TITLE: Measurement of Extracellular Vesicles with Tunable Resistive Pulse Sensing
AUTHORS: J. Nathaniel Diehl, Amelia Ray, Lauren B. Collins, Andrew Peterson, Kyle C. Alexander, John S. Ikonomidis, Adam W. Akerman

[DESCRIPTION]
This protocol details the steps necessary to quantify the concentration and size distribution of plasma-derived extracellular vesicles (EVs). This protocol utilizes a qNano Gold Tunable Resistive Pulsing Sensing (TRPS) measurement system from IZON Science. This is intended to serve as the second step in the workflow for EV quantification along with “Human Sample Processing and Isolation of Extracellular Vesicles with Size Exclusion Chromatography.”

[BEFORE_START]
Ensure IZON software has been installed on computer that will be used to run qNano instrument.
Reagents should be room temperature when used on the qNano. Allow adequate acclimation to ambient temperature prior to stretching the nanopore or beginning the experiment.

[GUIDELINES]
All human sample handling and processing should follow the Occupational Safety and Health Administration (OSHA) blood borne pathogens procedures to prevent possible pathogen transmission.
This protocol details the measurement of EVs from plasma samples. All human subjects research should be reviewed and approved by the site-specific Institutional Review Board (IRB) prior to proceeding.
This protocol uses the IZON qNano Gold TRPS instrument. If other TRPS measurement systems are used (e.g. IZON Exoid), this workflow will likely need to be adjusted slightly to incorporate alternate equipment.

Citations:

IZON qNano User Manual  https://f.hubspotusercontent30.net/hubfs/4136435/Manuals,%20Technical%20Notes%20and%20Customer%20Support/qNano/qnano-user-manual-QN1-OQ-014.pdf.
IZON qNano Control Suite Software  https://support.izon.com/qnano-control-suite-software.

Additional Notes:

2x PBS is preferred for measuring exosomes (30-150 nm) because it decreases the background noise.
Strategies for nanopore opening: These troubleshooting strategies can be used in any combination to facilitate the stabilization or opening of a nanopore. These methods can also be used to help unclog a nanopore when a run is paused.

        Tapping → With the VPM nozzle in place, use your index finger to firmly tap the shielding cap 3-5 times. This can disrupt bubbles and assist with pore opening.

        Clicking → Remove pressure from system and open pressure valve. Remove VPM nozzle. Raise the shielding and set it on the nanopore such that the posts of the shielding cap are resting on the arms of the nanopore. Apply downward pressure and begin to rotate until the shielding cap abruptly “clicks” downward into place.

        Pressure application device (PAD) use → Remove pressure from system and open pressure valve. Remove VPM nozzle and the shielding cap. Center PAD over upper fluid cell and give 10 plunges. Repeat as necessary to unclog nanopore or reduce RMS noise.

        Cleaning upper fluid cell → Remove pressure from system and open pressure valve. Remove VPM nozzle. Remove shielding cap and upper fluid cell. Clean upper fluid cell with filtered water and dry completely. Gently wipe top of nanopore with edge of Kimwipe. Replace upper fluid cell and shielding cap. Carefully pipette 2x ME or sample into upper fluid cell, avoiding introduction of bubbles. Proceed with run.

        Nanopore cleaning → Remove pressure from system and open pressure valve. Remove VPM nozzle. Remove shielding cap and upper fluid cell. Clean upper fluid cell with filtered water and dry completely. Reduce stretch on nanopore to 42 mm and remove from posts. Rinse each side of nanopore with 500 μL 70% ethanol followed by 500 μL filtered water. Carefully dry each side of nanopore with edge of Kimwipe. Dry lower fluid cell with Kimwipe. Replace nanopore on the posts and rotate knob clockwise to achieve desired stretch. Pipet 75 μL 2x ME carefully into the lower fluid cell, avoiding the introduction of bubbles. Replace upper fluid cell and shielding cap. Carefully pipette 35 μL 2x ME into upper fluid cell and click “Turn On”. Monitor current and RMS noise, proceed with other troubleshooting strategies as necessary. 

        Adjusting pore stretch → once a pore has been used, it is possible that it will need to be stretched >47 mm. Test nanopore stability at increased stretch values (maximum recommended stretch is 48 mm).

[STEPS]
SECTION: Reagent and sample preparation
1. Prepare 2x phosphate-buffered saline (2x PBS).
 

SECTION: Reagent and sample preparation
3. Dilute calibration particles (CPC100) 1:1500 in 2x ME. Store at  4 °C 1-2 weeks.
SECTION: Reagent and sample preparation
4. Thaw sample(s) at Room temperature and centrifuge 10000 x g for 10 min.
 

SECTION: Reagent and sample preparation
5. Dilute samples 1:10 in 2x ME and centrifuge 10000 x g for 10 min.
 

