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Izon qNano Скачать руководство пользователя страница 17

Calibrating particle size

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.

Blockade magnitude is proportional to the volume of the particle passing
through the nanopore, giving very high resolution of particle diameter.

For each sample particle passing through the nanopore:

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Содержание qNano

Страница 1: ...www izon com qNano USER MANUAL...

Страница 2: ...g individual nanoscale particles requires a level of due diligence and understanding often overlooked in other laboratory equipment Here we will help guide you through this process to get the best pos...

Страница 3: ...t operation 34 Key skills for qNano operation 35 Reference guide to nanopore selection 36 Behaviour of nanopores at different stretches 37 Nanopore set up and system optimisation 38 Troubleshooting 47...

Страница 4: ...GETTING STARTED 4 www izon com...

Страница 5: ...module may impair the safety protection provided Do not operate the instrument outside its rated supply voltages or environmental range Failure to read these instructions and use the instrument in th...

Страница 6: ...this will increase the risk of dropping and damaging the equipment The following is included with your qNano system Callipers USB to USB Mini B Cable qNano with Pressure Reading Module PM2 permanently...

Страница 7: ...Grounded Protected Outlets ONLY Make sure the power supply box is positioned away from fluids To prevent heat build up do not cover the power supply box Position unit so it can easily and quickly be d...

Страница 8: ...ort Windows Home Edition is not suitable for the installation of the Control Suite software Ensure that the computer is installed with Windows Professional Edition Install the Izon Control Suite Softw...

Страница 9: ...al Power Voltage 12 V DC At input jack on instrument Current 130 mA Power consumption 1 5 W PSU Input AC 90 to 264 Vac 50 60 Hz ac Use only Izon supplied TRG45A120 21E11 Output DC 12 V nom Output curr...

Страница 10: ...THEORY OF OPERATION 10 www izon com...

Страница 11: ...ecified detectable particle size range An accurate size distribution of these particles ideally plotted as a histogram of concentration vs particle diameter or volume The effective surface charge of i...

Страница 12: ...le particles are driven through the nanopore by applying a combination of voltage and pressure from the variable pressure module or VPM and each particle translocation event causes a resistive pulse o...

Страница 13: ...e zeta potential values by calibrating with particles of known size and surface charge Charge measurements The blockade duration changes with the velocity of the particle and can be used to calculate...

Страница 14: ...charges 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 gr...

Страница 15: ...nd the applied voltage Electrophoretic forces can dominate particle velocity in small nanopore systems when the applied voltage is increased Electro osmosis Voltage dependent force The second electrok...

Страница 16: ...anslocating through the nanopore from the upper to lower fluid cell Blockade magnitude and frequency can be controlled using three parameters FORCES EXPLANATION NOTES Applied Stretch S mm Optimise the...

Страница 17: ...ameter nm Figure 5 The Izon CSS is able to convert blockade magnitude into a particle diameter by using calibration particles of a known size Blockade magnitude is proportional to the volume of the pa...

Страница 18: ...derived at 2 or more pressure steps using calibration particles of a known concentration Figure 6 The particle rate observed should increase linearly with applied pressure This rate plot is used to c...

Страница 19: ...t be recorded under identical system settings which includes the electrolyte characteristics which affect the baseline current and blockade magnitude When possible always use the same electrolyte Adju...

Страница 20: ...asurement needs and can be set up to allow the user to easily and repeatedly obtain consistent measurements The Classic Capture tool manual signal optimisation and recording The classic capture tool a...

Страница 21: ...135 540 nm The smallest particle can be adjusted within the nanopore size range to suit your sample Once this has been edited the largest detectable particle size will be calculated automatically but...

Страница 22: ...on of the sample will be reported 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 resoluti...

Страница 23: ...ge The mean size of the calibration particle used The software will show the recommended size of calibration particles to use The relative particle size is an indication of particle size relative to t...

Страница 24: ...r the Relative Particle Speed plot ms 1 is an indication of particle velocity through the nanopore calculated from the blockade duration of individual particles Figure 9 The applied pressure can be al...

Страница 25: ...MEASUREMENT PLANNING 25 www izon com...

Страница 26: ...et particles and suspension conditions are fundamentally compatible with TRPS Sample particles must be between 45 10 000 nm in size suspended in a buffer and of sufficient concentration for analysis 4...

Страница 27: ...analytical Develop theoretical understanding Do some feasibility studies for novel samples Identify suitable protocols and consumables Method verification identify controls needed Analytical Optimise...

Страница 28: ...SAMPLE PREPARATION 28 www izon com...

