qnano User Manual QN1-OQ-013D Page 1

Transcription

1 qnano User Manual QN1-OQ-013D Page 1

2 WELCOME TO THE IZON FAMILY At Izon Science, we are committed to helping you get an accurate measurement of your particles. Measuring individual nanoscale particles requires a level of due diligence and understanding often overlooked in other laboratory equipment. Izon commit to supporting you every step of the way with: A practical training certification to help you learn and master the key skills required for accurate data collection Expert advice, available by or Skype A growing library of step-by-step application and technical notes, third party publications and SOPs A range of complementary products that help to eliminate common measurement errors and allow standardised measurements producing high quality data Software that is constantly evolving to address customer needs Keep reading, and get in touch if you have any questions: support@izon.com izonsupport This document can be printed as an A4 booklet Page 2

3 Contents WELCOME TO THE IZON FAMILY Have You Got Everything You Need? Hardware and Cables Computer Software Installation Training Kit Lab Equipment/Consumables (Customer to Supply) Instrument Components and Assembly The Fundamentals of TRPS What is TRPS? How does TRPS work? Theory of Operation Forces Acting on Nanoparticles What is "Tunable" and why? Applied Stretch, S Applied Pressure, P Applied Voltage, V Calibrating Particle Size Calibrating Particle Concentration Key Concepts within the Izon Control Suite Software (CSS) Resolution Range Analysis Size Fraction (or Concentration Fraction) Green Zone Key Skills and Maintenance Disassembling and Cleaning the Fluid Cells Stretching the Nanopore Variable Pressure Module (VPM) Operation Applying a PRESSURE with the VPM Applying a VACUUM with the VPM Importance of Sample Preparation Forward Pipetting Reverse Pipetting Reference Guide to Nanopore Selection Practical Training and Beyond Page 3

4 1. Have You Got Everything You Need? 1.1. Hardware and Cables qnano Gold base unit with pressure-reading module, fluid cell (3 parts) Variable Pressure Module (VPM) 2 x Airlines Power transformer Local power lead with earthed (3 pin) plug USB to mini USB lead 1.2. Computer The computer may be supplied by Izon OR the customer. The MINIMUM specification is shown below: Earthed (3 pin) power supply Current version of Windows (7 onwards) i7 processor 8 GB RAM, dedicated graphics processor and memory (1GB) 500 GB hard drive with at least 50 GB of free space USB 2.0 por Note: Apple TM MAC operating systems not currently supported. Page 4

5 Software Installation To install the software you will need the Izon USB drive which is located in the training kit that came with your qnano system. 1. Plug the USB drive into your computer. 2. Run the file Install.exe in the root folder of the USB drive (you should get an autorun prompt when you plug the USB into your computer). 3. An installation wizard will run. The wizard will guide you through installation of the software. 4. Close the software before connecting the qnano Training Kit A training kit contains: Control Suite Software and training material on a USB drive Nanopores for training Pipette tips Eppendorf and falcon tubes 0.22 µm filters Calibration particles and reagents for training (store these at 4 C) Page 5

6 1.4. Lab Equipment/Consumables (Customer to Supply) Micropipettes - 1 µl to 1 ml (do not use filtered tips) A vortex mixer Clean micro-centrifuge tubes (1.5mL) for sample preparation (high quality) Filtered deionised water (for cleaning) Compressed nitrogen (for drying) - clean compressed air spray acceptable and lint-free tissues (eg. Kimwipes) Standing racks for tubes Powder-free gloves Page 6

7 2. Instrument Components and Assembly Airline to Fluid Cell The SMA Connector screws onto the base of the fluid cell Airline to VPM Pressure Reading Module Power indicator light Base Instrument The TEETH hold nanopore in place during a measurement Power Connection Stretch Adjustment Handle Serial Number 1. Connect the power cable to the power supply. 2. Plug the DC power supply into the base of the instrument and connect the other end to an earthed power outlet 3. Turn the power on at the wall. The power indicator light will glow green. 4. If using a laptop, ensure that the power cable is connected and power turned on. 5. Using the USB cable, connect the base instrument to your computer. Your computer will automatically go through the process of installing the device software. 6. Upon completion, a confirmation popup will be displayed in the taskbar. Page 7

