Measurement of Extracellular Vesicles with Tunable Resistive Pulse Sensing

Prepare 2x phosphate-buffered saline (2x PBS).

Completely dissolve one PBS tablet in 100mL deionized water.

Prepare and filter (0.22 µm ) 2x Measuring Electrolyte (2x ME) daily.

Dilute 30µL Wetting Concentrate in 10mL 2x PBS. Filter (0.22 µm ) prior to use.

Dilute calibration particles (CPC100) 1:1500 in 2x ME. Store at 4°C 1-2 weeks.

Thaw sample(s) at Room temperature and centrifuge 10000x g,0h 0m 0s for 0h 10m 0s.

Dilute samples 1:10 in 2x ME and centrifuge 10000x g,0h 0m 0s for 0h 10m 0s.

Plug-in qNano instrument and open the Izon Control Suite. Select “Classic capture.”

Remove the shielding cap and upper fluid cell and set aside on kimwipe.

Wet each side of the nanopore (NP100) with 500µL 70% ethanol.

Rinse nanopore with 500µL filtered water to remove ethanol. Pat dry with kimwipe, careful not to apply pressure to the central nanopore.

Wet the bottom electrode with 75µL 70% ethanol, allow it to sit for 0h 1m 0s and then dry completely with kimwipe.

Rinse bottom electrode with 75µL filtered water and dry completely with kimwipe. Proceed immediately to next step.

Fit nanopore onto the posts with the serial number facing upwards and the long arm towards the user.

Stretch the nanopore by turning the knob clockwise and measure the stretch using the digital calipers. For initial measurement, stretch to 47 mm and enter this value in the software interface.

Measure distance between three-prong posts opposite one another.

Once the initial stretch is confirmed and recorded, the software will track additional adjustments.

Begin recording the duration of time that the nanopore has been stretched. Nanopores typically begin to destabilize or overstretch when used >6-7 hours.

To wet the nanopore, slowly pipet 75µL 2x ME into the lower fluid cell being careful not to introduce bubbles.

Clean the upper fluid cell with 500µL filtered water and dry completely by tapping firmly on a kimwipe or drying directly with a kimwipe.

Fit the upper fluid cell on the qNano above the nanopore and add 35µL 2x ME into the central well of the upper fluid cell, being careful to avoid introducing bubbles.

Place shielding cap over the upper fluid cell and fit the Variable Pressure Module (VPM) nozzle into the upper fluid cell.

Set voltage to 0.10 V and select “Turn On”. Apply 20 mbar pressure by closing the pressure valve and adjusting the pressure bar. Allow to run for 0h 4m 0s -0h 5m 0s for pore opening/wetting.

Ensure that the nanopore is open completely. See “Additional notes ->Strategies for pore opening” for strategies to promote pore opening and stabilization.

Close the pressure valve and increase the pressure with the pressure bar to 8 mbar-10 mbar.

Slowly increase the voltage in a step-wise manner until a current of 110-135 nA is reached.

Once the current is stable at ~120 nA and the RMS noise is <10, remove the shielding cap and upper fluid cell.

Clean upper fluid cell by tapping firmly on kimwipe and/or drying with kimwipe directly.

Gently use the edge of a kimwipe to dry the top of the nanopore.

Gently use the edge of a kimwipe to dry the top of the nanopore.

Replace upper fluid cell and shielding cap, add 35µL of diluted calibration particles (careful not to introduce bubbles during pipetting).

Replace VPM nozzle, close pressure valve, and set pressure bar to 3 mbar (‘Working Pressure 1’).

Observe particle size and adjust pore stretch and voltage accordingly.

Note the voltage, stretch, RMS noise, and current. These should be kept constant for all calibration and sample readings.

Start recording calibration particles at ‘Working Pressure 1’ (e.g. 3 mbar). Permit recording of a minimum of 500 particles before stopping the recording.

Remove pressure by pulling out pressure bar completely and then label the sample. An example label is included below:

• Investigation: Experiment name and date.
• Nanopore ID: serial number.
• Nanopore Part#: NP100.
• Sample ID: calibration#1_pressure3_dilution.1.1500.
• Raw concentration (calibration only): 1.7e13.
• Dilution: 1500.
• Pressure: 3 (the software resets this value, include pressure setting in Sample ID).
• Electrolyte ID: 2x ME.

