ISO/TS 21412:2020

Nanotechnologies - Nano-object-assembled layers for electrochemical bio-sensing applications - Specification of characteristics and measurement methods

This document specifies the characteristics to be measured of nano-object-assembled layers on electrodes by means of a solution process and of nano-objects constituting the layers for electrochemical applications such as nano-biosensor or diagnosis applications. It also provides measurement methods for determining the characteristics. It does not apply to: - the requirements of nanostructures by top-down nanomanufacturing; - the subsequent coating of materials such as biomaterials onto nano-object-assembled layers; - specific health and safety requirements during manufacturing; - the experimental conditions of electrochemical sensing; - the packaging, labelling, expiratory dates and transport of nano-object-enhanced electrochemical electrodes.

Nanotechnologies — Couches nanostructurées pour des applications de biodétection électrochimique — Spécification des caractéristiques et des méthodes de mesure

General Information

• Status: Published
• Publication Date: 27-Apr-2020
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229/WG 4 - Material specifications
• Current Stage: 9093 - International Standard confirmed
• Start Date: 04-Oct-2023
• Completion Date: 13-Dec-2025

Overview

ISO/TS 21412:2020 - Nanotechnologies: Nano-object-assembled layers for electrochemical bio-sensing applications - defines which characteristics to measure and how to measure them for nano-object-assembled layers deposited on electrodes by solution processes. The Technical Specification targets nano-enhanced electrochemical platforms (for example, nano-biosensors and diagnostic electrodes) and provides standardized measurement methods to support predictable performance and commercial transactions between electrode fabricators and instrument manufacturers.

This document covers:

• Characterization of the constituent nano-objects (nanoparticles, nanofibres, nanoplates).
• Characterization of the assembled nano-object layer on flat electrodes.
• Measurement and reporting requirements to document reproducible electrode quality.

It explicitly does not cover top‑down nanomanufacturing, subsequent biomaterial coatings, health & safety during manufacture, electrochemical test protocols, or packaging/transport requirements.

Key Topics

• Constituting nano-object characteristics (Table 1):

  • Surface chemical composition
  • Mean size and size distribution
  • Mean primary crystallite size (if crystalline)
  • Mean length/length distribution (nanofibres)
  • Number of walls for nanotubes
  • Surface functional groups
    Measurement methods for these are described in Annex B and may be adopted from supplier specifications when unchanged by assembly.

• Nano-object-assembled layer characteristics (Table 2):

  • Mass per unit area (mg/cm²) - calculated for drop-cast layers (Annex C) or determined by weighing for other deposition methods
  • Root mean square (RMS) height - surface topography metric (Annex B)
  • Specific electrochemically active surface area (ECSA) - electrochemical surface characterization (Annex D)

• Reporting and traceability:

  • Test report elements and required measurement results (Clause 5; Annex F example)
  • Value chain considerations for nano-object-modified electrodes (Annex A)

Applications

ISO/TS 21412:2020 is practical for:

• Manufacturers of nano-enhanced electrochemical electrodes who need to specify product attributes.
• Instrument makers integrating nano-object-modified electrodes into biosensors and diagnostic platforms.
• R&D and quality teams establishing reproducible deposition processes (e.g., drop-casting, solution-based assembly).
• Procurement and regulatory teams requiring standardized characterization data for supplier qualification.

Use of this specification improves comparability, repeatability and commercial interoperability of nano-biosensor components.

Related Standards

• ISO/TS 80004 series (nanotechnology vocabulary) - referenced normative definitions.
• ISO/TS 12805 - guidance on manufactured nano-object characteristics (informative link in the specification).
• Consult these related ISO documents for terminology and complementary measurement guidance.

Keywords: ISO/TS 21412:2020, nanotechnologies, nano-object-assembled layers, electrochemical bio-sensing, nano-biosensor, ECSA, mass per unit area, RMS height, measurement methods.