8 2.1. Assembly With VPM 3 1 Figure 1: Fully assembled qnano with airlines attached to VPM and fluid cell. The USB and power supply connect into the pressure module 1. Attach the airlines to the VPM and to the pressure reading module. 2. The lines attach by pressing over the barb and finger tightening the nut. 3. Attach the second airline to pressure reading module to be applied to the fluid cell when pressure is required. Page 8

9 3. The Fundamentals of TRPS 3.1. What is TRPS? Tunable Resistive Pulse Sensing (TRPS) is very simple and enables measurements of nanoparticles suspended in electrolytes. TRPS is the only technology that delivers: The concentration of particles in the fluid as a number of particles per unit volume of fluid, across a specified detectable particle size range. An accurate size distribution of these particles ideally plotted as a histogram of concentration v particle diameter (or volume). The surface charge of individual nanoparticles (up to 800nm in specified electrolyte - PBS or ITB) 3.2. How does TRPS work? TRPS technology uses the Coulter principle on the nanoscale. Voltage is applied across the fluid cell via the silver-silver (Ag/AgCl) electrodes. When ions move between the electrodes through the nanopore, it creates a baseline current. A temporary decrease in current is detected as particles pass though the nanopore which allows for the sizing and counting of particles in electrolyte solution. Page 9

10 The above figure illustrates how TRPS works. Sample particles are driven through the nanopore by applying a combination of pressure (from the VPM) and voltage, and each particle causes a resistive pulse or "blockade" signal that is detected and measured by the application software. Blockade magnitude is directly proportional to the volume of each particle. Blockade frequency is used to determine particle concentration. Blockade duration changes with the velocity of the particle and can be used to calculate the surface charge of each particle. Magnitude, frequency and duration values are converted into particle measurements by calibration with particles of known size, concentration and surface charge. Visit Izon s website to watch an introductory video demonstrating TRPS technology: Page 10

11 3.3. Theory of Operation Forces Acting on Nanoparticles A variety of forces influence the movement of particles through the nanopore: Fluid velocity (convection) Electrophoretic mobility Electro-osmosis Fluid velocity (Convection) Convection is the fluid flow caused by the pressure difference across the nanopore. There is always a static pressure head due to gravitational force on the fluid. An additional pressure or vacuum can be applied through use of the VPM. Electrophoretic Mobility Electrophoretic mobility is one of the two electro-kinetic forces that influence particle movement. It relates to the movement of charged nanoparticles through an electrolyte solution towards an oppositely-charged electrode. It is proportional to particle charge (ζ-potential) and the applied voltage. Electro-osmosis The second electro-kinetic force is electro-osmosis. Electro-osmosis relates to the fluid flow caused by the charge on the surface of the nanopore. It is proportional to the nanopore surface charge (ζpotential) and the applied voltage. Page 11

12 What is "Tunable" and why? Nanoparticle suspensions are complex systems. Characterising them fully requires an optimised setup, which involves tuning of the applied stretch, pressure and voltage. When a particle passes through the nanopore, it creates a temporary decrease in the baseline current (I) which results in a "blockade event", shown on the signal trace below: Each blockade events corresponds to a single particle going through the nanopore. The size of the blockades can be controlled using these three parameters: Applied Stretch, S Tuning of the nanopore size, by adjusting the stretch applied to the nanopore, allows for the blockade magnitude (di) to be optimised. For applied stretch the following concept applies: di 1 S Blockade magnitude, di, becomes larger as the stretch is decreased, and vice versa. An optimum signal-to-noise ratio is crucial to ensure that blockades will be detected. Too small and they may get lost in the RMS noise and not be counted. If the blockades are too large, it can cause partial blockages causing the baseline current to drop. Page 12

