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Work Package 4

Validated methods for simultaneous physical and chemical characterisation of nanomaterials in biological systems

This work package aims to provide traceable simultaneous measurements for the determination of size, total element concentration and size-related elemental speciation of inorganic nanoparticles (as defined in WP1) in biological samples such as cell media and cell extracts. This will be achieved by using a combination of methodology based on complementary measurement techniques including on-line hyphenations of field flow fractionation (FFF) with MALS and ICP-MS and X-ray based techniques such as SAXS (Small Angle X-ray Scattering), ASAXS (Anomalous SAXS) and XRF (X-ray Fluorescence analysis). The development of analytical methods such as total and speciated isotope dilution mass spectrometry (IDMS) for the accurate quantification of total element and size-based elemental concentrations in the biological system is needed for quality control of existing assays intended for nano-toxicology and other health applications and to produce reference materials.

There are no traceable methods for the determination of size, shape, total elemental and size-based elemental composition in complex samples such as cell media and cell systems. Ideally, characterisation of nanomaterials should be done at the time of exposure in the medium or in the organism or model cell component being exposed to the material. Using a combination of FFF with MALS and element-specific detection by ICP-MS will be used to determine size, size distribution and elemental composition of nanoparticles in the range of 1 to several 100 nm.

IDMS methodology will be developed for accurate total element determination in cell media and cell lysates. Speciated FFF-ICP-IDMS methodology will be developed and validated for real-time size-based element speciation. For this purpose, the use of internal standardisation (e.g. by using an isotopically enriched silica nanoparticle spike) will be investigated.

Develop and validate methodology for total element determination

The aim of this task is to develop and validate methodology for the accurate determination of total element concentration in biological samples/fractions. This task will address optimisation of sample preparation versus efficient introduction of nanomaterials into ICP-MS for their accurate quantitation in biological samples. It will also address improvement of on-line interfacing of fractionation techniques (e.g. FFF) with elemental detectors (e.g. ICPMS), needed to deliver task 4.2 (Develop and validate combined methodology for size and size-based element determination by on-line FFF coupled to MALS and ICP-MS).  The activities are:

  • Development of improved sample introduction of nanomaterials for ICP-MS
  • Development and validation of an IDMS methodology for total element quantitation of dispersed nanoparticles in biological samples by ICP-MS

Develop and validate combined methodology for size and size-based element determination by on-line FFF coupled to MALS and ICP-MS

The aim of this task is to investigate the potential of on-line FFF coupled with ICP-MS and MALS to accurately determine the size distribution, shape and the elemental concentration of each size fraction for dispersions of inorganic nanomaterials in cell media and extracts. Depending on the results obtained using FFF-MALS in WP2 and WP4 for cell media or serum, the capability will be extended and validated for cell extracts.

Develop combined X-ray techniques for simultaneous determination of size, size distribution and chemical composition of nanomaterials in biological systems

The aim of this task is the simultaneous determination of dimensional quantities like size, size distribution and shell thickness in case of core-shell nanoparticles, and of the chemical elemental composition of nanomaterials in biological systems by using synchrotron-radiation based X-ray techniques. To obtain the required information, SAXS, ASAXS and XRF measurements have to be combined. The measurements will be performed at the four-crystal monochromator beamline of PTB at the synchrotron radiation source BESSY II. This beamline covers the entire photon energy range from 1.75 keV to 10 keV, thus the K-edges of the biologically relevant elements as well as K and L edges of the metals in the metal-oxide nanoparticles are accessible.