Izon qNano User Manual
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Izon qNano User Manual

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  • Page 1 USER MANUAL www.izon.com...
  • Page 2 Here, we will help guide you through this process to get the best possible data from the Izon qNano, to obtain precise results with a high level of accuracy. www.izon.com...

  • Page 3: Table Of Contents

    Recovering an unstable baseline and maintaining nanopore stability ....48 Resolving common qNano issues in different situations .......... 49 Mitigating contributing factors to RMS noise ..............51 Further advice on the most common issues with the qNano........52 Repair and servicing of your Izon instrument ..............53 Further Support ..........................54...

  • Page 4: Getting Started

    G E T T I N G S TA R T E D www.izon.com...
  • Page 5 READ BEFORE OPERATING IZON INSTRUMENTATION Follow all directions in this document and complete the Izon Training Module before attempting to operate the instrument, they contain important safety and operational information. Use of this instrument in a manner not specified by the instructions in this guide and the training module may impair the safety protection provided.
  • Page 6 Assembling the Hardware Unbox your instrument, being careful not to pick anything up by moving parts e.g. the qNano stretch handle, as this will increase the risk of dropping and damaging the equipment. The following is included with your qNano system: —...
  • Page 7 Place the qNano in the recessed area of the base plate on the VPM. Connect the VPM-PM2 and PM2-Fluid Cell tubing by pushing the tubing over the correct barb and screwing the knurled collar on to be finger tight. DO NOT use tools for this operation as there is a risk of damaging the equipment.
  • Page 8 Suite software. Ensure that the computer is installed with Windows Professional Edition. Install the Izon Control Suite Software (CSS) by inserting the USB drive from the Training Kit into a computer that meets the minimum specifications. View the files and double click on “Install.exe”, the installation wizard will then take you through installing the software.
  • Page 9 1 Under electrical interference noise may increase but will return to normal operation on end of interference. Position equipment away from electrical switching gear and interfering equipment or noise may increase under sample running conditions. 2 PSU module rating. Izon Instruments running normally are approx. less than 1.6 W. www.izon.com...

  • Page 10: Theory Of Operation

    T H E O RY O F O P E R AT I O N www.izon.com...

  • Page 11: Fundamentals Of Trps

    PBS or KCl based electrolytes). How does TRPS work? TRPS technology uses the Coulter particle counting principle on the nanoscale. UPPER FLUID CELL LOWER FLUID CELL Figure 1. A schematic representation of how TRPS works within the Izon system. www.izon.com...
  • Page 12 Blockade magnitude is directly proportional to the particle diameter. — Blockade frequency is used to determine particle concentration. Figure 2. Magnitude and frequency measurements are converted into particle size and concentration by calibrating with particles of known size and concentration. www.izon.com...
  • Page 13 The blockade duration changes with the velocity of the particle and can be used to calculate the relative surface charge of each particle. Figure 3. Blockade duration values are converted into particle zeta potential values by calibrating with particles of known size and surface charge. www.izon.com...
  • Page 14 A particle with a larger negative surface charge will experience a greater attraction towards the positively charged electrode in the lower fluid cell and will travel through the pore at a greater speed than a more neutral particle. www.izon.com...
  • Page 15 (good by currents of solvated zeta capable pores) ions (ions surrounded by water molecules) moving along the surface of the nanopore. It is proportional to the nanopore surface charge (ζ-potential) and the applied voltage www.izon.com...
  • Page 16 Optimise the particle rate (blockade frequency, (f) P (mbar) and blockade duration by adjusting the pressure applied by the VPM. Applied Voltage, Optimise the signal- to-noise ratio of the V (V) system by increasing the electrophoretic force acting on charged particles. www.izon.com...
  • Page 17 As the software records blockades in nA, calibration particles of a known size are used to convert blockade magnitude (nA) into a diameter (nm). Figure 5. The Izon CSS is able to convert blockade magnitude into a particle diameter by using calibration particles of a known size.
  • Page 18 Figure 6. The particle rate observed should increase linearly with applied pressure: This rate plot is used to check for system stability. This is crucial as the concentration value is measured by comparing the gradient of the rate plot of sample to calibration measurements. www.izon.com...
  • Page 19 Sample and calibration particles should be recorded at the same time – one immediately after the other, or close together as possible if the system is stable enough to record 2-3 samples before the system needs to be recalibrated. www.izon.com...

