ISO/TS 23459:2021

TECHNICAL ISO/TS
SPECIFICATION 23459
First edition
2021-01
Nanotechnologies — Assessment of
protein secondary structure during an
interaction with nanomaterials using
ultraviolet circular dichroism
Nanotechnologies — Évaluation de la structure secondaire des
protéines durant une interaction avec des nanomatériaux à l'aide du
dichroïsme circulaire ultraviolet
Reference number
©
ISO 2021
© ISO 2021
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ii © ISO 2021 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Nanomaterial protein interactions . 2
6 Sample preparation . 3
6.1 General . 3
6.2 Desired properties of the UV-CD quartz cell. 3
6.3 Preparation of protein solution . 3
6.4 Instrumental setting condition . 4
6.5 Recording UV-CD spectra procedure. 4
6.5.1 General. 4
6.5.2 Buffer . 4
6.5.3 Protein sample . 4
6.5.4 Stability of NP suspension in the protein solution . 5
6.6 Preparation of protein-NPs conjugated suspension . 5
6.7 UV-CD spectra measurement . 5
6.8 Calculation of molar ellipticity . 6
6.9 Data analysis . 6
7 Test report . 7
Annex A (informative) Typical UV-CD spectra of proteins . 8
Annex B (informative) Literature survey on structural changes of NOAA and proteins .9
Annex C (informative) Description of buffers that can be used for protein solubility .15
Annex D (informative) Unit conversions in CD measurements .19
Annex E (informative) Calculating the concentration range of the sample .20
Annex F (informative) Methods for estimation of secondary structures of protein .21
Annex G (informative) Typical data of UV-CD used for estimation of secondary structures of
protein .22
Bibliography .23
Foreword
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This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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iv © ISO 2021 – All rights reserved

Introduction
Nano-objects and their aggregates and agglomerates (NOAA) are currently produced in large mass
quantities globally and used in a variety of applications. However, there is concern about their
interaction with biological systems, including proteins, which could lead to reversible or irreversible
alterations in their secondary structure. The latter could affect the functionality and conformation
of protein, which in turn might affect the overall bio-reactivity of the proteins. The monitoring of the
occurrence of such alterations could thus provide important information on the interaction of NOAAs
with biological systems.
The process of folding of polypeptides in biological media produces the secondary structure of proteins
which determines their bioactivity. The important features of this structure include hydrogen bonds
between the amine hydrogen and carbonyl oxygen atoms in the peptide backbone and disulfide bonds
between two cysteine residues.
The protein secondary structure could be affected by exposing it to certain metallic ions and bioactive
compounds. Furthermore, it is also influenced by different buffer ionic strength, pH values, and
[1]
temperature . Alterations in the functionality and conformation of proteins can be attributed to
reorganization (so-called misfolding) and changes of the overall molecular dimension that accompany
the folding process. Some diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s and
[2]
Parkinson’s, are a consequence of misfolded proteins .
There are several standard techniques for determining the molecular structures/conformations and
folding process of proteins and upon their interaction with NOAAs. These include high-field nuclear
magnetic resonance (NMR), Fourier-transform infrared (FT-IR), Raman spectroscopy and ultraviolet
[3][4][5][6]
circular dichroism (UV-CD) spectroscopies . In addition, a novel technique synchrotron
radiation circular dichroism (SRCD) spectroscopy is a sensitive method to provide information on
[7]
protein secondary structures and folding .
TECHNICAL SPECIFICATION ISO/TS 23459:2021(E)
Nanotechnologies — Assessment of protein secondary
structure during an interaction with nanomaterials using
ultraviolet circular dichroism
1 Scope
This document specifies measurement protocols and test conditions to determine alterations to
protein secondary structure induced by their interaction with nanomaterials using ultraviolet circular
dichroism (UV-CD) spectroscopy.
This document does not apply to the characterization of conformational changes of disordered proteins.
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 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-4, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials
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 80004-2,
ISO/TS 80004-4, 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
nanoparticle
NP
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as
“nanofibre” or “nanoplate” may be preferred to the term “nanoparticle”.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.2
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
Note 2 to entry: See also “engineered nanomaterial”, “manufactured nanomaterial” and “incidental nanomaterial”.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.3
circular dichroism
optical effect of the differential absorption of left- and right-handed circularly polarized light
Note 1 to entry: Ultraviolet circular dichroism spectroscopy is used to investigate the secondary structure of
proteins.
4 Abbreviated terms
Ag-NP silver nanoparticle
Au-NP gold nanoparticle
AU absorbance unit
BSA bovine serum albumin
CD circular dichroism
DLS dynamic light scattering
HSA human serum albumin
MRE mean residue ellipticity
MWCNT multiwall carbon nanotubes
NOAA nano-objects and their aggregates and agglomerates
PAA-GNP poly (acrylic acid)-coated gold nanoparticles
SRCD synchrotron radiation circular dichroism
SWCNT single-wall carbon nanotubes
UV-CD ultraviolet circular dichroism
UV-Vis ultraviolet-visible
5 Nanomaterial protein interactions
In a biological environment, NOAA can easily interact with proteins such as apolipoproteins, fibronectin,
[5]
human serum albumin (HSA), vitronectin, etc. The layers of b
...