Many users face the most difficulty during the initial nanopore setup. This is especially prevalent with the smaller nanopores. Izon has developed a coating procedure to reduce the impact of surface modification due to interaction with biological constituents. The coating procedure uses Izon Coating Solution (ICS) which treats the nanopore and minimizes non-specific binding of particles with the pore material. This step is compulsory for work with biological samples on a small nanopore.
For effective coating procedure, ICS must have not expired and must have been stored at 4°C. Beyond the recommended storage time, the reagent will deteriorate and cause instability problems.
Preparation of Izon Coating Solution
All solutions must be prepared fresh daily. To prepare the day’s solution (enough for a day’s use), filter 6 mL of ICS solution through a 0.22 μm filter to use in the Nanopore Coating step. 15 mL of Measurement Electrolyte is usually sufficient for a day’s worth of measurement.
Stock ICS must be stored in the fridge when not in use.
Nanopore Coating Procedure
In Assistant-based measurements, users are guided to coat the nanopore for 10 minutes. Follow these steps to achieve complete nanopore coating:
- Load upper and lower fluid well with filtered ICS.
- Connect the VPM nozzle to the upper fluid cell and apply a maximum pressure for 10 minutes
- Once the coating step is completed, proceed to equilibrating nanopore’s electrolyte.
- 10 mins is the minimum time required for a successful pore coating step. Better stability can be achieved with longer coating time e.g. 15-20 mins.
- A combination of longer coating time in both directions i.e. 15 mins (with pressure applied) followed by another 15 mins (with vacuum applied) would ensure maximum stability.