ISO/TS 5094:2023

TECHNICAL ISO/TS
SPECIFICATION 5094
First edition
2023-02
Nanotechnologies — Assessment of
peroxidase-like activity of metal and
metal oxide nanoparticles
Nanotechnologies — Evaluation de l'activité de type peroxidase des
nanoparticules métalliques et d’oxydes métalliques
Reference number
ISO/TS 5094:2023(E)
© ISO 2023

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ISO/TS 5094:2023(E)
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Published in Switzerland
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ISO/TS 5094:2023(E)
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 . 2
4 Principle . 2
5 Physicochemical characterization of metal or metal oxide NPs . 3
6 Apparatus and reagents .4
6.1 Apparatus and appliances . 4
6.2 Reagents . 4
7 Solution preparation .5
7.1 General requirements . 5
7.2 TMB solution . 5
7.3 Buffer solution . 5
7.4 Nanoparticle dispersion solution . 5
7.5 Additional control solution . 5
8 Measurement procedure . 6
8.1 Measurement condition . 6
8.2 Measurement procedure . 6
8.3 Measurement of reagent blank absorption . 7
8.4 Positive control measurement . . 7
8.5 Additional control measurement . 7
9 Data analysis . 7
10 Measurement uncertainties . 8
11 Test report . 9
Annex A (informative) Measurement for the mass from the tested metal and metal oxide
nanoparticles .10
Annex B (informative) Example of calculation and uncertainty evaluation from the
peroxidase-like activity of iron oxide nanoparticles .11
Bibliography .17
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ISO/TS 5094:2023(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
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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).
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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.
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ISO/TS 5094:2023(E)
Introduction
Enzymes are the biological catalysts that control biochemical reactions. The enzyme peroxidase is a
metalloenzyme with many isoforms. It catalyses the oxidation of various organic substrates by hydrogen
peroxide, which is used extensively in biochemistry applications. Metal and metal oxide nanoparticles
have a wide range of applications in biomedicine, environment protection, and some other fields, such
as magnetic separation, detection, anti-bacterial, degradation of contaminants, medical imaging and
tumour therapy. In recent years, an intrinsic peroxidase-like activity was observed in some metal and
metal oxide nanoparticles, which means that these metal and metal oxide nanoparticles can catalyse
the oxidation of substrates of natural peroxidase by hydrogen peroxide under mild reaction conditions
in comparable efficiency and kinetics. Iron oxide (Fe O ) nanoparticles are one representative
3 4
material, and cobalt oxide (Co O ) nanoparticles, copper oxide (CuO) nanoparticles, manganese oxide
3 4
(MnO ) nanoparticles, vanadium oxide (V O ) nanoparticles, gold (Au) nanoparticles and platinum (Pt)
2 2 5
nanoparticles have been reported to have the peroxidase-like activity as well. These findings extend
enzyme mimics from organic compounds to inorganic nanomaterials.
Certain metal and metal oxide nanoparticles can catalyse the transfer of electrons from H O to
2 2
colorimetric indicator under physiological condition. This phenomenon is like the colorimetric reaction
mediated by peroxidase and thus is called as peroxidase-like catalysis. Such catalytic property can
be used to produce colorimetric, chemiluminescent or electrochemical signals which have great
potential applications in biosensors, electrochemical sensors and immunoassays. The nanoparticles
with peroxidase-like activity may have anti-tumour, antibacterial or antioxidant functions in biological
system. In addition, the nanoparticles with such activity can have potential impacts on health, safety
and the environment. Therefore, it is important to assess the peroxidase-like activity of a nanoparticle
in practical applications.
The peroxidase-like activity of nanoparticles strongly depends on multiple factors including the
composition, size, surface chemistry and crystal structure of the nanoparticles, as well as the
measurement conditions. Therefore, it is important to establish a standard method for assessing the
peroxidase-like activity of metal and metal oxide nanoparticles.
This document provides a specification for the assessment of peroxidase-like activity of metal and
metal oxide nanoparticles. This protocol is useful to enterprises, research laboratories or institutions
and metrological organizations that are working on nanomaterials used in biomedical applications and
environment protection.
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TECHNICAL SPECIFICATION ISO/TS 5094:2023(E)
Nanotechnologies — Assessment of peroxidase-like
activity of metal and metal oxide nanoparticles
1 Scope
This document specifies a method for assessing the peroxidase-like activity of metal and metal oxide
nanoparticles by spectrophotometry. This document can serve as a reference for the measurements of
peroxidase-like activities in other types of nanoparticles.
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 18153:2003, In vitro diagnostic medical devices — Measurement of quantities in biological samples —
Metrological traceability of values for catalytic concentration of enzymes assigned calibrators and control
materials
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms, definitions and abbreviated terms
For the purposes of this document, the terms and definitions given in ISO/TS 80004-2 and the following
apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1 Terms and definitions
3.1.1
nanoparticle
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
[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — Note 1 to entry has been removed.]
3.1.2
catalytic activity
property of a component corresponding to the catalysed substance rate of conversion of a specified
chemical reaction, in a specified measurement system
Note 1 to entry: In this document, the “component” is one kind of metal or metal oxide nanoparticles.
Note 2 to entry: In this document, the catalytic activity is the peroxidase-like activity of metal and metal oxide
nanoparticles.
−1
Note 3 to entry: The coherent derived SI unit is “katal” (kat), equal to “mole per second” (mol·s ).
[SOURCE: ISO 18153:2003, 3.2, modified — Notes 1, 2 and 3 to entry have been added.]
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ISO/TS 5094:2023(E)
3.1.3
specific catalytic activity
catalytic activity per unit mass of metal or metal oxide in nanoparticles
−1
Note 1 to entry: Specific catalytic activity is expressed as kat·kg .
