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Fabrication of Nanoscale BLM Biosensors

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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

Composed of a hydrophilic polar head andhydrophobic 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.)

Bilayer Lipid Membranes

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Why use a BLM/protein system?

Biosensors based on natural receptors

(proteins) with BLMs provide a sensitive andselective 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 assignal tranduction properties.

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Electrochemical ImpedanceSpectroscopy (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

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Every circuit element has a transfer function

Transfer functions are used to derive the resistance andcapacitance 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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The most basic circuit

model utilized is

Zel

This circuit has a function of

ZelZmZt

RjwC

RZm

jwCRZm

1

11

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Assuming some knowledge of the circuit

structure, a transfer function can be derivedand the circuit parameters can be extracted.

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Unfortunately, these systems can be far more

complicated due to a variety of other parasiticinteractions

A primary source of these complications is the Si

substrate itself which is highly conductive. This

presents a low conductance, high capacitancepathway when combined with the membrane.

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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

Si3N4

Si

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Etch a 180 micron x 180 micron square

window on the backside of the substrate

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Use KOH as a wet etchant to etch through the Si substrate

KOH preferentially etches crystal plane, resulting in a V-

groove

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Evaporate a thin layer of Al onto the front side

of the substrateAl

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Anodize the aluminum

Al(metal) Al2O3

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Etch the backside to remove the Si2N3

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Alumina film characteristics can be adjusted by use of phosphoricacid and anodization conditions

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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

0.1 Hz 1 Hz 10 Hz

Quartz plus oxide 88 mm2 46.25 G 14.02 G 1.67 G

Silicon, N-type

0.005-0.02 -cm

88 mm2 1.51 M 173 k 21.32 k

Silicon plus oxide 88 mm2 559.6 M 53.58 M 5.66 M

Silicon/Nitride/Alumina (no H2PO4

etching)

88 mm2 25.21 M 4.197 M 494 k

Silicon/Nitride/Alumina (no H2PO4

etching)

12.6 mm2 18.91 M 3.85 M 503 k

Silicon/Nitride/Alumina (H2PO4 etch

20 min)

88 mm2 1.63 M 133 k 25.02 k

Silicon/Nitride/Alumina (H2PO4 etch

20 min)

12.6 mm2 3.26 M 488.5 k 72.32 k

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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 theenvironment 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 patchclamp 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 apatterned 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)
http://www.cnf.cornell.edu/index.html

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