1、BSI Standards Publication Nanotechnologies Determination of silver nanoparticles potency by release of muramic acid from Staphylococcus aureus PD ISO/TS 16550:2014National foreword This Published Document is the UK implementation of ISO/TS 16550:2014. The UK participation in its preparation was entr
2、usted to Technical Committee NTI/1, Nanotechnologies. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The Bri
3、tish Standards Institution 2014. Published by BSI Standards Limited 2014 ISBN 978 0 580 74491 4 ICS 07.030 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published under the authority of the Standards Policy and Strategy Committee on 30
4、September 2014. Amendments/corrigenda issued since publication Date Text affected PUBLISHED DOCUMENT PD ISO/TS 16550:2014 ISO 2014 Nanotechnologies Determination of silver nanoparticles potency by release of muramic acid from Staphylococcus aureus Nanotechnologies Dtermination de lefficacit des nano
5、particules dargent par la libration de lacide muramique du Staphylococcus aureus TECHNICAL SPECIFICATION ISO/TS 16550 First edition 2014-05-15 Reference number ISO/TS 16550:2014(E) ISO/TS 16550:2014(E)ii ISO 2014 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2014 All rights reserved. Unless o
6、therwise specified, 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 addre
7、ss 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 Switzerland PD ISO/TS 16550:2014 ISO/TS 16550:2014(E) ISO 2014 All rights reserved ii
8、i Contents Page Foreword iv Introduction v 1 Scope . 1 2 T erms and definitions . 1 3 Symbols and abbreviated terms . 3 4 Principles . 3 5 Sample preparation for the determination of MA . 4 5.1 Bacterial growth and AgNP treatment . 4 5.2 Hydrolysis of cell wall . 6 5.3 Derivatization of cell wall 6
9、5.4 Gas chromatography-mass spectrometry instrumentation . 6 6 Data analysis and results interpretations 7 7 The test method for silver nanoparticle sample . 8 Annex A (informative) Gas chromatography-mass spectrometry 9 Annex B (informative) Internal standard .10 Annex C (informative) Preparation o
10、f laboratory-prepared silver nanoparticles reference materials 11 Annex D (informative) Silver nanoparticle suspension preparation.12 Annex E (informative) Treatment control conditions; positive, negative, and bacterial suitability controls 13 Bibliography .14 PD ISO/TS 16550:2014 ISO/TS 16550:2014(
11、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 through ISO technical committees. Each member body interested in a subject for which
12、a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matt
13、ers 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 do
14、cument 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
15、 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
16、 does not constitute an endorsement. For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary in
17、formation. The committee responsible for this document is ISO/TC 229, Nanotechnologies.iv ISO 2014 All rights reserved PD ISO/TS 16550:2014 ISO/TS 16550:2014(E) Introduction The antimicrobial properties of silver nanoparticles (AgNPs) are being increasingly utilized in consumer products. It has been
18、 widely used for development of many biological and pharmaceutical processes, products, and applications such as coating material for medical devices, orthopaedic or dental graft materials, topical aids for wound repair, clothing, underwear and socks, textile products, and even washing machines. 1In
19、dustries are producing wide varieties of AgNPs as an antibacterial agents with different physical properties and with little low concern to their side effects. Moreover, there is a risk of enhanced bioavailability of the nanoparticles in the different media 23which might have impact on the beneficia
20、l soil flora, plants, animals, and humans. From this point of view, the potency of AgNPs should be evaluated and classified according to its final biological activities. This Technical Specification does not describe use of this Technical Specification to evaluate what properties of silver nanomater
21、ial are to be assessed. ISO 2014 All rights reserved v PD ISO/TS 16550:2014 Nanotechnologies Determination of silver nanoparticles potency by release of muramic acid from Staphylococcus aureus 1 Scope This Technical Specification provides a test method for evaluating potency of silver nanoparticles
