NanoSight

Engineered Nanoparticles

Engineered Nanoparticles

Size Determination of Gold Colloid by Nanoparticle Tracking Analysis

Introduction
Background
Measuring Mixtures of Materials
Aggregation of Au Nanoparticles Following Dilution
Aggregation of Functionalized Au Nanoparticles on Addition of Binding Ligand

Introduction

Gold nanoparticles can be utilized in a variety of different applications. These include:

Transmission Electron Microscopy (TEM)/Scanning Electron Microscopy (SEM) analysis, as antibody/protein labels for immunoassays and biosensors, for catalysis and, when combined with polymeric materials, as biological scaffolds.

Background

Nanoparticle Tracking Analysis (NTA) is a unique methodology for visualizing nanoparticles in liquid suspension, high resolution particle size versus concentration distributions can be derived through image analysis. The technique overcomes the inherent weaknesses in ensemble techniques such as Dynamic Light Scattering (DLS or Photon Correlation Spectroscopy), in which particle size distributions are heavily skewed towards the larger particles within a sample. This is due to the fact that DLS is strongly influenced by the intensity of light scattered from a particle, which varies with the 6^th power of the particle radius. As such, larger particles scatter significantly more light than smaller particles and will obscure the signal from the smaller particles. As NTA works by tracking the movement of each particle on a particle-by-particle basis and relating the degree of movement to the sphere-equivalent hydrodynamic diameter, this biasing is largely overcome.

Figure 1: Image of suitably diluted suspension of Au nanoparticles in water. Inset is of tracks followed from each particle on which size determination is based.

Measuring Mixtures of Materials

As particles of similar size but different material move at the same rate (the rate of Brownian motion is independent of mass), the technique is not influenced by material density. Similarly as the technique does not use light scattering intensity in any calculation, it is also independent of particle refractive index. The technique does however record how bright a particle is (even though this isn’t used to measure the size of a particle), particle size, relative scattering intensity and particle concentration can then be plotted on a 3D graph (see fig.3) allowing particles of a similar size but different refractive index to be discriminated. Fig 2. shows the size distribution generated from a mixture of 100nm polystyrene spheres and 30nm and 60nm gold particles.

Figure 2. Particle Size distribution of a mixture of 100nm polystyrene, 60nm gold and 30nm gold particles.

Figure 3. Showing 3d plot of Size vs particle intensity vs particle concentration for a mixture of 100nm polystyrene, 60nm gold and 30nm gold particles. Despite their smaller size, the 60 nm Au can be seen to scatter more than the 100 nm PS and the 3 populations of particles are easily resolved.

The three populations can now be clearly defined by the three separate peaks indicated. The three particle types can be clearly seen in the 3D Size vs. Intensity vs. Number plot confirming indications of a tri-modal given in the normal particle size distribution plot. Despite their smaller size, the 60 nm Au can be seen to scatter more than the 100 nm PS.

Aggregation of Au Nanoparticles Following Dilution

Colloidal materials are notoriously prone to aggregation in sub-optimal conditions. The following example is of a NIST 30nm Au colloid, and shows the importance of ensuring high quality purity of diluents when handling these types of materials.

Calibrated 30nm gold particles (NIST) were diluted into three types of water: tap, de-ionised and 18MΩ water (all free from contaminant nanoparticles) then analysed with the same concentration using the Nanosight system. The plots show that the degree of aggregation depends on water purity with only the pure 18MΩ water causing no aggregation.

For further information on this type of study, please see the NanoSight application note on aggregation studies

Aggregation of Functionalized Au Nanoparticles on Addition of Binding Ligand

The above shows a suspension of a mixture of 60nm 3’- and 5’-oligonucleotide-functionalised Au nanoparticles before (top) and after (below) addition of a DNA sample which bound to the 20-mer oligonucleotides immobilised on the Au nanoparticles. Mean size was seen to increase from 61nm to 81nm following dimerisation. The quantities of DNA ligand added to induce this detectable level of aggregation were extremely low and potentially provides an alternative to fluorescence based assays or signal amplification procedures such as PCR, in nucleic acid diagnostics.