NanoSight

Nanoparticle Standards

Size Determination of Gold Colloid by Nanoparticle Tracking Analysis

Introduction

Gold nanoparticles are commonly used in a variety of applications such as: 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

NanoSight instruments provide a unique ability to directly visualise and simultaneously size individual nanoparticles in a liquid suspension. This particle-by-particle visualisation and analysis overcomes inherent problems associated with techniques such as Photon Correlation Spectroscopy (PCS, or Dynamic Light Scattering) in which the intensity of light scattered from a ?/10 nanoparticle varies as the 6th power of the particle radius. The average particle size produced by PCS is therefore heavily weighted towards small numbers of larger particles. Other techniques such as Electron Microscopy require time consuming sample preparation and only view a small area and therefore a possibly non-representative sample.

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.

The NanoSight technique calculates a sphere equivalent hydrodynamic radius based on analysis of the Brownian motion of separate particles within a population. Each individual particle is simultaneously tracked over multiple frames, results being independent of both particle and solvent refractive index. This leads to an ability to resolve polydisperse mixtures at far higher resolution than is possible with ensemble averaging techniques such as DLS.

Au Colloid Mixture Analysis

Because each particle is tracked separately, it is possible to measure not only the size of the particle through its dynamic behaviour but also the relative amount of light it scatters. Because of the strong power law associated with such scattering, it is possible to resolve even closely sized particles with high resolution.

The following example is of an analysis of a mixture of 30nm and 60nm calibration-quality gold particles mixed with a suspension of 100nm polystyrene particles.

In this mixture of 30 nm and 60 nm gold nanoparticles mixed with 100 nm polystyrene, 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.

From the previous mixture of 30nm and 60nm Au mixed with 100nm polystyrene latex, the high resolving power of the NanoSight technique can be seen. The following plot is from NanoSight’s Nanoparticle Tracking Analysis NTA2.0 programme in which can be seen the 2 Au peaks at 34nm and 61nm and the polystyrene peak at 106nm.

Also shown overlaid is the cumulative number distribution plot for the Au particles.

Aggregation of Au Nanoparticles Following Dilution

Au colloid can be notoriously prone to aggregation in sub-optimal conditions. The following example is of a NIST-standard quality 30nm Au colloid shows the importance of ensuring high quality purity of diluents when handling Au colloids.

Calibrated 30 nm gold particles (NIST) were diluted into the same three types of water: tap, de-ionised and 18M? water (all free from 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 18M? water causing no aggregation.

Aggregation of Functionalised 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.

Contact Details

For further information, contact NanoSight or your local distributor, listed at www.nanosight.com