Nanotechnologies — Magnetic nanomaterials — Part 2: Specification of characteristics and measurement methods for nanostructured magnetic beads for nucleic acid extraction

This document specifies characteristics to be measured of magnetic beads in suspension and powder forms for nucleic acid extraction applications. This document deals with magnetic beads that contain a substantial amount of magnetic nanoparticles (which can be superparamagnetic). Potential applicable measurement methods are listed for the individual characteristics. Specifically, this document lists critical characteristics of magnetic beads and suspensions, and additional characteristics to describe the magnetic beads and the suspension for nucleic acid extraction. Health, safety and environmental aspects of magnetic beads are not within the scope of this document.

Nanotechnologies — Nanomatériaux magnétiques — Partie 2: Spécification des caractéristiques et des méthodes de mesure pour les billes magnétiques nanostructurées pour l’extraction d’acide nucléïque

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TECHNICAL ISO/TS
SPECIFICATION 19807-2
First edition
2021-10
Nanotechnologies — Magnetic
nanomaterials —
Part 2:
Specification of characteristics
and measurement methods for
nanostructured magnetic beads for
nucleic acid extraction
Nanotechnologies — Nanomatériaux magnétiques —
Partie 2: Spécification des caractéristiques et des méthodes de mesure
pour les billes magnétiques nanostructurées pour l’extraction d’acide
nucléïque
Reference number
ISO/TS 19807-2:2021(E)
© ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 19807-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on

the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below

or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 19807-2:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Abbreviations .......................................................................................................................................................................................................... 3

5 Characteristics to be measured and measurement methods .............................................................................. 4

5.1 General ........................................................................................................................................................................................................... 4

5.2 Descriptions of characteristics and their measurement methods ........................................................... 5

5.2.1 Bead mass concentration ............................................................................................................................................ 5

5.2.2 Bead size distribution .................................................................................................................................................... 5

5.2.3 Nucleic acid binding capacity .................................................................................................................................. 5

5.2.4 Remanent mass magnetization ............................................................................................................................. 6

5.2.5 Surface functional group type ............................................................................................................................... 7

5.2.6 Saturation mass magnetization ............................................................................................................................ 7

5.2.7 Initial magnetic mass susceptibility ................................................................................................................. 8

5.2.8 Iron ion concentration ................................................................................................................................................... 8

5.2.9 Mass specific surface area ......................................................................................................................................... 8

5.2.10 Size of primary magnetic nanoparticles ....................................................................................................... 9

5.2.11 Surface functional group density ........................................................................................................................ 9

6 Sample preparation .......................................................................................................................................................................................10

7 Test report ...............................................................................................................................................................................................................10

Annex A (informative) Schematics of magnetic beads ..................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

iii
© ISO 2021 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/TS 19807-2:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment,

as well as information about ISO's adherence to the World Trade Organization (WTO) principles in the

Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
A list of all parts in the ISO/TS 19807 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 19807-2:2021(E)
Introduction

Magnetic beads are composed of a large number of magnetic nanoparticles immobilized within a

nonmagnetic matrix with a size range between tens of nanometres and hundreds of micrometres (see

Annex A). The immobilization matrix is typically based on silica or organic polymers. The beads are

commonly supplied while dispersed in a liquid suspension, for example, ethanol, isopropanol, sodium

azide solutions, pure water. Magnetic beads in liquid suspension have become one of the most widely

used nanomaterials in the biological and chemical fields, due to their unique magnetic properties and

interactions with applied magnetic fields.

When the size of a magnetic object is small enough, it will form a single magnetic domain, behaving as

[1]

a single large macrospin. At yet smaller sizes (for iron oxide, typically less than 30nm ), the thermal

energy of the object can be sufficient to result in frequent reorientations of the magnetization direction

of the object. If the timescale of these reorientations is shorter than the timescale of the measurement,

the term ‘superparamagnetism’ is used to describe this behaviour and the magnetic nano-objects are

said to be superparamagnetic. In large non-interacting ensembles of such particles, the thermally

induced switching events will result in the average magnetization of the ensembles being zero in the

absence of an applied magnetic field. In the presence of an applied large field, the ensemble of magnetic

nano-objects is observed to acquire a large net magnetization, as the magnetic field overcomes the

thermal fluctuations and aligns the macrospins of the individual magnetic nano-objects within the

ensemble. Beads, if incorporating a large fraction of magnetic nano-objects which exhibit this behaviour,

are often referred to as “superparamagnetic beads”. However, as the beads may not themselves be

superparamagnetic, they are referred to as “magnetic beads” herein.
[2]

Magnetic beads have been applied in many fields, especially in biosensing applications such as in vitro

[3]-[5] [6] [7]

diagnostics, targeted drug delivery , magnetic resonance imaging , bioseparation , and genetic

[8]

engineering , among others. For example, nucleic acids, which carry genetic information, can be

extracted or isolated from blood, saliva, faeces, urine, leaves, viral lysates, using suitably functionalized

magnetic beads.

