Many users face the most difficulty during the initial nanopore setup. This is especially prevalent with the smaller nanopores, due to the smaller nanopore size, where establishing a connection between the upper and lower fluid cells can be challenging. The nanopore must be thoroughly prepared before any particles are introduced.
Full wetting of the nanopore, without any trapped air bubbles, is critical to achieve stable and repeatable measurements.
The Izon Wetting Concentrate is supplied at 10% concentration (available from TRPS Reagent Kit). A 100-fold dilution of Izon Wetting Concentrate (resulting in a final concentration of 0.1%) in PBS results in Izon Wetting Solution.
The nanopore is made of a hydrophobic thermoplastic polyurethane (TPU). Instructions on how to wet the nanopore are already included within the software (Capture Assistants). Below are some extra tips to ensure proper wetting:
- Stretch nanopore to 47 mm.
- Load 75 μL of wetting solution into the lower fluid cell.
- Fit the upper fluid cell and load 35 μL of wetting solution into the upper fluid cell.
- Connect the VPM nozzle to the upper fluid cell; apply a maximum positive pressure for 2 minutes.
- Once completed, remove applied pressure. A stable baseline current should now have been established.
- If a stable baseline has not been established, you can use the PAD (Pressure Application Device) to apply a plunging motion to the upper fluid cell up to 10 times.
- If a stable baseline has still not been established, the shielding cap can be gently clicked by placing it on the pore arms and then twisting it into place, as demonstrated in the video below:
- The wetting solution can then be removed from both fluid cells and replaced with electrolyte.
Once there is a stable baseline current, proceed to the next stage of nanopore preparation (Coating Procedure).