ISO/TS 23034:2021
(Main)Nanotechnologies — Method to estimate cellular uptake of carbon nanomaterials using optical absorption
Nanotechnologies — Method to estimate cellular uptake of carbon nanomaterials using optical absorption
This document describes a near-infrared optical absorption method to estimate the in vitro cellular uptake of carbon nanomaterials including both internalized and/or tightly adhered to the cell membrane from liquid dispersions. This is a simple method to screen carbon nanomaterials uptake; additional analysis using a different technique can be required if quantification is desired.
Nanotechnologies — Méthode d'estimation de la captation cellulaire des nanomatériaux carbonés par mesure d'absorption optique
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TECHNICAL ISO/TS
SPECIFICATION 23034
First edition
2021-08
Nanotechnologies — Method to
estimate cellular uptake of carbon
nanomaterials using optical
absorption
Nanotechnologies — Méthode d'estimation de la captation cellulaire
des nanomatériaux carbonés par mesure d'absorption optique
Reference number
©
ISO 2021
© 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
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Abbreviated terms . 1
4 Method overview . 2
4.1 General . 2
4.2 Optical absorption of carbon nanomaterials . 3
4.3 Optical absorption of biomolecules . 4
4.4 Determination of the concentration of CNMs in dispersion by absorbance . 4
4.5 Case studies . 4
5 Materials and apparatus . 5
5.1 Materials . 5
5.1.1 Chemicals . 5
5.1.2 Cell line . 5
5.2 Apparatus . 5
5.2.1 UV-Vis-NIR spectrometer . 5
5.2.2 Cuvette for optical absorption measurement . 5
5.2.3 Incubator, 37 °C, humidified, 5 % CO /air. . 5
5.2.4 Culture dishes, single-well or multi-well plates can be used. 6 multi-well
plates with flat bottom are recommended. . 5
5.2.5 Centrifuge. . 5
5.2.6 Homogenizer. . 6
5.2.7 Cell counter. . 6
6 Cell-uptake testing protocol . 6
6.1 General . 6
6.2 Sample preparation . 6
6.3 Preparation of calibration curve of CNM dispersions . 6
6.4 Cell-seeding . 6
6.5 Treatment of cells with testing suspension . 7
6.6 Cell counting. 7
6.7 Washing cells and preparation of the cell lysate . 7
6.7.1 General. 7
6.7.2 For adherent cells . 7
6.7.3 For floating cells . 8
6.8 Absorbance measurement of the cell lysate . 8
7 Sources of variability . 8
8 Data output . 9
8.1 General . 9
8.2 Data analysis and reporting . 9
8.3 Data sheet format . 9
Annex A (informative) Case study with SWCNTs .10
Annex B (informative) Case study of CNHs .16
Annex C (informative) Case study of MWCNTs .20
Bibliography .24
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 of the voluntary nature of standards, 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 www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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 © ISO 2021 – All rights reserved
Introduction
Owing to their unusual physical and chemical properties, carbon nanomaterials (CNMs), such as
carbon nanotubes, carbon black, graphene, and carbon nanohorns, have been considered for various
applications such as in the fields of electronics, energy, nanotechnology, and biology. With the increase
of CNM-based products on the market, the public concern regarding possible toxicities has also
increased. Estimation of the amount of CNM associated with the targeted cells is useful for an initial
[1][2][3][4]
toxicological screening of CNMs and for developing applications in medicine .
Fluorescent dyes and/or radioactive isotopes have been routinely used to measure cellular uptake.
Because CNMs absorb light in near infrared (NIR) region, where the bio-components such as protein
and water in cells or tissues have relatively low light absorption, the cellular uptake of CNMs can be
[5][6][7][8]
estimated from the absorbance of cell-lysate .
TECHNICAL SPECIFICATION ISO/TS 23034:2021(E)
Nanotechnologies — Method to estimate cellular uptake of
carbon nanomaterials using optical absorption
1 Scope
This document describes a near-infrared optical absorption method to estimate the in vitro cellular
uptake of carbon nanomaterials including both internalized and/or tightly adhered to the cell
membrane from liquid dispersions. This is a simple method to screen carbon nanomaterials uptake;
additional analysis using a different technique can be required if quantification is desired.
