Nanoparticle Tracking Analysis (NTA) for Exosome Characterization
Exosomes are increasingly attracting the attention of life science and pharmaceutical industry research, and people are increasingly interested in exosomes, so it is necessary to develop new protocols to improve exosome characterization as a precursor for downstream research.
Nanoparticle tracking analysis (NTA) can determine the size distribution and relative concentration of microvesicles, including exosomes, in cultured cells and biological fluid supernatants. It can be used to quantify exosomes in biological samples and is widely recognized as a useful diagnostic tool for detecting disease.
What is NTA?
NTA is a powerful characterization technique that combines the properties of laser light scattering microscopy and Brownian motion. Allows direct, individual visualization and real-time counting of exosomes and microvesicles in the 50-1000nm range in liquid suspensions. Particle motion is tracked by light scattering to assess the mean square displacement of moving particles under Brownian motion in the sample chamber. The tracking of the particles allows for the calculation of the diffusion constant, which is used in the Stokes-Einstein equations to calculate the hydrodynamic diameter. The equation also takes into account the temperature and viscosity of the suspension. Particle concentration calculations are based on estimating the number of particles tracked in the illuminated volume.
Figure 1. Overview of the NTA method using the particle tracking instrument. (Comfort N, et al., 2021)
How does NTA Work?
1) Particles in a liquid suspension are loaded into a sample chamber which is illuminated by a specially shaped laser beam. The particles in the path of the beam scatter the laser light, which is easily collected by the microscope objective and observed with a digital camera. Then, the camera captures a video of the particles moving under Brownian motion.
2) NTA provides high-resolution nanoparticle size, concentration, and aggregation measurements based on counting. Fluorescence modes provide specific results for appropriately labeled particles. Subtle changes in particle population characteristics are provided by real-time monitoring, all of which are confirmed by visual validation of the analysis.
3) The NTA software can analyze many particles individually or simultaneously and calculate their hydrodynamic diameters by using the Stokes-Einstein equations.
Figure 2. Scheme of the NTA working principle. (Urso M, et al., 2022)
What is Fluorescent NTA and How is it Different from Traditional NTA?
Fluorescent NTA optimizes exosome NTA research tools by collecting data only on exosomes present in heterogeneous samples. More accurate EV/exosome data can be obtained, ignoring protein aggregates, membrane fractions, and other background particles to provide specific particle sizes and concentrations. Fluorescent reagents specifically and efficiently bind membranes of intact exosomes. During fluorescent NTA data collection, only light scattering from fluorescently labeled exosomes/microvesicles is obtained, resulting in a specific signal. As a result, using fluorescent NTA analysis provides data that more accurately reports the specific exosomes in a sample rather than all particles in the sample.
There are two options for analyzing exosomes using fluorescent NTA: Use a universal cell membrane dye to label exosomes and detect them by fluorescent NTA; The antibody-based approach using a Qdot-conjugated antibody against the target exosome marker to label the exosome, followed by quantification of the marker-positive exosome using fluorescent NTA.
| Cat No. | Product Name | Source |
| LEV-F665-LnCAP | HQExo™ Microvesicles-665-LnCAP | Fluorescent labeled microvesicles derived from human prostate adenocarcinoma (LnCAP cell line) |
| Exo-F488-A549 | HQExo™ Exosome-488-A549 | Fluorescent labeled exosome derived from human non-small cell lung cancer cell line (A549 cell line) |
| Exo-F488-B16F10 | HQExo™ Exosome-488-B16F10 | Fluorescent labeled exosome derived from murine melanoma cell line (B16F10 cell line) |
| Exo-F488-BLCL | HQExo™ Exosome-488-BLCL | Fluorescent labeled exosome derived from EBV transformed lymphoblastoid B cells (BLCL21 cell line) |
| Exo-F488-BPH | HQExo™ Exosome-488-BPH | Fluorescent labeled exosome derived from human benign prostatic hyperplasia-1 (BPH-1 cell line) |
| Explore All Fluorescent Exosomes/Microvesicles | ||
Applications
- Exosome, microvesicle, and extracellular vesicle characterization
- Nanotoxicology
- Viral vaccine development
- Protein aggregation research
- Drug delivery system development
NTA Future Developments
- Improvement of detection accuracy and resolution
NTA has been able to realize real-time detection and analysis of nanoparticles, but how to improve the detection accuracy and resolution has been the research hotspot of NTA technology. In the future, NTA can improve detection accuracy and resolution by improving the focusing method of the laser beam and the calculation method of the particle trajectory.
- Realization of multi-parameter joint analysis
NTA technology can realize the measurement of nanoparticle size distribution and analyze the particle trajectory and velocity. In the future, NTA can realize multi-parameter joint analysis to improve the precise positioning and in-depth understanding of the nature of nanoparticles.
Why Choose NTA for Exosome Characterization?
NTA is a powerful tool for characterizing exosomes for several reasons.
- Size distribution - NTA provides high-resolution measurements of exosome size distribution. This is essential because size and size distribution are essential parameters that define the biological function of exosomes.
- Concentration measurement - NTA can accurately measure the concentration of exosomes. This is essential for understanding their biological activity, as the number of exosomes can affect the interpretation of scientific research results.
- Real-time analysis - NTA uses the principles of Brownian motion and light scattering to allow real-time analysis of nanoparticles like exosomes. This allows dynamic measurement of the particle size distribution.
- Visual verification - NTA provides a video recording of the particles being analyzed, allowing for visual verification of the results, thus providing greater confidence in the data obtained.
- Quantifiable data - NTA provides direct, reliable quantifiable data that is essential for robust statistical analysis and reproducibility.
Based on our proven technology and experienced scientists, Creative Biostructure is committed to developing an NTA platform for exosome characterization services. Clients are required to provide prepared exosomes or samples containing exosomes (supported by our exosome isolation service). We will select a personalized exosome characterization service for you based on the purpose of your project. Please feel free to contact us for more information.
References
- Comfort N, et al. Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles. J Vis Exp. 2021. (169): 10.3791/62447.
- Urso M, et al. Trapping and detecting nanoplastics by MXene-derived oxide microrobots. Nat Commun. 2022. 13(1): 3573.
- Parsons MEM, et al. A Protocol for Improved Precision and Increased Confidence in Nanoparticle Tracking Analysis Concentration Measurements between 50 and 120 nm in Biological Fluids. Front Cardiovasc Med. 2017. 4: 68.
