IZON Exoid Training Programme Forefront of Bio-Nanoparticle Analysis Instruction Manual

IZON Exoid Training Programme Forefront of Bio-Nanoparticle Analysis

TRAINING PROGRAMME OVERVIEW

Before starting this training programme, make sure you have:

Read through and understood the Exoid User Manual and the Reagent Kit Technical Note.

There are two sample types run in the training programme:

1. Monomodal: Uniform, simple sample.
2. Bimodal: More diverse, complex sample with two populations.

RECORDING VS MEASUREMENT
In order to understand this document properly it is important to know these two key definitions:

• A recording is defined as an individual data recording.
• A measurement is a collection of multiple recordings. A complete size measurement includes both sample and calibration recordings.

OVERVIEW

• Complete a pre-defined measurement plan
• Edit sample details
• Complete a basic size and concentration measurement
• Apply filters to measured data
• Self-assess your results (guidance provided) and repeat if required
• Reagent Preparation
  • Prepare solutions in accordance with instructions provided in the lzon reagent kit
• Sample Preparation
  • Dilute sample and calibration particles
• TRPS Setup
  • Nanopore setup
  • Establish a stable baseline
  • Optimise stretch and pressure using calibration particles
• Data Recording
  • Recording data for size and concentration analysis
  • Monomodal
  • Bimodal
• Data Analysis
  View data, charts and reports

PREPARE REAGENTS

Prepare reagents following the protocol in the Reagent Kit Tech Note.

SAMPLE PREPARATION

Prepare Solution S (Sample) and TKP200 (Calibration) at a dilution factor of 51 by putting 20 µL of the particles into 1000 µL of ME in a 1.5 mL centrifuge tube. Prepare a bimodal sample by mixing 200 µL of each of your diluted TKP200 and Solution S and mix them together in a clean 1.5 mL centrifuge tube. Remember to label your vials accordingly.

PREPARING THE SYSTEM

Connect the power and two USB cables before opening the Exoid Control Suite (ECS).

• Enter Investigation Details.
• Pipette 75 µL of ME onto the lower fluid cell.
• Remove the ME but leave the surface wet.

NANOPORE SETUP

Follow the on-screen instructions to prepare the fluid cell, calibrate the nanopore stretch, wet the nanopore, coat the nanopore, establish a stable baseline, insert the calibration particles, and optimise the stretch and pressure.
If manual wetting of the nanopore is required, first remove the APS nozzle and attempt to use the Izon Pressure Application Device (PAD) to apply pulses of pressure to the upper fluid cell up to 10 times. If further encouragement is required, use a combination of increased stretch, high positive or negative pressure, and tapping the top of the APS nozzle. This is done by inserting the APS nozzle, applying a pressure of 2500 Pa and hitting the top of the APS nozzle with the back of the PAD to provide a short, sharp mechanical stimulation.

DEF INITION OF A STABLE BASE LINE CURRENT

A stable baseline current is one that drifts by less than 0.5 nA in 60 seconds and has an RMS noise of <10 pA. An unstable baseline current will produce unreliable measurements and should not be used under any circumstances.

NANOPORE CALIBRATION

Once the nanopore has been calibrated with calibration particles according to the on-screen instructions, the ECS will output a display message announcing the resolvable particle range with that particular nanopore. If this range does not cover 200 and 350 nm then set up a new nanopore as it will not resolve the training particles.

PART ICLE SIZE AND CONCENTRATION ANALYS IS

Monomodal Sample

Select “Start Analysis” and then “Size and Concentration.” The baseline current established earlier during nanopore setup should be visible. Enter the sample details, making sure that
the dilution factor entered is correct before proceeding.
Use the slider bar to select a stretch that will allow a target ananlysis size range that encapsulates 200 nm and 350 nm particles. Select yes for “Would you like to run a calibration at these settings?” Enter the calibration details, making sure the dilution factor and size are entered correctly before proceeding. Select yes for “Does the upper fluid cell contain calibration particles?”
After the system has optimised and entered the “Ready to (pre)calibrate” screen* (Fig 1), edit the pressure and voltage values by clicking on the displayed number (Fig 2). Apply a voltage that gives a current of ~100 nA, and choose a pressure that will give particle rates of 500-1500 particles/min. This pressure will be the middle pressure value, the ECS will go 200 Pa below and 400 Pa above for the other two pressures. Figure 1. A screen capture from the ECS, showing that the user is prompted to optimise their settings.

After this stage, these values will not be able to be changed without canceling the measurement, with the exception of troubleshooting the system while a recording is paused.
Run the calibration when prompted by the ECS. During this time there will be acccess to several functions during data recording outlined on the following page.
Following calibration collection, clean the nanopore according to the on- screen instructions.
Once the nanopore is cleaned, follow the
on-screen instructions regarding insertion of Solution S as the sample, and any consecutive cleaning protocols.
Select no for “do you want to run a calibration at these settings?”
Select yes for “Would you like to run another measurement?”
*If “no” was originally selected for “Would you like to run a calibration at this stage?” then the system settings will need to be optimised at the “Great! You’re good to go.” screen.

Bimodal Sample

Follow the on-screen prompts to run another calibration and another sample using exactly the same process as for the monomodal sample. The second sample will be the bimodal sample prepared earlier.
Once again, select no for “Do you want to run a calibration at these settings”
Select no for “Would you like to run another measurement?”
Follow the on-screen instructions for cleaning the nanopore before proceeding to data analysis. Figure 2. A screen capture from the ECS, demonstrating the location of the applied pressure (Pa), voltage (mV), and stretch (mm).

