1. Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI) Xinquin Jiang (Cornell)

2. Presentation Contents Objectives Background Fabricated devices Signal Processing Current Goals

3. Objectives Fabrication of a stable platform for transducing signals through artificial BLMs –Allow for the most stable BLM possible Analysis of BLM impedance characteristics –Including signals produced with proteins Packaging of a sensor with analytic capabilities on-chip

4. BLMs Composed of a hydrophilic polar head and hydrophobic non polar tail 5nm thickness with.5nm 2 area / lipid molecule BLM’s have high resistances and high capacitances An Artist's conception of ion channels in a lipid bilayer membrane (taken from Hille, B., 1992. Ionic Channels of Excitable Membranes. Sinauer, Sunderland, Massachusetts.) Bilayer Lipid Membranes

5. Why use a BLM/protein system? Biosensors based on natural receptors (proteins) with BLMs provide a sensitive and selective method of sensing chemical species (ions or molecules) Upon binding with analytes, transport proteins change their transport behavior across BLMs These types of sensors are unique in that they have molecular recognition as well as signal tranduction properties.

6. Electrochemical Impedance Spectroscopy (EIS) A small amplitude sinusoidal voltage is applied across the device The frequency dependant impedance is measured as a magnitude and phase angle device electrodes

7. Electrochemical Impedance Spectroscopy (EIS)

9. Every circuit element has a transfer function Transfer functions are used to derive the resistance and capacitance of the system ComponentCurrent Vs.VoltageImpedance resistorE= IRZ = R inductorE = L di/dtZ = jwL capacitorI = C dE/dtZ = 1/jwC

10. Electrochemical Impedance Spectroscopy (EIS) The most basic circuit model utilized is Z el This circuit has a function of

11. Electrochemical Impedance Spectroscopy (EIS) Assuming some knowledge of the circuit structure, a transfer function can be derived and the circuit parameters can be extracted.

12. Electrochemical Impedance Spectroscopy (EIS) Unfortunately, these systems can be far more complicated due to a variety of other parasitic interactions –A primary source of these complications is the Si substrate itself which is highly conductive. This presents a low conductance, high capacitance pathway when combined with the membrane.

13. Electrochemical Impedance Spectroscopy (EIS)

14. Fabrication Requirements Hold a stable membrane –Smooth and clean surface Preferably oxide surface –Porous surface Allow for signals to be passed through membrane/proteins Pore size should be small to increase the stability of suspended region and prevent lipids from forming conformally to the surface

15. Fabrication Requirements Measure signals with a high S/N ratio –Need a high resistance, low capacitance substrate Prevents capacitive coupling, capacitive signal leakage High resistance allows for signals to be measured only through the membrane area –Good electrode placement i.e. Ag/AgCl electrodes for Cl- measurement

16. Porous alumina substrates Designed by Xinquin Jiang (Spencer group) –Utilizes porous alumina formed

17. Porous Alumina Substrate Fabrication Use LPCVD (Low Pressure Chemical Vapor Deposition) to coat a 4” DSP (Double sided polish) wafer with Silicon Nitride Si 3 N 4 Si

18. Porous Alumina Substrate Fabrication Etch a 180 micron x 180 micron square window on the backside of the substrate

19. Porous Alumina Substrate Fabrication Use KOH as a wet etchant to etch through the Si substrate –KOH preferentially etches crystal plane, resulting in a “V- groove”

20. Porous Alumina Substrate Fabrication Evaporate a thin layer of Al onto the front side of the substrate Al

21. Porous Alumina Substrate Fabrication Anodize the aluminum –Al(metal) Al2O3

22. Porous Alumina Substrate Fabrication Etch the backside to remove the Si 2 N 3

23. Porous Alumina Substrate Fabrication Alumina film characteristics can be adjusted by use of phosphoric acid and anodization conditions

24. Porous Alumina Substrate Fabrication BLM can then be deposited

25. Signals obtained from this system Our results are comparable to state of the art systems The results do require some amount of interpretation –This is because the systems on which the BLMs reside are not identical.

26. Si substrates have a much lower resistance and higher capacitance than quartz substrates SampleAREAImpedance 0.1 Hz1 Hz10 Hz Quartz plus oxide88 mm 2 46.25 GΩ14.02 GΩ1.67 GΩ Silicon, N-type 0.005-0.02 Ω-cm 88 mm 2 1.51 MΩ173 kΩ21.32 kΩ Silicon plus oxide88 mm 2 559.6 MΩ53.58 MΩ5.66 MΩ Silicon/Nitride/Alumina (no H 2 PO 4 etching) 88 mm 2 25.21 MΩ4.197 MΩ494 kΩ Silicon/Nitride/Alumina (no H 2 PO 4 etching) 12.6 mm 2 18.91 MΩ3.85 MΩ503 kΩ Silicon/Nitride/Alumina (H 2 PO 4 etch 20 min) 88 mm 2 1.63 MΩ133 kΩ25.02 kΩ Silicon/Nitride/Alumina (H 2 PO 4 etch 20 min) 12.6 mm 2 3.26 MΩ488.5 kΩ72.32 kΩ

27. Proposed Structure Change of Silicon substrate for SiO 2 Difficulty in etching through the wafer –HF wet etch is isotropic –Dry etching of SiO 2 has a maximum rate of 100nm/minute which is 5000 minutes for a 500um wafer.

28. Proposed Structure Cut 100um diameter holes in a quartz substrate with a micromachining laser Quartz

29. Proposed Structure Cut 100um diameter holes in a quartz substrate with a micromachining laser

30. Proposed Structure Anodize the aluminum –Al(metal) Al 2 O 3

31. Proposed Structure Coat the surface with a polymer (polyimide or adhesive wax)

32. Proposed Structure Adhere the Si and quartz surfaces (hot press)

33. Proposed Structure Dry etch the Si wafer (Bosch etch process) at a rate of 1um/minute. Dry etch polymer (RIE)

34. Proposed Structure BLM can then be deposited

35. The Next Step Addition of proteins –The proteins are the mechanism by which the environment is actually measured –Measurements will be made at a single frequency that is chosen to maximize sampling while remaining in the resistive regime –Optimally this frequency will be in the kHz range

36. Hirano from Nihon University used a patch clamp to measure current openings from a single gramicidin protein in response to different concentrations of ferritin avidin

37. Opening percentage vs. FA concentration

38. Conclusion We have developed a system to hold membranes at a high resistance over a patterned substrate Current readings are feasible and should generate readable results due to the larger number of measurement proteins

39. Wadsworth Center (State of NY)