1. DSO DARPA Integrated Nanoscale Ion-Channel Sensor

2. DSO DARPA 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 AgCl Electrode Oxide SU-8 Resist Si Lipid Bilayer with Ion Channels Important building blocks of a fully integrated biosensor with on-chip sensing and signal processing

3. DSO DARPA Technical Approach 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) Challenges we are facingFor the fabrication … impedance analysis of bilayers current-voltage measurements of bilayers and porin channels studying the influence of surface modification layers on bilayer Gigaseal formation Experiments involve …

4. DSO DARPA Summary sheet 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 MilestonesAccomplishments 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. DSO DARPA Summary sheet 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 Demonstration of ResultsTechnology Transition 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. DSO DARPA Microfabrication Details (ASU)

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

8. DSO DARPA 250  m deep silicon etch process that is optimized on high etch rate (4.7  m/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  m/min with planar bottom profiles below 100  m ridge width Process optimization

9. DSO DARPA 250  m switch to double-side polished 100 mm (4”) wafer with 380  m 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 Process optimization

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

11. DSO DARPA 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 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  Bilayer Torus Substrate

12. DSO DARPA Lipid Bilayer Experiments (Rush)

13. DSO DARPA 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). Lipid Bilayer Experiments Hole diameter = 150  m PTFE coated surface

14. DSO DARPA 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. DSO DARPA Ag/AgCl Electrodes (ASU)

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

17. DSO DARPA Integrated AgCl Electrodes AgCl Electrode Potential, Single substrate Simulation Measurement 012345 -100 -80 -60 -40 -20 0 Potential difference (mV) KCl Molarity difference (M) 0.1M KCl Reference solution no notable difference between electrodes on oxide and epoxy good potential stability of the microstructured electrodes minimal difference between the expected and measured Nernst potential variation with KCl concentration 0.5M (trans) and 0.6M (cis) KCl Test solutions AgCl layer, chloridized in 5% NaOCl Potential difference (mV) 012345 -10 -8 -6 -4 -2 0 2 4 6 8 10 Time (h)

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

19. DSO DARPA Making a Calcium Channel (Rush)

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

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

22. DSO DARPA Zero-current potential or reversal potential = measure of ion selectivity Henk Mediema Wim Meijberg Ca 2+ over Cl - selectivity (P Ca /P Cl ) recorded in 1 : 0.1 M CaCl 2 IV-Plot

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

24. DSO DARPA Conclusions 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 Future work under Phase IAccomplishments 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

25. DSO DARPA 1) Project Details Title: Integrated Nanoscale Ion Channel Sensor Start Date: December 15 th 2003 End Date: December 31 st 2004 (Phase I) Partners: Marco Saraniti (IIT) Bob Eisenberg (Rush) Steve Goodnick (ASU) Trevor Thornton (ASU) Plus: Dr. J. Tang (Rush), Dr. M. Goryll (ASU), Dr. G. Laws (ASU), Mr. S. Wilk (ASU) and Mr. D. Marreiro (IIT) 2) Project Goals 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. 3) “Phase I’ Deliverables ▪ demonstrate ‘Gigaseal’ properties  ▪ demonstrate reversible electrodes  ▪ measure single channels with integrated device ▪ characterize stability of integrated device Si Bilayer 4) Future Plans - issues to be addressed membrane stabilization simulants detection identifying stochastic signatures ………..