Nanoparticle tracking and analysis technology (NTA) is one of the emerging nano-scale measurement technologies in recent years. The principle is shown in Figure 1. The nanoparticles undergo random Brownian motion in the suspension by the impact of surrounding solution molecules, and then through the Stokes-Einstein equation, the movement speed of these particles in unit time (ts) (2) There is a quantitative relationship between the particle size (dh) itself, the viscosity (?) of the solution, and the temperature (T). Therefore, by observing the trajectories of the particles in the solution, the particle size data associated therewith is obtained. At the same time, each of the observed particles is tracked and analyzed by the high-speed camera and software built into the instrument, and finally the particle size distribution and particle concentration analysis results different from those of the conventional particle size analyzer are provided.


Figure 1 (left) Nanoparticles are subjected to solution molecules to make random Brownian motion (right) Stokes-Einstein equation

In the era of optical microscopy, the size of particles or targets that can usually be observed in biomedicine generally ranges from a few microns to tens of microns, but before the introduction of nanometers, people realized that future research would tend to be more Microscopic perspectives, exosomes, viruses and phage, as well as biological products such as antibodies and vaccines, are much smaller than the optical microscope's observation range; at the same time, conventional biomedical detection methods (eg, virus titer testing, viral plaques) Tests, Elisa, etc.) can only be expressed relatively broadly on a macro level. Therefore, this paper hopes to further understand how the new nanotechnology plays an increasingly important role in the biomedical field by simply sharing the application of nanoparticle tracking and analysis technology in recent years.

Prospective application of NTA in the field of exosomes, viruses and vaccines

Exosomes were first discovered in the supernatant of sheep red blood cells cultured in vitro. They are vesicle-like bodies with a relatively uniform size and a diameter of 30-100 nm and a density of 1.10~1.18 g/ml. With the continuous development of molecular technology, the exploration of exosomes in the biological community has become increasingly deep. In 2013, three foreign scientists were awarded the Nobel Prize in Physiology or Medicine for their breakthrough in the study of cell membrane transport mechanisms. As a result, exosome research has reached a new height.

In recent years, more and more evidence has shown the importance of exosomes for clinical treatment. Exosomes are intercellular messengers that provide strong evidence for further clinical diagnosis through changes in their numbers and biochemical composition. It carries a variety of proteins and miRNAs, is involved in cell signal transduction, cell migration, angiogenesis and tumor cell growth, and may become a natural carrier of drugs for tumor detection or clinical treatment.

At present, the detection and observation of exosomes are mainly completed by electron microscopy. The electron microscopy describes the particle size of the exosomes and can clearly show the specific structure of the exosomes by direct measurement comparison. Of course, the shortcomings of this method are also obvious. Since the range that can be observed at one time is limited, the obtained particle size distribution data is often not representative. At the same time, electron microscopy samples often need to be pretreated by different methods such as drying, fixing and freezing, which will cause certain damage to the structure of the biological sample, which will ultimately affect the observation effect.

In contrast, the Malvern Nanoparticle Tracking Analyzer (NanoSight) has a solution state (in situ test) that provides excellent structural and functional protection for exosomes and allows for Exosome particles are measured closer to their original state, ensuring the authenticity and validity of the measured data. In addition, its high-precision particle size distribution test can even distinguish particles with a relative particle size difference of 1:1.5 times, especially in biological samples, the distribution of particles is extremely uneven and wide, and some particle sizes are small. A small number of particles are easily overlooked by conventional measurement methods in the analysis, and the quantitative distribution particle size test provided by Malvern NanoSight effectively solves this problem. In addition, the unique concentration measurement technology directly provides exogenous researchers with reliable exosome concentration data. It has been demonstrated that the changes in exosome concentration are related to different cell growth states and species under different physiological conditions.

In traditional virology, the concentration of viral particles in a viral solution is typically tested by physical or biological methods. Among them, the physical method measures the absorption of light by viral DNA, and the result is very stable but cannot distinguish the type of virus particles. Biological methods such as GTU, PFU and TCID50 also have limitations. If the test results are unstable, it is difficult for different operators to obtain similar results.

By analyzing the Brownian motion of viral particles in viral suspensions, NTA technology uses the Stokes-Einstein equation to quickly and accurately detect the quantitative distribution of virus particle size. At the same time, a unique concentration test system provides real-time concentration information for the sample. More importantly, Malvern NanoSight's unique fluorescence system provides an extremely convenient platform for distinguishing the types of virus particles. Researchers can design fluorescent dyes with different absorption wavelengths according to their own needs, so as to distinguish different types of virus particles. . There are currently four fluorescence wavelengths available, 405nm, 488nm, 532nm and 635nm, respectively, with corresponding filters to measure fluorescent samples, which are then individually tested by NTA technology to protect against complex samples (such as serum). , urine, etc.) environmental impact.

