Caterina Minelli

Quantitation of IgG protein adsorption to gold nanoparticles using particle size measurement

The attachment of proteins, and other biomolecules, to nanoparticles is of critical interest both in the development of medical products using nanoparticles and in understanding the behaviour and fate of nanoparticles in biological systems. Measuring the amount of protein attached to the particles is a fundamental step in these regards and there are a variety of methods available for this purpose. In this work, we compare the use of three methods which measure particle diameter: Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Differential Centrifugal Sedimentation (DCS). The choice of gold particles also permits measurement of the amount of adsorbed protein through a shift in plasmon frequency in UV-visible spectroscopy. When the protein layer is complete, the results from all methods are consistent to within ∼20% scatter and suggest that IgG adsorption on these 20 nm to 80 nm nanoparticles is rather similar to adsorption on flat gold surfaces with a water content of ∼60% by volume. We note an excellent correlation between plasmon frequency shift and DCS sedimentation times which indicates that both DCS and analytical ultracentrifugation can provide precise measurement of the thickness of complete protein shells on dense nanoparticles, but also show that these methods will fail for particles with a density of ∼1.38 g cm−3. In the low protein coverage regime, the measured amount of protein depends upon the technique: NTA and DLS provide, as expected, similar values that also correlate well with plasmon frequency shift. DCS analysis underestimates protein shell thicknesses in this regime and this may be explained through redistribution of the protein shell which reduces the frictional force during sedimentation. Crown copyright ©2013. Reproduced with the permission of the controller of HMSO.

Reference materials and representative test materials to develop nanoparticle characterization methods: the NanoChOp project case

This paper describes the production and characteristics of the nanoparticle test materials prepared for common use in the collaborative research project NanoChOp (Chemical and optical characterization of nanomaterials in biological systems), in casu suspensions of silica nanoparticles and CdSe/CdS/ZnS quantum dots (QDs). This paper is the first to illustrate how to assess whether nanoparticle test materials meet the requirements of a “reference material” (ISO Guide 30, 2015) or rather those of the recently defined category of “representative test material (RTM)” (ISO/TS 16195, 2013). The NanoChOp test materials were investigated with small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and centrifugal liquid sedimentation (CLS) to establish whether they complied with the required monomodal particle size distribution. The presence of impurities, aggregates, agglomerates, and viable microorganisms in the suspensions was investigated with DLS, CLS, optical and electron microscopy and via plating on nutrient agar. Suitability of surface functionalization was investigated with attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR) and via the capacity of the nanoparticles to be fluorescently labeled or to bind antibodies. Between-unit homogeneity and stability were investigated in terms of particle size and zeta potential. This paper shows that only based on the outcome of a detailed characterization process one can raise the status of a test material to RTM or reference material, and how this status depends on its intended use.

A comparison of techniques for size measurement of nanoparticles in cell culture medium

Plain and aminated silica nanoparticles dispersed in purified water, in 50 mM Tris–HCl buffer and in cell culture medium were measured using dynamic light scattering (DLS), centrifugal liquid sedimentation (CLS), small-angle X-ray scattering (SAXS), and particle tracking analysis (PTA). The test samples were measured by all methods immediately after dispersion and after incubation at room temperature for 24 h. The effect of the biological dispersion medium on the modal value of the particle size distribution was compared for each method taking into account the estimated uncertainty. For the methods based on light scattering, DLS and PTA, the size distributions obtained were significantly altered due to the formation of a protein corona and induced agglomeration effects. With SAXS and CLS, the measured size of the primary particles was mostly unchanged. While SAXS offers excellent precision and traceability to the SI unit system if the model fitting approach is used for data analysis, CLS provides detailed size distributions from which additional information on the agglomeration state can be deduced.

