Rapid conductance based ion channel analysis
Ligand-ion channel interactions are analyzed via electrophysiological methods having rapid temporal response and high sensitivity, thereby reducing the collection time and enabling monitoring of dynamic processes. This protocol allows quantification of ligand concentrations in the sub-millisecond to ms range, as compared to s-min for traditional approaches. Moreover, the method can be easily integrated into existing patch clamp analysis packages and allow for monitoring of rapid, dynamic chemical processes in a feasible manner.
1. A method of obtaining ion channel measurements from an ion channel (IC) functionalized sensor platform, said method comprising:
a. providing the IC-functionalized sensor platform comprising a pipette and a lipid membrane suspended on a pipette aperture of the pipette, wherein a plurality of ion channels is embedded in the lipid membrane, wherein the IC-functionalized sensor platform is operatively connected to an amplifier;
b. placing the IC-functionalized sensor platform in a solution such that the lipid membrane is disposed in the solution;
c. continuously applying a voltage set to the IC-functionalized sensor platform via the amplifier, wherein the voltage set comprises a plurality of consecutive voltages that are applied in a step-wise pattern so as to alternate between decreasing and increasing voltages, wherein each voltage is rapidly applied for a short duration pulse; and
d. measuring a conductance of the lipid membrane; wherein a net time required for measuring the conductance is about 0.5 msec to about 50 msec.
2. The method of claim 1 , wherein a diameter of the pipette aperture is about 1-5 μm.
3. The method of claim 1 , wherein the voltage set comprises about 2-5 increasing voltage steps and about 2-5 decreasing voltage steps.
4. The method of claim 3 , wherein each voltage step ranges from about 5-15 mV.
5. The method of claim 1 , wherein the short duration pulse ranges from about 5-15 ms.
6. The method of claim 1 , wherein the lipid membrane comprises at least about 5 consistently active ion channels.
7. The method of claim 1 , wherein the pipette aperture is silanized.
8. The method of claim 1 , wherein the pipette aperture is coated with a siloxane compound.
9. The method of claim 1 , further comprising a step of adding an analyte to the solution, wherein measuring the conductance comprises measuring a first conductance after the analyte is added to the solution.
10. The method of claim 9 , wherein measuring the conductance further comprises measuring a second conductance before the analyte is added to the solution.
11. A method of obtaining ion channel measurements from an ion channel (IC) functionalized sensor platform, said method comprising:
a. providing the IC-functionalized sensor platform comprising an apparatus with an aperture and a lipid membrane suspended on the aperture, wherein a plurality of ion channels is embedded in the lipid membrane, wherein the IC-functionalized sensor platform is operatively connected to an amplifier;
b. placing the IC-functionalized sensor platform in a solution such that the lipid membrane is disposed in the solution;
c. continuously applying a voltage set to the IC-functionalized sensor platform via the amplifier, wherein the voltage set comprises a plurality of consecutive voltages that are applied in a step-wise pattern so as to alternate between decreasing and increasing voltages, wherein each voltage is rapidly applied for a short duration pulse; and
d. measuring a conductance of the lipid membrane; wherein a net time required for measuring the conductance is about 0.5 msec to about 50 msec.
12. The method of claim 11 , wherein a diameter of the aperture is about 1-5 μm.
13. The method of claim 11 , wherein the voltage set comprises about 2-5 increasing voltage steps and about 2-5 decreasing voltage steps.
14. The method of claim 13 , wherein each voltage step ranges from about 5-15 mV.
15. The method of claim 11 , wherein the short duration pulse ranges from about 5-15 ms.
16. The method of claim 11 , wherein the lipid membrane comprises at least about 5 consistently active ion channels.
17. The method of claim 11 , wherein the aperture is silanized.
18. The method of claim 11 , wherein the aperture is coated with a siloxane compound.
19. The method of claim 11 , further comprising a step of adding an analyte to the solution, wherein the measuring the conductance comprises measuring a first conductance after the analyte is added to the solution.
20. The method of claim 19 , wherein the measuring the conductance further comprises measuring a second conductance before the analyte is added to the solution.