When working with smaller nanopores, there are a few parameters to consider before you can measure your samples.
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.
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).
2. Nanopore Preparation
- 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.
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:
- How do I isolate EV from urine using qEV?
- How do I isolate EV from cell culture media using qEV?
- How do I isolate EV from blood products using qEV?
Please note that all biological are complex and the user must optimise the protocols for their own biological particles systems.