1、 ISO 2017 Nanotechnologies Electron spin resonance (ESR) as a method for measuring reactive oxygen species (ROS) generated by metal oxide nanomaterials Nanotechnologies Rsonance paramagntique lectronique (RPE) pour la mesure des espces ractives de loxygne (ROS) gnres par des nanomatriaux sous forme
2、doxyde mtallique TECHNICAL SPECIFICATION ISO/TS 18827 Reference number ISO/TS 18827:2017(E) First edition 2017-06 ISO/TS 18827:2017(E)ii ISO 2017 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2017, Published in Switzerland All rights reserved. Unless otherwise specified, no part of this publi
3、cation 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 ISOs member body in the count
4、ry of the requester. ISO copyright office Ch. de Blandonnet 8 CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax +41 22 749 09 47 copyrightiso.org www.iso.org ISO/TS 18827:2017(E)Foreword v Introduction vi 1 Scope . 1 2 Normative references 1 3 T erms, definitions and abbr e viati
5、ons 1 3.1 Terms and definitions . 1 3.2 Abbreviations . 2 4 Principle 2 4.1 General . 2 4.2 Spin trapping method . 2 4.2.1 General 2 4.2.2 DMPO . 2 4.2.3 BMPO . 3 4.2.4 TPC . 3 4.3 Positive control for generating free radicals . 3 4.3.1 Fenton reaction 14. . 3 4.3.2 Hypoxanthinexanthine oxidase syst
6、em 15. 3 4.3.3 Rose bengal photosensitization 16174 5 Reagents 4 6 Apparatus . 4 7 Sampling 5 7.1 Preparation of test sample (metal oxide nanomaterial suspension) . 5 7.2 Preparation of solution for generating the hydroxyl radical . 5 7.2.1 FeSO 4solution 5 7.2.2 H 2 O 2solution 5 7.3 Preparation of
7、 solution for generating the superoxide anion radical . 5 7.3.1 Phosphate buffer 5 7.3.2 Hypoxanthine solution . 5 7.3.3 Xanthine oxidase solution 5 7.4 Preparation of solution for generating the singlet oxygen 5 7.5 Preparation of spin trapping agent . 6 7.5.1 General 6 7.5.2 DMPO stock solution 6
8、7.5.3 BMPO stock solution. 6 7.5.4 TPC stock solution 6 7.6 Reaction of test sample and spin trapping agent 6 7.6.1 General 6 7.6.2 DMPO reaction . 6 7.6.3 BMPO reaction . 7 7.6.4 TPC reaction . 7 7.7 Reaction of positive control and spin trapping agent 7 7.7.1 DMPO radical adduct form (DMPO/OH) . 7
9、 7.7.2 BMPO radical adduct form (BMPO/OOH) 7 7.7.3 TPC radical adduct form (TPC/ 1 O 2 ) 7 7.8 Preparation of the standard sample for spin calculation . 7 8 Interferences 8 8.1 Sampling . 8 8.2 Sampling time 8 9 Procedure. 8 9.1 General . 8 9.2 Injection of sample 9 ISO 2017 All rights reserved iii
10、Contents Page ISO/TS 18827:2017(E)9.3 ESR measurement .10 10 Examples of results .16 10.1 DMPO/OH .16 10.2 BMPO/OOH .16 10.3 TPC/ 1 O 216 10.4 TEMPOL .17 Bibliography .18 iv ISO 2017 All rights reserved ISO/TS 18827:2017(E) Foreword ISO (the International Organization for Standardization) is a world
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17、l. This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies. ISO 2017 All rights reserved v ISO/TS 18827:2017(E) Introduction Recently, the use of metal or metal oxide-based nanomaterials has dramatically increased in biomedical and industrial applications. However, the scienti
18、fic basis for the cytotoxicity and genotoxicity of most manufactured nanomaterials are not fully understood. An important mechanism of nanotoxicity is the generation of reactive oxygen species (ROS). The study on the hazardous effects of metal oxide nanomaterials is still in its initial stage. The a
19、bility to generate ROS is one main source of toxicity of metal oxide nanomaterials. Overproduction of ROS can induce oxidative stress, resulting in cells failing to maintain normal physiological redox-regulated functions. This in turn may lead to DNA damage, unregulated cell signalling, change in ce
20、ll motility, cytotoxicity, apoptosis and cancer initiation. There are critical determinants that can affect the generation of ROS. The critical determinants include size, shape, particle surface, surface positive charges, surface-containing groups, particle dissolution, metal ion release from nanome
21、tals and nanometal oxides, UV light activation, aggregation, mode of interaction with cells, inflammation and pH of the medium 1 . Thus, to detect and quantify ROS formation on the surface of metal oxide nanomaterials, this document suggests the electron-spin-resonance (ESR) method. Amongst ROS, the