13 Applied Pressure, P The applied Pressure "P" can be tuned using the Variable Pressure Module (VPM) to adjust the fluid flow through the nanopore - allowing adjustment of both blockade frequency and duration. Measurements at more than one pressure are required to calculate particle concentration, and very fine pressure control is required for single particle charge analysis. For applied pressure the following concept applies: di 1 P Applied Voltage, V The applied Voltage V" can be tuned to attract particles of different surface charge or polarity through the nanopore, and optimise the signal to noise ratio of the system. Measurements at more than one voltage are required to calibrate single particle charge values. For voltage the following concept applies: I di V Note: in an Assistant-based measurement, the software automatically tunes the applied voltage. For accurate particle sizing, the sample and calibration particles must be recorded under identical system settings, which include: Measurement Electrolyte. Electrolyte characteristics will affect baseline current and blockade magnitude. When possible, always use the same electrolyte. Nanopores are not identical, so the same nanopore must be used. Voltage, Stretch and Pressure must be identical for sample and calibration. Adjusting any one of these parameters between the sample and calibration will affect blockade magnitude and particle rate, and thus give invalid results. Sample and calibration particles should be recorded at the same time one immediately after the other. Page 13

14 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). Blockade magnitude is proportional to the volume of the particle passing through the nanopore, giving very high resolution of particle diameter. nanopore: Particle Volume = For each sample particle passing through the di (sample) x Calibration Volume (mean) di (calibration, mean) Calibrating Particle Concentration Particle concentration is proportional to the change in blockade rate per unit of applied pressure. Accurate concentration values are typically derived at 2 or more pressures using calibration particles of a known concentration. The Figure below shows the rate increase with pressure at 5 and 8 mbar: Calibration Sample For each of the particle count v time measurements shown in the plot above, particle rate is calculated and plotted against pressure Gradient used to calculate sample concentration Page 14

15 This rate plot is used to check for system stablilty. This is crucial as the concentration value is measured by comparing the gradient of the rate plot of sample to calibration measurement. Sample Conc = Sample Gradient x Cal Conc Cal Gradient If concentration is calculated at a single pressure the software will calculate a gradient based on the fitted line passing through the origin. This can introduce significant errors, especially with smaller nanopores (NP200 and below) where a large proportion of particles are driven through the nanopore due to applied voltage V. 4. Key Concepts within the Izon Control Suite Software (CSS) Izon Control Suite Software (CSS) has data capture assistants which assist in the collection of sample data and help ensure that the best measurement practice is followed. An assistant allows the user to define a sample measurement plan and then guides the user through the measurement process including system optimisation, finally delivering calibrated measurement data to the user. The CSS Assistants are able to accommodate a wide range of measurement needs and can be set up to allow the use to easily and repeatedly obtain consistent measurements. There are a few key concepts that user needs to understand to complete an Assistant-based measurement: 4.1. Resolution Range When performing an Assistant-based measurement, the user will be asked to enter measurement details, one of which will be the Resolution Range: Page 15

16 The Resolution Range is an indication of the size range that can be observed at a single stretch. The default resolution range for each nanopore is at mid-range stretch (e.g. NP250 default range is nm) The smallest particle can be adjusted within the nanopore size range to suit your sample. Once this has been edited, the largest particle will be calculated automatically but cannot be larger than the upper nanopore size range. 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 Analysis Size Fraction (or Concentration Fraction) The Analysis Size Range is the size range over which the total concentration 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 Analysis Size Range will default to being the same as the Resolution Range. Page 16

17 4.3. Green Zone The Green Zone assists users to achieve optimised measurement settings for the quoted Resolution Range. Calibration particles must lie within the Green Zone to ensure that the smallest particle can be detected. This can be achieved by changing the applied nanopore stretch. The calculated/location of the Green Zone depends on: The smallest particle diameter (set in the Resolution Range), AND 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 the nanopore for individual particles. Change the applied stretch to optimise the relative particle size. At a constant stretch, the larger particle size would produce larger blockades; therefore, large blockade magnitude. During calibration recording, any particles that are significantly outside of the Green Zone are assumed to be noise or contamination and are automatically removed from the calibration calculations. Green Zone for the Relative Particle Speed plot (1/ms) is an indication of particle velocity through the nanopore (calculated from the blockade duration of individual particles). Change the applied pressure to optimise the relative particle speed. Page 17

18 5. Key Skills and Maintenance 5.1. Disassembling and Cleaning the Fluid Cells To keep the fluid cell in good working order, clean it before and after use as shown below: 1. Lift off the Fluid Cell Cap 2. Rotate and lift the Upper Cell 3. Rinse fluid contact areas only of the lower cell using Milli-Q Water or 70% Ethanol 4. Rinse fluid contact areas only of upper cell also using Milli-Q Water or 70% Ethanol. 6. Dry Upper and Lower Fluid Cells with compressed gas. Do NOT blow dry biohazardous materials! To avoid damage, do NOT fully immerse components in fluid Page 18

