ISO/TS 19807-2:2021
(Main)Nanotechnologies — Magnetic nanomaterials — Part 2: Specification of characteristics and measurement methods for nanostructured magnetic beads for nucleic acid extraction
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
ii
© 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
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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
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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
v
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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
1
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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".]
2
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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
3
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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 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]
IR
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
4
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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.
5
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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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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]
IR
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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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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