The nucleic acids (DNA) and ribonucleic acid (RNA) carry the key information that organisms use to

build or maintain their biostructures. Correctly identifying DNA offers immensely valuable information

on health. In recent years, in the human blood stream, scientists have not only found circulating cell

free DNA (cfDNA), but also circulating tumour DNA (ctDNA). Now ctDNA extraction is one of the most

widely used liquid-biopsy methods to determine cancer or track cancer development. However, the

content of ctDNA is only 1 % or less of the total cfDNA amount. The concentration of cfDNA is very

low, generally 5 ng/ml blood to 30 ng/ml blood. Therefore, the development of reliable methods for

extracting the ctDNA is critical. The proper description of physicochemical characteristics of magnetic

beads for DNA extraction is both valuable for developers of extraction kits and for users applying them

for DNA analysis.

Nucleic acid binding to magnetic beads relies on electrostatic interactions, hydrophobic interactions,

hydrogen bonding or specific binding mechanisms to the bead surface. Once DNA or RNA from cell or

tissue lysate is released into the solution, then nucleic acids can bind to surface-modified magnetic

[9]-[19]
beads to form a “nucleic acid-magnetic bead complex”.

Then, the complex can be separated under a proper combination of magnetic field and magnetic field

gradient. The eluate can wash away the residual impurities. Finally, the nucleic acids to be extracted

[9]-[19]
can be obtained from the beads after desalination and purification.

The different forms of magnetic beads and dispersing media for the extraction of nucleic acid will have

different physicochemical characteristics such as specific surface area, bead concentration etc. All

[9]-[19]

these characteristics will affect their performance to extract nucleic acid to varying extents.

In common with other nanostructured materials, the manufacturing and material specification of

composite magnetic beads are complex. Small variations in the synthesis conditions during bead

manufacturing and functionalization can lead into dramatic shifts in the properties and binding

capacities of the manufactured beads. This requires these products to have high manufacturing

consistency. Currently, different manufacturers provide different characteristics and most of them

© ISO 2021 – All rights reserved
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ISO/TS 19807-2:2021(E)

never provide the measurement methods, so it is difficult for consumers or regulators to compare

different products or to verify the characteristics, which increases the difficulty of further development

of the application. Universally accepted material specification and test reports for magnetic beads are

a requirement in order to ensure customer confidence and the quality of the nucleic acid extraction

products.
© ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/TS 19807-2:2021(E)
Nanotechnologies — Magnetic nanomaterials —
Part 2:
Specification of characteristics and measurement methods
for nanostructured magnetic beads for nucleic acid
extraction
1 Scope

This document specifies characteristics to be measured of magnetic beads in suspension and powder

forms for nucleic acid extraction applications. This document deals with magnetic beads that contain a

substantial amount of magnetic nanoparticles (which can be superparamagnetic). Potential applicable

measurement methods are listed for the individual characteristics. Specifically, this document lists

critical characteristics of magnetic beads and suspensions, and additional characteristics to describe

the magnetic beads and the suspension for nucleic acid extraction.

Health, safety and environmental aspects of magnetic beads are not within the scope of this document.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms

ISO/TS 80004-6, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-6

and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
bead mass concentration

ratio of the mass of magnetic beads (3.6) to the total volume of a magnetic beads sample in suspension

or powder form
3.2
bead size

effective outer diameter of a magnetic bead (3.6) determined by using the specified measurement

method
© ISO 2021 – All rights reserved
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ISO/TS 19807-2:2021(E)
3.3
bead size distribution
distribution of beads as a function of bead size (3.2)

Note 1 to entry: Bead size distribution may be expressed as cumulative distribution or a distribution density

(distribution of the fraction of beads in a size class, divided by the width of that class).

3.4
dispersing medium
liquid in which magnetic beads (3.6) are suspended
3.5
initial magnetic mass susceptibility

differential ratio of the change in mass magnetization of a material to the amplitude of a magnetic field

change at a sufficiently small absolute magnetic field

Note 1 to entry: A magnetic beads (3.6) sample is assumed to be magnetically isotropic and its initial magnetic

mass susceptibility is indicated as a scalar.
3.6
magnetic bead

small round piece containing a large number of magnetic nanoparticles which can be superparamagnetic

and are immobilized within a non-magnetic matrix

Note 1 to entry: The size range of magnetic beads for DNA extraction spans from a few tens of nanometres to

several micrometres.
3.7
mass-specific surface area
absolute surface area of the sample divided by sample mass

[SOURCE: ISO/TS 80004-6:2021, 4.6.1, modified — Note 1 to entry has been removed.]

3.8
nucleic acid

macromolecule that is the medium for genetic information or acts as an agent in expressing the

information
Note 1 to entry: There are two types of nucleic acid, DNA and RNA.
[SOURCE: ISO 17822:2020, 3.32]
3.9
nucleic acid binding capacity

mass of nucleic acid (3.8) bound to the surfaces of magnetic beads (3.6) per unit mass of the magnetic

beads under specified conditions
3.10
operational time

maximum time after the start of the extraction process where the suspension of magnetic beads (3.6) is

ready for use to extract nucleic acid (3.8)

Note 1 to entry: the operational time is usually recommended by the manufacturer.