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 21412
First edition
2020-04
Nanotechnologies — Nano-object-
assembled layers for electrochemical
bio-sensing applications —
Specification of characteristics and
measurement methods
Nanotechnologies — Couches nanostructurées pour des applications
de biodétection électrochimique — Spécification des caractéristiques
et des méthodes de mesure
Reference number
©
ISO 2020
© ISO 2020
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
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Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Characteristics and measurement methods . 3
4.1 General . 3
4.2 Characteristics of constituting nano-objects . 3
4.3 Characteristics of nano-object-assembled layer . 4
4.3.1 General. 4
4.3.2 Mass per unit area . 4
4.3.3 Root mean square height . 5
4.3.4 Specific electrochemically active surface area (ECSA) . 5
5 Test report . 6
5.1 General . 6
5.2 General information on nano-object-modified electrode . 6
5.3 Measurement results of characteristics . 6
Annex A (informative) Value chain of nano-object-modified electrochemical electrode .8
Annex B (informative) Measurement methods for characteristics . 9
Annex C (informative) Calculation of mass per unit area using drop-casting method .13
Annex D (informative) Measurement of specific ECSA of nano-object-assembled layer .14
Annex E (informative) General information on nano-object-modified electrode .15
Annex F (informative) Example of test report .18
Bibliography .20
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
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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 2020 – All rights reserved

Introduction
Electrochemical electrodes can exhibit nano-enhanced performance after the deposition of nano-
objects on the electrode surface. The increased surface area, orientation, the assembled density
and ability to control the bio-receptor of the nano-object layer improves the performance of nano-
biosensors.  Nano-biosensor sensitivity, selectivity and reliability can be enhanced with specific nano-
[22][25][26] [24] [37] [38]
objects, e.g. gold nanoparticles , carbon nanotubes , CuS nanorods and silver or
[23]
palladium nanoplates .
Currently, most of the nano-enhanced electrochemical electrodes are fabricated by researchers in
order to achieve predictable performance in their own programs without mass-production. However,
the technology is maturing into a commercial phase. Fabricators are offering nano-enhanced
electrodes to instrument manufacturers as a platform to add additional coatings for specific sensing
applications. This document supports the development of material specifications for the transaction
between electrode fabrications and instrument manufacturers to allow the purchase of electrodes with
predictable performance.
This document is intended to help address this issue. It is also relevant to the process of qualification,
specification and use of nano-object-modified electrodes. The standardization of protocols to specify
various types of nano-object-modified electrodes related to electrochemical detection will be used by
most manufacturers or business owners of electrochemical electrodes products. This document focuses
on the nano-object-assembled layer on electrodes by means of a solution process for electrochemical
applications.
In this document, the specifications for a nano-object constituting an assembled layer are provided,
based on ISO/TS 12805, which describes the characteristics of manufactured nano-objects and their
measurement methods (see Annex A). In addition, the characteristics of nano-object-assembled layer
for enhanced electrochemical bio-sensing applications and their measurement methods are provided
in detail.
TECHNICAL SPECIFICATION ISO/TS 21412:2020(E)
Nanotechnologies — Nano-object-assembled layers for
electrochemical bio-sensing applications — Specification
of characteristics and measurement methods
1 Scope
This document specifies the characteristics to be measured of nano-object-assembled layers on
electrodes by means of a solution process and of nano-objects constituting the layers for electrochemical
applications such as nano-biosensor or diagnosis applications. It also provides measurement methods
for determining the characteristics.
It does not apply to:
— the requirements of nanostructures by top-down nanomanufacturing;
— the subsequent coating of materials such as biomaterials onto nano-object-assembled layers;
— specific health and safety requirements during manufacturing;
— the experimental conditions of electrochemical sensing;
— the packaging, labelling, expiratory dates and transport of nano-object-enhanced electrochemical
electrodes.
2 Normative references
The following documents are referred to 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/TS 80004-2:2015, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-4:2011, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials
ISO/TS 80004-8:2013, Nanotechnologies — Vocabulary — Part 8: Nanomanufacturing processes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-2, ISO/TS 80004-4,
ISO/TS 80004-8 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
nanoscale
size range from approximately 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size will typically, but not exclusively, be
exhibited in this size range. For such properties the size limits are considered approximate.
Note 2 to entry: The lower limit in this definition (approximately 1 nm) is introduced to avoid single and small
groups of atoms from being designated as nano-objects (3.2) or elements of nanostructures, which might be
implied by the absence of a lower limit.
[SOURCE: ISO/TS 80004-4:2011, 2.1]
3.2
nano-object
material with one, two or three external dimensions in the nanoscale (3.1)
Note 1 to entry: Generic term for all discrete nanoscale objects.
[SOURCE: ISO/TS 80004-4:2011, 2.2]
3.3
particle
minute piece of matter with defined physical boundaries
[SOURCE: ISO/TS 80004-2:2015, 3.1, modified — The notes to entry have been deleted.]
3.4
agglomerate
collection of weakly bound particles (3.3), aggregates (3.5) or a mixture of the two where the resulting
external surface area is similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example van der Waals forces or
simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4, modified — “weakly bound particles, aggregates or a mixture of
the two” has replaced “weakly or medium strongly bound particles”.]
3.5
aggregate
particle (3.3) comprising strongly bonded or fused particles where the resulting external surface area
is significantly smaller than the sum of surface areas of the individual components
Note 1 to entry: The forces holding an aggregate together are strong forces, for example covalent or ionic bonds,
or those resulting from sintering or complex physical entanglement, or otherwise combined former primary
particles.
Note 2 to entry: Aggregates are also termed secondary particles and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.5]
3.6
nanoparticle
nano-object (3.2) with all three external dimensions in the nanoscale (3.1)
Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically
by more than three times), the terms nanofibre (3.7) or nanoplate (3.8) are intended to be used instead of the
term nanoparticle.
[SOURCE: ISO/TS 80004-4:2011, 2.4]
3.7
nanofibre
nano-object (3.2) with two similar external dimensions in the nanoscale (3.1) and the third dimension
significantly larger
Note 1 to entry: A nanofibre can be flexible or rigid.
2 © ISO 2020 – All rights reserved

