October 11, 2005Sensor workshop

Membrane/ion-channel biosensors

Wadsworth CenterAlbany, NY

Mary Rose BurnhamJames TurnerDavid Martin

Cornell UniversityIthaca, NY

Tad KaburakiXingqun JiangMichael Spencer

Membrane biosensors for chemical and biological agentsWhat can we use Membrane biosensors for?

LEVEL I agents: bind directly to ion channel proteins

CHEMICALS / NERVE AGENTS *Soman lethal dose is half that of sarin; liquid, volatile, inhaled/dermal

(contact)/ingested.*VX approx. 10-fold more toxic than sarin, v = very long; stable,primary mode of contact is dermal.TMPP (trimethylolpropane phosphate): convulsive agent; generated by pyrolysis of certain military turbine engine lubricantsStrychnine convulsant, plant origin, adsorbtion/ingestion of contaminated water or food.

TOXINS/Neurotoxins*Saxitoxin Schedule I chemical agent; toxic shellfish (mollusks), generated by marine dinoflagellates and blue green algae; cause of paralytic shellfish poisoning (PSP)*Tetrodotoxin puffer fish, also blue-green algaeBrevetoxin-2, -3 toxic mollusks/marine dionflagellatesAnatoxin- blue-green algaeVanatridineAcomtine Plant toxinsGrayanotoxinsBungarotoxin Snake venom*Batrachotoxin frog (poison arrow toxin)Tityustoxin Scorpion venomDendrodotoxin green mamba snake

Membrane biosensors for chemical and biological agents

LEVEL II agents:Would require molecularly engineered binding sites built into a natural ion

channel

SarinTabinParaoxonParathioneMalathioneEchothiophatePhosdrinOrganophosphorous nerve agents and pesticides

LEVEL III agents:Would require synthetic ion channels with genetically engineered recognition sites

RicinAbrinAflatoxinsBotulinum toxiodsCholera toxinSEB (streptavidin)SalmonellaAnthrax

Level I agents are detected by different types of ion channel proteins

Sodium (Na++) ion channelSaxitoxin (neosaxitoxin, gonyautoxin)TetrodotoxinBatrachotoxinBrevetoxinPlant toxinsalkaloid toxinsmu-conotoxinx and neurotoxins

GABA ion channel (Cl- )TMPPbarbituratesbenzodiazapines

nAcetylcholine (nACH) ion channel (Na++ and Ca++)Anatoxin- SomanBungarotoxin VXepibatadineconotoxins -neurotoxins

K+ ion channelTityustoxinCharybdotoxinNoxiustoxinDendrotoxinsAlkaloid toxins

Glycine ion channelStrychnine

O.D. 80 ÅI. D. 18 ÅPore D. 5.1 Å

O.D.

I.D.

A.Karlin, 2002

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.

Properties of single GABA ion channels

R = receptorA = analyte

D. Colquhoun, 1999

Current amplitude of 0.5 pA-70 mV holding potentialSymmetrical chloride concentration (145 mM Cl)

Bormann and Feigenspan, 1995

7 ± 0.8 pS

150 mS

SIGNAL = change in conductance/resistance

Target

XXNon-target molecules

Receptor (ion channel protein in a lipid membrane)

Sensor design: affinity-based amperometric sensor

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).

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

4

32

1

IGABA

Device design

Device Design: Paint Cell

Device Design: enclosed BLM cell

siliconnitride

porous Alumina

100 um2 window

1.2 cm2

100 um2

Chip Design

Pore size of the porous alumina scaffold can be controlled by acid treatment

(10 nm)

(40 nm)

FRAP analysis tells us that lipid bilayers with appropriate fluidity will form on porous alumina substrates

Pre-bleach

Pre-bleach

t=0

t=0

t=20 min

t=20 min

25 min acid

40 min acid

Gamry Femtostat: Impedance analyzerTraditional electrophysiology set-up for studying lipid membranes and ion

channels

Electronic measurements (conductance changes) of lipid membranesand ion channels

siliconnitride

porous Alumina

Impedance analysis of porous alumina

Impedance changes associated with the formation of a lipid membrane on the porous alumina scaffold

BLUE =before lipid membraneRED = lipid membrane

Single frequency monitoring of the lipid membrane (no ion channels present)Voltage bias = 10 mV AC

Impedance analysis of lipid membranes

Increased impedance correlates with Lipid membrane sealing the pores in the porous alumina membrane

BLUE = porous alumina (BEFORE)RED = lipid membrane (AFTER)

BEFORE

AFTER

Impedance analysis reveals the time-dependent formation of the lipid bilayer and associated changes in phase angle

Single frequency monitoring of lipid membrane plus ion channel (constitutively open ion channel)

Impedance changes associated with ion channel insertion into the lipid membrane

BLUE = porous alumina (before lipid membrane)RED = lipid membrane alonePURPLE = after ion channels have inserted into the membrane

Current traces of lipid membrane, and lipid membrane plus ion channel

Ion channel causes a change in the BASELINE conductance across the membrane. Usually transient, usually much smaller than what is shown here.

9 nA

-80 nA

C = 90 nA

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)

Different lipid compositionsMembrane 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?