3.11
remanent mass magnetization

value of the mass magnetization remaining in a magnetized body when, in the absence of a self-

demagnetizing field, the applied magnetic field strength is brought to zero

[SOURCE: IEC 60050:1990, 221-02-40, modified — "magnetization" has been changed to "mass

magnetization".]
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ISO/TS 19807-2:2021(E)
3.12
saturation mass magnetization

limiting value of the mass magnetization of a liquid or dried sample with increasing applied magnetic

field strength

Note 1 to entry: The saturation mass magnetization of magnetic beads (3.6) is indicated for the dried matter of a

bead suspension sample or for the dried sample in the case of beads in powder form.

3.13
shelf life

recommended time period by manufacturer during which a product (suspension or powder) can be

stored, throughout which the defined quality of specified characteristics of the product remains

acceptable under expected (or specified) conditions of distribution, storage, display and usage

Note 1 to entry: Defined characteristics should be measured after fixed time intervals.

[SOURCE: ISO/TS 19807-1:2019, 3.37, modified — the manufacturer has been specified and the powder

product has been added.]
3.14
surface functional group density
mass of surface functional groups per unit mass of magnetic beads (3.6)
3.15
surface functional group type

chemical type of substituents or moieties on the surface of magnetic beads (3.6) that are responsible for

a specific chemical reaction
4 Abbreviations
For the purposes of this document, the following abbreviations apply:
BET method Brunauer–Emmett–Teller method
DLS Dynamic light scattering
DNA Deoxyribonucleic acid
ICP-OES Inductively coupled plasma optical emission spectrometry
IR Infrared
PCR Polymerase chain reaction
RNA Ribonucleic acid
SEM Scanning electron microscopy
SQUID Superconducting quantum interference device
TEM Transmission electron microscopy
UV-Vis spec- Ultraviolet-visible spectrometry
trometry
VSM Vibrating sample magnetometry
XPS X-ray photoelectron spectroscopy
© ISO 2021 – All rights reserved
---------------------- Page: 9 ----------------------
ISO/TS 19807-2:2021(E)
5 Characteristics to be measured and measurement methods
5.1 General

The critical characteristics listed in Table 1 of magnetic beads products supplied for nucleic acid

extraction shall be measured. The additional characteristics listed in Table 2 are frequently measured

in industrial communities depending on the application. However, whether to provide these additional

characteristics is optional for the supplier. The selection criteria for the first table are the critical

characteristics of magnetic beads and suspensions. They determine fundamentally the extraction

performance, independent of the nucleic acid sample type or the subsequent extraction process.

The additional characteristics can influence the extraction performance, depending on the specific

application or process.

Measurement methods and relevant standards for these methods are listed in Tables 1 and 2. Listed

measurement methods can be alternatively used. However, other measurement methods may also be

used as agreed between supplier and purchaser. Any characteristic from these tables shall be reported

by stating its value and the measurement method used. The listed ISO documents for measurements

have been generally applied to measurements for characteristics of non-magnetic objects. However,

it should be noted that these ISO documents have not yet been fully validated for the application to

magnetic beads.

Tables 1 and 2 provide alternative measurement methods for some characteristics. It should be noted

that the values of characteristics obtained by a measurement method can deviate to some extent from

that obtained by another measurement method.
Table 1 — Critical characteristics of magnetic beads to be measured
Characteristics Measurement method Relevant standards
[20]
Bead mass concentration Gravimetry and oven drying ISO 11358-1
[21]-[24] [25]
DLS ISO 22412
[21],[23] [26]
SEM ISO 19749
[21]-[23] [27]
TEM ISO 21363
Bead size distribution
[29]
ISO 20998-1
[28]
Ultrasonic attenuation spectroscopy
[30]
ISO 20998-3
[31] [32]
Electrical sensing zone ISO 13319-1
[33],[34] [35]
UV-Vis spectrometry ISO 21571
Nucleic acid binding
[36],[37] [35]
Real-time PCR ISO 21571
capacity
[38] [35]
Agarose gel electrophoresis ISO 21571
[39],[40]
SQUID magnetometry
Remanent mass magneti-
zation [21],[22],[41]
VSM
[21],[24],[42]
Surface functional group
[43]
type
XPS ISO 20903
SQUID magnetometry
Saturation mass magnet-
ization
VSM
Table 2 — Additional characteristics of magnetic beads to be measured
Characteristics Measurement method Relevant standards
VSM
Initial magnetic mass
susceptibility
SQUID magnetometry
[44] [45]
Iron ion concentration ICP-OES ISO 11885
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ISO/TS 19807-2:2021(E)
Table 2 (continued)
Characteristics Measurement method Relevant standards
[48]
ISO 9277
[46],[47]
Mass specific surface area Gas adsorption method
[49]
ISO 18757
Size of primary magnetic
[27]
TEM ISO 21363
nanoparticles
Surface functional group
Conductometric titration
density
5.2 Descriptions of characteristics and their measurement methods
5.2.1 Bead mass concentration

The bead mass concentration of a sample of magnetic beads in suspension or in powder form is the

ratio of the mass of the magnetic beads to the total volume of the sample.