22、 most biologically relevant and widely studied are hydroxyl radical (OH), superoxide anion radical (O 2 - ), singlet oxygen ( 1 O 2 ) and hydrogen peroxide (H 2 O 2 ). However, direct detection of some free radicals (e.g. superoxide anion and hydroxyl radical) is very difficult or impossible 2in sol
23、ution at room temperature. ESR spin trapping is a valuable tool in the study of transient free radicals 3 . Spin trapping is a technique, developed in the late 1960s, where a nitrone or nitroso compound (a spin trap) reacts with a target free radical to form a stable and distinguishable free radical
24、 (spin adducts) to be detected by ESR spectroscopy. Spin adducts can be observed directly by ESR spectroscopy. The ESR spectra of these spin adducts are unique and provide a fingerprint for the presence of ROS. This document specifies methods of detection by ESR of 5,5-dimethyl-1-pyrroline-N-oxide (
25、DMPO) hydroxyl adduct, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) superoxide adduct and 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC) singlet oxygen adduct formation from metal oxide nanomaterials. This document provides a method to assess ROS generation on the metal oxide nanom
26、aterials in a cell free condition. This method may provide valuable information for the prediction of ROS-mediated cytotoxicity without cytotoxicity assay at physico-chemical evaluation phase.vi ISO 2017 All rights reserved Nanotechnologies Electron spin resonance (ESR) as a method for measuring rea
27、ctive oxygen species (ROS) generated by metal oxide nanomaterials 1 Scope This document provides a procedure for the detection of ROS (OH, O 2 - , 1 O 2 ) generated by metal oxide nanomaterials in aqueous solution with a reactive oxygen species-specific spin trapping agent using ESR, but excludes ES
28、R procedures that do not use a spin trapping agent. 2 Normative references There are no normative references in this document. 3 T erms, d efinitions and abbr e viations For the purposes of this document, the following terms and definitions apply. ISO and IEC maintain terminological databases for us
29、e in standardization at the following addresses: IEC Electropedia: available at h t t p :/ www .electropedia .org/ ISO Online browsing platform: available at h t t p :/ www .iso .org/ obp 3.1 T erms and definiti ons 3.1.1 nanomaterial material with any external dimension in the nanoscale or having i
30、nternal 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 ISO/TS 80004-1:2015, 2.8 to 2.10. SOURCE: ISO/TS 80004-1:2015, 2.4 3.1.2 test sample material, device, device portion, compone
31、nt, extract or portion thereof that is subjected to biological or chemical testing or evaluation SOURCE: ISO/TS 10993-5:2009, 3.5 3.1.3 zero baseline control equivalent of the positive control where no radicals are detected Note 1 to entry: For example, zero baseline control for the positive control
32、 of fenton reaction will be H 2 O 2and DMPO in the absence of iron; for hypoxanthinexanthine oxidase (HX-XO) system, it will be hypoxanthine and BMPO in the absence of HX-XO; for rose bengal photosensitization, it will be rose bengal and TPC in the absence of light. TECHNICAL SPECIFICATION ISO/TS 18
33、827:2017(E) ISO 2017 All rights reserved 1 ISO/TS 18827:2017(E) 3.1.4 positive control well-characterized material or substance that, when evaluated by a specific test method, demonstrates the suitability of the test system to yield a reproducible, appropriately positive or reactive response in the
34、test system SOURCE: ISO/TS 10993-12:2012, 3.12 3.2 Abbreviations ROS reactive oxygen species ESR electron spin resonance DMPO 5,5-dimethyl-1-pyrroline-N-oxide BMPO 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide TPC 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide DTPA diethylenetriaminepentaacetic
35、acid OH hydroxyl radical OH- hydroxide ion O 2 superoxide anion radical 1 0 2 singlet oxygen TEMPOL 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl 4 Principle 4.1 General In most atoms and molecules, electrons are paired. The paired electrons do not give an ESR signal while atoms and molecules with
36、 unpaired electrons give an ESR signal. When an atom or molecule with an unpaired electron is placed in a magnetic field, the spin of the unpaired electron can align either in the same direction or in the opposite direction as the field. These two alignments of electron spin have different energies.
