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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
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)
• 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
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)
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
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