THE S IGNAL TRACE

The signal trace is where the user can observe the baseline current and any particle blockades, as well as real time values for RMS noise, blockade count and particle rate.
The user can adjust the signal trace baseline zoom at any stage using the controls in the top left-hand corner of the signal trace window, while the time period zoom can be changed on the bottom right. The user can also expand (or collapse if already expanded) the signal trace window by pressing the symbol in the top right.
This is where the baseline should be monitored closely for stepping down, drifting, or becoming noisy at which point the recording should
first be paused and recovered, or cancelled and restarted if recovery is not possible. The baseline current should not differ by more than 5% between data recordings. The particle rate also requires monitoring and should have a particle rate between 500-1500 particles per minute, the rate should also remain constant over the course of a recording.

DATA RECORDING

During data recording there are four recording features available to the user:

Advance
Saves the recording and advances to the next pressure step before the required particle count has been reached. This is available in the un-paused data collection screen (Fig 3).

Pause Analysis
Pauses the recording so that the user can manually troubleshoot the system. This is available in the un-paused data collection screen.

Cancel**
Cancels the recording and allows the user to return to the previous step without saving the current data set. This is available in the un-paused data collection screen. Figure 4. A screen capture from the ECS, showing the un-paused data collection screen with the Advance, Pause Analysis, and Cancel buttons available.

Resume Analysis
Resumes the recording once system troubleshooting has been successfully completed while the recording was paused. This is available in the paused data collection screen (Fig 4).

Restart Run
Restarts the current pressure recording. This is available in the paused data collection screen. Figure 5. A screen capture from the ECS, showing the paused data collection screen with the Resume Analysis and Restart Run buttons available.

**This is not the same as the cancel button located at the top right of the instructions screen. The top right cancel button will exit sample analysis, causing data loss if pressed during data collection, and return the user to the home screen.

DATA ANALYSIS

Below is a guide to useful functions in the Izon Data Suite (IDS). “Settings” will allow you to change the bin size and axis scales among other things. You can check mark multiple recordings to display more than one on the same chart at one time.

Assess the quality of the data:

• Is there any contamination present?
• Is there one distinct peak observed in the calibration and monomodal size histogram and two peaks in the bimodal size histogram?
• Is the average baseline current similar between recordings ±5%?
• Are the rate plots linear, with the higher pressure having a higher particle rate?

Using a filter
If there is contamination present, you can filter this out by hovering over the grey bar in the very bottom left corner. This will open the information for all the individual data points, at which point you can select the funnel icon, which will add a line for you to enter your filters e.g. if you have contamination less than 300 nm diameter then put >300 as the filter. This will include all data points larger than 300 nm and will exclude all others. You can also “deselect” individual data points by unticking the checkbox to the left under the “Include” header. You can select a range of values by using the – symbol e.g. 280-400 will display all data points between 280 and 400 nm.

NOTE: Blockade Magnitude (nA) will change to Particle Diamter (nm) in a calibrated data set. Once you are happy with your filters, select one of the monomodal samples for viewing and click preview in the upper tool ribbon to generate a measurement report which can be saved as a PDF. Check the acceptance criteria:

Mean Measured Particle Size = Labelled Particle Size ± 5%
Measured Raw Concentration = Labelled Concentration ± 20%

F I LTER DATA FOR THE B IMODAL SAMPLE

Small Population

1. Edit filter settings:
   • Keep all three sample files ticked for viewing.
   • Enter a filter in the particle diameter column (should be similar to 150-280 nm).
   • Select “Apply filters to all viewed records.”
   • Close the filter pane.
2. Particle concentration will no longer be displayed.
3. Edit dilution factor.
   • Click on “Sample Details.”
   • Change the dilution to 102 for all three sample files.
4. Recalibrate sample to update concentration.
   • Re-tick the box in the “calibrated” column.
   • Go to the “Multi-Pressure Calibration” tab.
   • Select all three sample files, all calibration files associated with the sample files will be populated.
   • Select all three calibration files to calibrate with.
5. Produce a group report for this population and save as a PDF for submission.
   • Right click on the group name.
   • Save group as “Small Population”.
   • Right click and select “Group Report”
   • Save as PDF.

Large Population
Follow the steps for filtering for a small population, except the size filter should be about 280-400 nm . Save this group as “Large Population“ before producing the group report.

Prepare Group File for Assessment
Remove all the filters and create a compressed copy of the data files which were used to produce the group report. Right click on the group name “TK- Section2” and select “Compress Group,” this will save a compressed copy named “TK-Section2compressedCopy.” Navigate to the Exoid Data folder in Documents and find your compressed copy. Send this, along with the two group report PDFs to [email protected] for evaluation.
Please use TK-Section 2 Assessment(NAME)_(INSTITUTE) as the subject line.

PACK ING UP

Once this section is complete and a good quality data set has been sent to lzon, the system can be shut down for the day.
Remove the nanopore, rinse it with DI water and dry it with compressed gas or a lint free tissue before storing it back inside its ziplock bag. It is a good idea to write on the ziplock bag what the pore has been used for and how long it was in use, this way if someone wishes to re-use the pore then they know the pore’s history.

Fluid Cell Cleaning and System Shutdown Procedure
Rinse the fluid cell cap, the upper fluid cell, and the lower fluid cell liquid contact areas with DI water and blow dry with compressed gas. Reassemble the fluid cell in the top of the Exoid. Dry the APS nozzle with a kimwipe and connect it to the fluid cell. See lzon online resources for more rigorous system maintenance.

PROCEEDING WITH UNKNOWN SAMPLES

Once a TRPS certificate has been received from lzon, then use the lzon support portal at support.izon.com for more information on how to process your experimental samples.
www.izon.com

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