1、 ISO 2012 Surface characterization of gold nanoparticles for nanomaterial specific toxicity screening: FT-IR method Caractrisation de surface des nanoparticules dor pour criblage de toxicit spcifique de nanomatriau: mthode FT-IR TECHNICAL SPECIFICATION ISO/TS 14101 First edition 2012-11-01 Reference
2、 number ISO/TS 14101:2012(E) ISO/TS 14101:2012(E)ii ISO 2012 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2012 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopy
3、ing and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerl
4、and ISO/TS 14101:2012(E) ISO 2012 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references 1 3 Terms and definitions . 1 4 Symbols and abbreviated terms . 3 5 Sample preparation mode 3 5.1 Removal of unbound molecules . 3 5.2 Dehydration . 6 5.3 Screening t
5、est for impurities in DW from sample tubes . 6 6 FT-IR measurement procedure . 7 6.1 General . 7 6.2 ATR method . 7 6.3 Transmission method . 8 6.4 Determination of time required for complete purge . 9 6.5 Linear range of IR band intensity versus concentration 10 6.6 LOD and LOQ determination 11 6.7
6、 Repeatability determination 12 7 Application examples .12 7.1 Degrees of ligand exchange .12 7.2 Qualitative measurement of biomolecular binding 13 Annex A (informative) Case study for validation of ligand exchange 15 Annex B (informative) Case study for qualitative measurement of biochemical moiet
7、ies binding to the AuNP surface .17 Annex C (informative) Selection guide for window materials 20 Bibliography .21 ISO/TS 14101:2012(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing
8、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 liaiso
9、n with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of tec
10、hnical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. In other circums
11、tances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in an ISO working group and is accepted for pub
12、lication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it is approved by 2/3 of the members of the committee ca
13、sting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which tim
14、e it must either be transformed into an International Standard or be withdrawn. 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. ISO/TS 14101 was prep
15、ared by Technical Committee ISO/TC 229, Nanotechnologies.iv ISO 2012 All rights reserved ISO/TS 14101:2012(E) Introduction Gold nanoparticles (AuNPs) can be controlled with regard to size, shape and surface ligands, making them ideal for the study of relationships between their physicochemical prope
16、rties and cytotoxicity on living bodies 123 . Among the various properties of AuNPs, surface ligand characteristics, such as the chemical composition, molecular structure and quantity of bound molecules, were found to play an important role in determining the behaviour of AuNPs, e.g. the degree of a
17、ggregation or agglomeration in solution, binding with biomolecules in cell culture media and cytotoxicity to living cells 456789 101112 . On the other hand, surface ligand modification is not always successful in the synthesis step, and the degree of ligand exchange should be identified prior to the
18、 property specific cytotoxicity test of AuNPs in order to obtain reliable and consistent results. FT-IR (Fourier transform infrared) absorption spectroscopy is one of the most useful tools of NP surface ligand identification and quantification. By using the FT-IR method, the structures and relative
19、quantities of ligand molecules bound to NP surfaces can be analysed 1314151617181920 . However, the low concentrations and aqueous environment of synthesized AuNPs will complicate the interpretation of measurement results. Low concentrations of AuNPs result in small absorbance values, which can easi
20、ly be influenced by background noise or the absorbance of trace impurities. Since cytotoxicity tests are performed in aqueous environments, we should analyse what is on the surface of AuNP in aqueous solutions if we want to study the effect of the surface characteristics on cytotoxicity of AuNPs. Ho
21、wever, water molecules strongly absorb IR light over a wide frequency range, disabling IR absorption analysis on the solutes in very low concentrations. It is necessary to develop measurement guidelines by which the above issues can be minimized. In this project, we seek to develop a Technical Speci
22、fication (TS) for the observation of chemical moieties bound to the synthetic AuNP in the form of dehydrated films, which can deliver the information about the molecular species bound to AuNPs when they were in aqueous solutions. Although the standardization of FT-IR measurement procedures will be t
23、he basis for this Technical Specification, a great deal of weight will also be given to the sample preparation procedure for correct FT-IR analysis. ISO 2012 All rights reserved v Surface characterization of gold nanoparticles for nanomaterial specific toxicity screening: FT-IR method 1 Scope This T
24、echnical Specification provides guidelines for the identification of the surface bound molecules using FT-IR of dehydrated gold nanoparticle (AuNPs) films both before and after nanomaterial (NM) cytotoxicity testing. NOTE 1 AuNPs may have surface bound ligands prior to testing and may be additionall
25、y covered (or coated) with organic- or bio-molecules during the cytotoxicity test. NOTE 2 Nucleic acids, amino acids, lipids or membrane components binding to AuNPs can be observed by FT- IR spectroscopy by detection of absorption bands corresponding to phosphodiester, amine or lipid, respectively,
26、although the type of nucleic acids, proteins or lipid cannot be identified in detail based on IR spectra. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, th
27、e latest edition of the referenced document (including any amendment) applies. ISO/TS 27687, Nanotechnologies Terminology and definitions for nano-objects Nanoparticle, nanofibre and nanoplate 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO/TS 27687
28、and the following apply. 3.1 attenuated total reflection mode ATR Mode instrumental mode of operation in which the incident angle of IR light on the crystal is adjusted to be higher than the critical angle NOTE The light is completely reflected by the upper surface of the crystal, and the intensity
