Izon qNano User manual

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qNano
USER MANUAL

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. 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.
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Getting Started................................................................................................................................ 4
Theory of operation..................................................................................................................... 10
Fundamentals of TRPS................................................................................................................11
Key concepts within the Control Suite Software........................................................... 20
Measurement planning............................................................................................................. 25
The importance of thoughtful study design.................................................................... 26
Variables to control at different analytical stages .......................................................27
Sample preparation................................................................................................................... 28
Key skills for sample preparation ......................................................................................... 29
Essential Considerations.......................................................................................................... 30
Instrument 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
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
TABLE OF CONTENTS
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GETTING STARTED
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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.
Do not operate the instrument outside its rated supply voltages or
environmental range.
Failure to read these instructions and use the instrument in the manner
specified may result in impaired performance and damage to the equipment.
The following symbols are used on the instrument and in this manual:
1 /
Earth (ground) terminal
Direct Current (DC).
Voltage polarity of Jack.
USB Connection.
Caution – risk of danger
or impaired operation, if
instructions not followed.
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CONSUMABLES ORDERING
For ordering of nanopores, calibration particles, and other consumables,
please see our online store at store.izon.com
SETTING UP THE SYSTEM
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:
— Callipers
— USB to USB Mini-B Cable
— qNano with Pressure Reading Module (PM2) permanently attached
— Fluid Cell
— Power Supply Unit
— Regional Power Cable (This should be correct for your region, please
contact your local Izon office or supplier if this is not the case)
— Izon Training Kit
— Variable Pressure Module (VPM)
— VPM Nozzle Kit
2 /
3 /
PM2-Fluid Cell Tubing
VPM-PM2 Tubing
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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. The
correct tubing layout is outlined below:
Check that the fluid cell is connected to the system with the SMA cable
screwed in properly by gently pulling the entire fluid cell assembly upwards.
Once this has been ensured, click the fluid cell back into place by pushing down
firmly in the correct orientation.
Powering Your Instrument
— Plug into Earth 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 disconnected from
mains power.
This instrument has a universal input range and will operate from a nominal
115 V or 230 V mains supply without adjustment. Check that the local supply
meets the AC Input requirement given in the Specification.
Izon Instruments are only to be operated with Izon supplied leads and power
supplies. Failure to use the correct power supply may result in
invalid operation.
Do not place the power supply or operating computer in a position where it
can come into contact with spills or moisture. Make sure the power supply is
placed away and to the rear of the instrument to avoid spills during operation.
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Connecting to a Computer
Devices that can be connected to the equipment should be compliant with a
relevant safety standard such as IEC 60950-1 for IT equipment or IEC 61010-
1 for laboratory equipment and should provide double or reinforced insulation
from hazardous voltage sources. Always use an Izon supplied USB cable to
connect to your instrument.
Computer Minimum Specifications:
— Windows (7 onwards) Professional Edition (64-bit)
— i7 processor (i5 minimum)
— 8 GB RAM
— Dedicated graphics processor and memory (1 GB). Intel HD Graphics 620
or higher on an i7 processor is acceptable
— Hard drive with at least 50 GB free space for software installation and data
— USB port
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 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.
Once the CSS and instrument are both successfully installed, complete reading
the User Manual and proceed to undertake the training course described in the
“Izon’s TRPS Training Module” PDF document on the USB drive.
General Operating Precautions
Izon Instrumentation is designed for indoor use only and is to be used
within the rated conditions outlined in the system specification table on the
following page.
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System Specifications
ITEM SPECIFICATION COMMENT
Environment Indoor use at altitudes
up to 2000 m, Pollution
Degree 2
20% to 80% RH.
-
Operating Temperature 5 °C to 25 °C -
RMS Noise Performance Typical < 10 pA For +/- 200 nA range
NP200
Data Sampling Rate 50,000 samples per second -
USB Standard
connector
USB Mini-B Cable must be to a relevant
safety standard such as IEC
60950-1 for IT equipment
or IEC 61010-1 for
laboratory equipment and
should provide double or
reinforced insulation from
hazardous voltage sources
Max input
voltage
5.00 ± 0.25 V
Electrical
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
current max
3.75 A
Output
power max
45 W2
Nanopore holding stage
extension range
Min. 41 mm
Max. 61 mm
Measured across the outside
of opposing holding arms
Electrical safety EMC IEC/EN 61010-1
IEC/EN 61326-1
-
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.