The mass of the beads after drying shall be measured by the oven drying method. For oven drying of

magnetic beads in suspension, a certain volume of the suspension shall be washed by deionized water

and separated by magnetic separation several times to remove any soluble ingredients in the dispersing

medium. This makes sure that the soluble content in the dispersing medium in the bead suspension is

neglectable.

Then, the sample in powder or suspension form shall be dried until a constant mass is reached which

is determined by weighing. The temperature used for drying shall induce the evaporation of the liquid

compartments of the sample but not lead to decomposition of the beads. For water-based suspensions,

the drying temperature is typically 105 °C ± 2 °C.
The bead mass concentration is expressed in the unit kg/l.

NOTE 1 The bead mass concentration is correctly measured when solid materials other than the magnetic

beads are negligible in the sample. Otherwise, the measurement result includes the mass of the other solid

materials.

NOTE 2 If the mass of the dried beads is divided by the total sample mass measured before drying, the result

is called dry matter content and it is expressed in the unit kg/kg.
5.2.2 Bead size distribution

The bead size distribution can have an impact on their extraction performance, which makes the

measurement of the size distribution necessary. The bead size distribution shall be measured by

an appropriate measurement method. The recommended methods are DLS, SEM, TEM, ultrasonic

attenuation spectroscopy and electrical sensing zone method. The measurement results are expressed

in the unit of nm or µm.

The bead size distribution of magnetic beads for nucleic acid extraction should be performed according

to the procedures described in the relevant ISO documents mentioned in Table 1. These documents

explain the measurement, the data analysis and the expression of results in detail.

5.2.3 Nucleic acid binding capacity

For the measurement of nucleic acid binding capacity, a binding experiment between a magnetic beads

sample and a reference sample of suspended nucleic acid is performed. Due to the large variety of

magnetic beads for nucleic acid extraction and their different application scenarios, it is not possible to

define a harmonized protocol for the binding experiment. The bead manufacturer should establish its

own specified protocol to perform the binding experiment. Bead users and other interested parties can

also develop their own protocol to perform the binding experiment. It is also possible that the details of

the binding experiment are established by negotiations between bead manufacturer and bead user or

other interested parties.
© ISO 2021 – All rights reserved
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ISO/TS 19807-2:2021(E)

The purpose of the reference sample is to provide target nucleic acid under known conditions for

binding by the magnetic beads. It should be made sure that the reference sample contains only target

nucleic acids and no proteins or other confounding compartments.

The magnetic beads are mixed with the reference sample and the nucleic acid will bind to the magnetic

beads. The established protocol shall contain procedures on how to perform the mixing and how to

handle the mixed suspension including the time of the binding phase and the temperature.

After the binding phase, the magnetic beads are fixated on a solid surface by a magnetic field gradient

and washed to remove unbound nucleic acid from the suspension medium. In a second washing step,

the bound nucleic acids are separated from the magnetic bead surfaces according to the established

protocol. The magnetic beads and the suspension containing now only the previously bound nucleic

acid are then separated by magnetic separation. The mass of previously bound nucleic acid in the

suspension shall be quantitatively determined by an appropriate measurement method.

The possible measurement methods include UV-Vis spectrometry, agarose gel electrophoresis, and

real-time PCR. The principles of these measurement methods are:

a) UV-Vis spectrometry: ultraviolet and visible light is absorbed by the sample. The concentration of

nucleic acids in solution is derived from the absorptivity and the optical length of the sample.

b) Agarose gel electrophoresis: nucleic acid molecules are separated by applying an electric field to

move the negatively charged molecules through a matrix of agarose. After electrophoresis, the

nucleic acid will be concentrated in bands that are characteristic for their length and charge. The

light absorption of these bands is compared to that of a reference sample with known concentration

of the same nucleic acid type and thus the amount of nucleic acid can be quantified.

c) Real-time PCR: nucleic acid molecules are stained by ethidiumbromide which enhances their

fluorescence signal. Then the nucleic acid molecules are amplified in a polymerase chain reaction

and the fluorescence is measured over different cycles. The intensity of the fluorescence signal is

proportional to the amount of reaction products. Thus, the initial amount of nucleic acid can be

calculated.
[35]

ISO 21571 specifies procedures of those measurement methods for foodstuffs applications which

can also be applied here.

The magnetic beads which have been involved in the experiment are washed again and dried, and their

mass is measured by weighing.

The nucleic acid binding capacity is determined by the ratio of the mass of the bound nucleic acid and

the mass of magnetic beads.

The measurement results of nucleic acid binding capacity and measurement conditions shall be reported

according to Clause 7 d); the type of nucleic acid used for the reference sample, concentration of the

nucleic acids in the reference sample, concentration of magnetic beads in the mixture and measurement

temperature.

The result of the measurement for nucleic acid binding capacity is expressed in the unit kg/kg.

