Izon TRPS instruments allow for the voltage, pressure and stretch of the system to be tuned by the user, with each parameter having different effects on your measurement:
Voltage (V) - Determines the strength of the electrophoretic forces acting on the particles and the amplitude of the baseline current. Increasing the voltage will increase the baseline current, which will give relatively larger blockades and increase resolution. The trade off is a higher baseline current is more noisy and small blockades may be lost.
∆I ∝ V
I ∝ V
Measurements should be aimed to be recorded at a baseline of ~80-140 nA.
Pressure (Pa) - Controls the magnitude of the convection force through the pore, which allows for the particle rate to be tuned by increasing or decreasing the pressure. The blockade duration is also affected by stronger pressures, with high pressures causing many quick blockades. The particle rate is also proportional to particle concentration.
Rate ∝ P
Pressure should be tuned to give a particle rate of 200-1500 blockades per minute.
Stretch (mm) - Controls the size of the hole in the nanopore, which determines the size of particles allowed through and their relative blockade magnitude. Increasing the stretch increases the aperture size and will make blockades smaller as there is less resistance on the particles as they traverse the nanopore, it will also allow for larger particle sizes to be measured. As the blockade magnitude decreases, smaller particles can be lost in the baseline upon increasing stretch.
If smaller particles are becoming lost in the baseline, decreasing the stretch will increase the blockade magnitude and improve the signal-to-noise ratio, the lower the stretch, the smaller the particle size that can be measured.
The baseline current, I, will also increase when stretch increases, often requiring the applied voltage to be lowered at high stretches to achieve stable working baseline currents.
I ∝ S
Stretch should be set to between 44-49 mm to maintain pore stability.