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.
Dilute 25% EtOH + 1% Izon Wetting Concentrate (available from TRPS Reagent Kit) in PBS. The Izon Wetting Concentrate is supplied at 10% concentration.
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 47.5 mm (in the Assistants, software states a conservative number of 47 mm however, depending on the pore size, working at higher stretch is also possible).
- Load 75 μL of wetting solution into the lower fluid cell ONLY.
- Fit the upper fluid cell and connect the VPM nozzle to the upper fluid cell; apply a maximum vacuum for 4-5 minutes.
- Click the metal shielding cap 5-10 times to mechanically assist the nanopore wetting.
- Once completed, remove applied vacuum.
- Remove the wetting solution from the lower fluid cell.
Establish Baseline Current
To ensure that the wetting procedure is successful:
- Load 75 μl of electrolyte into lower fluid cell.
- Fit the upper fluid cell and load 35 μL of electrolyte.
- Apply 0.1V and maximum pressure.
- Verify there is a stable baseline current in the system (I > 10 nA, RMS noise <15pA), if not repeat Wetting Procedure.
- Once there is a stable baseline current, proceed to the next stage of nanopore preparation (Coating Procedure).