Reagent Kit for TRPS Analysis -...

REAGENT KIT TECHNICAL NOTE WWW.IZON.COM

1 March 2018

Reagent Kit for TRPS Analysis

Specifications and Operation Guide

Total Measurement Solution

Izon Science's Reagent Kit is designed as part of a total measurement solution for qEV and TRPS

analysis .

qEV Size Exclusion Columns (SEC) isolate extracellular vesicles (EVs) from bio-fluids providing

high-purity vesicles.

Reagent kits provide the components and instructions for the preparation of Nanopore coating

solution and contaminant-free reagents with matched pH and conductivity to ensure reliable

and repeatable measurements using Izon’s qNano.

Izon Control Suite Software (CSS) provides assistants that aid in experimental planning,

capturing the required particle ranges and performing measurements with the attention to

detail necessary at the nanoscale.

Figure 1: The Izon Total Measurement Solution ensures rapid and reliable measurements:

The components of the system combine to produce high quality results.

Reagent Kit

Enhance the reliability and reproducibility of

measurements

Biological particle isolation using qEV

SEC columns

Rapid & effective purification

TOTAL

MEASUREMENT

SOLUTION

qNano with Izon CSS

Repeatable experiment planning and execution.

http://www.izon.com/


2 March 2018

Applicable for TRPS analysis of all samples and highly beneficial for biologicals1

For example EVs derived from the following: Blood-derived samples (serum, plasma)

Saliva

Urine

Cell culture media

Cerebrospinal fluid

Advantages of using Izon Science's Reagent Kits

Reliable and consistent results; measurements on different Nanopores are comparable and

accurate

Stable operation, reduces non-specific binding to Nanopores

Contaminant-free solutions

Higher throughput

Safety Data Sheets (SDS)

Safety data sheets for Reagent Kit components can be viewed and downloaded:

http://www.izon.com/products/sds/

Safety Precautions

Always use appropriate personal protection such as gloves, lab coats, and safety glasses when

handling reagents or qEV columns.

There is a trace amount of sodium azide in the wetting solution only. 0.05% w/v

sodium azide is used as an anti-bacterial agent in the wetting solution

concentrate only. Sodium azide in higher concentrations is toxic. Direct contact

with skin or eyes should be avoided.

Waste reagents should be disposed of in a safe manner.

Biological samples can be hazardous; consult your laboratory safety officer for

information on safe handling of your sample when using a qEV column or qNano.

Storage 1Contact Izon Support for assistance with protocols.



3 March 2018

Store the Wetting Solution Concentrate and prepared solutions at +4 to +8 °C. All other Reagent

Kit components should be stored at room temperature. Avoid leaving prepared reagent bottles

open - always replace lids after use and return to the fridge as soon as possible. Observe carefully

the recommended use time of prepared reagents (1 week max) and the expiry date of the wetting

solution.

Operating Instructions in Izon CSS

Standard instructions and measurement protocols for using reagent kits are contained within the

CSS measurement assistants. Contact Izon Support if you need to upgrade or require further

information.

Best Measurement Practices

Careful preparation of reagents and samples for the qNano is critical. Key considerations to get

accurate and reproducible results are:

Use contaminant-free tubes and clean glassware

Wear gloves to avoid contaminating reagent kit components

Dilution errors are a common form of inaccuracy; use calibrated pipettes for dilution

Use a new un-filtered pipette tip each time a reagent is used. This avoids contamination and

ensures the longest life of the reagents

Filter reagents daily using the 0.22 m filters provided, to remove unwanted large

contaminants

Mix samples gently to avoid introduction of bubbles that can interfere with qNano

measurements

Use Izon qEV separation columns to isolate particulates from biofluids, for example

exosomes, microvesicles, viruses etc. to ensure quality TRPS data on biological particles of

interest

Reagent Kit Components

The reagents are tailored to each stage of the measurement. They are designed to control the pH

and electrolyte concentration in order to ensure that measurements on the TRPS system are stable

and physiological. Mixed pH or electrolyte levels can cause instability in measurements. The kit

contains enough components to make four batches of reagents. The reagents included in the kit

are as follows:


http://www.izon.com/products/qev/


4 March 2018

Coating Solution (4 x 20mL, provided as a powder to be prepared)

A critical part of the Reagent Kit is to minimise the non-specific binding (NSB) of biological

molecules/particles to the Nanopore that can interfere with EV measurements. In TRPS, NSB can

interfere with measurements in two ways – firstly, contaminating molecules can bind to the pore,

altering the membrane properties, and secondly, EVs themselves can bind to the membrane,

causing it to block and preventing further measurements.

qEV purification greatly reduces the problems caused by NSB; however, pre-treatment of the

Nanopore is necessary to prevent NSB of low-level contaminants that may remain after

purification.

