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 minimises non-specific binding of particles with the pore material. This step is compulsory for work with samples of a biological nature 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
- Add 15 mL of Measurement Electrolyte to a 50 mL tube and place in a warm water bath.
- Accurately weigh out 2.0 g of the ICS powder and slowly add to the warmed Measurement Electrolyte. Fit the lid tightly and mix vigorously until the lump of powder has disappeared. The solution should remain in the water bath until the solution is clear.
- When the solution is clear, top up with Measurement Electrolyte resulting in a total volume of 20 mL.
- Filter with a 0.45 μm filter into a clean labelled tube, along with the date. ICS should be stored at 4°C when not in use.
Daily Preparation of Previously made up ICS
All solutions must be prepared fresh daily. To prepare the day’s solution (enough for a day's use), filter 1-2 mL of ICS solution through a 0.22 μm filter to use in the Nanopore Coating step. Each coating procedure requires 110 μL.
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 cell with filtered ICS with 35 μL and 75 μL, respectively.
- Connect the pressure 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.