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

Dec 16, 2015

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

Fabrication of Nanoscale BLM Biosensors

Tadahiro Kaburaki (Cornell)MR Burnham (Wadsworth Postdoc)

M.G. Spencer (Cornell PI)James Turner (Wadsworth PI)

Xinquin Jiang (Cornell)

Page 2: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Presentation Contents

• Objectives• Background• Fabricated devices• Signal Processing• Current Goals

Page 3: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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

Page 4: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

BLMs

• Composed of a hydrophilic polar head and hydrophobic non polar tail

• 5nm thickness with .5nm2 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

Page 5: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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.

Page 6: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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

Page 7: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Electrochemical Impedance Spectroscopy (EIS)

Page 8: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Electrochemical Impedance Spectroscopy (EIS)

Page 9: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Electrochemical Impedance Spectroscopy (EIS)

• Every circuit element has a transfer function• Transfer functions are used to derive the resistance and

capacitance of the system

Component Current Vs.Voltage

Impedance

resistor E= IR Z = R

inductor E = L di/dt Z = jwL

capacitor I = C dE/dt Z = 1/jwC

Page 10: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Electrochemical Impedance Spectroscopy (EIS)

• The most basic circuit model utilized is

Zel

• This circuit has a function of

ZelZmZt

RjwC

RZm

jwCRZm

1

11

Page 11: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Electrochemical Impedance Spectroscopy (EIS)

• Assuming some knowledge of the circuit structure, a transfer function can be derived and the circuit parameters can be extracted.

Page 12: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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.

Page 13: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Electrochemical Impedance Spectroscopy (EIS)

Page 14: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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

Page 15: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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

Page 16: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous alumina substrates

• Designed by Xinquin Jiang (Spencer group)– Utilizes porous alumina formed

Page 17: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Use LPCVD (Low Pressure Chemical Vapor Deposition) to coat a 4” DSP (Double sided polish) wafer with Silicon Nitride

Si3N4

Si

Page 18: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Etch a 180 micron x 180 micron square window on the backside of the substrate

Page 19: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Use KOH as a wet etchant to etch through the Si substrate– KOH preferentially etches <100> crystal plane, resulting in a “V-

groove”

Page 20: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Evaporate a thin layer of Al onto the front side of the substrate

Al

Page 21: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Anodize the aluminum– Al(metal) Al2O3

Page 22: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Etch the backside to remove the Si2N3

Page 23: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• Alumina film characteristics can be adjusted by use of phosphoric acid and

anodization conditions

Page 24: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Porous Alumina Substrate Fabrication

• BLM can then be deposited

Page 25: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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.

Page 26: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

• Si substrates have a much lower resistance and higher capacitance than quartz substrates

Sample AREA Impedance

0.1 Hz 1 Hz 10 Hz

Quartz plus oxide 88 mm2 46.25 GΩ 14.02 GΩ 1.67 GΩ

Silicon, N-type

0.005-0.02 Ω-cm

88 mm2 1.51 MΩ 173 kΩ 21.32 kΩ

Silicon plus oxide 88 mm2 559.6 MΩ 53.58 MΩ 5.66 MΩ

Silicon/Nitride/Alumina (no H2PO4

etching)

88 mm2 25.21 MΩ 4.197 MΩ 494 kΩ

Silicon/Nitride/Alumina (no H2PO4

etching)

12.6 mm2 18.91 MΩ 3.85 MΩ 503 kΩ

Silicon/Nitride/Alumina (H2PO4 etch 20 min)

88 mm2 1.63 MΩ 133 kΩ 25.02 kΩ

Silicon/Nitride/Alumina (H2PO4 etch 20 min)

12.6 mm2 3.26 MΩ 488.5 kΩ 72.32 kΩ

Page 27: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• Change of Silicon substrate for SiO2

• Difficulty in etching through the wafer– HF wet etch is isotropic

– Dry etching of SiO2 has a maximum rate of 100nm/minute which is 5000 minutes for a 500um wafer.

Page 28: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• Cut 100um diameter holes in a quartz substrate with a micromachining laser

Quartz

Page 29: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• Cut 100um diameter holes in a quartz substrate with a micromachining laser

Page 30: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• Anodize the aluminum– Al(metal) Al2O3

Page 31: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• Coat the surface with a polymer (polyimide or adhesive wax)

Page 32: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• Adhere the Si and quartz surfaces (hot press)

Page 33: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

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

Page 34: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Proposed Structure

• BLM can then be deposited

Page 35: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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

Page 36: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

• 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

Page 37: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

• Opening percentage vs. FA concentration

Page 38: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

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

Page 39: Fabrication of Nanoscale BLM Biosensors Tadahiro Kaburaki (Cornell) MR Burnham (Wadsworth Postdoc) M.G. Spencer (Cornell PI) James Turner (Wadsworth PI)

Wadsworth Center (State of NY)