3.2 Abbreviated terms
ABTS 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt)
DMSO dimethyl sulfoxide
H O hydrogen peroxide
2 2
HRP horseradish peroxidase
IONPs iron oxide nanoparticles
NPs nanoparticles
OPD o-phenylenediamine
TMB 3,3',5,5'-tetramethylbenzidine
4 Principle
The HRP reaction can be expressed by Formula (1):
Horseradish Peroxidase
SubstrateH+→OS ubstrate + HHO (1)
reduced 22 ox 2
To evaluate the activity of HRP, chromogenic substrates are often employed, such as TMB, OPD, ABTS,
among these, TMB can be the most widely used one.
Some metal and metal oxide NPs can also catalyse the substrates of horseradish peroxidase in the
presence of H O , which is referred as peroxidase-like activity in this specification. For the substrate
2 2
TMB, the chemical reaction can be expressed by Formula (2):
metal and metal oxide NPs
TMBH+→OT MB + HO (2)
22 ox 2
The reaction of Formula (2) generates a blue colour oxidized product that has a characteristic absorption
peak at a wavelength of 650 nm. The absorbance is measured as a function of time at (37 ± 1) °C (see
Figure 1). The measurement time can be determined based on the linear range of the progressing
curve of the peroxidase reaction. During the initial phase of the reaction within the first few percent
progression towards total completion, there is a linear phase of the reaction. It is recommended to
record the absorbance within the linear phase of the reaction. To determine the enzymatic activity, it is
sufficient to calculate from the change in absorbance per unit of time during this linear phase.
Usually, metal or metal oxide NPs show peroxidase-like activity under acidic conditions and the
activity is weak or even vanishes under neutral or base conditions. The relevance of determining the
peroxidase-like activity under acidic environment is considered as the low pH exists ubiquitously in
biological systems including lysosomes, tumours, wounds, stomach and in environmental systems
including polluted water. At acidic conditions, the dissolution of ions is possible and therefore inclusion
of an additional control is needed (see 7.5).
The solubility of TMB is significantly decreased in solutions of pH ≥ 5,5. This document is applicable for
suspensions with a pH value in the range of 3,5 to 5,5.
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ISO/TS 5094:2023(E)
Key
X time, in s A TMB + H O
2 2
Y absorbance B TMB + H O
ox 2
Figure 1 — Schematic of peroxidase-like activity measurement for metal or metal oxide NPs
The number of peroxidase-like activity units of metal or metal oxide NPs is calculated according to the
Lambert-Beer law:
−1
The initial change rate of absorbance (min ) is obtained from the slope of the early, linear phase, of the
experiment, as shown in Figure 1. After deducting the reagent blank rate, the number of peroxidase-
like activity units of metal or metal oxide NPs is calculated according to Formula (3).
V ΔA
b = × (3)
nano
ε×l Δt
where
b is the number of enzyme activity units of metal or metal oxide NPs, in kat;
nano
V is the total volume of the reaction solution, in l;
−1 −1
Ɛ is the molar attenuation coefficient of the TMB derivative, which is 39 000 mol ·l·cm ;
l is the optical path length of the cuvette, in cm;
∆A/∆t is the initial change rate of absorbance of the reaction solution after correcting with a reagent
−1
blank rate, in s .
The specific catalytic activity of NPs, a , is calculated by dividing b by the mass of the tested
nano nano
NPs.
The peroxidase-like activity of metal or metal oxide NPs is calculated according to Formula (4).
b
nano
a = (4)
nano
m
M
where
−1
a is the peroxidase-like activity of metal or metal oxide NPs, in kat·mg ;
nano
m is the mass of metal or metal oxide NPs, in mg.
M
NOTE See Annex A for the example from measurement and calculation of the mass.
5 Physicochemical characterization of metal or metal oxide NPs
Prior to the assessment of peroxidase-like activity of metal or metal oxide NPs, the size (distribution),
shape, composition, surface chemistry and crystal structure of the nanoparticles should be
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ISO/TS 5094:2023(E)
characterized according to Reference [1], as the peroxidase-like activity strongly depends on these
factors.
6 Apparatus and reagents
6.1 Apparatus and appliances
6.1.1 Spectrophotometer.
[2]
A calibrated standard spectrophotometer covering visible wavelength range shall be used. The
spectrophotometer shall be turned on 1 h prior to the measurement to allow the baseline to stabilize.
6.1.2 Thermometer, with an accuracy equal to or under ±1,0 °C.
6.1.3 pH meter, with a resolution equal to or under 0,01 and an accuracy of ±0,002.
6.1.4 Thermostatic water bath.
The thermostatic water bath of the spectrophotometer is used to control the tank of cuvette at a
constant temperature of (37 ± 1) °C.
6.1.5 Electronic balance, with a precision of 0,01 mg and a repeatability (calibration weight) of
≤0,015 mg (5 g).
6.1.6 Adjustable pipette, of 200 μl (uncertainty, u < 0,3 %) and 1 000 μl (u < 0,3 %).
6.1.7 Volumetric flask, of volume (10 ± 0,04) ml and (100 ± 0,2) ml.
6.1.8 Cuvette, with an optical path length of (10 ± 0,05) mm.
6.2 Reagents
All essential reagents for the assay are listed in Table 1.
Table 1 — Reagents
Classification Chemical name Name
a
Anhydrous sodium acetate (AR) sodium acetate
a
Anhydrous acetic acid (AR) glacial acetic acid
a
Reagents 3,3',5,5'-Tetramethylbenzidine (AR) TMB
a
30 % hydrogen peroxide (AR) 30 % H O
2 2
−6
Horseradish peroxidase (≥4,167 × 10 kat/mg solid) HRP
Double distilled water, grade 2, in accordance w
...