Qnano gold system

NTA was performed using the TRPS technique and analyzed on a qNano Gold system (Izon, Medford, MA) to determine the size and concentration of EV particles. With the qNano instrument, an electric current between the two fluid chambers is disrupted when a particle passes through a nanopore NP150 with an analysis range of 70 nm to 420 nm, causing a blockade event to be recorded. The magnitude of the event is proportional to the amount of particles traversing the pore, and the blockade rate directly relates to particle concentration that is measured particle by particle. The results can be calibrated using a single-point calibration under the same measurement conditions used for EV particles (stretch, voltage, and pressure) (80 (link)). CPC200 calibration particles (Izon) were diluted in filtered Izon-supplied electrolyte at 1:500 to equilibrate the system prior to measuring EVs and for useused as a reference. Isolated extracellular vesicle samples were diluted at 0, 1:10, 1:100, and 1:1,000. For the measurements, a 35 µl sample was added to the upper fluid chamber and two working pressures, 5 mbar and 10 mbar, were applied under a current of 120 nA. Particle rates between 200 and 1500 particles/min were obtained. The size, density, and distribution of the particles were analyzed using qNano software (Izon).

Particle concentration and size distribution were measured using the qNano Gold system (Izon Science) and an NP150 Nanopore (Izon Science). Extracellular vesicles samples were diluted 1:100 in filtered PBS and CPC100 beads (Izon Science) were used as the calibration standard. Particles were measured using 46.0 mm stretch with a voltage of 0.6-1.4 V and a pressure between 4.46 and 7.33 mbar. The number of particles analysed per sample was at least 1000. The blockade magnitude of the calibration particles was above 0.2 nA and a new Nanopore was used in every different measurement day. Data were processed with Izon Control Suite software version 3.3 (Izon Science).