Is your material "Nano"?Can we make Safer Nanomaterials by Design?

Is your material 'Nano'?

What is the definition of "Nanomaterials"?

According to the EU definition of a nanomaterial in 2011
(Recommendation on the definition of a nanomaterial(2011/696/EU))


Making Decision based on
"Good Measurement & Analysis Practice : TEM"

  • TEM Sample Prep.

    Dispersion & TEM sample prep. Protocol

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  • TEM measurement

    TEM measurement Protocols (NanoReg, ACEnano)

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  • TEM Image Analysis

    TEM Image Analysis in s2nano.org

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  • Decision on "NANO"

    Decision-Making on "Nanomaterial" using TEM

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TEM Sample Prep.

Dispersion & TEM sample prep. Protocol

ISO NanoREG ACEnano S2NANO
Method
  • Prepare suspension solution of NPs
  • Drop suspension solution of NPs (5uL) on TEM grid & dry
  • Pre-wetting sample using Ethanol
  • Disperse sample in Bovine serum albumin-water (2.56 mg/ml) using probe sonication
  • Drop & dry sample on TEM grid
  • Drop suspension solution of NPs on TEM grid & dry for 0.5–1h
  • Prepare suspension solution from NPs powder using water/ ethanol
  • Drop suspension solution (5-10uL) of NPs on TEM grid & dry for 2-3h
Instrument
  • Copper grid
  • Inter-laboratory
  • Copper grid: 400 mesh
  • Inter-laboratory
  • Copper grid: 300 mesh
  • JEOL 1200EX TEM (accelerating voltage 80 kV)
  • Copper grid: 300 mesh
  • JEM-2010 TEM (accelerating voltage 200 kV)

TEM measurement

TEM measurement Protocols (NanoReg, ACEnano)

Summary of TEM Technique :
Principle :
  • A high velocity electron beam is generated and accelerated under high vacuum, it is focused by condenser lens (electromagnetic bending of electron beam) onto specimen.
  • Electrons are transmitted through the specimen.
  • On the way through the specimen some parts of the material stop or deflect electrons more than other parts.
  • The electrons are collected from below the sample onto a phosphorescent screen or through a camera for viewing images. The regions in which electrons do not pass through the specimen the image is dark.
  • Where electrons are un-scattered, the image is brighter, and there are a range of greys in between depending on the way the electrons interact with and are scattered by the sample.

TEM Image Analysis

TEM Image Analysis in s2nano.org

ISO NanoREG ACEnano S2NANO
Method
  • Set the measurement scale
  • Crope image to remove image artifacts
  • Thresholding grey scale
  • Apply despeckle to remove possible artifacts
  • Set the list of measurands desired (such as area, shape descriptors…)
  • Analyze the particles
  • Export data
  • Setting and adjusting the threshold value
  • Defining the detection area Setting the detection parameters
  • Detection of the primary particles in aggregated NM
  • Selection of the primary particle parameters
  • Defining the classification schemes
  • Classification of the particles according the selected parameters
  • Exporting of results in excel spreadsheets and storage of the (annotated) images in the NM Database
  • Obtain are and diameter of >100 particles / 1 sample to plot size histogram
  • Set measurement scale
  • Separate particles & background using grey scale threshold
  • Define circularity of NPs
  • Obtain area, diameter… of each particle
  • Export data to excel format
  • Collect data for >300 particles
Software
  • Image J
  • IgorPro
  • DigitalMicrograph
  • Image J
  • Image J
  • Image J

Decision on "NANO"

Decision-Making on "Nanomaterial" using TEM


Making Decision based on
"Good Measurement & Analysis Practice : DLS"

  • DLS Sample Prep.

    DLS sample prep. Protocol

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  • DLS measurement

    DLS measurement Protocols (NanoReg, ACEnano)

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  • DLS Data Interpretation

    DLS Data Analysis in s2nano.org

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  • Decision on "NANO"

    Decision-Making on "Nanomaterial" using DLS

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DLS Principle

DLS Data Analysis in s2nano.org

Summary of Technique :
  • The technique determine the size distribution profile of small particles in suspension or polymers in solution.
  • A monochromatic light source is shot through a polarizer and into a sample. The scattered light goes through a second polarizer and is collected by a photomultiplier.
  • By repeating the process in short-time interval, the scattered pattern can be analyzed by an autocorrelator to analyze the light intensity profile of each spot.

DLS Sample Prep.

DLS sample prep. Protocol

ACEnano NanoREG S2NANO
NM dispersions: analyze as received. Powder NM samples were dispersed by means of specific dispersion protocol Dispersion stability: conduct the standard measurement Perform 10 measurements to reach automatic optimization of the software Prepare suspension solution of NPs
Size: 1 mL of the liquid was injected into the polystyrene cuvette. Minimum of 5 measurements are required. High-accuracy size-data: determine the viscosity of the sample after DLS measurement for data-correction Appropriate sample volume to measure DLS is 3 mL and the minimum volume is 1 mL.
Zeta Potential: 1 mL of the liquid was injected into the zeta potential cuvette. Minimum of 3 measurements are required.

DLS Standard Measurement

DLS measurement Protocols (NanoReg, ACEnano)

Factors ACEnano NanoREG S2NANO
Instrument Malvern Zetasizer (nano ZS) 633-nm laser Malvern Zetasizer (nano ZS) 633-nm laser Malvern Zetasizer (nano ZS) 633-nm las
Scattering angle Back-scatter angle 173o Back-scatter angle 173o Side-scatter angle 173o
Temperature 20oC 25oC
37oC
(in vitro/in vivo exposure media)
25oC
Replication 5 consecutive measurements 10 consecutive measurements 3 consecutive measurements

DLS Data Interpretation

DLS Data Analysis in s2nano.org

ACEnano NanoREG S2NANO
Output Average size distribution Analysis of relative intensity Average size distribution (Z average)
Zeta potential graph Analysis of average zeta-size Intensity-based diameter
Volume-based diameter
Number-based diameter
Analyze the evolution in relative scattered light intensity and zeta-size as a function of time

Decision on "NANO"

Decision-Making on "Nanomaterial" using DLS