Nanoparticle tracking analysis (NTA) allows for live and direct tracking, sizing, and counting of particles suspended in a liquid with sizes ranging from 10 nm to 1 mm.1 NTA helps determine both the size and concentration of nanoparticles per milliliter.2
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The technique exploits the principles of Brownian motion to quickly reveal particle size distribution with a high resolution.1 Due to direct counting of the events in the field of view, NTA can reveal the exact particle concentration.4 With appropriate instrument settings, the technique finds applications in the study of proteins, viruses, vaccines, orthopedic implants, metal and silica nanoparticles, etc.1
Principles and Instrumentation for NTA
NTA mainly uses the principle of scattering of light by a particle to locate it in real-time. The particle movements are tracked, and the size is measured using a high-speed video recording.2 A laser beam is used to illuminate particles suspended in a liquid. Since the size of these particles is generally much smaller than the laser light wavelength, they act as light-scattering agents and their Brownian motion can be visualized through a conventional microscope fitted with an objective lens of appropriate magnification.1
The hydrodynamic radius of a sphere is calculated using the captured data and solvent information, and the sphere diffuses at the same rate as the particle being tracked.2
Depending upon the manufacturer, different configurations of NTA instruments are available. The optical microscope is generally fitted with a complementary metal-oxide semiconductor (CMOS) digital camera, which can record a video of the particles illuminated by the laser beam at a speed as fast as 30 frames per second.
The visual tracking of each particle helps in building a data set containing information on every individual particle encountered by the light path. Direct analysis of a few thousand particles helps generate an accurate histogram that represents the particle distribution in the sample.2
NTA instruments are generally set up in two ways: flow-cell and cuvette-based; however, the data recording, analysis, and accuracy are the same. In a flow-cell NTA system, the sample is passed through the laser path using a fluidic channel whose flow is thermally controlled, and the scattered light is collected by an objective placed directly above it.
Such systems have longer data recording periods and multiple recordings. On the other hand, in cuvette-based systems, the objective is focused on a particular volume within the sample, which is agitated and replaced with a fresh sample after some time. The total number of recordings is higher in such systems.2
Data Analysis and Interpretation
The Brownian motion of particles is recorded by the NTA software, which identifies and tracks the center of each particle’s movement in a 2D frame. After tracking every particle for a fixed time, the average distance covered and diffusion coefficient are calculated.1 Next, by substituting these values in the Stokes-Einstein equation along with the solvent viscosity and sample temperature, an equivalent (hydrodynamic) particle diameter is obtained. Finally, particle size distribution per unit concentration of the solution is generated in the form of a histogram.
NTA can also help compare the refractive index of nanoparticles and allow sub-sample classification. Light scattering varies with particles of different materials because of their varying refractive indexes. The presence of particles with different refractive indexes is evident in an NTA data plot between particle concentration, particle size, and light scattering intensity.1
Some existing NTA instruments allow the analysis of inherently fluorescent or fluorescently labeled nanoparticles. Such systems use red (642 nm), green (532 nm), blue (488 nm), or violet (405 nm) laser for exciting fluorescence within particles. Bandpass filters are used in the optical path of the instrument so that only fluorescent particles can be visualized by the system. This NTA mode is useful for studying suspensions with a high background material like blood plasma, where conventional light scattering mode may not be effective.1
Advantages of NTA
Nanoparticles have various applications in academics and industries due to their properties and potential.1 A size change of a few nanometers can greatly influence their properties. For instance, in drug delivery applications, the size of nanoparticles can significantly impact the availability of the drug at the desired location.1
Some commonly used methods for nanoparticle analysis include analytical disk centrifugation, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Among these, DLS is widely used for analyzing liquid suspensions of nanoparticles. However, its accuracy is limited to highly monodispersed (only one type of nanoparticle) systems.1
Alternatively, the main strength of NTA is its applicability to samples with a wide polydispersity or an unknown particle population. In other words, the presence of particles of size 400 nm does not affect its potential to measure a subpopulation of 40 nm particle size.2 Moreover, NTA does not require any complex sample preparation. Hence, NTA is an advantageous multiparameter analytical technique for continuous nanoparticle tracking and sizing.1
Applications of NTA
NTA systems find applications in many industries but have become essential in biopharmaceuticals. A recent study in Nanomedicine: Nanotechnology, Biology and Medicine demonstrated the use of NTA and Raman spectroscopy for label-free characterization of the size and morphology of urinary extracellular vesicles. The data correlates with the metabolic parameters of patients and has the potential to help in the stratification of diabetic patients at different stages of chronic kidney disease.3
Another recent review article in Fundamental Research presented NTA as a characterization tool for lipid-based nanomedicines (LBNMs) with single-particle resolution. LBNMs include extracellular vesicles, lipid nanoparticles, and liposomes and are used in the treatment of cancer and the development of messenger ribonucleic acid (mRNA) vaccines.
NTA helps in the biochemical and physical characterization of LBNMs. Additionally, the homogeneity as well as structural stability of liposomes can be confirmed using NTA data. NTA can measure the size and surface potential of extracellular vesicles during several drug delivery processes.4
NTA also finds applications in environmental protection, as demonstrated by a recent study in Environmental Science & Technology. The researchers used NTA to segregate and track magnetite nanoparticles from anthropogenic sources in an urban atmosphere. The particle size distribution derived from NTA clearly distinguished between the magnetite nanoparticles originating from combustion and non-combustion sources. Such distinction is important to control the release of magnetite particles in the air, which are highly toxic and can cause neurodegenerative diseases.5
In conclusion, nanoparticle tracking analysis has proved to be a powerful tool for quantitative nanoparticle characterization in various colloidal solutions.
Using Nanoparticle Tracking Analysis (NTA) to Assess Nanoparticle Toxicity in Waste Water
References and Further Reading
- Griffiths, D., et al. (2020) Nanoparticle Tracking Analysis for Multiparameter Characterization and Counting of Nanoparticle Suspensions. Methods in Molecular Biology, pp. 289–303. https://doi.org/10.1007/978-1-0716-0319-2_22
- Campbell, J., et al. (2020) Nanoparticle characterization techniques. In Nanoparticles for Biomedical Applications, pp. 129-144. Elsevier. doi.org/10.1016/B978-0-12-816662-8.00009-6
- Roman, M., et al. (2022) Raman spectroscopy of urinary extracellular vesicles to stratify patients with chronic kidney disease in type 2 diabetes. Nanomedicine: Nanotechnology, Biology and Medicine, 39, pp. 102468–102468. doi.org/10.1016/j.nano.2021.102468
- Chen, C., et al. (2023) Characterization of lipid-based nanomedicines at the single-particle level. Fundamental Research, 3(4), pp. 488-504. doi.org/10.1016/j.fmre.2022.09.011
- Zhang, Q., et al. (2020) Separation and tracing of anthropogenic magnetite nanoparticles in the urban atmosphere. Environmental Science & Technology, 54(15), pp. 9274-9284. doi.org/10.1021/acs.est.0c01841