  • Page 20: Key Concepts Within The Control Suite Software

    KEY CONCEPTS WITHIN THE IZON CONTROL SUITE SOFTWARE (CSS) The Izon Control Suite Software (CSS) has two different data capture modes: — The Custom Planner tool – automatic data recording The custom planner capture assistant allows the user to define a sample measurement plan and then guides the user through the measurement process, including sample preparation, system optimisation, and calibration.
  • Page 21 The resolution range ensures that the software guides the user to the optimised instrument setting for the selected range of particles. It also allows the user to measure the same size range every time regardless of instrument use or nanopore size. www.izon.com...
  • Page 22 Only particles in the analysis size range will be used to calculate the sample concentration. If not defined, the custom analysis range will be the same as the resolution range. Figure 7. Different resolution ranges possible for a given NP250 pore. www.izon.com...
  • Page 23 The green zone is only shown in the custom planner tool and is not available in the classic capture tool. Figure 8. A visual representation of the importance of the green zone and how particles are excluded above and below. www.izon.com...
  • Page 24 Figure 9. The applied pressure can be altered to optimise the relative particle speed so that particles fall within the green zone. If using the classic capture tool, refer to the instrument set-up and signal optimisation section for advice. www.izon.com...

  • Page 25: Measurement Planning

    M E A S U R E M E N T P L A N N I N G www.izon.com...

  • Page 26: The Importance Of Thoughtful Study Design

    Determine how you will optimise the instrument operating conditions to maximise data quality (optimise the signal-to-noise ratio). Ensure that you have the correct consumables. www.izon.com...

  • Page 27: Variables To Control At Different Analytical Stages

    Identify suitable protocols and consumables — Method verification – identify controls needed Analytical — Optimise instrument operation conditions — Collect high-quality data — Adjust method if needed Post-analytical — Analyse data quality — Identify study limitations and suggest improvements www.izon.com...
  • Page 28 S A M P L E P R E PA R AT I O N www.izon.com...

  • Page 29: Sample Preparation

    With the plunger still depressed, pipette down into the remove the pipette from the fluid. top of a pipette tip in the holder. Figure 10.Forward or standard pipetting should be used when diluting sample particles. www.izon.com...

  • Page 30: Essential Considerations

    Small nanopores especially are easily blocked by large precipitates, aggregates, and microbial contaminants. All samples and reagents that come into contact with nanopores must be filtered with a 0.22 µm syringe filter daily. www.izon.com...
  • Page 31 Microorganisms and Store samples at -80 °C only after their secretions/by- centrifugation and SEC steps have been products completed, flush qEVs in an antimicrobial agent before storage, do not use bench PBS – always make up buffers from powder www.izon.com...
  • Page 32 (SDS) and destructive enzymes. It is important to maintain the suspension conditions throughout the sample cleaning process. It is also important to remove cells from samples, as both living and dead cells can release destructive agents into the suspension. www.izon.com...
  • Page 33 Always vortex samples (except delicate biologicals, which should be mixed with a pipette) before diluting, to avoid concentration errors. For particles larger than 500 nm, we recommend vortexing particles before each recording, and sonicating samples for 5-10 minutes if necessary. www.izon.com...

  • Page 34: Instrument Operation

    I N S T RU M E N T O P E R AT I O N www.izon.com...

  • Page 35: Key Skills For Qnano Operation

    Release the plunger to rest beaded on the outside Depress the plunger to the position. surface of the pipette tip second stop. Discard the pipette tip. Figure 11. Reverse pipetting is used when introducing any fluid into the qNano fluid cells. www.izon.com...

  • Page 36: Reference Guide To Nanopore Selection

    200 nm in diameter, we recommend ordering pore sizes slightly smaller than your average estimated particle size and using these smaller nanopores at higher stretches. Figure 12. Guide to Izon Nanopore categories and their corresponding appropriate calibration particles sizes and target concentrations. www.izon.com...

  • Page 37: Behaviour Of Nanopores At Different Stretches

    STRETCHES The stretch of the nanopore being used can be altered by winding the stretching mechanism on the qNano up or down. Rotating clockwise will increase the applied stretch, while anti-clockwise will decrease it. Choosing a suitable operating stretch is critical, as this will have an impact on the stability of the pore.