Note 2 to entry: The two similar external dimensions are considered to differ in size by less than three times and
the significantly larger external dimension is considered to differ from the other two by more than three times.
Note 3 to entry: The largest external dimension is not necessarily in the nanoscale.
[SOURCE: ISO/TS 80004-4:2011, 2.5, modified — The original note has been deleted and Notes 1, 2
and 3 to entry have been added.]
3.8
nanoplate
nano-object (3.2) with one external dimension in the nanoscale (3.1) and the two other dimensions
significantly larger
Note 1 to entry: The smallest external dimension is the thickness of the nanoplate.
Note 2 to entry: The two significantly larger dimensions are considered to differ from the nanoscale dimension
by more than three times.
Note 3 to entry: The larger external dimensions are not necessarily in the nanoscale.
[SOURCE: ISO/TS 80004-4:2011, 2.6]
3.9
top-down nanomanufacturing
processes that create structures at the nanoscale (3.1) from macroscopic objects
[SOURCE: ISO/TS 80004-8:2013, 3.13]
4 Characteristics and measurement methods
4.1 General
This clause describes the characteristics of nano-object-assembled layers and constituting nano-
objects on flat substrate electrodes for electrochemical application. Because electrochemical biosensing
requires an efficient electron transfer and a stable immobilization of biomolecules retaining their
bioactivity, the characteristics given in 4.2 are selected for constituting nano-objects if they logically
and/or experimentally affect the high electron conducting pathway, high surface energy, high binding-
[25] to [30]
site density and high functioning ability of the nano-object . From the perspective of the
assembled layer, nano-objects may not be evenly assembled on a flat substrate electrode but produce
surface topography throughout the whole area of the substrate. Because electrochemical biosensing
requires an electrochemically active surface and a robust conducting layer, the characteristics in 4.3,
such as mass per unit area and root mean square height, are selected to describe how thick and evenly
the assembled layer is formed.
4.2 Characteristics of constituting nano-objects
The characteristics given in Table 1 shall be measured to describe the raw materials of nano-objects
constituting the nano-object-assembled layer. Measurement methods for the individual characteristics
in Table 1 are described in Annex B. The measured value of the characteristics in Table 1 may be adopted
from the material specifications by the provider of the nano-object in suspension form if the intrinsic
dimensional characteristics are unchanged after assembling.
Table 1 — Characteristics required to describe constituting nano-objects
Measurement
Characteristic Nanoparticles Nanofibres Nanoplates Units
a
method
b
Surface chemical Yes Yes Yes See 2.3 in Table B.2
composition
c
Mean size and Yes Yes Yes nm For nanoparticles, see
d
size distribution 1.1 in Table B.1 and
2.1 in Table B.2
For nanofibres and
nanoplates, see 2.4 in
Table B.2
Mean primary Yes Not applicable Yes nm See 1.2 in Table B.1
crystallite size (if crystalline) (if crystalline) and 2.2 in Table B.2
e
Mean length and Not applicable Yes Not applicable nm See 2.5 in Table B.2
d
length distribution
Number of walls, Not applicable Yes Not applicable N/A See 2.6 in Table B.2
i.e. single, double or (if nanotubes)
multi-walled
Surface functional Yes Yes Yes N/A See 2.7 in Table B.2
f
group
a
The measurement is conducted on the powder sample or the suspension sample depending on the characteristics.
b
Surface chemical composition is the elemental composition and usually expressed as atomic percent.
c
Mean size is mean particle size for nanoparticles or mean diameter for nanofibres and nanoplates.
d
Size distribution is provided in histogram, percentile plot or standard deviation.
e
This characteristic is exceptionally not applicable to any bundled, branched and/or entangled nanofibres
f
The type of this characteristic is required while the content is not required.
4.3 Characteristics of nano-object-assembled layer
4.3.1 General
The characteristics given in Table 2 shall be measured to describe a nano-object-assembled layer for
electrochemical application. Measurement methods for the individual characteristics in Table 2 are
described in Annexes B, C and D.
Table 2 — Characteristics required to describe a nano-object-assembled layer
Characteristic Units Measurement method
Mass per unit area mg/cm Calculation (if drop casted, see Annex C)
Weighing (for other deposition methods)
Root mean square height µm See 2.8 in Table B.2
Specific electrochemically active surface cm /g Cyclic voltammetry
area (ECSA)
See Annex D
4.3.2 Mass per unit area
Generally, the mass of the coated or adhered layer can be determined by one of two methods if the layer
is thick enough:
a) weighing the test specimen before and after dissolving the assembled layer and taking the
difference;
b) dissolving the substrate and weighing the assembled layer directly.
4 © ISO 2020 – All rights reserved