NOTE The numeric value of the nucleic acid binding capacity is characteristic for the applied protocol of

the binding experiment. It can be used for quality control of the magnetic beads and it gives an assessment of

the binding performance of the beads under the specified conditions. The nucleic acid binding capacity obtained

in a specific application can differ from the reported value, if the conditions of the application differ from the

procedure which led to the reported value.
5.2.4 Remanent mass magnetization

The remanent mass magnetization of a magnetic bead for nucleic acid extraction is directly

proportional to the mechanical force acting on the bead when i
...

TECHNICAL ISO/TS
SPECIFICATION 19807-2
First edition
Nanotechnologies — Magnetic
nanomaterials —
Part 2:
Specification of characteristics
and measurement methods for
nanostructured magnetic beads for
nucleic acid extraction
Nanotechnologies — Nanomatériaux magnétiques —
Partie 2: Spécification des caractéristiques et des méthodes de mesure
pour les billes magnétiques nanostructurées pour l’extraction d’acide
nucléïque
PROOF/ÉPREUVE
Reference number
ISO/TS 19807-2:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 19807-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 19807-2:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Abbreviations........................................................................................................................................................................................................... 3

5 Characteristics to be measured and measurement methods ................................................................................ 4

5.1 General ........................................................................................................................................................................................................... 4

5.2 Descriptions of characteristics and their measurement methods ............................................................. 5

5.2.1 Bead mass concentration ......................................................................................................................................... 5

5.2.2 Bead size distribution ........................................................................................................................................... ....... 5

5.2.3 Nucleic acid binding capacity ............................................................................................................................... 5

5.2.4 Remanent mass magnetization ........................................................................................................................... 6

5.2.5 Surface functional group type .............................................................................................................................. 7

5.2.6 Saturation mass magnetization .......................................................................................................................... 7

5.2.7 Initial magnetic mass susceptibility ................................................................................................................ 8

5.2.8 Iron ion concentration ................................................................................................................................................ 8

5.2.9 Mass specific surface area ........................................................................................................................................ 8

5.2.10 Size of primary magnetic nanoparticles ...................................................................................................... 9

5.2.11 Surface functional group density ....................................................................................................................... 9

6 Sample preparation ........................................................................................................................................................................................10

7 Test report ................................................................................................................................................................................................................10

Annex A (informative) Schematics of magnetic beads ....................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 19807-2:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO's adherence to the World Trade Organization (WTO)

principles in the Technical Barriers to Trade (TBT) see the following URL: www .iso .org/ iso/ foreword

.html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
A list of all parts in the ISO/TS 19807 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 19807-2:2021(E)
Introduction

Magnetic beads are composed of a large number of magnetic nanoparticles immobilized within a

nonmagnetic matrix with a size range between tens of nanometers and hundreds of micrometers (see

Annex A). The immobilization matrix is typically based on silica or organic polymers. The beads are

commonly supplied while dispersed in a liquid suspension, for example, ethanol, isopropanol, sodium

azide solutions, pure water. Magnetic beads in liquid suspension have become one of the most widely

used nanomaterials in the biological and chemical fields, due to their unique magnetic properties and

interactions with applied magnetic fields.

When the size of a magnetic object is small enough then it will form a single magnetic domain, behaving

[1]

as a single large macrospin. At yet smaller sizes (for iron oxide, typically less than 30nm ), the thermal

energy of the object can be sufficient to result in frequent reorientations of the magnetization direction

of the object. If the timescale of these reorientations is shorter than the timescale of the measurement,

the term ‘superparamagnetism’ is used to describe this behaviour and the magnetic nano-objects are

said to be superparamagnetic. In large non-interacting ensembles of such particles, the thermally

induced switching events will result in the average magnetization of the ensembles being zero in the

absence of an applied magnetic field. In the presence of an applied large field, the ensemble of magnetic

nano-objects is observed to acquire a large net magnetization, as the magnetic field overcomes the

thermal fluctuations and aligns the macrospins of the individual magnetic nano-objects within the

ensemble. Beads, if incorporating a large fraction of magnetic nano-objects which exhibit this behaviour,

are often referred to as “superparamagnetic beads”. However, as the beads may not themselves be

superparamagnetic, they are referred to as “magnetic beads” herein.
[2]

Magnetic beads have been applied in many fields, especially in biosensing applications such as in vitro

[3]-[5] [6] [7]

diagnostics, targeted drug delivery , magnetic resonance imaging , bioseparation , and genetic

[8]

engineering , among others. For example, nucleic acids, which carry genetic information, can be

extracted or isolated from blood, saliva, faeces, urine, leaves, viral lysates, using suitably functionalized

magnetic beads.

