DSO DARPA

Integrated

Nanoscale Ion-

Channel Sensor

DSO DARPA

Project Goals

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

AgCl Electrode

OxideSU-8 Resist

Si

Lipid Bilayer with

Ion Channels

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

DSO DARPA

Technical Approach

• 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 bilayer attachment

• stability of the integrated reversible Ag/AgCl electrodes

• manufacturability of the sensor

• usability issues (reusability, cleaning, automation)

Challenges we are facing For the fabrication …

• 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 …

DSO DARPA

Summary sheet

• 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 inte- grated Ag/AgCl electrodes

Milestones Accomplishments

• design and process flow-chart for a silicon bilayer support chip

• working proof-of-concept in form of a silicon chip as a direct Teflon membrane replacement

• Gigaseal formation proven

• channel insertion succeeded

• PTFE layers deposited by plasma CVD facilitate bilayer formation

• planar AgCl electrodes exhibit desired properties

DSO DARPA

Summary sheet

• 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 Technology Transition

• construct a silicon-based sensor template (reusable if possible) along with a fixture to allow easy bilayer formation and protein insertion

• development of a procedure to reproducibly create bilayers with Gigaseals

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

DSO DARPA

Microfabrication

Details

(ASU)

DSO DARPA

Small Hole Etching

825 Resist, 1m thickness

AZ 4330 Resist, 2.6m thickness

Si Substrate

50m

300m

SU-8 Resist

Si

1 mm250m

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

Process Flow

DSO DARPA

250 m

• deep silicon etch process that is optimized on high etch rate (4.7 m/min), good selectivity (220:1) and a concave bottom profile

• etch process that exhibits vertical sidewalls and a low aspect ratio dependent etch rate of 3.7 m/min with planar bottom profiles below 100 m ridge width

Process optimization

DSO DARPA

250 m

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

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

• optimized backside alignment re- sults in good centering of the hole

Process optimization

DSO DARPA

250 m

• conventional hole preparation using electrical discharge to create an aperture in a PTFE sheet of 25 m thickness

• using deep silicon dry etching and back side alignment photo- lithography a small hole (150 m) was created inside a recess

Sample comparison

DSO DARPA

PTFE Surface Modification• 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

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

• using Plasma CVD is a novel method that provides an easy way to deposit thick PTFE layers

Bilayer

Torus

Substrate

DSO DARPA

Lipid Bilayer

Experiments

(Rush)

DSO DARPA

• 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).

Lipid Bilayer Experiments

Hole diameter = 150 m

PTFE coated surface

DSO DARPA

Lipid Bilayer Experiments

• physiological behavior of OmpF

• response is indistinguishable from channels in Teflon supported membranes

• reproducibility of measurements and voltage dependence indicates that switching is not an artifact but real channel activity

DSO DARPA

Ag/AgCl

Electrodes

(ASU)

DSO DARPA

Integrated AgCl ElectrodesAgCl ring on SU-8

(chloridized)

• chloridization in 5% NaOCl for 30 sec

• measurements are performed using 0.1M or 0.5M KCl reference solutions

AgCl ring on oxide

3 mm

Schematic view of the electrode layout

• silver is evaporated on both sides of the wafer (> 500 nm)

• layer patterning by photo- lithography and etching

DSO DARPA

Integrated AgCl ElectrodesAgCl Electrode Potential, Single substrate

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)

0.1M KClReference

solution

• no notable difference between electrodes on oxide and epoxy

• good potential stability of the microstructured electrodes

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

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)

DSO DARPA

AgCl Electrode

• difference between expected and measured potential due to partially chloridized surface

• longterm failure mechanism: AgCl gets dissolved in the KCl electrolyte

AgCl layer before measurement AgCl layer after 5 h measurement

DSO DARPA

Making a Calcium

Channel

(Rush)

DSO DARPA

Make a Calcium Channelby Site-directed Mutagenesis

Theory, Simulation, Experiment show

Crowded Charge Selectivity

George Robillard, Henk Mediema, Wim Meijberg

BioMaDe Corporation, Groningen, Netherlands

DSO DARPA

Strategy

Use site-directed mutagenesis to put in extra glutamates

and create an EEEE locus in the selectivity filter of OmpF

Site-directed

mutagenesis

R132

R82E42

E132

R42 A82

Wild type WT EAE mutant

E117 E117

D113D113

George Robillard, Henk Mediema, Wim MeijbergBioMaDe Corporation, Groningen, Netherlands

DSO DARPA

-100 -50 50 100

-150

-50

50

150

ECa

WT

EAE

Current (pA)

Voltage (mV)

Cis Trans

1 M CaCl2 0.1 M CaCl2

Ca2+

Ca2+

IV-PLOT

Cis Trans Cis Trans

IV-plot EAE: current reverses at equilibrium potential of Ca2+ (ECa),

indicating the channel can discriminate between Ca2+ and Cl-

Zero-current potentialor reversal potential = measure of ion selectivity

Henk MediemaWim Meijberg

Ca2+ over Cl- selectivity (PCa/PCl)recorded in 1 : 0.1 M CaCl2

IV-Plot

DSO DARPA

Selectivity arises from Electrostatics and Crowding of Charge

Precise Arrangement of Atoms is not involved

Make a Calcium Channelby constructing the right

Charge, Volume, Dielectric

DSO DARPA

Conclusions

• 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

DSO DARPA

1) Project DetailsTitle: Integrated Nanoscale Ion Channel SensorStart Date: December 15th 2003End Date: December 31st 2004 (Phase I)Partners: Marco Saraniti (IIT)

Bob Eisenberg (Rush)Steve Goodnick (ASU)Trevor Thornton (ASU)

Plus: Dr. J. Tang (Rush), Dr. M. Goryll (ASU), Dr. G. Laws (ASU), Mr. S. Wilk (ASU) and Mr. D. Marreiro (IIT)

2) Project Goals

resist

silicon

sealingring

electrode 1

membrane channel

electrode 2 electrode 2

• embed channels in a membrane device that maintains stable potential across them and allows recording of stable, artifact free current through them.

• Simulants will be found that bind and transiently block conduction of ions through ompF.

3) “Phase I’ Deliverables

▪ demonstrate ‘Gigaseal’ properties ▪ demonstrate reversible electrodes ▪ measure single channels with integrated device ▪ characterize stability of integrated device

Si

Bilayer

4) Future Plans - issues to be addressed

• membrane stabilization

• simulants detection

• identifying stochastic signatures

• ………..