GB/Z 42840-2023 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 to cell wall degradation of Staphylococcus aureus and muramic acid release as quantified by a gas chromatography-mass spectrometry (GC-MS).
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
antibacterial activity
property of compound or substance that kills (bactericidal) or slows down (bacteriostatic) the growth of bacteria
3.2
dynamic light scattering; DLS
photon correlation spectroscopy
technique in physics that can be used to determine the size distribution profile of small particles in suspension or polymers in solution
3.3
gas chromatography–mass 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)
3.4
Gram positive bacteria
bacteria that are stained/resistant to bleaching by organic solvent during Gram staining
3.5
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 20752:2014, 3.2]
3.6
limit of detection; LOD
minimum amount or concentration of 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 8196-1:2009, 3.4.3]
3.7
limit of quantification; 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 other validation
[SOURCE: ISO/TS 15216-1:2017, 3.18]
3.8
linear dynamic range; LDR
regression curve of Y on X is a straight line and the regression is called LDR
Note: The coefficient of linear regression of Y on X is the coefficient of x (slope) in the equation of the regression line.
3.9
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[12].
Note 1: 3-O-α-carboxyethyl-D-glucosamine.
Note 2: See Figure 1 b).
3.10
peptidoglycan; PGN
murein
polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria forming the cell wall
Note: The peptidoglycan is a specific and essential element in the cell wall of almost all bacteria. Its amount in cell walls of Gram positives is substantial, see Figure 1 a)[12].
3.11
silver nanoparticle potency
reaction of silver nanoparticles with a bacterium’s cell wall as measured indirectly by muramic acid release
3.12
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
3.13
trace
µg/ml concentration or lower
3.14
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: GB/T 30543-2014, 3.8, modified]
3.15
ultraviolet visible spectroscopy; UV-Vis
absorption spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region
Note: This means it uses light in the visible and adjacent [near-UV and near-infrared (NIR)] ranges.
3.16
x-ray power diffraction; XRD
method in which the elastically scattered intensity of X-rays is measured
[SOURCE: GB/T 30544.6-2016, 5.2.1]
4 Symbols and abbreviated terms
The following symbols and abbreviated terms apply to this document.
AgNP Silver Nanoparticle
MA Muramic Acid
MHB Mueller Hinton Broth
OD600 Optical Density at 600 nm
SIM Selected Ion Monitoring
4 Principles
PGN is a specific and essential component in the cell wall of bacteria.[12] Its main structural features 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 β-1-4 bonds, and the peptide chain is composed of L-alanine, D-glutamine, L-lysine, and D-alanine in the case of S. aureus [(see Figure 1 a)][12][13]. It 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[12][14].
AgNPs as the active entity of colloidal AgNPs could have various sizes, surface features, shapes, zeta potential, and concentrations in different trade products. The activity of AgNPs depends on the mentioned characteristics, which could have synergistic or antagonistic cross-effects[14]. Distinguishing among the effects caused by each of these parameters would require the preparation of a large number of samples 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[14][15][16][17][18].
This Technical Specification utilizes a very sensitive measure of a single analyte, muramic acid as an indirect measure of a single mode of action, cell wall degradation. This method’s sensitivity allows for muramic acid quantification below the minimum inhibitory concentration (MIC)[19][20]. However, the effect of silver nanoparticles detected through 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 duality; 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 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 these considerations, characterization data for the laboratory-prepared silver nanoparticle calibration and system validation reference material can be found in Annex C.