The nucleic acids (DNA) and ribonucleic acid (RNA) carry the key information that organisms use to

build or maintain their biostructures. Correctly identifying DNA offers immensely valuable information

on health. In recent years, in the human blood stream, scientists have not only found circulating cell

free DNA (cfDNA), but also circulating tumour DNA (ctDNA). Now ctDNA extraction is one of the most

widely used liquid-biopsy methods to determine cancer or track cancer development. However, the

content of ctDNA is only 1 % or less of the total cfDNA amount. The concentration of cfDNA is very

low, generally 5 ng/ml blood to 30 ng/ml blood. Therefore, the development of reliable methods for

extracting the ctDNA is critical. The proper description of physicochemical characteristics of magnetic

beads for DNA extraction is both valuable for developers of extraction kits and for users applying them

for DNA analysis.

Nucleic acid binding to magnetic beads relies on electrostatic interactions, hydrophobic interactions,

hydrogen bonding or specific binding mechanisms to the bead surface. Once DNA or RNA from cell or

tissue lysate is released into the solution, then nucleic acids can bind to surface-modified magnetic

[9]-[19]
beads to form a “nucleic acid-magnetic bead complex”.

Then, the complex can be separated under a proper combination of magnetic field and magnetic field

gradient. The eluate can wash away the residual impurities. Finally, the nucleic acids to be extracted

[9]-[19]
can be obtained from the beads after desalination and purification.

The different forms of magnetic beads and dispersing media for the extraction of nucleic acid will have

different physicochemical characteristics such as specific surface area, bead concentration etc. All

[9]-[19]

these characteristics will affect their performance to extract nucleic acid to varying extents.

In common with other nanostructured materials, the manufacturing and material specification of

composite magnetic beads are complex. Small variations in the synthesis conditions during bead

manufacturing and functionalization can lead into dramatic shifts in the properties and binding

capacities of the manufactured beads. This requires these products to have high manufacturing

consistency. Currently, different manufacturers provide different characteristics and most of them

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ISO/TS 19807-2:2021(E)

never provide the measurement methods, so it is difficult for consumers or regulators to compare

different products or to verify the characteristics, which increases the difficulty of further development

of the application. Universally accepted material specification and test reports for magnetic beads are

a requirement in order to ensure customer confidence and the quality of the nucleic acid extraction

products.
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TECHNICAL SPECIFICATION ISO/TS 19807-2:2021(E)
Nanotechnologies — Magnetic nanomaterials —
Part 2:
Specification of characteristics and measurement methods
for nanostructured magnetic beads for nucleic acid
extraction
1 Scope

This document specifies characteristics to be measured of magnetic beads in suspension and powder

forms for nucleic acid extraction applications. This document deals with magnetic beads that contain a

substantial amount of magnetic nanoparticles (which can be superparamagnetic). Potential applicable

measurement methods are listed for the individual characteristics. Specifically, this document lists

critical characteristics of magnetic beads and suspensions, and additional characteristics to describe

the magnetic beads and the suspension for nucleic acid extraction.

Health, safety and environmental aspects of magnetic beads are not within the scope of this document.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms

ISO/TS 80004-6, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization

3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-6

and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
bead mass concentration

ratio of the mass of magnetic beads (3.6) to the total volume of a magnetic beads sample in suspension

or powder form
3.2
bead size

effective outer diameter of a magnetic bead (3.6) determined by using the specified measurement

method
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ISO/TS 19807-2:2021(E)
3.3
bead size distribution
distribution of beads as a function of bead size (3.2)

Note 1 to entry: Bead size distribution may be expressed as cumulative distribution or a distribution density

(distribution of the fraction of beads in a size class, divided by the width of that class).

3.4
dispersing medium
liquid in which magnetic beads (3.6) are suspended
3.5
initial magnetic mass susceptibility

differential ratio of the change in mass magnetization of a material to the amplitude of a magnetic field

change at a sufficiently small absolute magnetic field

Note 1 to entry: A magnetic beads (3.6) sample is assumed to be magnetically isotropic and its initial magnetic

mass susceptibility is indicated as a scalar.
3.6
magnetic bead

small round piece containing a large number of magnetic nanoparticles which can be superparamagnetic

and are immobilized within a non-magnetic matrix

Note 1 to entry: The size range of magnetic beads for DNA extraction spans from a few tens of nanometers to

several micrometers.
3.7
mass-specific surface area
absolute surface area of the sample divided by sample mass

[SOURCE: ISO/TS 80004:2021-6, 4.6.1, modified — Note 1 to entry has been removed.]

3.8
nucleic acid

macromolecule that is the medium for genetic information or acts as an agent in expressing the

information
Note 1 to entry: There are two types of nucleic acid, DNA and RNA.
[SOURCE: ISO 17822:2020, 3.32]
3.9
nucleic acid binding capacity

mass of nucleic acid (3.8) bound to the surfaces of magnetic beads (3.6) per unit mass of the magnetic

beads under specified conditions
3.10
operational time

maximum time after the start of the extraction process where the suspension of magnetic beads (3.6) is

ready for use to extract nucleic acid (3.8)

Note 1 to entry: the operational time is usually recommended by the manufacturer.

