Integrated

Nanoscale Ion-

Channel Sensor

Project Goals

• develop conceptual design for an all-silicon chip that allows freestanding lipid bilayer support

• fabricate a prototype of that chip

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

• test channel insertion into supported membrane

• evaluate properties of planar integrated AgCl electrodes

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

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

Experiments involve …

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 flowchart 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

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

Project Goals

• develop conceptual design for an all-silicon• chip that allows lipid bilayer support

• fabricate a prototype of that chip

• investigate the influence of surface modi- fication layers on bilayer Gigaseal formation

• test channel insertion into membrane

• evaluate properties of planar integrated AgCl electrodes

• relation between the size of the 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 Ag/AgCl electrodes

• manufacturability of the sensor

Challenges

• chip design and process flowchart

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

• Gigaseal formation and channel insertion succeeded

• PTFE layers deposited by plasma CVD facilitate bilayer formation

• planar AgCl electrodes exhibit desired properties

Accomplishments Outlook

• usability issues (reusability, cleaning, auto- mation) have to be investigated regarding the technology transfer

• the influence of local electric fields of a sealing ring on membrane stability will be studied

• cooperate with DARPA and other groups within the MOLDICE network to incorporate ion channels that show desired properties and to finally test the sensor

Microfabrication

Details

(ASU)

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

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

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

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

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

PTFE Plasma Deposition at ASU

• 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

Inde

x of

re

frac

tion

(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

,

(de

gre

es)

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)

Lipid Bilayer

Experiments

(Rush)

• 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

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

Ag/AgCl

Electrodes

(ASU)

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

AgCl Electrode

• planar Ag/AgCl electrode shows only minimal voltage offset when compared to theoretical value

• good potential stability, drift of approx. 0.65 mV/h

0.1M KCl Test solution

AgCl layer, chloridized in 5% NaOCl

0 1 2 3 4 50

5

10

15

20

25

30

35

40

45

50

Vol

tage

vs.

Sa

tur.

Cal

omel

Ele

ctro

de (

mV

)

Time (h)

0 1 2 3 4 5

-60

-40

-20

0

20

40

60

Simulation Measurement

ESCE = 0.2412 V

AgCl layer, chloridized in 5% NaOCl

Vol

tage

vs.

Sa

tur.

C

alom

el E

lect

rode

(m

V)

KCl Molarity (M)

Electrode Design Topview

Integrated AgCl ElectrodeAgCl ring on SU-8

• layer structuring by photo- lithography and etching

• potential difference measurement in Teflon cell using 1M KCl

1 mm

• planar Ag/AgCl electrodes on SU-8 epoxy show similar KCl concentration dependence compared to electrodes on oxide

AgCl Electrode Potential, Patterned Electrode

0 1 2 3 4 5

-60

-40

-20

0

20

40

60

Simulation Experiment

Pot

entia

l diff

eren

ce (

mV

)KCl Molarity (M)

AgCl ring on oxide

3 mm

Making a Calcium

Channel

(Rush)

Make a Calcium Channelby Site-directed Mutagenesis

Theory, Simulation, Experiment show

Crowded Charge Selectivity

George Robillard, Henk Mediema, Wim Meijberg

BioMaDe Corporation, Groningen, Netherlands

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

-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

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

Conclusions

• usability issues have to be investigated regarding the technology transfer

• the influence of local electric fields of a sealing ring on mem- brane stability will be studied

OutlookAccomplishments

• 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

AgCl Electrodes

Sealing ring