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THEORY OF
OPERATION
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THE FUNDAMENTALS OF TRPS
What is TRPS?
Tunable Resistive Pulse Sensing (TRPS) enables the characterisation of
nanoparticles suspended in electrolytes. TRPS is the only technology that
can deliver:
— The concentration of particles in the fluid as 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 vs. particle diameter (or volume).
— The effective surface charge of individual nanoparticles (up to 800 nm in
PBS or KCl based electrolytes).
How does TRPS work?
TRPS technology uses the Coulter particle counting principle on the nanoscale.
1 /
Figure 1. A schematic representation of how TRPS works within the Izon system.
UPPER
FLUID CELL
LOWER
FLUID CELL
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Voltage is applied across the fluid cell via the silver/silver chloride (Ag/
AgCl) electrodes. When electrolyte 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 suspension.
Size and concentration measurements
Sample 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 or “blockade” signal
that is detected and measured by the application software.
— 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.
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Figure 3. Blockade duration values are converted into particle 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 the relative surface charge of each particle.
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Figure 4. Displaying duration data in the relative particle speed plot using an inverted unit (ms-1)
allows for intuitive comparisons of particle speed.
A particle speed plot with two different speed signals implies that the
suspension contains particles with different surface 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 greater speed than a more neutral particle.
The FWHM, or “full width at half maximum” is the blockade width (duration
in milliseconds) measured at the point of half of the maximum blockade
magnitude, shown as the horizontal pink bar in the centre of the blockade
above.
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FORCES EXPLANATION NOTES
Convection
Pressure-dependent force
There is always a static
pressure head due to
gravitational force on the
fluid. An additional pressure
or vacuum can be applied
with the variable pressure
module (VPM)
Convective forces tend to
dominate particle velocity in
larger pore systems
Electrophoresis
Voltage-dependent force
Electrophoretic mobility
relates to the movement
of charged nanoparticles
through an electrolyte
solution towards an
oppositely charged
electrode. It is proportional
to particle surface charge
(ζ-potential) and 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 electrokinetic
force is electro-osmosis.
Electro-osmosis relates
to the fluid flow caused
by currents of solvated
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
Electro-osmosis is stronger
in some nanopore systems
(non-zeta capable pores),
but weak in others (good
zeta capable pores)
Summary of dominant forces at play in a nanopore system
Forces influencing the speed at which nanoparticles travel through the
nanopore are:
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What is “tunable” and why?
Nanoparticle suspensions are complex systems. Characterising them fully
requires an optimised setup, which involves tuning the applied stretch,
pressure and voltage. When a particle passes through the nanopore, it
creates a temporary decrease in the baseline current, which results in
a blockade event. Each blockade event corresponds to a single particle
translocating 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 blockade
magnitude (dI) and sample
resolution by adjusting the
nanopore size (stretch)
Applied Pressure,
P (mbar)
Optimise the particle rate
(blockade frequency, (f)
and blockade duration
by adjusting the pressure
applied by the VPM.
Applied Voltage,
V (V)
Optimise the signal-
to-noise ratio of the
system by increasing the
electrophoretic force acting
on charged particles.
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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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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 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 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.
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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.
Important
For accurate and precise results, the sample and calibration particles must
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.
Adjusting any of the parameters between the sample and calibration
recordings will affect blockade magnitude and particle rate, invalidating the
sample-calibration pairing.
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.
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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.
The CSS assistant accommodates a wide range of measurement 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 allows the user to tune the system in real time, rapidly
respond to changes in the system, remeasure and calibrate different samples.
This manual data capture tool is used most often for quick feasibility studies,
and as the default tool for experienced instrument users who do not need the
measurement process prompts.
There are a few key concepts that the user needs to understand to complete
an assistant-based measurement.
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