Reviewing the Use of Nanoparticle Tracking Analysis (NTA) for Nanomaterial Characterization

Nanoscale materials, in the form of nanoparticles, are playing an important and growing role across a range of different applications and industries which seek to exploit the unique properties exhibited by these materials, such as their very high surface area to volume ratio and high number. The stability and properties of various end products typically rely on the capacity to create particle populations within fine tolerances and with minimal aggregates and impurities.

Particle concentration within a suspension may also impact the preferred outcome of a product. In order to gain a better understanding of the link between the formulation and the overall properties of the materials it is important to define a wide range of properties during nanoparticle analysis.

This article demonstrates the key role of high resolution measurement to determine particle size and concentration in nanoparticle research. In addition it also describes and compares the nanoparticle tracking analysis (NTA) technique against other characterization techniques, cites comparative papers and summarizes a number of studies with reference to the application and value of the NTA technique.

Analysis of Particle Size and Size Distribution

The most standard techniques available for studying particle size and size distribution are atomic force microscopy (AFM), dynamic light scattering (DLS), analytical ultracentrifugation (AUC), and electronmicroscopy (EM). Each of these methods have certain advantages and limitations. In the case of AFM and EM, users can obtain particle images with high resolution data about the size and morphology of existing particles. However, these methods take a significant amount of time in sample preparation and therefore extend a user’s analysis time (Syvitski, 1991).

The AUC technique also gives high resolution data regarding the particle size in a sample, but requires knowledge about the material’s composition and takes a considerable amount of time. Also the equipment for AUC can be costly (Mächtle, 2006).

Light scattering-based methods are suitable for examining monodisperse systems, but cannot analyze polydisperse systems. DLS, also known as quasielastic light scattering (QELS) or photon correlation spectroscopy (PCS) is the foremost method for nanoparticle analysis. A widely assessed technique (Pecora, 1985), DLS, uses a digital correlator to examine fluctuation timescales in the intensity of light dispersed by particle suspension moving under Brownian motion. Analysis of the ensuing exponential autocorrelation function makes it possible to measure the polydispersity index and average particle size. Since the link between particle size and the amount of light scattered by these particles strongly differs as a function of radius, the outcomes are relatively biased towards the bigger and higher scattering particles within the sample.

The advanced NTA method makes it possible to view the size and the number of nanoparticles in liquid suspension. It is capable of analyzing nanoparticle population on an individual basis. this means it is perfect for real-time analysis of polydispersed systems spanning between 10 and 30 nm up to 1 to 2 µm in size based on the type of particle. With more parameters, users can obtain data on the relative intensity of light scattered and the nanoparticle concentration to view and study particles that are fluorescently labeled (NanoSight, 2011, Carr et al. 2009).

Nanoparticle Tracking Analysis: Principles and Methodology

Brownian motion and light scattering properties are used by the NTA technique to acquire the samples’ particle size distribution in a liquid suspension. When passing a laser beam via the sample chamber the particles incident on the beam path and scatter light making it possible to view them through a 20x magnification microscope equipped with a camera. The camera records a video file of the particles under Brownian motion within the 100 µm x 80 µm x 10 µm field of view (Figure 1). Figure 2 shows the NTA particle sizing process.

Schematic of the optical configuration used in NTA.

Figure 1. Schematic of the optical configuration used in NTA.

The NTA particle sizing process.

The NTA particle sizing process.

The NTA particle sizing process.

The NTA particle sizing process.

Figure 2. The NTA particle sizing process.

Measuring Concentration

In the case of NTA particle sizing, particles need to be tracked for several consecutive frames to establish their size consistently, while particle concentration can be determined by examining just a single video frame. This technique removes concerns that the same particle would be counted by NTA several times in cases where it had been absent and then appeared again. When a particle is observed a number of times it will eventually give a contribution based on the overall proportion of frames in which it was viewed in the video.

The concentration obtained is measured by taking the average count and then dividing it by the interrogated volume in which the particle number is determined. With a depth of 10 µm, the interrogated volume is approximate to a cuboid of 100 µm x 80 µm. Utilizing a graticule in the microscope view, the cuboid’s height and width are directly determined. The cuboid’s depth is defined by the depth of the laser beam as well as the depth of field collected by the lens. They are roughly 10 µm.

