Transcript Fabrication of Nanoscale BLM Biosensors

Fabrication of Nanoscale BLM Biosensors
Tadahiro Kaburaki (Cornell)
MR Burnham (Wadsworth Postdoc)
M.G. Spencer (Cornell PI)
James Turner (Wadsworth PI)
Xinquin Jiang (Cornell)
Presentation Contents
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Objectives
Background
Fabricated devices
Signal Processing
Current Goals
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
BLMs
Bilayer Lipid Membranes
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.)
• 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
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.
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
Electrochemical Impedance
Spectroscopy (EIS)
Electrochemical Impedance
Spectroscopy (EIS)
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
Electrochemical Impedance
Spectroscopy (EIS)
• The most basic circuit
model utilized is
• This circuit has a function of
Zel
1
1
  jwC
Zm R
R
Zm 
RjwC  1
Zt  Zm  Zel
Electrochemical Impedance
Spectroscopy (EIS)
• Assuming some knowledge of the circuit
structure, a transfer function can be derived
and the circuit parameters can be extracted.
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.
Electrochemical Impedance
Spectroscopy (EIS)
•
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
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
Porous alumina substrates
• Designed by Xinquin Jiang (Spencer group)
– Utilizes porous alumina formed
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
Porous Alumina Substrate Fabrication
• Etch a 180 micron x 180 micron square
window on the backside of the substrate
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 “Vgroove”
Porous Alumina Substrate Fabrication
• Evaporate a thin layer of Al onto the front side
of the substrate
Al
Porous Alumina Substrate Fabrication
• Anodize the aluminum
– Al(metal)
Al2O3
Porous Alumina Substrate Fabrication
• Etch the backside to remove the Si2N3
Porous Alumina Substrate Fabrication
•
Alumina film characteristics can be adjusted by use of phosphoric
acid and anodization conditions
Porous Alumina Substrate Fabrication
• BLM can then be deposited
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.
• 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
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.
Proposed Structure
• Cut 100um diameter holes in a quartz
substrate with a micromachining laser
Quartz
Proposed Structure
• Cut 100um diameter holes in a quartz
substrate with a micromachining laser
Proposed Structure
• Anodize the aluminum
– Al(metal)
Al2O3
Proposed Structure
• Coat the surface with a polymer (polyimide or
adhesive wax)
Proposed Structure
• Adhere the Si and quartz surfaces (hot press)
Proposed Structure
• Dry etch the Si wafer (Bosch etch process) at
a rate of 1um/minute. Dry etch polymer (RIE)
Proposed Structure
• BLM can then be deposited
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
• 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
• Opening percentage vs. FA concentration
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
Wadsworth Center
(State of NY)