22、to cell wall degradation of Staphylococcus aureus and muramic acid release as quantified by a gas chromatography-mass spectrometry (GC-MS). 2 T erms a nd definiti ons For the purposes of this document, the following terms and definitions apply. 2.1 antibacterial activity property of compound or subs
23、tance that kills (bactericidal) or slows down (bacteriostatic) the growth of bacteria 2.2 dynamic light scattering photon correlation spectroscopy DLS technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution 2.3 gas chr
24、omatographymass spectrometry GC-MS method that combines the features of gas-liquid chromatography and mass spectrometry to qualitatively and quantitatively analyse volatile compounds within a test sample (Annex A) 2.4 Gram positive bacteria bacteria that are stained/resistant to bleaching by organic
25、 solvent during Gram staining 2.5 internal standard compound of known concentration added to a sample to facilitate the qualitative identification and/or quantitative determination of the sample components (Annex B) SOURCE: ISO 20752, 3.2 2.6 limit of detection LOD minimum amount or concentration of
26、 the analyte in a test sample which can be detected reliably, but not necessarily quantified, as demonstrated by a collaborative trial or other appropriate validation SOURCE: ISO 8196-1, 3.4.3 TECHNICAL SPECIFICATION ISO/TS 16550:2014(E) ISO 2014 All rights reserved 1 PD ISO/TS 16550:2014 ISO/TS 165
27、50:2014(E) 2.7 l i m i t o f q u a n t i f i c a t i o n LOQ lowest concentration of target in a test sample that can be quantitatively determined with acceptable level of precision and accuracy under the experimental conditions specified in the method, as demonstrated by a collaborative trial or ot
28、her validation SOURCE: ISO/TS 15216-1, 3.18 2.8 linear dynamic range LDR regression curve of Y on X is a straight line and the regression is called LDR SOURCE: ISO 3534-1, 1.34 Note 1 to entry: The coefficient of linear regression of Y on X is the coefficient of x (slope) in the equation of the regr
29、ession line. 2.9 muramic acid form of sugar amide which chemically is the ether conjugate between lactic acid and glucosamine. It occurs naturally as an N-acetyl derivative in peptidoglycan 8 Note 1 to entry: 3-O-carboxyethyl-D-glucosamine. Note 2 to entry: See Figure 1 b). Figure 1 Structure of a)
30、peptidoglycan layer, b) muramic acid 2.10 peptidoglycan (murein, PGN) polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria forming the cell wall Note 1 to entry: The peptidoglycan is a specific and essential element in the cell wall of alm
31、ost all bacteria. Its amount in cell walls of Gram positives is substantial, see Figure 1 a). 3 2.11 silver nanoparticle potency reaction of silver nanoparticles with a bacteriums cell wall as measured indirectly by muramic acid release2 ISO 2014 All rights reserved PD ISO/TS 16550:2014 ISO/TS 16550
32、:2014(E) 2.12 Staphylococcus aureus S. aureus facultative anaerobic, Gram positive coccus, coagulase positive, which appears as grape-like clusters when viewed through a microscope, able to grow in a hypersaline medium with manitol as the sole carbon source producing a yellow pigment 2.13 trace g/ml
33、 concentration or lower 2.14 transmission electron microscopy TEM instrument that produces magnified images or diffraction patterns of the sample by an electron beam which passes through the sample and interacts with it SOURCE: ISO/TS 10797, 3.8 2.15 ultraviolet visible spectroscopy UV-Vis absorptio
34、n spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region Note 1 to entry: This means it uses light in the visible and adjacent near-UV and near-infrared (NIR) ranges. 2.16 x-ray power diffraction XRD method in which the elastically scattered intensity of X-rays is measur
35、ed SOURCE: ISO/TS 80004-6, 3.2.4 3 Symbols and abbreviated terms AgNP Silver Nanoparticle MA Muramic Acid MHB Mueller Hinton Broth OD Optical Density at 600 nm SIM Selected Ion Monitoring 4 Principles PGN is a specific and essential component in the cell wall of bacteria. 8Its main structural featur