Characterisation of antibody conjugated particles and their influence on diagnostic assay response

The batch-to-batch assay performance ‘activity’ of antibody conjugated particles is often variable, leading to poor reproducibility between different production batches. We therefore sought to quantify the properties of such particles using differential centrifugal sedimentation (DCS) to see how they influence assay performance, with the aim to improve the reproducibility of the conjugation reaction. The DCS high resolution size distributions of the antibody conjugated particles allowed us to examine the thickness of the antibody corona on the particles and to quantify the amount of ‘contaminating’ particle oligomers produced via carbodiimide chemistry. The DCS data was correlated with the assay response of the resulting conjugate using an interleukin 6 (IL6) lateral flow assay developed in house. We prepared a series of antibody conjugates using various carbodiimide reaction conditions and analysed the size, antibody corona and oligomerisation profile of the resulting particles using both dynamic light scattering (DLS) and DCS. Both the amount of antibody bound to the particle and the presence of higher order particle oligomers produced conjugates that when applied in an IL6 lateral flow assay were associated with an enhanced fluorescent signal. Both the amount of particle oligomers and antibody bound to the particle was found to be positively correlated with increased assay response in the lateral flow. The DCS estimation of protein corona thickness for each carbodiimide condition tested was found to correlate with the amount of antibody coupled to the particles, as assessed using the bicinchoninic acid (BCA) assay. We have shown the novel application of DCS for the analysis of antibody-particle conjugates. DCS analysis provides a quantitative method to characterise particles and provides a rationale for variable assay performance observed from batch-to-batch production.

Effect of fluorescent staining on size measurements of polymeric nanoparticles using DLS and SAXS

The influence of fluorescence on nanoparticle size measurements using dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) was investigated. For this purpose, two series of 100 nm-sized polymer nanoparticles stained with different concentrations of the fluorescent dyes DY555 and DY680 were prepared, absorbing/emitting at around 560 nm/590 nm and 695 nm/715 nm, respectively. SAXS measurements of these particle series and a corresponding blank control (without dye) revealed similar sizes of all particles within an uncertainty of 1 nm. DLS measurements carried out at three different laboratories using four different DLS instruments and two different laser wavelengths, i.e., 532 nm and 633 nm, revealed also no significant changes in size (intensity-weighted harmonic mean diameter, Z-Average) and size distribution (polydispersity index, PI) within and between the two dye-stained particle series and the blank sample. Nevertheless, a significant decrease of the detected correlation coefficients was observed with increasing dye concentration, due to the increased absorption of the incident light and thus, less coherent light scattering. This effect was wavelength dependent, i.e. only measurable for the dye-stained particles that absorb at the laser wavelength used for the DLS measurements.

Measuring the size and density of nanoparticles by centrifugal sedimentation and flotation

The successful translation of nanoparticle-based systems into commercial products depends upon the ability to reliably measure important physical and chemical properties of these particles. The density of nanoparticles is one such property, because it provides important information about the composition of the material. In this work, an analytical centrifugation approach based on line-start centrifugal sedimentation and flotation measurements is described. The two independent measurements permit both the size and the density of these nanoparticles to be determined with excellent precision. A set of monodisperse polystyrene nanoparticles of different sizes is used to demonstrate this method. The density and size measurements are validated by comparison to accurate Small Angle X-ray Scattering (SAXS) analysis for particles within the size range of SAXS, i.e. less than ∼300 nm in diameter. Both sedimentation and flotation measurements produce consistent high resolution size distributions of the particles and the measured size and density values are identical, within experimental uncertainty, to the SAXS results. This approach has the potential to provide useful characterisation of a range of particles of interest, for example, for medical application, such as liposomes and polymeric drug carriers.

Measuring the relative concentration of particle populations using differential centrifugal sedimentation

The factors that affect the accuracy and precision of differential centrifugal sedimentation (DCS) for the analysis of nanoparticle concentration are described. Particles are separated by their sedimentation rate and detected using light absorption. In principle, the relative concentration of particles in different populations can be found, but the uncertainty in such measurements is unclear. We show that the most appropriate measurement of particle concentration using this technique is the mass concentration, rather than the number concentration. The relative mass concentration of two discrete populations can be measured with reasonable precision, usually without resorting to complicated data analysis. We provide practical approaches to find the relative mass concentrations for two cases: spherical particles of different materials and agglomerated particles of the same material. For spherical particles made of different materials, naive analysis of the results can provide relative mass concentrations that are many orders of magnitude in error. Correction factors can be calculated that reduce the error to less than 50%. In the case of agglomerated particles we show that errors of less than 20% are possible and demonstrate, in the case of gold particles, that a combination of UV-visible spectroscopy and DCS enable practical values of mass and number based particle concentrations to be obtained.

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.