3.11
remanent mass magnetization

value of the mass magnetization remaining in a magnetized body when, in the absence of a self-

demagnetizing field, the applied magnetic field strength is brought to zero

[SOURCE: IEC 60050, 221-02-40, modified — "magnetization" has been changed to "mass

magnetization".]
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ISO/TS 19807-2:2021(E)
3.12
saturation mass magnetization

limiting value of the mass magnetization of a liquid or dried sample with increasing applied magnetic

field strength

Note 1 to entry: The saturation mass magnetization of magnetic beads (3.6) is indicated for the dried matter of a

bead suspension sample or for the dried sample in the case of beads in powder form.

3.13
shelf life

recommended time period by manufacturer during which a product (suspension or powder) can be

stored, throughout which the defined quality of specified characteristics of the product remains

acceptable under expected (or specified) conditions of distribution, storage, display and usage

Note 1 to entry: Defined characteristics should be measured after fixed time intervals.

[SOURCE: ISO/TS 19807-1:2019, 3.37, modified — Specified manufacturer and added powder product.]

3.14
surface functional group density
mass of surface functional groups per unit mass of magnetic beads (3.6)
3.15
surface functional group type

chemical type of substituents or moieties on the surface of magnetic beads (3.6) that are responsible for

a specific chemical reaction
4 Abbreviations
For the purposes of this document, the following abbreviations apply:
BET method Brunauer–Emmett–Teller method
DLS Dynamic light scattering
DNA Deoxyribonucleic acid
ICP-OES Inductively coupled plasma optical emission spectrometry
IR Infrared
PCR Polymerase chain reaction
RNA Ribonucleic acid
SEM Scanning electron microscopy
SQUID Superconducting quantum interference device
TEM Transmission electron microscopy
UV-Vis spec- Ultraviolet-visible spectrometry
trometry
VSM Vibrating sample magnetometry
XPS X-ray photoelectron spectroscopy
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ISO/TS 19807-2:2021(E)
5 Characteristics to be measured and measurement methods
5.1 General

The critical characteristics listed in Table 1 of magnetic beads products supplied for nucleic acid

extraction shall be measured. The additional characteristics listed in Table 2 are frequently measured

in industrial communities depending on the application. However, whether to provide these additional

characteristics is optional for supplier. The selection criteria for the first table are the critical

characteristics of magnetic beads and suspensions. They determine fundamentally the extraction

performance, independent of the nucleic acid sample type or the subsequent extraction process.

The additional characteristics can influence the extraction performance, depending on the specific

application or process.

Measurement methods and relevant standards for these methods are listed in Tables 1 and 2. Listed

measurement methods can be alternatively used. However, other measurement methods may also be

used as agreed between supplier and purchaser. Any characteristic from these tables shall be reported

by stating its value and the measurement method used. The listed ISO standards for measurements

have been generally applied to measurements for characteristics of non-magnetic objects. However,

it should be noted that these ISO standards have not yet been fully validated for the application to

magnetic beads.

Tables 1 and 2 provide alternative measurement methods for some characteristics. It should be noted

that the values of characteristics obtained by a measurement method can deviate to some extent from

that obtained by another measurement method.
Table 1 — Critical characteristics of magnetic beads to be measured
Characteristics Measurement method Relevant standards
[20]
Bead mass concentration Gravimetry and oven drying ISO 11358-1
[21]-[24] [25]
DLS ISO 22412
[21],[23] [26]
SEM ISO 19749
[21]-[23] [27]
TEM ISO 21363
Bead size distribution
[29]
ISO 20998-1
[28]
Ultrasonic attenuation spectroscopy
[30]
ISO 20998-3
[31] [32]
Electrical sensing zone ISO 13319-1
[33],[34] [35]
UV-Vis spectrometry ISO 21571
Nucleic acid binding
[36],[37] [35]
Real-time PCR ISO 21571
capacity
[38] [35]
Agarose gel electrophoresis ISO 21571
[39],[40]
SQUID magnetometry
Remanent mass magneti-
zation [21],[22],[41]
VSM
[21],[24],[42]
Surface functional group
[43]
type
XPS ISO 20903
SQUID magnetometry
Saturation mass magnet-
ization
VSM
Table 2 — Additional characteristics of magnetic beads to be measured
Characteristics Measurement method Relevant standards
VSM
Initial magnetic mass
susceptibility
SQUID magnetometry
[44] [45]
Iron ion concentration ICP-OES ISO 11885
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ISO/TS 19807-2:2021(E)
Table 2 (continued)
Characteristics Measurement method Relevant standards
[48]
ISO 9277
[46],[47]
Mass specific surface area Gas adsorption method
[49]
ISO 18757
Size of primary magnetic
[27]
TEM ISO 21363
nanoparticles
Surface functional group
Conductometric titration
density
5.2 Descriptions of characteristics and their measurement methods
5.2.1 Bead mass concentration

The bead mass concentration of a sample of magnetic beads in suspension or in powder form is the

ratio of the mass of the magnetic beads to the total volume of the sample.

The mass of the beads after drying shall be measured by the oven drying method. For oven drying of

magnetic beads in suspension, a certain volume of the suspension shall be washed by deionized water

and separated by magnetic separation several times, to remove any soluble ingredients in the dispersing

medium. This makes sure that the ionic content in the dispersing medium in the bead suspension is

neglectable.