SECTION: Reagent and sample preparation
2. Prepare and filter ( ) 2x Measuring Electrolyte (2x ME) daily.
SECTION: Reagent and sample preparation
2.1. Dilute 30 µL Wetting Concentrate in 10 mL 2x PBS. Filter ( ) prior to use.
SECTION: Reagent and sample preparation
1.1. Completely dissolve one PBS tablet in 100 mL deionized water.
SECTION: Preparing and Wetting the nanopore
6. Plug-in qNano instrument and open the Izon Control Suite. Select “Classic capture.”
SECTION: Preparing and Wetting the nanopore
7. Remove the shielding cap and upper fluid cell and set aside on kimwipe.
SECTION: Preparing and Wetting the nanopore
8. Wet each side of the nanopore (NP100) with 500 µL 70% ethanol.
 

SECTION: Preparing and Wetting the nanopore
9. Rinse nanopore with 500 µL filtered water to remove ethanol. Pat dry with kimwipe, careful not to apply pressure to the central nanopore.
SECTION: Preparing and Wetting the nanopore
10. Wet the bottom electrode with 75 µL 70% ethanol, allow it to sit for 1 min and then dry completely with kimwipe.
SECTION: Preparing and Wetting the nanopore
11. Rinse bottom electrode with 75 µL filtered water and dry completely with kimwipe. Proceed immediately to next step.
SECTION: Preparing and Wetting the nanopore
12. Fit nanopore onto the posts with the serial number facing upwards and the long arm towards the user.
SECTION: Preparing and Wetting the nanopore
13. Stretch the nanopore by turning the knob clockwise and measure the stretch using the digital calipers. For initial measurement, stretch to   and enter this value in the software interface.
SECTION: Preparing and Wetting the nanopore
13.1. Measure distance between three-prong posts opposite one another.
SECTION: Preparing and Wetting the nanopore
13.2. Once the initial stretch is confirmed and recorded, the software will track additional adjustments.
SECTION: Preparing and Wetting the nanopore
13.3. Begin recording the duration of time that the nanopore has been stretched. Nanopores typically begin to destabilize or overstretch when used >6-7 hours.
SECTION: Preparing and Wetting the nanopore
14. To wet the nanopore, slowly pipet 75 µL 2x ME into the lower fluid cell being careful not to introduce bubbles.
 

SECTION: Preparing and Wetting the nanopore
15. Clean the upper fluid cell with 500 µL filtered water and dry completely by tapping firmly on a kimwipe or drying directly with a kimwipe.
SECTION: Preparing and Wetting the nanopore
16. Fit the upper fluid cell on the qNano above the nanopore and add 35 µL 2x ME into the central well of the upper fluid cell, being careful to avoid introducing bubbles.
 

SECTION: Preparing and Wetting the nanopore
17. Place shielding cap over the upper fluid cell and fit the Variable Pressure Module (VPM) nozzle into the upper fluid cell.
SECTION: Preparing and Wetting the nanopore
18. 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 4 min -5 min for pore opening/wetting.
SECTION: Preparing and Wetting the nanopore
19. Ensure that the nanopore is open completely. See “Additional notes ->Strategies for pore opening” for strategies to promote pore opening and stabilization.
 

SECTION: Preparing and Wetting the nanopore
20. Close the pressure valve and increase the pressure with the pressure bar to 8 mbar-10 mbar.
SECTION: Preparing and Wetting the nanopore
21. Slowly increase the voltage in a step-wise manner until a current of 110-135 nA is reached.
 

SECTION: Recording calibration particles
22. Once the current is stable at ~120 nA and the RMS noise is <10, remove the shielding cap and upper fluid cell.
SECTION: Recording calibration particles
23. Clean upper fluid cell by tapping firmly on kimwipe and/or drying with kimwipe directly.
SECTION: Recording calibration particles
24. Gently use the edge of a kimwipe to dry the top of the nanopore.
SECTION: Recording calibration particles
26. Replace upper fluid cell and shielding cap, add 35 µL of diluted calibration particles (careful not to introduce bubbles during pipetting).
 

SECTION: Recording calibration particles
27. Replace VPM nozzle, close pressure valve, and set pressure bar to 3 mbar (‘Working Pressure 1’).
 

SECTION: Recording calibration particles
28. Observe particle size and adjust pore stretch and voltage accordingly.
 

SECTION: Recording calibration particles
29. Note the voltage, stretch, RMS noise, and current. These should be kept constant for all calibration and sample readings.
SECTION: Recording calibration particles
30. Start recording calibration particles at ‘Working Pressure 1’ (e.g. 3 mbar). Permit recording of a minimum of 500 particles before stopping the recording.
 