Without Coating Nanopore With Izon Coating Solution

Figure 2: Contact with biological fluids alters the properties of the Nanopore: Exposure of the pore to

serum proteins shows that the IV curve (a measure of the pore performance) is significantly altered. When the

Nanopore is pre-coated its performance is unaltered leading to repeatable calibrated measurements.

The coating solution is used to pre-coat the Nanopore prior to sample analysis. It is also added to

the measurement electrolyte at a lower concentration, to maintain a low volume of coating reagent

throughout the measurement process.

Store the powder well sealed at room temperature.

Shelf life: 1 week when made up as a solution, if stored at 4-8°C

Glassware for mixing PBS must be highly clean and devoid of particulates, washed with de-ionized (DI) water. Use high-quality plasticware for reagent mixing and sample preparation e.g. 1.5 mL, 15 mL, 50 mL plastic tubes, we recommend the use of Axygen

Scientific tubes.



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PBS tablets (4 tablets)

The supplied PBS tablets are used to prepare stock PBS solutions solely for TRPS and qEV columns.

Reagent quality is highly important for TRPS. Do not use general purpose lab PBS. The stock PBS

is used to prepare the measurement electrolyte which is used extensively through the

measurement process. The measurement electrolyte is also used as the diluent for samples and

calibration particles.

If using a buffer other than the PBS buffer provided substitute the alternate buffer where PBS stock

solution is mentioned in the steps below.

Store at room temperature.

Shelf life: 1 week when made up as a solution, if stored at 4-8°C

Wetting Solution Concentrate (4 mL)

Wetting solution concentrate is added to the measurement stock solution as a surfactant.

Store the at 4-8°C

Shelf life: 6 months once opened, if stored at 4-8°C

Syringe Filter Unit, 0.22 µm (20 pieces) and 0.45µm (4 pieces)

Single use 0.45 µm filters are provided to filter the coating solution when it is first prepared (Step

2 below).

Single use 0.22 µm syringe filters are provided to filter flushing water, refilter an aliquot of the

coating solution and measurement electrolyte (in this sequence) before use each day (Step 4

below). A critical part of the measurement of nanoparticles is high-purity reagents. It is necessary

to filter all solutions to remove any large particles which will block the Nanopore.

Store well sealed at room temperature.

Solution Preparation

All unused stock solutions must be stored in the fridge and discard after one week. Follow the

steps below to prepare the measurement solutions.



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Particle Preparation

Calibration Particles

All calibration particles must be diluted from concentrated stocks immediately before use, using

Measurement Electrolyte. Each Nanopore size has an associated target particle concentration.

Consult the Nanopore Reference Guide in the qNano User Manual for further details.

•Stock PBS Solution must be prepared weekly and unused solution should be discarded after one week.

•To make the Stock Solution: •Completely dissolve one PBS tablet in 200 mL of DI water using clean

glassware. •Add 0.6 mL of Wetting Solution Concentrate to the resultant solution

and mix evenly. •Seal container and label as Stock PBS Solution. •Store in the fridge and discard after 1 week. •Allow solutions to warm up to room temperature before use.

1. Prepare Stock PBS Solution

•Add 10 mL of Stock PBS Solution to a 50 mL graduated plastic tube. •Accurately weigh out 2.0g of the coating solution powder and add this to

the 50mL tube. •Add Stock PBS Solution to bring the volume to 20 mL. Apply the lid. •Place the 50 mL tube and contents into a 50 °C water bath or beaker

containing warm water.

•Once warm mix by swirling at regular intervals until the powder is totally dissoved.

•When the solution is clear, check the volume and top up to 20 mL with Stock PBS Solution if necessary.