Then, the sample in powder or suspension form shall be dried until a constant mass is reached which

is determined by weighing. The temperature used for drying shall induce the evaporation of the liquid

compartments of the sample but not lead to decomposition of the beads. For water-based suspensions,

the drying temperature is typically 105 °C ± 2 °C.
The bead mass concentration is expressed in the unit kg/l.

NOTE 1 The bead mass concentration is correctly measured when solid materials other than the magnetic

beads are negligible in the sample. Otherwise, the measurement result includes the mass of the other solid

materials.

NOTE 2 If the mass of the dried beads is divided by the total sample mass measured before drying, the result

is called dry matter content and it is expressed in the unit kg/kg.
5.2.2 Bead size distribution

The bead size distribution can have an impact on their extraction performance, which makes the

measurement of the size distribution necessary. The bead size distribution shall be measured by

an appropriate measurement method. The recommended methods are DLS, SEM, TEM, ultrasonic

attenuation spectroscopy and electrical sensing zone method. The measurement results are expressed

in the unit of nm or µm.

The bead size distribution of magnetic beads for nucleic acid extraction should be performed according

to the procedures described in the relevant ISO standards mentioned in Table 1. These standards

explain the measurement, the data analysis and the expression of results in detail.

5.2.3 Nucleic acid binding capacity

For the measurement of nucleic acid binding capacity, a binding experiment between a magnetic beads

sample and a reference sample of suspended nucleic acid is performed. Due to the large variety of

magnetic beads for nucleic acid extraction and their different application scenarios, it is not possible to

define a harmonized protocol for the binding experiment. The bead manufacturer should establish its

own specified protocol to perform the binding experiment. Bead users and other interested parties can

also develop their own protocol to perform the binding experiment. It is also possible that the details of

the binding experiment are established by negotiations between bead manufacturer and bead user or

other interested parties.
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ISO/TS 19807-2:2021(E)

The purpose of the reference sample is to provide target nucleic acid under known conditions for

binding by the magnetic beads. It should be made sure that the reference sample contains only target

nucleic acids and no proteins or other confounding compartments.

The magnetic beads are mixed with the reference sample and the nucleic acid will bind to the magnetic

beads. The established protocol shall contain procedures on how to perform the mixing and how to

handle the mixed suspension including the time of the binding phase and the temperature.

After the binding phase, the magnetic beads are fixated on a solid surface by a magnetic field gradient

and washed to remove unbound nucleic acid from the suspension medium. In a second washing step,

the bound nucleic acids are separated from the magnetic bead surfaces according to the established

protocol. The magnetic beads and the suspension containing now only the previously bound nucleic

acid are then separated by magnetic separation. The mass of previously bound nucleic acid in the

suspension shall be quantitatively determined by an appropriate measurement method.

The possible measurement methods include UV-Vis spectrometry, agarose gel electrophoresis, and

real-time PCR. The principles of these measurement methods are:

a) UV-Vis spectrometry: ultraviolet and visible light is absorbed by the sample. The concentration of

nucleic acids in solution is derived from the absorptivity and the optical length of the sample.

b) Agarose gel electrophoresis: nucleic acid molecules are separated by applying an electric field to

move the negatively charged molecules through a matrix of agarose. After electrophoresis, the

nucleic acid will be concentrated in bands that are characteristic for their length and charge. The

light absorption of these bands is compared to that of a reference sample with known concentration

of the same nucleic acid type and thus the amount of nucleic acid can be quantified.

(c) Real-time PCR: nucleic acid molecules are stained by ethidiumbromide which enhances their

fluorescence signal. Then the nucleic acid molecules are amplified in a polymerase chain reaction

and the fluorescence is measured over different cycles. The intensity of the fluorescence signal is

proportional to the amount of reaction products. Thus, the initial amount of nucleic acid can be

calculated.

ISO 21571 specifies procedures of those measurement methods for foodstuffs applications which can

also be applied here.

The magnetic beads which have been involved in the experiment are washed again and dried, and their

mass is measured by weighing.

The nucleic acid binding capacity is determined by the ratio of the mass of the bound nucleic acid and

the mass of magnetic beads.

The measurement results of nucleic acid binding capacity and measurement conditions shall be reported

according to Clause 7 d); the type of nucleic acid used for the reference sample, concentration of the

nucleic acids in the reference sample, concentration of magnetic beads in the mixture and measurement

temperature.

The result of the measurement for nucleic acid binding capacity is expressed in the unit kg/kg.

NOTE The numeric value of the nucleic acid binding capacity is characteristic for the applied protocol of

the binding experiment. It can be used for quality control of the magnetic beads and it gives an assessment of

the binding performance of the beads under the specified conditions. The nucleic acid binding capacity obtained

in a specific application can differ from the reported value, if the conditions of the application differ from the

procedure which led to the reported value.
5.2.4 Remanent mass magnetization
The remanent mass magnetization of
...

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