19 5.2. Stretching the Nanopore DECREASE STRETCH ANTICLOCKWISE INCREASE STRETCH CLOCKWISE Page 19

20 Load/Unload Nanopore 40 to 41.5 mm For the qnano the nanopore serial number should be facing upwards as shown. Calibrate Stretch At 45 mm stretch approximately. Once calibrated the Control Suite Software will give a stretch readout and the calipers are no longer required*. Wet the Nanopore At 47 mm stretch approximately. *Calipers should not be required here use the readout in the software. Check calipers display in mm and read zero when closed. Page 20

21 5.3. Variable Pressure Module (VPM) Operation The VPM is configured to allow for two pressure stages allowing standard and low pressure operation: Standard pressure range is 0 to 20mBar (or cmh 2 0 equivalent). This is the typical mode of operation and is used for sizing and concentration measurements. 1 unit = 1mBar Low pressure range is 0 to 2mBar (or 0-20mm H 2 0 equivalent). This range is only used for Zeta potential measurements. 1 unit = 0.1mBar The pressure stage knob (C) adjusts from standard pressure to low pressure. Twist the knob clockwise while lifting to shift to the low pressure range. Ensure the system is disconnected from the qnano while changing the pressure stage. Page 21

22 Applying a PRESSURE with the VPM The order of applying pressure/a vacuum is important. Every time the system is vented or the nozzle is disconnected, repeat from Step 1. Rotate the plunger until the PRE pressure scale is uppermost Vent valve OPEN 2. Plug in VPM nozzle 3. Check Pressure PRE Scale is set to 4 ZERO 3 4. Close the vent valve 5. Push in the plunger to apply a pressure 5 Page 22

23 Applying a VACUUM with the VPM The order of applying vacuum is important. Every time the system is vented or the nozzle is disconnected repeat from step 1. Rotate the plunger until the VAC vacuum scale is uppermost Vent valve OPEN 2. Plug in VPM nozzle. 3. Vacuum VAC Scale is set to ZERO Close the vent valve 5. Pull out the plunger to apply a vacuum 5 Page 23

24 6. Importance of Sample Preparation Sample preparation is one of the most crucial aspects in any measurement. Different samples have different requirements. Time spent on good sample preparation will save time and frustration during particle measurement. A badly prepared sample can yield imprecise and inaccurate results! Tips and recommendations: All electrolyte MUST be filtered immediately before use to remove unwanted contaminants (recommended: Millipore Millex-GS 0.22µm membrane filters that are supplied in the training kit) Prepare fresh electrolyte weekly as this is prone to bacterial growth. Store electrolyte in the fridge when not in use. All particles must be diluted immediately before use. Forward pipetting should be used for sample preparation to give the most accurate dilutions and avoid wastage of sample and calibration fluids. Take care to mix fluids before and after dilution. Incorrect pipetting technique or using a non-calibrated pipette would affect the final concentration of the prepared sample. Other tips: o Check that no droplets are transferred on the outside of the pipette tip. o Use pipettes that are calibrated and well maintained o Use tips that are clean and do not have filters in them. Page 24

25 6.1. Forward Pipetting Forward pipetting should be used in ALL sample preparation. Page 25

26 6.2. Reverse Pipetting Air bubbles are the most common sources of problems. Practice reverse pipetting for dispensing liquid into the fluids cells to minimise issues with nanopore setup and sample changeover. With reverse pipetting you draw out more fluid than you dispense, so the risk of pipetting out a bubble of air is reduced. Page 26

27 7. Reference Guide to Nanopore Selection Use this reference table to select nanopores and calibration particles Page 27

28 8. Practical Training and Beyond Your training kit contains the equipment and instructions required to complete your practical training this training makes sure that you are able to get good, repeatable data from your qnano system. Please complete your training and submit your results to Izon support. We are here to help so please contact us if you require any support. To order additional consumables visit our online store at store.izon.com izonsupport Page 28