  • Page 38: Nanopore Set-Up And System Optimisation

    Fluid Cell Cap Upper Fluid Cell Lower Fluid Cell Figure 13.Schematic of the fluid cell assembly and key parts for using the qNano system. Order of operation: 1 / Prepare the fluid cell and stretch the nanopore 2 / Wet the nanopore...
  • Page 39 If a baseline does not establish, disconnect the PM2 nozzle, remove the fluid cell cap, and apply pulses of pressure with the Izon PAD up to 10 times. If this is still not successful, then try clicking the fluid cell cap by putting pressure down on the nanopore arms and twisting until it clicks into place.
  • Page 40 If the history of the pore is unknown, a quick size check can be easily performed with PBS (with stretch = 47 mm, voltage = 0.1 V). APPROXIMATE BASELINE NANOPORE SIZE (NP) CURRENT AT 0.1 V (NA) 7-35 10-38 13-47 17-53 20-63 27-80 65-138 97-148 www.izon.com...
  • Page 41 5 / Nanopore coating protocol: When working with biological samples, it is strongly recommended to use the Izon reagent kit and coat nanopores. The Izon coating solution is used to prevent the non-specific binding of protein from biological samples to the nanopore.
  • Page 42 35 L of the next sample before connecting the µ PM2 nozzle and applying your chosen pressure. In order to obtain high quality data, certain parameters can be optimised. These are the stretch, the Voltage, and the Pressure. www.izon.com...
  • Page 43 Average particle rate: (blockade frequency, f) P (mbar) Ideally: 500–1500 p/min and blockade duration (acceptable range: 200- by adjusting the pressure 1500 p/min) applied by the VPM. Pressure ranges recommended: 1-15 mbar Concentrate samples or adjust dilution if necessary. www.izon.com...
  • Page 44 90% of your polydisperse sample. Use a smaller nanopore if you cannot properly resolve the sample at the lowest recommended stretch (44.5 mm). Concentrate samples if the particle rate is below 200 particles per minute at a pressure of 10 mbar. www.izon.com...
  • Page 45 If the baseline or particle rates drop by more than 5%, or the noise increases past the acceptable range of 5–9 pA, this indicates a partial nanopore blockage. Consult the troubleshooting guide and restore the system stability before repeating the recording. www.izon.com...
  • Page 46 Do the same with the upper and lower fluid cells, and make sure the inside of the fluid cell cap is also clean and dry. — Re-assembled the fluid cell components on the qNano and put the clean, dry nanopore in its ziplock bag. IMPORTANT NOTES: —...

  • Page 47: Troubleshooting

    T RO U B L E S H O OT I N G www.izon.com...

  • Page 48: Recovering An Unstable Baseline And Maintaining Nanopore Stability

    Put a droplet of electrolyte on top of the pore, take the nanopore off the qNano and wipe the bottom gently with a lint free tissue before remounting on the qNano the applying the same stretch as before.

  • Page 49: Resolving Common Qnano Issues In Different Situations

    Cannot establish — Unplug and replace USB cable to restore the baseline current connection between the qNano and the PC. Ensure voltage is switched on and non-zero. — Use reverse pipetting (as described in the instrument operation section) to prevent bubbles forming on top of the pore and blocking the fluid flow.
  • Page 50 — Pore is partially blocked by a bubble or an aggregate. Unblock pores by gently replacing fluid in lower well and flushing the pore with filtered deionised water at 47 mm stretch as shown on the previous page. www.izon.com...

  • Page 51: Mitigating Contributing Factors To Rms Noise

    50/60 kHz noise from nearby laboratory equipment and ungrounded power supplies can interfere with the baseline current signal. To reduce influence from external noise, do not operate the qNano in close proximity to large laboratory appliances that draw a lot of power, and ensure the fluid cell cap is used during data recording to minimise noise on the baseline current and obtain better blockade resolution.

  • Page 52: Further Advice On The Most Common Issues With The Qnano

    FURTHER ADVICE ON THE MOST COMMON ISSUES WITH THE QNANO Measurement planning Before analysing samples on the qNano, run some quick preliminary tests using other nanoparticle analysis techniques to determine the approximate size and concentration of your particles. Ensure you have purchased the correct nanopore and calibration particle sizes for your sample.

  • Page 53: Repair And Servicing Of Your Izon Instrument

    0.22 µm filter. REPAIR AND SERVICING OF YOUR IZON INSTRUMENT There are no user-serviceable parts. Return equipment to Izon for service. All replacement parts, cables, mains lead, power supply, Nanopores, Fluid Cells must be obtained via Izon Science Ltd.

  • Page 54: Further Support

    This document is not part of any sales contract between Izon Science Ltd. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents.
  • Page 55 Izon Science Ltd PO Box 39168 Burnside Christchurch 8053 New Zealand Telephone: +64 3 357 4270 Email: info@izon.com Website: www.izon.com FCC Declaration of Conformance This device complies with part 15 of the FCC Rules. Operation is subject to the following two conditions: (1) This device may not cause harmful interference, and (2) this device must accept any interference received, including interference that may cause undesired operation.
  • Page 56 www.izon.com...

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