DSO DARPASilicon-based Ion Channel SensorM. Goryll1, S. Wilk1, G. M. Laws1, T. J. Thornton1, S. M. Goodnick1, M. Saraniti2, J. Tang3, R. S. Eisenberg3,

1 Arizona State University, Dept. of Electrical Engineering, Tempe, AZ 85287 , 2 Illinois Institute of Technology, Dept. of Electrical and Computer Engineering,

Chicago, IL 60616

Process Flow

Project Goals Technical

Approach

Achievements150 m wide aperture in a silicon wafer,

back side view

AgCl Electrode

OxideSU-8 Resist

Si

Important building blocks of a fully integrated biosensor with on-chip sensing and signal processing

Small Hole Etching

825 Resist, 1m thickness

AZ 4330 Resist, 2.6m thickness

Si Substrate

50m

300m

SU-8 Resist

Si

1 mm 250m

Si

150m

150mSi

Thermally Grown Oxide, d = 500 nm

Si

150m

Si

Photoresist

SU-8 Resist

Si

AgCl

Hydrophobic Layer

SU-8 Resist

Si

AgCl

Bilayer

Resist for Initial Hole Etching

Thermal Oxidation

Resist for Small Hole Etching

Large Hole Etching

SU-8 Resist (Epoxy)

Surface Modification Layer

AgCl Electrode

AgCl Electrode, up to 1m thickness

SU-8 Resist

Si

Lipid Bilayer Attachment

• Experiment showing the opening of a single OmpF porin channel. The vertical lines through the red current trace are an artifact from stirring of the bath to facilitate the insertion of porin into the bilayer membrane.

• Plot showing the different levels of OmpF porin (Trimer). Level 1 is not shown. All the traces in the above plot are from the same OmpF porin bilayer experiment using the silicon wafer coated with PTFE (Teflon).

Hole diameter = 150 m

PTFE coated surface

For the fabrication …

• coating the oxide surface with a Teflon film changes its properties from hydrophilic to hydrophobic (small to large contact angle)

• Plasma CVD provides an easy way of depositing several nm thick PTFE layers

• good agreement between model and experimental ellipsometric data allows a reliable thickness measurement

• dispersion curve indicates a high density PTFE polymer layer similar to bulk material

• “stackable” layers

Bulk PTFE DuPont : n = 1.35MIT : n = 1.38

PTFE layer on Si

Ind

ex o

f re

frac

tio

n (

n)

400 450 500 550 600 650 700 750 8001.350

1.355

1.360

1.365

1.370

1.375

1.380

1.385

Wavelength (nm)

900 Å layer 600 Å layer

PTFE on Si: d = 598 Å ± 2 Å, n = 1.377 ± 2E-3

,

(

deg

rees

)

400 450 500 550 600 650 700 750 80020

30

40

50

60

70

Wavelength (nm)

Model Fit ( in degrees) Model Fit ( in degrees)

Exp -E 75° ( in degrees) Exp -E 75° ( in degrees)

• Blanket silver deposition by thermal evaporation on oxidized Si wafer

• Chloridization in 5% NaOCl

• minimal difference between the expected and measured Nernst potential variation with KCl concentration

• good potential stability of the microstructured electrodes

Summary

Silver Chloride Electrode

• the stability of the lipid bilayer is related to the contact angle between the bilayer and the supporting substrate

• water contact angle measure- ments can be used to determine the substrate’s surface energy

Bilayer

Torus

PTFE Surface Modification

3Rush Medical College, Dept. of Molecular Biophysics and Physiology , Chicago, IL 60612

Plasma CVD is an interesting novel method to provide PTFE surface modification layers for lipid bilayer attachment to solid supports

0.5M (trans) and 0.6M (cis)

KCl Test solutions

AgCl layer, chloridized in 5% NaOCl

Pot

entia

l diff

eren

ce (

mV

)

0 1 2 3 4 5-10

-8

-6

-4

-2

0

2

4

6

8

10

Time (h)

Simulation Measurement

0 1 2 3 4 5-100

-80

-60

-40

-20

0

Pot

entia

l diff

eren

ce (

mV

)

KCl Molarity difference (M)

AgCl Electrode Potential, Single substrate

• response is indistinguishable from channels in Teflon supported membranes, shows physiological behavior of OmpF• reproducibility of measurements and voltage dependence indicates that switching is not an artifact but real channel activity

• switch to double-side polished 100 mm (4”) wafer with 380 m thickness allows the fabrication of multiple samples per run with identical geometry

• optimized backside alignment results in good centering of the hole

• front and backside have a smooth surface and the etching does not roughen the lower surface

150 m wide aperture in a silicon wafer,back side view (FESEM closeup)

150 m wide aperture in a silicon wafer,front side view

• measure single channels in an integrated device

• study the relation between the size of the lipid bilayer and the signal-to-noise ratio

• find optimal surface treatment for bilayer attachment

• find simulants that bind and transiently block conduction of ions through ompF

• work with DARPA and other groups MOLDICE groups to incorporate ion channels that show desired properties

Future work under Phase IAccomplishments

• a silicon bilayer support chip has been constructed and successful Gigaseal formation has been demonstrated

• channel insertion succeeded

• first milestones have been achieved

• integration of the reversible electrodes demonstrated

• PTFE layers deposited by plasma CVD exhibit excellent properties

Goal 1: Embed channels in an integrated device that maintains stable potential across them and allows recording of stable, artifact free current through them.

Goal 2: Find simulants that bind and transiently block conduction of ions through OmpF.*

* we shall work with DARPA and other groups within the MOLDICE network to incorporate ion channels that show desired properties

• silicon substrates are used

• layers are structured by conventional optical lithography

• the aperture that supports the bilayers is constructed using deep silicon dry etching

• relation between the size of the lipid bilayer and its stability and the signal-to-noise ratio of the ion channel response

• ultimate limit for the size scaling of the sensor

• optimal surface treatment for lipid bilayer attachment

• stability of the integrated reversible Ag/AgCl electrodes

• manufacturability of the sensor

• usability issues (reusability, cleaning)

Challenges we are facing

• impedance analysis of bilayers

• current-voltage measurements of bilayers and porin channels

• studying the influence of surface modification layers on bilayer Gigaseal formation

Experiments involve …

• maintain stable potential (± 1 mV for 1 hour) across a single channel of OmpF porin

• recording of stable, artifact - free current voltage curves (± 100 pA for 1 hour) from a single channel of OmpF porin using external electrodes

• recording stable current voltage curves using integrated Ag/AgCl electrodes

Milestones

• measure sealing resistance on samples with different geometries and surface properties

• measure Nernst potential of Ag/AgCl electrodes

• measure DC potential across porin

• measure current through porin

Demonstration of Results