During Exoid setup, you are required to set the voltage to produce a stable baseline current between 100-140 nA. The blockade magnitude for a given particle size is dependent on this baseline current (Figure 1).
- A lower baseline current will result in a smaller blockade magnitude than a larger baseline current for the same calibration standard.
- A larger baseline current will result in stronger signals, which increase the resolution of size measurements and allow detection of smaller particle sizes but also produce more noise.
The recommended setup process provides an optimum trade-off between these two factors.
Figure 1: The relationship between baseline current and blockade magnitude for a fixed particle size.
Calibration establishes the relationship between blockade magnitude and particle size under a particular set of experimental conditions—stretch, pressure and voltage—and at a stable baseline current. When you analyse a particle of unknown size under the same conditions, the Exoid measures the magnitude of the blockade it produces, and the Izon Data Suite (IDS) software uses the relationship between blockade and particle size from the calibration standard to calculate the size of the particle. It uses the following equation, where ∆I is the blockade magnitude:
If the baseline current drifts significantly from calibration:
- Lower currents → Blockades shrink → Particle size may be underestimated, and small particles may be lost in background noise, lowering concentration measurements.
- Higher currents → Blockades increase → Particle size may be overestimated, and noise spikes could be miscounted as particles, potentially increasing concentration measurements.
If there is a significant shift in the baseline current despite experimental conditions being kept the same, this could indicate a problem with the nanopore. Baseline current is dependent on nanopore hole size and unexpected changes can be caused by blockages or tears. Changes to hole size will cause opposite effects to those described above:
- Smaller hole → lower currents but bigger blockades
- Larger hole → higher currents but smaller blockades
To ensure accurate size and concentration data, keep the baseline current within 10% of the calibration measurement – the closer, the better!