NOTE Guidance on the terms, definitions and determination of mass per unit area is given in ISO 10111.
However, in case of a nano-object-assembled layer where the amount of adhered nano-objects on flat
substrate by solution-based deposition process is too small, determining the mass per unit area by using
the weighing methods described above may be not appropriate because the weighing error becomes
similar to the mass of the layer itself. To address this problem, the calculation of the mass per unit area
by the drop-casting method has been widely used as an alternative method. In this calculation method,
the mass per unit area of nano-object-assembled layer can be determined by dividing the loaded mass
of nano-objects on the electrode by the area size of the substrate electrode. The detailed calculation
procedure of mass per unit area is described in Annex C.
As many nano-objects are reactive, their chemical properties can be affected by the sampling point and
their storage environment. However, their physical properties, such as mass, are very unlikely to vary
during the solution-based deposition process, thus this theoretical approach using the drop-casting
method is applicable and advantageous to avoid troublesome weighing.
4.3.3 Root mean square height
Nano-object-assembled layers by the solution-based deposition process may not yield a flat topographic
surface in macroscopic millimetre range, even if the nano-object-assembled layer still looks flat in the
microscopic range. In this case, the uneven surface of the assembled layer can affect the electrochemical
performance, especially near the edge of the electrode. The major cause of uneven topography is
the strong surface tension of solvent, which drags and piles nano-objects during drying. As a result,
although the nano-object-assembled layers have the same mass per unit area, their electrochemical
performance can be very different from one another if the loaded nano-objects are assembled on a
partial part of electrode; in the worst cases, revealing the substrate electrode.
Unevenness of the nano-object-assembled layer can be assessed using surface roughness characteristics
such as root mean square height (S ), maximum height of surface (S ) and arithmetical mean height
q z
(S ). Among these parameters, root mean square height is the most widely used in various industries.
a
Therefore, low root mean square height can convince users to consider that the nano-objects are evenly
assembled layer on the entire surface area of electrode.
NOTE Guidance on the terms, definitions and root mean square height is given in ISO 25178-2.
The measurement of the root mean square height using an atomic force microscope is not adequate for
an uneven surface area of the nano-object-assembled layer because the maximum scan area and the
Z range of the piezo-scanner do not exceed the scale of uneven topography. Surface area metrologies
such as a contact profiler and optical profiler should be used to measure the root mean square height
of the nano-object-assembled layer. If a contact profiler is selected, care should be taken to put an
adequate load so as not to damage the assembled layer on the electrode surface in order to guarantee
the integrity of the electrode surface.
4.3.4 Specific electrochemically active surface area (ECSA)
ECSA specifies the order of electrochemical enhancement by nano-object-modified electrochemical
electrodes. Electrochemical enhancement is generally accomplished by an enlarged surface area of
electrodes. Unlike a flat surface area of pristine electrodes, however, a whole enlarged surface area
of the nano-object-assembled layer does not point to electrochemical enhancement because some
of the enlarged surface area is not active for electrochemical reaction. Therefore, electrochemical
enhancement cannot be specified by a specific surface area, but it can be assessed by ECSA, which is
[31] to [36]
widely used in the electrochemical industry .
Electrochemical methods for determining ECSA fall into two general categories:
— the first type uses a surface-limited adsorption of gas;
— the second type uses a well-characterized and reversible redox reaction such as the reduction of
3− 4−
ferricyanide Fe()CN  to ferrocyanide Fe()CN  .
 6  6
As the method using surface-limited adsorption is not suitable for small mass of nano-object on
electrode, ECSA should be measured by the cyclic voltammetry method using a redox reaction to get
a precise and accurate measured value. In the redox reaction, ECSA is determined by the calculation
of Randles-Sevcik equation after obtaining a cyclic voltammogram using a redox couple in aqueous
solution. The measurement procedure of a specific ECSA of a nano-object-assembled layer is described
in Annex D.
5 Test report
5.1 General
A user prefers specific synthetic properties of nano-object-assembled layer according to their bio-
sensing application and experimental conditions. To meet the preference of users, besides the
characteristics of the nano-object-assembled layer, additional specifications are required to provide
the information about the synthetic procedure of the nano-object-assembled layer such as substrate
type and deposition process. All the characteristics and relevant information of the nano-object-
assembled layer shall be concisely arranged for effective delivery to stakeholders. This clause describes
the formation and the order of specifications. An example of a test report is given in Annex F. The test
report does not require to use the tables but may list the items in three groups.
5.2 General information on nano-object-modified electrode
General information on the nano-object-modified electrode given in Table 3 shall be provided to
describe the synthetic procedure of the nano-object-assembled layer in the first of all tables. This is
because the characteristics of the nano-object-assembled layer could become meaningless when they
are not matched to the preferences of the user’s application.
Table 3 — General information on nano-object-modified electrode
Item Information
Name of substrate material of electrochemical electrode See Table E.1
Material name and type of constituting nano-objects Nominal description
(e.g. gold nanoparticles)
Name of deposition process See Table E.1
Name of adhesion method See Table E.1
5.3 Measurement results of characteristics
In accordance with Clause 4, relevant characteristics are categorized into two tables. The characteristics
of nano-objects given in Table 4 shall be listed if the characteristics are applicable, next to Table 3.
Measurement methods for the individual characteristics in Table 4 are described in Annex B.
Characteristics of the nano-object-assembled layer given in Table 5 shall be provided, next to Table 4.
Measurement methods for the individual characteristics in Table 5 are described in Annexes B, C and D.
6 © ISO 2020 – All rights reserved