Concentration Ranges Measurable

NTA measures each particle individually irrespective of others. It is critical to ensure that an adequate number of particles is studied within the sample time so the information obtained is statistically sound. A particle concentration of 106 to 109 particles mL-1 provides users with statistically robust and repeatable particle size distributions within a timescale of 30 to 60s.

NTA reported concentration vs. actual sample concentration for 100 nm latex particles.

Figure 3. NTA reported concentration vs. actual sample concentration for 100 nm latex particles.

Under standard conditions when studying optimal concentrations of nanoparticles showing identical optical properties concentration precisions can be as high as ±5% to 10% if the sample is diluted to an appropriate concentration range. Figure 3 shows the NTA reported concentration versus actual sample concentration for 100 nm latex particles.

Particle Size Determination Combined with Particle Light Scattering

A unique feature of the NTA method is that it can determine the quantity of light it scatters (IScat) and plot it against particle size. This makes it possible to distinguish particles which could have identical size yet different refractive index or composition.

It is possible to resolve a combination of 100 nm polystyrene, 60 nm Au and 30 nm Au in a 3D plot of size against intensity vs. number (Figure 4). Here, the smaller yet high refractive index 60 nm Au particles scatter a greater amount of light when compared to the larger 100 nm polystyrene.

A 3D plot of 30 nm and 60 nm gold, and 100 nm polystyrene particles in which the smaller but higher refractive index 60 nm gold particles can be seen to scatter more light than the larger 100 nm polystyrene.

Figure 4. A 3D plot of 30 nm and 60 nm gold, and 100 nm polystyrene particles in which the smaller but higher refractive index 60 nm gold particles can be seen to scatter more light than the larger 100 nm polystyrene.

Assessment of NTA

In an analysis of precise particle size distribution determination by NTA methodology based on 2D Brownian dynamics simulation a physical model was presented by Saveyn et al. (2010). The model replicates the standard step length distribution during the course of NTA experiments as a function of the distribution of the number of steps within the tracks and the particle size distribution. They demonstrated that the replication of a step length distribution made it possible to consistently estimate the particle size distribution, thus minimizing artificial broadening of the distribution to a large extent.

A variation of this modeling step, as mentioned above, is integrated within the NTA algorithm as a 'finite track length adjustment' that retains the actual distribution width of narrow distributions of monodisperse nanoparticle suspensions with calibration quality.

In order to obtain standardization of results (ASTM E2834-12, 2012), a standard guide has been published by the American Society for Testing and Materials (ASTM) to determine particle size distribution of nanomaterials in suspension through the NTA method.

Drug Delivery and Targeting

Nanoparticles are increasingly being used in drug delivery applications. Year on year a lower number of novel drugs are entering the market. As a result versatile and multifunctional structures of nanoparticles have attracted significant attention for drug delivery. These particles provide improved pharmacokinetic properties, allow sustained and controlled release, and target particular organs, tissues or cells. These aspects could enhance the safety, efficacy and bioavailability of current drugs (Malam et al., 2011). In this regard, nanoparticles have been referred to as submicron sized colloidal systems that can be produced from different materials.

When developing targeted drug delivery with nanoparticles to particular locations molecular structures with an affinity towards a particular cells surface biomarkers are often added. By using such biomarkers the target cell types accumulate the drug-containing nanoparticle. However, adding capture molecules on the surface of a drug delivery nanoparticle structure can pose a challenge. Factors like adequate loading, activity retention, and reduced aggregation are critical for optimum performance.

NTA can easily detect slight changes in hydrodynamic diameter as soon as macromolecules are added to nanoparticles and can also identify and measure any aggregate which could occur during such changes.

As a result the NTA method has been applied in various drug delivery analysis including one that demonstrated the impact of a conjugating polymer-alendronate-taxane complex to target bone metastases (Miller et al., 2009).

While studying the trends and challenges in the development of nanomedicines Wei et al. (2012) identified different requirements including sound techniques for precise characterization of shape, size and composition of nanoparticles. He also developed particle engineering to improve stability during storage and sustain low levels of nonspecific cytotoxicity.