29、of the light is attenuated through absorption by materials covering the upper surface of the crystal. The frequency of IR light absorbed is used to identify the absorbed chemical moiety, and the fraction of light that is absorbed is used to quantitate the amount of that moiety present. 3.2 dialysis
30、process by which small molecules or ions diffuse through a membrane, thus causing their separation from larger molecules in solution and from suspended matter ISO 6107-2:2006, definition 38 3.3 Fourier transform infrared spectroscopy FT-IR analytical chemical technique based on absorption of infrare
31、d radiation by chemical moieties in the specimen, used to identify and quantitate the absorbing chemical moieties TECHNICAL SPECIFICATION ISO/TS 14101:2012(E) ISO 2012 All rights reserved 1 ISO/TS 14101:2012(E) 3.4 limit of detection LOD measured quantity value, obtained by a given measurement proce
32、dure, for which the probability of falsely claiming the absence of a component in a material is , given a probability of falsely claiming its presence NOTE 1 Adapted from ISO/IEC Guide 99:2007, definition 4.18. NOTE 2 LOD may be determined as 2,776 times the standard deviation of the measurements of
33、 5 replicate blanks under conditions of repeatability with IUPAC recommended values of 0,05 for both and . NOTE 3 See also ISO 17191. 3.5 limit of quantification LOQ lowest value of an analyte that can be determined with an acceptable level of accuracy and precision NOTE 1 LOQ may be determined as 1
34、0 times the standard deviation of the photometric noise, which will give relative precision A /A 10 % for the minimum signal level A. NOTE 2 See Reference 24. 3.6 molecular weight cut-off value MWCO molecular weight of solute that is retained by more than 90 % after 16 h dialysis NOTE See References
35、 25 and 26. 3.7 nano-object material with one, two or three external dimensions in the nanoscale ISO/TS 27687:2008, definition 2.2 NOTE Generic term for all discrete nanoscale objects. 3.8 nanoparticle NP nano-object with all three external dimensions in the nanoscale ISO/TS 27687:2008, definition 4
36、.1 NOTE If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically by more than three times), the terms nanorods or nanoplate are intended to be used instead of the term nanoparticle. 3.9 nanoscale size range from approximately 1 nm to 100 nm ISO/TS 27687:2
37、008, definition 2.1 NOTE 1 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 The lower limit in this definition (approximately 1 nm) is introduced to a
38、void single and small groups of atoms from being designated as nano-objects or elements of nanostructures, which might be implied by the absence of a lower limit.2 ISO 2012 All rights reserved ISO/TS 14101:2012(E) 3.10 pre-tested distilled water DW distilled water validated to be free from IR absorb
39、ing impurities by FT-IR measurement 3.11 relative centrifugal force RCF acceleration force relative to the Earths gravity 3.12 surface plasmon resonance band SPR range of frequencies of absorbed light, where the absorption is the result of the collective oscillation of electrons within the near-surf
40、ace region of a solid NOTE SPR occurs in thin metal films or metallic NPs. 4 Symbols and abbreviated terms AuNP gold nanoparticle IR infrared MW molecular weight SCM serum containing media UV /Vis ultraviolet/visible g Earths gravimetric acceleration as a reference unit for the relative centrifugal
41、force 5 Sample preparation mode 5.1 Removal of unbound molecules 5.1.1 General Since FT-IR absorption spectroscopy measures total molecular species in the sample film, all unbound molecules that are active IR absorbing species except solvents shall be removed from the solution before preparing the s
42、ample film in order to correctly identify the molecules bound to the surface of AuNPs. 5.1.2 Dialysis Dialysis is an efficient method for separating unbound molecules from AuNPs when membranes with adequate MWCOs are available. If used adequately, dialysis membranes reduce the concentration of unbou
43、nd molecules according to the volume ratio of the sample and dialysis solutions, generally retaining more than 90 % of NPs. It is recommended that MWCO is lower than half the MW of the species to be retained, and higher than three times the MW of the species intended to pass through. Because the eff
44、iciency of dialysis membrane depends on the charge and shape of molecules, the unbound molecular removal efficiency of a dialysis membrane shall be verified before the separation of AuNPs from unbound molecules. Prior to the efficiency test, the membrane shall be tested if it is free from IR absorbi
45、ng impurities. The procedure for impurity test is as follows: a) fill the dialysis bag with 0,5 ml to 3 ml of pretested DW; ISO 2012 All rights reserved 3 ISO/TS 14101:2012(E) b) seal the DW containing dialysis bag with a proper clip and dialyse 16 h in a bath containing pretested DW (600 ml in volu
46、me); c) clean the surface of ATR crystal or IR window by using a solvent moistened cotton swab; d) take a required volume (2 or 200 l) of liquid in the dialysis bag; NOTE Use 2 l for the ATR method and 200 L for the transmission method. e) drop the sampled liquid on an ATR crystal or IR window and d
47、ry it in a dehydration chamber (see 5.2); this procedure is called “drop and dry”; f) measure the FT-IR spectrum of any potential impurities dissolved in the sampled liquid by using the procedure in 6.2 or 6.3; g) if there is no IR band exceeding LOD in the frequency region of interest, the membrane
48、 is considered free from IR active impurities. The membrane that is free from IR active impurities can be used for the dialysis. The procedure for the test of dialysis efficiency is as follows: a) fill the dialysis bag with 0,5 ml to 3 ml of solution that contains only molecules to be removed by dia
49、lysis; molecules to be removed by dialysis correspond to surface ligands before and after exchange, or biomolecules in SCM or cell extracts for binding test; the concentration of sample solution is set to the maximal amount of surface ligands that can exist in the NP suspension. This value may be the same as the added amount of ligand molecules for exchange or estimated from the number of gold atoms on the surface, which can be calculated from the lattice constant 27a