Страница 29: ...r volume into the larger volume and pump the plunger 3 4 times before discarding the tip 1 Rotate the thumbwheel to the correct volume Attach pipette tip by firmly pressing the pipette down into the t...

Страница 30: ...have an ionic strength of at least 0 01 M Samples dissolved in pure water can be spiked with small amounts of concentrated PBS to rectify this issue However the requirement for solutions to be conduc...

Страница 31: ...secretions products Make up electrolyte from powder weekly filter sterilise daily store solutions at 2 8 C Trace chemicals Use reagent grade buffers and electrolytes fresh deionised water clean glassw...

Страница 32: ...way of preserving the functionality of all particles Biological samples Immunoassay techniques use membrane bound proteins as markers to distinguish between specific bio nanoparticles messenger or car...

Страница 33: ...ional to the temperature dependent rate of diffusion Keep samples below 25 C to reduce the diffusion rate and do not freeze calibration particles EV samples may be snap frozen and kept at 80 C Zeta po...

Страница 34: ...INSTRUMENT OPERATION 34 www izon com...

Страница 35: ...mbwheel to the correct volume Attach pipette tip by firmly pressing the pipette down into the top of a pipette tip in the holder Depress the plunger to the second stop Immerse the pipette tip into the...

Страница 36: ...s analysing particles between 50 and 200 nm in diameter we recommend ordering pore sizes slightly smaller than your average estimated particle size and using these smaller nanopores at higher stretche...

Страница 37: ...d may be more prone to blockages Operating at a stretch of 47 48 mm and greater may work well but the pore may become problematic for further use at lower stretches This is because the cruciform has u...

Страница 38: ...ise ratio for calibration particles 7 Adjust conditions for the sample if necessary and record data 8 Re run calibration measurements to ensure system stability 1 Prepare the fluid cell and stretch th...

Страница 39: ...he upper fluid cell pipette a droplet of electrolyte on top of the nanopore and replace the lower fluid with electrolyte prepared in accordance with the Reagent Kit tech note Wipe away the electrolyte...

Страница 40: ...more bubbles Apply full pressure on the VPM and tap the fluid cell cap to dislodge any bubbles trapped in the aperture Repeat the wetting process but use electrolyte instead of the wetting solution a...

Страница 41: ...coating Replace the electrolyte in the lower fluid cell with freshly filtered coating solution fit the upper fluid cell and dispense 35 L of coating solution into the upper fluid cell Fit the fluid c...

Страница 42: ...e onto the nanopore Rinse the inside of the upper fluid cell with deionised water and dry with compressed air Gently wipe the top of the nanopore dry with a lint free tissue fit the upper fluid cell a...

Страница 43: ...oise ratio of the system by increasing the electrophoretic force acting on charged particles Baseline current Ideally 100 140 nA Applied voltage Ideally 0 2 1 V Adjust electrolyte molarity if necessar...

Страница 44: ...icles To guarantee good resolution for your sample particles AND high quality data you must choose a nanopore size that will resolve your sample size distribution properly AND choose a calibration sta...

Страница 45: ...d be recorded for each run The measurement plan for multiple samples may look like this Calibration 1 P10 P6 P3 Current 131 nA Sample 1 P10 P6 P3 Current 131 nA Sample 2 P10 P6 P3 Current 136 nA Calib...

Страница 46: ...il you are satisfied that there are no significant particle blockades present Repeat with DI water and ensure that the current goes to 0 nA Remove the nanopore rinse with DI water and dry with compres...

Страница 47: ...TROUBLESHOOTING 47 www izon com...

Страница 48: ...he lower fluid cell periodically once every half hour to prevent bubbles from gathering around the pore If the baseline is very unstable there may be many invisible bubbles beneath the pore Put a drop...

Страница 49: ...ower fluid cell to remove any bubbles Running synthetic or inorganic particles ISSUE ACTIONS THAT MAY BE NEEDED Stable baseline current but very few particles Increase the sample concentration Use low...

Страница 50: ...ability Increase the sample concentration Use low pressures until you have a stable system otherwise you risk blocking the pore Pore is not the predicted size try a different calibration standard or p...

Страница 51: ...power and ensure the fluid cell cap is used during data recording to minimise noise on the baseline current and obtain better blockade resolution Short circuits Electrolyte can creep in between metal...

Страница 52: ...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 cali...

Страница 53: ...luid cell is removed it must be firmly patted dry When washing the fluid cell only wash the inner chamber do not immerse or wash the outside The most common cause of a wandering baseline is a fluid ce...

Страница 54: ...bject to change without notice All technical information in this document is for reference purposes only System configurations and specifications in this document supersede all previous information re...

Страница 55: ...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 th...

Страница 56: ...www izon com...

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