Fabrication of Nanoscale BLM Biosensors

Presentation Contents
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 BLMs 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
Quartz plus oxide Silicon, N-type 0.005-0.02 -cm Silicon plus oxide Silicon/Nitride/Alumina (no H2PO4 etching) Silicon/Nitride/Alumina (no H2PO4 etching) Silicon/Nitride/Alumina (H2PO4 etch 20 min) Silicon/Nitride/Alumina (H2PO4 etch 20 min)

AREA
0.1 Hz 88 mm2 88 mm2 88 mm2 88 mm2 12.6 mm2 88 mm2 12.6 mm2 46.25 G 1.51 M 559.6 M 25.21 M 18.91 M 1.63 M 3.26 M

Impedance
1 Hz 14.02 G 173 k 53.58 M 4.197 M 3.85 M 133 k 488.5 k 1.67 G 21.32 k 5.66 M 494 k 503 k 25.02 k 72.32 k 10 Hz

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)