Table 4 — Measurement results of characteristics for constituting nano-objects
a
Characteristic Measurement method Measurement result
Surface chemical composition Method name Quantity value
(graphical data may be added)
Mean size and size distribution Method name Quantity value
(graphical data may be added)
Mean primary crystalline particle size Method name Quantity value
Mean length and length distribution Method name Quantity value
Number of walls, i.e. single, double or mul- Method name Number
ti-walled
Surface functional group Method name Nominal functional type
a
The measurement result shall be given with the uncertainty of quantity value, the date of the test and name of the
testing laboratory.
Table 5 — Measurement results of characteristics for nano-object-assembled layer
a
Characteristic Measurement method Measurement result
Mass per unit area Method name Quantity value
Root mean square height Method name Quantity value
ECSA Method name Quantity value
a
The measurement result shall be given with the uncertainty of quantity value, the date of the test and name of testing
laboratory.
Annex A
(informative)
Value chain of nano-object-modified electrochemical electrode
In the value chain of a nano-object-modified electrochemical electrode, the buyers are the instrument
manufacturers and researchers who use the electrode for nano-enhanced electrochemical sensing of
biomolecules, and the sellers are the manufacturers who produce nano-object-modified electrochemical
electrodes. Currently, most of the nano-enhanced electrochemical electrodes are fabricated by
researchers in order to achieve predictable performance in their own programs without mass-
production. However, the technology is maturing into a commercial phase. To manufacture nano-object-
assembled layers on flat electrodes by means of solution processes, the sellers purchase nano-objects
as raw materials from a nano-object provider and measure the characteristics of nano-objects given
in Table 1. After the assembling of a constituting nano-object, the characteristics of the nano-object-
assembled layer given in Table 2 are measured for quality control. The sellers report the measurement
results in accordance with Clause 5 and provide a specification sheet as a test report with the product
...