What are the recommended protocols for measuring particles that are <100 nm in diameter?

When working with smaller nanopores, there are a few parameters to consider before you can measure the size and concentration of your samples.

1. Electrolyte

Reagents must be of the highest purity to prevent blockages and minimise RMS noise. Higher strength electrolyte is preferred when working with particles in the sub-100 nm region.

When measuring particles down to 40 nm Izon supplied electrolyte can be used, it may be required to use 2X PBS buffer (or an electrolyte with an equivalent conductivity) with surfactant.

DO NOT use PBS that is not fresh or has been left on the lab bench. In some instances some particles may have reduced stability in higher salt conditions.

Higher strength electrolyte allows for:

An increased signal-to-noise ratio - more ions to displace as particles go through the nanopore which would give a larger resistive pulse signal.
A lower applied voltage which helps with minimising RMS noise, stabilising baseline current, as well as minimising the electro-osmotic component (which moves in the opposite direction of electro-phoretic component).

The nanopore is made of TPU which is slightly hydrophobic. Addition of 0.03% surfactant in the electrolyte is necessary to aid in wetting of the nanopore.

2. Nanopore Preparation

The nanopore must be thoroughly prepared and wetted before any particles are introduced. Establishing a baseline current on smaller nanopores (NP200 and smaller) requires the use of Izon Wetting Solution. Wetting Solution is a component of the Reagent Kit which can be purchased from the Izon Store.

Izon Coating Solution (ICS) must always be used when working with biological samples as it minimizes non-specific binding of particles with the nanopore material. ICS is a component of the Reagent Kit which can be purchased from the Izon Store. 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

Tips for working with Izon Coating Solution (ICS)

Always keep bulk solution in fridge when not in use. Discard weekly.
ICS solution is quite viscous. Ensure that solutions are mixed thoroughly before taking aliquots out of the bulk solution.
Filter all solutions before use.

In Assistant-based measurements, users are guided to coat the nanopore for 10 minutes. Note that this is the minimum time that’s required for a successful pore coating step. Better stability may 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.

3. Voltage Polarity

Electro-osmosis component is dominant when using smaller nanopores coupled with a high applied voltage (>0.9 V).

If the Classic Capture mode is used, user can apply negative polarity to encourage particle travelling downwards, in the same direction of the electro-osmotic flow. This, combined with a high applied pressure (> 7 mbar) would allow stable measurement to be recorded.

When using negative polarity pore calibration is best achieved with neutral calibration particles rather than negatively charged calibration particles. Izon can supply the neutral particles. Contact Izon Support to purchase these.

4. Sample Preparation

Below are an example of a generic protocol for EV sample purification pre-TRPS measurement:

Complete guide on different sample types are also available:

Please note that all biological are complex and the user must optimise the protocols for their own biological particles systems.