Characterization of Colloidal Gold Using Dynamic Light Scattering

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Introduction 

Colloidal gold is a suspension of gold nanoparticles that shows interesting properties[1]. The color of the sample is determined by the size and shape of the gold particles[2]. Figure 1 shows suspensions of colloidal gold with various particle sizes. Particles smaller than 5nm appear yellow, 5nm~20nm red, and over 100nm appear blue. 

In aqueous solutions, gold particles are negatively charged, giving them a strong affinity for various biological macromolecules such as proteins and antibodies[3]. Due to this, colloidal gold is currently applied in various biotechnological areas such as DNA binding, imaging probes, and diagnostics[1,4,5]. Moreover, it is being developed for use in advanced electronics and coatings[6].

Characterizing the size of colloidal gold is crucial to confirm uniform particle diameter and the absence of aggregated particles in the suspension sample. Electron microscopy techniques are widely used for size characterization[1,2]. Figure 2 shows a transmission electron micrograph of a colloidal gold sample. While individual gold particles can be clearly seen, most exist as clusters of two or more particles.

Electron microscopy is an excellent method for visualizing particles, but from a statistical standpoint, it measures only a few dozen to hundreds of particles, making it inadequate for comprehensive analysis. However, by calculating particles of various sizes, it can be used to find the particle size distribution based on number.

Dynamic Light Scattering (DLS) is a non-invasive technique to measure the size of nanoparticles in dispersion. The method utilizes Brownian motion to measure the intensity of scattered light over time in a particle suspension. Analyzing fluctuations in this scattered light intensity allows determining the diffusion coefficient, from which the particle size can be calculated using the Stokes-Einstein equation.

This application note will explore the method of characterizing the size of colloidal gold using dynamic light scattering and the differences in results compared to those obtained using electron microscopy.

Experiment

All measurements in the application note were conducted at 25°C using the Zetasizer Nano S. The Nano S features a 4mW He-Ne laser with a wavelength of 633nm and an Avalanche Photodiode (APD) detector, detecting scattered light at an angle of 173°.

Results and Discussion

Figure 3 shows the intensity particle size distribution of the colloidal gold solution measured with the Nano S. The figure displays the relative proportion of light scattered by particles of various size groups (X axis) (Y axis). The resulting particle distribution has two peaks, with averages of 13.6nm and 339nm respectively. The analysis for intensity, volume, and number results of these peaks is shown in Table 1.

The measured intensity distribution indicates that there is a significant amount of aggregated particles in the sample. However, converting this intensity distribution into volume (or mass or weight) (Figure 4) reveals that aggregated particles exist at a low concentration. When converting intensity results to volume, Mie’s theory is applied, requiring values for the particle’s refractive index (n) and absorbance (k). Here, values of 0.2 (n) and 3.32 (k) were used[9]. The resulting volume distribution shows that over 90% of the sample by weight consists of small particles around 13nm.

Converting these results to a number distribution, shown in Figure 5, gives a single peak with an average of 12.4nm. This result shows that when characterizing the sample’s properties using a number-based method like electron microscopy, the majority of visible particles are small. The presence of larger particles is only evident when a sufficient number of measurements are taken. Based on numbers, there are very few aggregated particles in the sample, but these particles scatter a significant amount of light, dominating the intensity size peak. (Figure 3). Consequently, samples like this one can yield significantly different results when analyzed using dynamic light scattering and electron microscopy.

If the sample shown in Figure 2 is measured with dynamic light scattering, analyzing various particles (single particles, aggregates of two particles, aggregates of three, etc.) becomes extremely challenging. Therefore, dynamic light scattering is not recommended as an analytical method for samples consisting of three sizes.

As a result, mixtures of single particles and aggregates formed of two, three, or four particles will show a single broad peak due to the influence of large particles that scatter most of the light. The z-average diameter and polydispersity index values are sensitive to the presence of aggregates. The z-average diameter is the average hydrodynamic diameter, and the polydispersity index is an estimate of the distribution width. Both values are calculated according to the international standard for dynamic light scattering, ISO13321[10].

Conclusion

Dynamic light scattering is an appropriate method for determining the size of colloidal gold. This method is very sensitive to the presence of aggregates, allowing the use of z-average diameter and polydispersity index as methods for determining sample uniformity.

For monodisperse samples, results obtained by dynamic light scattering and electron microscopy should be very similar. However, for polydisperse samples, scattering by large particles results in larger particle size outcomes by dynamic light scattering than electron microscopy.

References

[1] M.A. Hayat (1989) Colloidal Gold:Principles, Methods and Applications,
Academic Press, New York.

[2] K. Miura and B. Tamamushi (1953)J. Electron Microscopy 1, 36-39.
[3] M. Horisberger and M.F. Clerc(1985) Histochem and Cell Biol. 82, 219-223.
[4] A. Csaki, R. Möller and W. Fritzsche (2002) Expert Rev. Mol. Diagn. 2, 89-94.
[5] R. Tanaka, T. Yuhi, N. Nagatani,T. Endo, K. Kerman, Y. Takamura and E. Tamiya (2006) Anal. Bioanal.Chem 385, 1414-1420.
[6] T. Sato and H. Ahmed (1997)Applied Phys. Letters 70, 2759-2761.
[7] A.N. Shipway, E. Katz and I Willner (2000) 1, 18-52.
[8] P. Mulvaney, M. Giersig and A.Henglein (1992) J. Phys. Chem. 96,
10419-10424.

[9] L. G. Shulz (1954) J. Opt. Soc. Am.44, 357-362 and 362-368.
[10] International Standard ISO13321 Methods for Determination of Particle
Size Distribution Part 8: Photon Correlation Spectroscopy, International Organization for Standardization (ISO) 1996.

Zetasizer Nano

Malvern Instruments’ Zetasizer Nano is the first commercially available device with hardware and software capable of measuring static, dynamic, and electrophoretic light scattering. The Nano device can measure variables such as particle size, molecular weight, and zeta potential.

The Zetasizer Nano is designed to meet the unique requirements of pharmaceuticals, biomolecule sectors with low concentration and small sample volumes, and high concentration colloidal applications. It achieves these through backscatter optical arrangements and new cuvette designs. Consequently, the particle size and concentration specifications of the Zetasizer Nano surpass other commercially available dynamic light scattering devices, with a particle size range of 0.6nm to 6um and a concentration range of 0.1ppm to 40% w/v.

The Zetasizer Nano System offers not only patent-pending hardware design but also excellent software for instrument control and data analysis. This software uses self-optimizing algorithms to automate the necessary optical settings for each sample and features a “one-click” measurement, analysis, and reporting system designed to minimize new users’ software learning curve.