2 Normative references
The following document is referenced in the text in such a way that some or all of their content
constitutes requirements 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 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO/TS 80004-3, Nanotechnologies — Vocabulary — Part 3: Carbon nano-objects
3 Terms, definitions and abbreviated terms
For the purposes of this document, the following terms, definitions and abbreviations as well as the
terms and definitions given in ISO/TS 80004-3 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .
...
TECHNICAL ISO/TS
SPECIFICATION 23034
First edition
2021-08
Nanotechnologies — Method to
estimate cellular uptake of carbon
nanomaterials using optical
absorption
Nanotechnologies — Méthode d'estimation de la captation cellulaire
des nanomatériaux carbonés par mesure d'absorption optique
Reference number
©
ISO 2021
© 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
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Abbreviated terms . 1
4 Method overview . 2
4.1 General . 2
4.2 Optical absorption of carbon nanomaterials . 3
4.3 Optical absorption of biomolecules . 4
4.4 Determination of the concentration of CNMs in dispersion by absorbance . 4
4.5 Case studies . 4
5 Materials and apparatus . 5
5.1 Materials . 5
5.1.1 Chemicals . 5
5.1.2 Cell line . 5
5.2 Apparatus . 5
5.2.1 UV-Vis-NIR spectrometer . 5
5.2.2 Cuvette for optical absorption measurement . 5
5.2.3 Incubator, 37 °C, humidified, 5 % CO /air. . 5
5.2.4 Culture dishes, single-well or multi-well plates can be used. 6 multi-well
plates with flat bottom are recommended. . 5
5.2.5 Centrifuge. . 5
5.2.6 Homogenizer. . 6
5.2.7 Cell counter. . 6
6 Cell-uptake testing protocol . 6
6.1 General . 6
6.2 Sample preparation . 6
6.3 Preparation of calibration curve of CNM dispersions . 6
6.4 Cell-seeding . 6
6.5 Treatment of cells with testing suspension . 7
6.6 Cell counting. 7
6.7 Washing cells and preparation of the cell lysate . 7
6.7.1 General. 7
6.7.2 For adherent cells . 7
6.7.3 For floating cells . 8
6.8 Absorbance measurement of the cell lysate . 8
7 Sources of variability . 8
8 Data output . 9
8.1 General . 9
8.2 Data analysis and reporting . 9
8.3 Data sheet format . 9
Annex A (informative) Case study with SWCNTs .10
Annex B (informative) Case study of CNHs .16
Annex C (informative) Case study of MWCNTs .20
Bibliography .24
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 of the voluntary nature of standards, 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 www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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 © ISO 2021 – All rights reserved
Introduction
Owing to their unusual physical and chemical properties, carbon nanomaterials (CNMs), such as
carbon nanotubes, carbon black, graphene, and carbon nanohorns, have been considered for various
applications such as in the fields of electronics, energy, nanotechnology, and biology. With the increase
of CNM-based products on the market, the public concern regarding possible toxicities has also
increased. Estimation of the amount of CNM associated with the targeted cells is useful for an initial
[1][2][3][4]
toxicological screening of CNMs and for developing applications in medicine .
Fluorescent dyes and/or radioactive isotopes have been routinely used to measure cellular uptake.
Because CNMs absorb light in near infrared (NIR) region, where the bio-components such as protein
and water in cells or tissues have relatively low light absorption, the cellular uptake of CNMs can be
[5][6][7][8]
estimated from the absorbance of cell-lysate .
TECHNICAL SPECIFICATION ISO/TS 23034:2021(E)
Nanotechnologies — Method to estimate cellular uptake of
carbon nanomaterials using optical absorption
1 Scope
This document describes a near-infrared optical absorption method to estimate the in vitro cellular
uptake of carbon nanomaterials including both internalized and/or tightly adhered to the cell
membrane from liquid dispersions. This is a simple method to screen carbon nanomaterials uptake;
additional analysis using a different technique can be required if quantification is desired.
2 Normative references
The following document is referenced in the text in such a way that some or all of their content
constitutes requirements 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 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO/TS 80004-3, Nanotechnologies — Vocabulary — Part 3: Carbon nano-objects
3 Terms, definitions and abbreviated terms
For the purposes of this document, the following terms, definitions and abbreviations as well as the
terms and definitions given in ISO/TS 80004-3 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .
...
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