Protein Aggregation

Detection and measurement of sub- visible particles measuring less than 100 nm in diameter in therapeutic protein products is a much debated topic. According to Carpenter et al. (2009) insufficient knowledge combined with the clinical significance of ignoring such particles may affect product quality. He concluded that existing USP particulate testing is not suitable for controlling the risk of bulk protein aggregates affecting protein immunogenicity.

Analytical techniques that evaluate the properties of particulates are important to devise robust methods to assess and reduce the risk to product quality induced by large protein aggregates.

It is obvious that lack of instrumentation with sufficient sensitivity widens the possible issues related to submicron aggregates in proteinaceous products. NTA, which is capable of visualizing, sizing and determining sub-micron particle concentration, has attracted a great deal of interest and has been evaluated and applied to the real-time analysis of proteinaceous aggregates and their occurrence in various applications.

NTA in Nanomaterials Regulation

A number of consumer products like cosmetics, food and food packaging include nanoparticles. However, detection and quantification of these particles in food is a complex process. The new definition set by the European Commission in October 2011 will have a major effect in different areas of legislation.

Calzolai et al. (2012) assessed techniques to determine nanoparticle size distribution in food and consumer products and Linsinger et al. (2012) studied the measurements requirements to implement the European Commission definition of the word 'nanomaterial'. When dealing with this latest definition of nanomaterials they mainly focused on how appropriate widely utilized methods were for nanoparticle size measurement and provided practical examples to overcome the issues when determining nanoparticles in food and consumer products. When compared to other techniques, they agreed that NTA was indeed effective when a combination of similarly sized particles need to be analyzed.

Monitoring and Treatment of Waste and Contamination

Sachse et al. (2012) used NTA to investigate the impact of nanoclay on dust production during mechanical drilling of polymer nanocomposites to track particle size quantity and distribution. Although adequate information was not available on the rates of nano and ultra-fine particle emission from these it was observed that the effect of nanoclay on drilling of PA6 composites will rapidly produce a higher number of particles measuring 175 to 350 nm in diameter.

Nanoparticles are produced from nanofiller-reinforced polymer nanocomposites during structural testing. Njuguna et al. (2011) studied this effect in detail. Rezic (2011) assessed analytical methods used for characterizing engineered nanoparticles on textiles. In this regard, the growing number of nanomaterial-based consumer products indeed raises concerns regarding their potential effect on the environment.

While reviewing the effluent from a silver nanowashing machine, Farkas et al. (2011) employed TEM and inductively coupled plasma mass spectrometry (ICP-MS) to substantiate the presence of 10 nm silver nanoparticles, and also used NTA to establish that 60 to 100 nm particles also exist. This effluent negatively affected the natural bacterial habitat. This means, if AgNPs-producing washing machines become a standard feature in all households, wastewater will have excess amounts of AgNPs that may severely impact the environment.


Schulz and Boulestreau performed filtration studies using the NTA technique to test the efficiency and performance of filter devices. While elucidating the analysis of nanoparticles to inhibit membrane fouling by a secondary effluent, Boulestreau et al. (2011a and 2011b) examined NTA with respect to reproducibility and reliability of the filter device besides the effect of prefiltration on the measurements performed.

They demonstrated that NTA can easily determine the particle size distribution as well as the absolute particle concentration of particles measuring between 100 and 1000 nm in secondary effluent. These outcomes demonstrated a link between filtration behavior and the amount of nanoparticles measuring less than 200 nm.

Boulestreau recently described the on-line usage of NTA to optimize the coagulation conditions and ozonation in a filter device. Rapid detection of absolute concentration and size of the nanoparticles enables users to detect the effect of coagulation and ozonation and it was concluded that NTA is an effective device for studying nanoparticles (Boulestreau et al., 2012).