37、 The application of a magnetic field to an unpaired electron lifts the degeneracy of its spin states. ESR spectroscopy measures the absorption of microwave radiation associated with the transition between these non-degenerate spin states 4 . 4.2 Spin trapping method 4.2.1 General Spin trapping is us
38、ed in ESR spectroscopy for detection and identification of short-lived free radicals. Ideally, the adduct formed between a spin trapping agent and a free radical has an ESR spectrum characteristic and specific to that free radical. Advanced ESR studies employing spin-trap agents were adopted to dist
39、inguish the different types of ROS. 4.2.2 DMPO DMPO has significant advantages over other nitrone spin traps. It is particularly useful for identifying oxygen-centred radicals, e.g. superoxide anion and hydroxyl radicals. The spin adduct formed between 2 ISO 2017 All rights reserved ISO/TS 18827:201
40、7(E) DMPO and the hydroxyl radical has an ESR signal consisting of a quartet with intensity ratio of 1:2:2:1 and hyperfine splitting of a N= a H= 1,49 mT to 1,5 mT, which is consistent with the DMPO-OH adduct 5 . 4.2.3 BMPO BMPO is suitable for the specific in vivo or in vitro detection of short -li
41、ved superoxide anions and hydroxyl radicals by forming distinguishable adducts measurable with ESR spectroscopy 6 . Other nitrone spin traps, such as DMPO, do not distinguish superoxide and hydroxyl radical easily because of spontaneous decay of DMPO-superoxide adduct (t1/2 = 0, 9 min to 1,3 min ) i
42、nto the D MPO-hydroxyl adduct. BMPO- superoxide adduct does not decay into a hydroxyl adduct and has a much longer half-life ( t1/2 = 8,5 min to 15,7 min) 7 . The BMPO-superoxide adduct were fitted with a N= 1,34, a H= 1,18 mT 89 . 4.2.4 TPC TPC has proper sensitivity and dynamic range for detecting
43、 the formation of singlet oxygen 10 . TPC- singlet oxygen adduct spectrum shows a triplet with 1:1:1 signal intensity 11 . The TPC/ 1 O 2adduct has a hyperfine splitting of a N= 0,172 mT 12 . NOTE 1 The hyperfine coupling constants of magnetic nuclei (a = the hyperfine splitting of the spectrum, ai
44、where i = type of nucleus, e.g. 1 H, 13 C, 14 N) and the pattern of an ESR spectrum contains the information about the structure and geometry of such radicals. NOTE 2 The width of spectral line is characteristic of resonance frequency-energy absorption conditions 13 . 4.3 Positive control for genera
45、ting free radicals Fenton reaction, hypoxanthinexanthine oxidase (HX-XO) system and rose bengal photosensitization are well-characterized systems that can generate hydroxyl radical, superoxide anions and singlet oxygen, respectively. These systems demonstrates the suitability of the spin trapping ag
46、ents to yield a reproducible and ESR signal patterns of the spin adducts such as intensity ratio and hyperfine splitting. 4.3.1 Fenton reaction 14 Transition metal ions can activate H 2 O 2to form hydroxyl radicals which are strong oxidants. This system is called the fenton reaction. Iron (II) (ferr
47、ous ion) is oxidized by hydrogen peroxide to produce iron (III) (ferric ion), a hydroxyl radical and a hydroxyl anion (Reaction 1). The hydroxyl radical produced in the fenton reaction might be then trapped by DMPO to yield the spin adduct, DMPO/OH (Reaction 2). Fe 2+ H 2 O 2 Fe 3+ OH +OH - Reaction
48、 1 OH + DMPO DMPO/OH Reaction 2 4.3.2 Hypoxanthinexanthine oxidase system 15 Hypoxanthinexanthine oxidase (HX-XO) system is a well-characterized system that can generate superoxide anions (Reaction 3 and Reaction 4). The superoxide anion can then be trapped by BMPO to form the spin adduct, BMPO/OOH
49、(Reaction 5). Hypoxanthine + H 2 O + O 2 Xanthine + H 2 O 2 Reaction 3 Xanthine + H 2 O + O 2 Uric acid + O 2- Reaction 4 O 2 -+ BMPO BMPO/OOH Reaction 5 ISO 2017 All rights reserved 3 ISO/TS 18827:2017(E) 4.3.3 Rose bengal photosensitization 1617 Rose bengal is known as a photosensitizer for generation of singlet oxygen. When photoexcited, rose bengal transfers its energy to oxygen producing singlet oxygen (Reaction 6