36、es are linear glycan strands cross-linked by short peptides. The glycan strands are made up of alternating N-acetylglucosamine and N-acetylmuramic acid, MA, residues linked by -14 bonds, and the peptide chain is composed of L-alanine, D-glutamine, L-lysine, and D-alanine in the case of S. aureus (se
37、e Figure 1 a). 89It has been shown that AgNPs will damage the bacterial cell wall and production of the PGN fragments, leading to increased MA concentration in the culture media. 812 AgNPs as the active entity of colloidal AgNPs could have various sizes, surface features, shapes, zeta potential, and
38、 concentrations in different trade products. The activity of AgNPs depends on the mentioned characteristics, which could have synergistic or antagonistic cross-effects. 12Distinguishing among the effects caused by each of these parameters would require the preparation of a large number of samples IS
39、O 2014 All rights reserved 3 PD ISO/TS 16550:2014 ISO/TS 16550:2014(E) and use of a great variety of instrumentations. Subsequently, monitoring the concentration of MA is one indicator of the effects of these characteristics on AgNPs potency. 1213141516 This Technical Specification utilizes a very s
40、ensitive measure of a single analyte, muramic acid as an indirect measure of a single mode of action, cell wall degradation. This methods sensitivity allows for muramic acid quantification below the minimum inhibitory concentration (MIC). 1718However, the effect of silver nanoparticles detected thro
41、ugh muramic acid liberation does not indicate lethality or cessation of cell growth. Furthermore, the method relies on a single laboratory synthesized calibration sample to establish a dose response curve for later use with a widely accepted reference. Nanoparticles possess a particle- chemical dual
42、ity; in that, the particle can be a reservoir for chemical release by dissolution processes. The protocol described in this Technical Specification does not offer information about broad spectrum bactericidal action of silver nanoparticles. An effect on Staphylococcus aureus cannot be predictive for
43、 other Gram positive organisms and would not be expected to apply to Gram negative organisms due to the profound structural difference between the two cell wall types with respect to peptidoglycan proportion. Thus, this is not a comprehensive survey of nanoparticle effects upon bacteria. In light of
44、 these considerations, characterization data for the laboratory-prepared silver nanoparticle calibration and system validation reference material can be found in Annex C. 5 Sample preparation for the determination of MA 5.1 Bacterial growth and AgNP treatment 5.1.1 Principle of the test method Fresh
45、 broth cultures of S. aureus A T C C 2 5 9 2 3 , i n t h e O D 600 , approximately 0,45 to 0,5 (CFU/ml approximately 1 - 2 10 9or the mid-logarithmic phase of growth) phase are treated with AgNPs and incubated for 18 h at 37 C with shaking (180 rpm to 200 rpm). Afterwards, the culture is clarified w
46、ith centrifugation and filtration prior to being dried using rotary evaporator and freeze drier instruments. In the next step, muramic acid concentration will be determined by using method described in this Technical Specificaition. It is essential that the cultures used in the experiment contain a
47、high titre of viable bacteria. In each experiment, the titre at least should be demonstrated either from historical control data on growth curves and through the determination of OD 600within a time range that could ensure the high titre of viable cells (OD 600approximately 0,45 to 0,5). 5.1.2 Descr
48、iption of the method 5.1.2.1 Materials S. aureus ATCC 25923 MHB medium 1)5.1.2.2 Test conditions 1) A fresh culture of bacterium should be prepared on nutrient agar (NA) plate, by striking of a single colony on the NA plate. 2) Triplicate flasks, each containing 25 ml sterile MHB medium (in 150 ml t
49、o 200 ml Erlenmeyer flasks) should be used for assay of each concentration of AgNPs. 1) MHB medium is available from Merck Co., Darmstadt, Germany. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.4 ISO 2014 All rights reserved PD ISO/TS 16550:2014 ISO/TS 16550:2014(E) 3) A pre-culture of bacterium should be prepared in the MHB medium about 2 h to 3 h before inoculation of Erlenmeyer fl