Theoretically, a bulk nanobubble must be less stable when compared to a nanobubble of equal volume at an interface. The nanobubble in bulk has a larger liquid or gas interface to facilitate gas diffusion from the bubble. Such nanobubbles generated by mechanical means resulted in high supersaturation, which were imaged from freeze-fracture replicas through scanning electron microscopy and were generated in large volumes which significantly reduced the bulk density of the solution. Kikuchi et al. (2011) and Takaya et al. (2011) illustrated the formation of nanobubbles through water electrolysis using NTA. Ioka at el (2011) studied the weight and stability of nanobubbles after establishing their size distribution with NTA.

Following TEM analysis of nanobubbles and their capture of contaminants in wastewater, Uchida et al. (2011) produced a nanobubble solution by adding pure O2 gas to highly purified water through an MNB generator and utilized NTA to give the resulting number concentration, which is believed to be on the order of 107 particles/mL of solution within the same sample preparation conditions.


Monodisperse round-shaped silica particles are available as building blocks for the chromatography stationary phase, photonic crystals, and drug support for sustained release. In such applications immobilization of a molecular-recognizable unit to the spherical particle surface is very important.

Okada et al. (2012) applied NTA in their analysis of swellable microspheres which included a layered silicate. The research demonstrated that a swellable layered silicate covers sub-micron-sized silica spheres. This layered silicate helped in accommodating cationic species.

Nanoparticulate Silver

NTA was used by Khaydarov et al. (2010) to test the aggregation properties of silver nanoparticles to devise a new technique of nonstop generation of silver nanoparticles using cellulose fibers. It was shown that the artificial colloidal dispersions had a distinct antibacterial effect, as seen by minimum inhibitory concentration values acquired for Bacillus subtilis, Staphylococcus aureus, and Escherichia coli cultures.

NTA was used to test the dispersions of the self-assembling, antimicrobial click monolayers developed by Hodges (2011) using silver nanoparticles for implantable medical devices.

Iron Oxide

Cheng et al. (2012) used NTA to measure porous, carbon-coated and water dispersive Fe3O4 nanocapsules measuring 120 nm and reported superior performance for heavy metal removal applications. In another analysis of the influence of four polymers on the surface chemistry, size, sedimentation behavior, and colloidal stability of nanoparticles of non zero valent irons.

Cirtiu et al. (2011) determined the values for iron nanoparticles post and pre-treatment. NTA and TEM images showed that the iron nanoparticles produced in the presence of polymers had larger diameters, with TEM mean diameters spanning between 84.5 and 189 nm in contrast to a mean diameter of 59.1 nm for bare non zero valent iron NPs, when produced with the same initial Fe2+ concentration.

Metallic Gold

Vogel et al. (2011) demonstrated a unique method for large-scale production of homogeneous metal nanoparticles in water using laser light-induced processes where NTA revealed that pulsed laser ablation from a gold plate immersed in water leads to significant amount of nanoparticles with a wide size distribution of sigma=31% and with radii in the range of R=75 nm. Using selective laser tailoring, the wide size distribution was narrowed in one irradiation step to sigma=20% without any major modification of the mean nanoparticle radius.

Carbon and Carbon Nanotubes

Kim et al. (2012) created nanosized water-based carbon colloid by an electro-chemical approach to evaluate the removal efficiency of formaldehyde using nanosized carbon colloid. This carbon colloid was utilized as a gaseous formaldehyde pollutant and NTA was applied to track carbon particle size in production. Furthermore, carbon nanotubes have highly asymmetric shapes but NTA was able to establish the sphere equivalent diameter to indicate monodispersity and behavior of the sample in different matrices.


NTA is built on the known standards of sizing by determining the rate of Brownian motion of particles to provide nanoparticle diffusion constant. A spherical hydrodynamic diameter can then be predicted from this diffusion constant. However, since the optical configuration used in the NTA technique enables nanoparticles to be monitored and examined individually, the ensuing data is a high resolution particle size distribution analysis.

This facilitates differentiation of a wide range of materials through their varied refractive indices and from which the concentration of particles can be retained. Also the ability to determine the fluorescent properties of a nanoparticle provides a rich profile of nanoparticle characteristics. The ability of visualizing the suspension directly is another unique feature of NTA that is advantageous to users.

This information has been sourced, reviewed and adapted from materials provided by Malvern Panalytical.

For more information please visit Malvern Panalytical.


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