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October 11, 2005 Sensor workshop Membrane/ion-channel biosensors Wadsworth Center Albany, NY Mary Rose Burnham James Turner David Martin Cornell University.

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Presentation on theme: "October 11, 2005 Sensor workshop Membrane/ion-channel biosensors Wadsworth Center Albany, NY Mary Rose Burnham James Turner David Martin Cornell University."— Presentation transcript:

1 October 11, 2005 Sensor workshop Membrane/ion-channel biosensors Wadsworth Center Albany, NY Mary Rose Burnham James Turner David Martin Cornell University Ithaca, NY Tad Kaburaki Xingqun Jiang Michael Spencer

2 Membrane biosensors for chemical and biological agents What 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 lubricants Strychnine 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 algae Brevetoxin-2, -3 toxic mollusks/marine dionflagellates Anatoxin-  blue-green algae Vanatridine Acomtine Plant toxins Grayanotoxins Bungarotoxin Snake venom *Batrachotoxin frog (poison arrow toxin) Tityustoxin Scorpion venom Dendrodotoxin green mamba snake

3 Membrane biosensors for chemical and biological agents

4 LEVEL II agents: Would require molecularly engineered binding sites built into a natural ion channel Sarin Tabin Paraoxon Parathione Malathione Echothiophate 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

5 Level I agents are detected by different types of ion channel proteins Sodium (Na++) ion channel Saxitoxin (neosaxitoxin, gonyautoxin) Tetrodotoxin Batrachotoxin Brevetoxin Plant toxins alkaloid toxins mu-conotoxinx and neurotoxins GABA ion channel (Cl - ) TMPP barbiturates benzodiazapines nAcetylcholine (nACH) ion channel (Na ++ and Ca++) Anatoxin-  Soman BungarotoxinVX epibatadine conotoxins  -neurotoxins K + ion channel Tityustoxin Charybdotoxin Noxiustoxin Dendrotoxins Alkaloid toxins Glycine ion channel Strychnine

6 O.D. 80 Å I. D. 18 Å Pore 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.

7 Properties of single GABA ion channels R = receptor A = analyte D. Colquhoun, 1999 Current amplitude of 0.5 pA -70 mV holding potential Symmetrical chloride concentration (145 mM Cl) Bormann and Feigenspan, 1995 7 ± 0.8 pS 150 mS

8 SIGNAL = change in conductance/resistance Target X X Non-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).

9 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 3 2 1 I GABA Device design

10 Device Design: Paint Cell

11 Device Design: enclosed BLM cell

12 silicon nitride porous Alumina 100 um 2 window 1.2 cm 2 100 um 2 Chip Design

13 Pore size of the porous alumina scaffold can be controlled by acid treatment (10 nm) (40 nm)

14 FRAP analysis tells us that lipid bilayers with appropriate fluidity will form on porous alumina substrates Pre-bleach t=0 t=20 min 25 min acid 40 min acid

15 Gamry Femtostat: Impedance analyzer Traditional electrophysiology set-up for studying lipid membranes and ion channels Electronic measurements (conductance changes) of lipid membranes and ion channels

16 silicon nitride porous Alumina Impedance analysis of porous alumina

17 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 Impedance analysis of lipid membranes

18 Increased impedance correlates with Lipid membrane sealing the pores in the porous alumina membrane BLUE = porous alumina (BEFORE) RED = lipid membrane (AFTER) BEFORE AFTER

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

20 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 alone PURPLE = after ion channels have inserted into the membrane

21 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

22 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 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?

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