•Filter through a 0.45 m filter into a clean tube labelled Coating Solution. •Store in the fridge and discard after 1 week.

2. Prepare Coating Solution

•Measurement Electrolyte should be made fresh daily. 15 mL of Measurement Electrolyte should suffice for a day's work.

•To make the Measurement Electrolyte : •Vortex the Coating Solution in short bursts for 20 seconds. •Using a new pipette tip, pipette 3 mL of Coating Solution into a clean

15mL tube. •Add 12 mL of Stock PBS Solution to the tube. •Vortex the resultant solution in short bursts for 20 seconds.

3. Prepare

Measurement Electrolyte

•Filter 15 mL of DI water into a clean 15 mL tube using a 0.22 m filter,

•Using the same 0.22 m filter, filter 0.5 mL of Coating Solution into a clean 1.5 mL tube labelled Coating Solution. (This aliquot will be used during the system set up and prevents contamination of the bulk solution).

•Finally using the same 0.22 m filter, filter the Measurement Electrolyte from step 3 into a clean 15 mL tube. Discard the filter.

•Discard these solutions after 1 day.

4. Filter

Solutions



7 March 2018

Sample Particles

Correct sample preparation will provide faster and more accurate results. For the best results Izon

Science recommends the use of qEV separation columns for biological samples. Each sample may

require slightly different preparation.

Follow the protocol outlined in the Izon qEV Technical Note to isolate the EVs from the raw

sample. When using qEV separation columns, reagent quality is critical. Izon recommends

the use of Sigma-Aldrich PBS tablets (Cat# P4417) with high-purity water.

Dilute the qEV fraction(s) in Measurement Electrolyte. The dilution should be optimised

to achieve a particle rate at the highest operating pressure of 1000-2000/min to avoid pore

blockage.

If an initial approximate concentration of the sample is unknown, a series of samples may

be prepared at different dilutions, e.g. 1:100, 1:10, 1:5. For EV samples from qEV purified

plasma we recommend 1:5 to 1:10 initially.

Highly Polydispersed samples: If the sample contains very large contaminants that create

problems for TRPS analysis, consider filtering the sample. Izon recommends Millipore spin

filters (Ultrafree –MC centrifugal filter units for exosome samples). Note as these remove

larger particulates the data will be biased to some degree.

Nanopore Setup Overview

Users should complete an Izon Training Programme before attempting to measure their own

samples. The chart below shows an overview of Nanopore setup:

Prepare Measurement

Electrolyte and Sample Particles

Prepare the Fluid Cell

Fit and calibrate Nanopore

Wetting Protocol1 Coating Protocol2 Equilibrate

Measurement Electrolyte3

Insert Calibration Particles

Optimise stretch, voltage, and

pressure

Sample and calibration Recordings



8 February 2018

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EUROPE

The Oxford Science Park Magdalen Centre, 1 Robert Robinson Ave, Oxford OX4 4GA, United Kingdom

Tel:+44-1865-784-630

Fax:+44-1865-784-631

Email:[email protected]

NORTH AMERICA

85 Bolton Street Cambridge, MA 02140 United States

Tel:+1-617-945-5936

Fax:+1-857-259-6623


ASIA PACIFIC

8C Homersham Place, PO Box 39168, Burnside, Christchurch 8053, New Zealand

Tel:+64 3 357 4270 Fax:+64 3 357 4273


Clicking is only recommended during Nanopore wetting.

Step Procedure

Wetting Protocol1

1. Load 35 µL of Measurement Electrolyte in the

upper fluid well ONLY.

2. Apply maximum pressure for 2 minutes.

3. Click the shielding lid 5 to 10 times to

mechanically assist wetting the Nanopore.*

4. Add 75 L of Measurement Electrolyte to the

lower fluid well and verify that the Nanopore is

wetted by applying an appropriate voltage and

checking for a stable open Nanopore current.

Coating Protocol2

1. Load filtered Coating Solution in the upper and

lower fluid well.


3. Apply maximum vacuum for 10 minutes.

Equilibrate Measurement

Electrolyte3

1. Flush the coating solution out of the upper and

lower fluid wells two to three times with

Measurement Electrolyte.



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