Fabrication of Nanoscale BLM Biosensors

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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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Presentation Contents

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

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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

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BLMs
Bilayer Lipid Membranes

• Composed of a hydrophilic polar head and
  hydrophobic non polar tail
• 5nm thickness with .5nm2 area / lipid molecule
• BLMs 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.)
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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.

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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

electrodes
device
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Electrochemical Impedance Spectroscopy (EIS)
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Electrochemical Impedance Spectroscopy (EIS)
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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
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Electrochemical Impedance Spectroscopy (EIS)

• The most basic circuit model utilized is

• This circuit has a function of

Zel
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Electrochemical Impedance Spectroscopy (EIS)

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

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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.

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Electrochemical Impedance Spectroscopy (EIS)

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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

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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

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Porous alumina substrates

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

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Porous Alumina Substrate Fabrication

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

Si
Si3N4
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Porous Alumina Substrate Fabrication

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

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Porous Alumina Substrate Fabrication

• Use KOH as a wet etchant to etch through the Si
  substrate
• KOH preferentially etches lt100gt crystal plane,
  resulting in a V-groove

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Porous Alumina Substrate Fabrication

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

Al
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Porous Alumina Substrate Fabrication

• Anodize the aluminum
• Al(metal) Al2O3

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Porous Alumina Substrate Fabrication

• Etch the backside to remove the Si2N3

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Porous Alumina Substrate Fabrication

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

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Porous Alumina Substrate Fabrication

• BLM can then be deposited

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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.

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

Sample AREA Impedance Impedance Impedance
0.1 Hz 1 Hz 10 Hz
Quartz plus oxide 88 mm2 46.25 GO 14.02 GO 1.67 GO
Silicon, N-type 0.005-0.02 O-cm 88 mm2 1.51 MO 173 kO 21.32 kO
Silicon plus oxide 88 mm2 559.6 MO 53.58 MO 5.66 MO
Silicon/Nitride/Alumina (no H2PO4 etching) 88 mm2 25.21 MO 4.197 MO 494 kO
Silicon/Nitride/Alumina (no H2PO4 etching) 12.6 mm2 18.91 MO 3.85 MO 503 kO
Silicon/Nitride/Alumina (H2PO4 etch 20 min) 88 mm2 1.63 MO 133 kO 25.02 kO
Silicon/Nitride/Alumina (H2PO4 etch 20 min) 12.6 mm2 3.26 MO 488.5 kO 72.32 kO
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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.

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Proposed Structure

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

Quartz
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Proposed Structure

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

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Proposed Structure

• Anodize the aluminum
• Al(metal) Al2O3

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Proposed Structure

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

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Proposed Structure

• Adhere the Si and quartz surfaces (hot press)

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Proposed Structure

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

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Proposed Structure

• BLM can then be deposited

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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

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• 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

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• Opening percentage vs. FA concentration

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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

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Wadsworth Center (State of NY)