. Wilk (ASU) and Mr. D. Marreiro (IIT) 3) “Phase I’ Deliverables 4) Future Plans - issues to be addressed • membrane stabilization • simulants detection • identifying stochastic signatures • ……….. ▪ demonstrate ‘Gigaseal’ properties ▪ demonstrate reversible electrodes ▪ measure single channels with integrated device ▪ characterize stability of integrated device