SECTION: Recording calibration particles
31. 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.
SECTION: Recording calibration particles
32. Set the pressure bar to ‘Working Pressure 2’ (6 mbar recommended). Repeat steps 29-30.
SECTION: Recording calibration particles
33. Set the pressure bar to ‘Working Pressure 3’ (9 mbar recommended). Repeat steps 29-30.
 

SECTION: Sample/calibration particle changeover
34. Remove the shielding cap and upper fluid cell.
SECTION: Sample/calibration particle changeover
35. Clean upper fluid cell by pipetting 500 µL filtered water into upper fluid cell and then tapping firmly on kimwipe and/or drying with kimwipe directly.
SECTION: Sample/calibration particle changeover
36. Gently use the edge of a kimwipe to dry the top of the nanopore.
SECTION: Sample/calibration particle changeover
37. Replace upper fluid cell and shielding cap.
SECTION: Sample/calibration particle changeover
38. Add 35 µL of 2x ME to upper fluid cell (careful not to introduce bubbles during pipetting).
SECTION: Sample/calibration particle changeover
39. Replace VPM nozzle, close pressure valve, and set pressure bar to 20 mbar (max pressure).

SECTION: Sample/calibration particle changeover
40. If particle rate is <15 particles/min, remove shielding cap and upper fluid cell.
SECTION: Sample/calibration particle changeover
41. Tap dry upper fluid cell and gently use a kimwipe to clean top of nanopore.
SECTION: Sample/calibration particle changeover
42. Replace upper fluid cell and shielding cap.
SECTION: Sample recording
43. Follow sample changeover protocol as above when switching from calibration particles to samples or between samples.
 

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

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

SECTION: Sample recording
47. 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.
SECTION: Sample recording
48. Set the pressure bar to ‘Working Pressure 2’ (e.g. 6 mbar). Repeat steps 40-41.
 

SECTION: Sample recording
49. Record 2-3 technical replicates per sample and average replicates post-hoc for more accurate concentration estimates.
 

SECTION: Sample recording
50. Follow “Sample changeover” procedures between samples.
SECTION: Nanopore and qNano Cleaning
51. Click “Turn off” to discontinue current across the nanopore.
SECTION: Nanopore and qNano Cleaning
52. Remove pressure by pulling out pressure bar completely followed by opening the pressure valve.
SECTION: Nanopore and qNano Cleaning
53. Remove VPM nozzle and follow the sample changeover process above.
SECTION: Nanopore and qNano Cleaning
54. Reduce the stretch to   by turning the knob counterclockwise.
SECTION: Nanopore and qNano Cleaning
55. Remove the nanopore and rinse the nanopore from both sides with 500 µL 70% ethanol followed by 500 µL deionized water. Gently dry with clean with kimwipe and secure Nanopore in respective pouch and record date and usage hours on pouch.
 

SECTION: Nanopore and qNano Cleaning
56. Rinse top fluid cell with 500 µL 70% ethanol followed by 500 µL deionized water and dry completely with kimwipe.
SECTION: Nanopore and qNano Cleaning
57. Remove 2x ME from bottom electrode, rinse with deionized water, and then dry with kimwipe.
SECTION: Nanopore and qNano Cleaning
58. Replace top fluid cell and shielding cap.
SECTION: Nanopore and qNano Cleaning
59. Unplug qNano until next use.
SECTION: Data processing and analysis
60. Select analyze data, which will take the user to a new interface.
SECTION: Data processing and analysis
61. Right click on “unprocessed files” -> select “process files”.
SECTION: Data processing and analysis
62. Select each sample/calibration particle reading and ensure all data entries are accurate.
 

SECTION: Data processing and analysis
63. Right click on sample and select “Save dataset”.
 

SECTION: Data processing and analysis
64. 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.
SECTION: Data processing and analysis
65. 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.
SECTION: Data processing and analysis
66. Repeat step 63 for all samples.
SECTION: Data processing and analysis
67. Select all samples by placing a check in all tick boxes along left most column of sample interface.
SECTION: Data processing and analysis
68. 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.
SECTION: Data processing and analysis
69. Select the funnel icon → select “Particle Diameter (nm) → enter “<300”.
SECTION: Data processing and analysis
70. Right click and save all datasets.
SECTION: Data processing and analysis
71. Apply concentration fraction right clicking on a dataset → select “Concentration Fraction” → set lower limit to  , upper limit to  , and check “apply to all datasets” → click “OK”.
SECTION: Data processing and analysis
72. Repeat step 63 to recalibrate all sample sets.
SECTION: Data processing and analysis
73. 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.
SECTION: Sample/calibration particle changeover
39.1. If the rate is below 15 particles/min after 10 s, proceed with the next sample/calibration. If the rate is above 15 particles/min after 10 s, repeat steps 33-38.
SECTION: Recording calibration particles
25. Gently use the edge of a kimwipe to dry the top of the nanopore.
 

SECTION: Sample recording
45. 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.