Set the pressure bar to ‘Working Pressure 2’ (6 mbar recommended). Repeat steps 29-30.

Set the pressure bar to ‘Working Pressure 3’ (9 mbar recommended). Repeat steps 29-30.

Remove the shielding cap and upper fluid cell.

Clean upper fluid cell by pipetting 500µL filtered water into upper fluid cell and then tapping firmly on kimwipe and/or drying with kimwipe directly.

Gently use the edge of a kimwipe to dry the top of the nanopore.

Replace upper fluid cell and shielding cap.

Add 35µL of 2x ME to upper fluid cell (careful not to introduce bubbles during pipetting).

Replace VPM nozzle, close pressure valve, and set pressure bar to 20 mbar (max pressure).

If the rate is below 15 particles/min after 0h 0m 10s, proceed with the next sample/calibration. If the rate is above 15 particles/min after 0h 0m 10s, repeat steps 33-38.

If particle rate is <15 particles/min, remove shielding cap and upper fluid cell.

Tap dry upper fluid cell and gently use a kimwipe to clean top of nanopore.

Replace upper fluid cell and shielding cap.

Follow sample changeover protocol as above when switching from calibration particles to samples or between samples.

Add 35µL dilute sample to upper fluid cell. Replace the VPM nozzle, close the pressure valve, and set pressure bar to desired working pressure.

Ensure that stretch and voltage settings are the same for as those used for the corresponding calibration particles. Additionally, ensure the current is within the 116-124 nA range and the RMS noise is <10.

Start recording sample at ‘Working Pressure 1’ (e.g. 3 mbar). Permit recording of a minimum of 500 particles before stopping the recording.

Remove pressure by pulling out pressure bar completely and then label the sample. An example label is included below:

• Investigation: Experiment name and date.
• Nanopore ID: serial number.
• Nanopore Part#: NP100.
• Sample ID: samplename#1_pressure3_dilution.1.10.
• Dilution: 10.
• Pressure: 3 (the software resets this value, include pressure setting in Sample ID).
• Electrolyte ID: 2x ME.

Set the pressure bar to ‘Working Pressure 2’ (e.g. 6 mbar). Repeat steps 40-41.

Record 2-3 technical replicates per sample and average replicates post-hoc for more accurate concentration estimates.

Follow “Sample changeover” procedures between samples.

Click “Turn off” to discontinue current across the nanopore.

Remove pressure by pulling out pressure bar completely followed by opening the pressure valve.

Remove VPM nozzle and follow the sample changeover process above.

Reduce the stretch to 42 mm by turning the knob counterclockwise.

Remove the nanopore and rinse the nanopore from both sides with 500µL 70% ethanol followed by 500µL deionized water. Gently dry with clean with kimwipe and secure Nanopore in respective pouch and record date and usage hours on pouch.

Rinse top fluid cell with 500µL 70% ethanol followed by 500µL deionized water and dry completely with kimwipe.

Remove 2x ME from bottom electrode, rinse with deionized water, and then dry with kimwipe.

Replace top fluid cell and shielding cap.

Unplug qNano until next use.

Select analyze data, which will take the user to a new interface.

Right click on “unprocessed files” -> select “process files”.

Select each sample/calibration particle reading and ensure all data entries are accurate.

Right click on sample and select “Save dataset”.

Ensure that each set of calibration and sample recordings have increased particle rates at higher pressures by selecting individual files in the left-most interface and then clicking the line graph icon.

Select the “Calibrated” tick-box next to the first sample. This will bring up a new window → select the sample recording at 2 pressures and the calibration particles at the equivalent pressure, stretch and voltage → click OK.

Repeat step 63 for all samples.

Select all samples by placing a check in all tick boxes along left most column of sample interface.

Hover over blockade summary at bottom left of screen → a tab interface should appear, pin it by clicking the push pin icon in the upper right corner of the new tab interface.

Select the funnel icon → select “Particle Diameter (nm) → enter “<300”.

Right click and save all datasets.

Apply concentration fraction right clicking on a dataset → select “Concentration Fraction” → set lower limit to 30 nm , upper limit to 300 nm , and check “apply to all datasets” → click “OK”.

Repeat step 63 to recalibrate all sample sets.

Select dropdown menu next to “Preview” button → select “Group Report” to generate a PDF containing a simplistic report summarizing all samples and their data values.