October 11, 2005

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October 11, 2005 Sensor workshop
Membrane/ion-channel biosensors
Cornell University Ithaca, NY Tad
Kaburaki Xingqun Jiang Michael Spencer
Wadsworth Center Albany, NY Mary Rose
Burnham James Turner David Martin
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Membrane biosensors for chemical and biological
agents
What can we use Membrane biosensors for? LEVEL I
agents bind directly to ion channel proteins
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Membrane biosensors for chemical and biological
agents
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LEVEL II agents Would require molecularly
engineered binding sites built into a natural ion
channel
Sarin Tabin Paraoxon Parathione Malathione Echothi
ophate Phosdrin Organophosphorous nerve agents
and pesticides
LEVEL III agents Would require synthetic ion
channels with genetically engineered recognition
sites
Ricin Abrin Aflatoxins Botulinum toxiods Cholera
toxin SEB (streptavidin) Salmonella Anthrax
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Level I agents are detected by different types of
ion channel proteins
Sodium (Na) ion channel Saxitoxin
(neosaxitoxin, gonyautoxin) Tetrodotoxin Batrach
otoxin Brevetoxin Plant toxins alkaloid
toxins mu-conotoxinx and neurotoxins GABA ion
channel (Cl- ) TMPP barbiturates benzodiazapine
s nAcetylcholine (nACH) ion channel (Na and
Ca) Anatoxin-a Soman Bungarotoxin VX epibata
dine conotoxins a-neurotoxins K ion
channel Tityustoxin Charybdotoxin Noxiustoxin
Dendrotoxins Alkaloid toxins Glycine ion
channel Strychnine
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Our receptor is the GABA ion channel

• Three GABA receptors
• GABA-C is a ligand-gated chloride ion channel
  (very similar the nACh ion channel)
• The GABA-C receptor consists of five identical
  subunits (rho-1)
• Binding of GABA to the receptor opens up a
  channel in the protein, allowing the passage of
  ions from one side of the membrane to the other.

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Properties of single GABA ion channels
7 0.8 pS
150 mS
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Sensor design affinity-based amperometric sensor
Receptor (ion channel protein in a lipid membrane)
Target
SIGNAL change in conductance/resistance
X
X
Non-target molecules
The choice of receptor will be determined by the
identity of the target. The conductance change
associated with target binding will have a
characteristic magnitude and duration (an
electronic fingerprint).
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Device design
4 LAYERS 1. Electrodes Detection 2. porous
Alumina/OTS structural (for attachment and
stabiliation of the lipid membrane) 3. Lipid
membrane provides the proper environment for
the receptor 4. GABA Receptor sensing
component
I
4
GABA
3
2
1
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Device Design Paint Cell
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Device Design enclosed BLM cell
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Chip Design
100 um2
1.2 cm2
porous Alumina
nitride
silicon
100 um2 window
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Pore size of the porous alumina scaffold can be
controlled by acid treatment
(10 nm)
(40 nm)
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FRAP analysis tells us that lipid bilayers with
appropriate fluidity will form on porous alumina
substrates
25 min acid
t0
t20 min
Pre-bleach
40 min acid
t20 min
Pre-bleach
t0
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Electronic measurements (conductance changes) of
lipid membranes and ion channels
Traditional electrophysiology set-up for studying
lipid membranes and ion channels
Gamry Femtostat Impedance analyzer
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Impedance analysis of porous alumina
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Impedance analysis of lipid membranes
Impedance changes associated with the formation
of a lipid membrane on the porous alumina
scaffold BLUE before lipid membrane RED lipid
membrane
Single frequency monitoring of the lipid membrane
(no ion channels present) Voltage bias 10 mV AC
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Increased impedance correlates with Lipid
membrane sealing the pores in the porous alumina
membrane
BEFORE
AFTER
BLUE porous alumina (BEFORE) RED lipid
membrane (AFTER)
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Impedance analysis reveals the time-dependent
formation of the lipid bilayer and associated
changes in phase angle
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Impedance changes associated with ion channel
insertion into the lipid membrane
Single frequency monitoring of lipid membrane
plus ion channel (constitutively open ion channel)
BLUE porous alumina (before lipid membrane) RED
lipid membrane alone PURPLE after ion
channels have inserted into the membrane
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Current traces of lipid membrane, and lipid
membrane plus ion channel
9 nA
D C 90 nA
-80 nA
Ion channel causes a change in the BASELINE
conductance across the membrane. Usually
transient, usually much smaller than what is
shown here.
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CHALLENGES/FUTURE DIRECTIONS
1. Stability, stability, stability,
stability Our lipid membranes last up to 20
hours with minimal to no agitation/movement
(highly controlled laboratory environment) Diffe
rent lipid compositions Membrane additives
(cholesterol) No data yet on the duration of
the ion-channel response 2. WHAT ARE WE GOING
TO DETECT? Using the GABA channel (TMPP, GABA,
Barbiturates, anti- convulsants) Do we want to
use other channels?