At present, nanoparticle tracking and analysis technology is being used more and more in the basic and clinical research of biomedicine. At the same time, we are increasingly seeing it in the production and quality inspection of some biological and pharmaceutical preparations. Figure. Of course, NTA technology has not only been applied in scientific research, but has also expanded into many production and quality inspections of biological and pharmaceutical products.

It is well known that in the production, transportation and storage of vaccines and antibodies, due to environmental changes and conditions, it is often impossible to fully guarantee that the product is always in the best environment. Subtle changes in the environment often lead to different levels of small particle agglomeration. During the use of vaccines and antibodies, if larger particles are produced, they are very likely to cause a strong rejection reaction, which may have extremely serious consequences.

Protein-based vaccines and antibody particles, when pH, temperature, and solution composition change, protein particles are prone to changes in structure and surface potential, resulting in agglomeration of particles. Malvern NanoSight provides a very comprehensive test platform, users have full freedom to simulate different situations. Since the platform can be simultaneously injected and tested, after adjusting the test environment of the particle solution, the user can quickly obtain the change data of the sample under the new condition.

In general, the most important environmental factor affecting protein products may be temperature. At sustained high temperatures, proteins undergo denaturation and agglomeration. In order to understand the effect of temperature on immunoglobulin IgG, with the automatic temperature control of Malvern NanoSight (platform controllable temperature is 18-55 ° C

The researchers did the following tests: The IgG solution was stored at a constant temperature of 55 ° C, and data and video were collected at 5 mins, 15 min, 20 mins, 30 mins, 60 mins, 120 mins, 3 hrs, 4 hrs, and 5 hrs, respectively. Through comparison and data analysis, the researchers found that at 55 ° C ambient temperature, IgG began to show significant aggregation in about 30 minutes (see Figure 2 for details). This also provides credible actual data for manufacturers of vaccines and antibodies to infer the time and requirements of products under different conditions, prevent product deterioration and improper use, and ensure the efficiency and safety of the use process.


Figure 2 The video collected at 5 °s, 15 min, 20 mins, 30 mins, 60 mins, 120 mins, 3 hrs, 4 hrs, and 5 hrs clearly shows the agglomeration process of the antibody at 55 °C.

More new feature development, fully responsive to customer needs

Of course, in the field of application, NanoSight is not only limited to the field of biomedicine, but also widely used in other fields, such as toxicology research, material molecular research, nanobubbles, ink, food and so on. In this regard, Malvern NanoSight product expert Mr. Mei Jie said: "It is not our experts who persuade customers, but Malvern's products themselves."

NanoSight, equipped with advanced NTA inspection technology, has the power that conventional particle size analyzers do not have: it can perform rigorous quality control, and by analyzing and calculating all the observed particles, the detailed distribution and concentration of sample size are given at the first time. Specific information. It is especially critical that the NTA works by illuminating a sample (a solution of suspended particles) by passing a beam of concentrated laser light through a glass prism, with chrome plating on the glass surface to minimize background signal, allowing researchers to pass the microscope. Directly observe the Brownian motion of the nanoparticles in solution, and take images, track and analyze the Brownian motion of each particle, and quickly and accurately calculate the hydrodynamic radius and concentration of the nanoparticles in the sample. Compared with other traditional techniques, NTA can fully characterize particles with a wide range of particle size distribution in suspension, and has a very high resolution. It can still accurately analyze particles with similar particle size, especially suitable for exogenous secretion. Research on complex systems such as body, protein aggregation, drug delivery, nanoparticle toxicology, viruses and vaccines.

In addition, NanoSight can also determine the number of particles, accurately indicate the particle volume per unit volume and the number of particles in the sample solution, and perform volume-based particle size testing and distribution testing. In the current various injections, the requirements for the number of suspension particles and the particle size have become stricter.

In terms of operation, NanoSight greatly reduces the time and sample size required for testing. Often, large instruments (such as flow cytometers) require complex warm-up and calibration preparations before use. Due to the calculation of the Stokes-Einstein equation, NanoSight does not need to do this, it can be used directly by connecting it to the power supply. And because NTA technology uses a hydrodynamic diameter measurement technique that does not require prior knowledge of mass, refractive power, and particulate material, the instrument can skip the cumbersome standard calibration and go directly into the research analysis, saving the staff time before the study.

Under normal circumstances, the average time to test a sample using flow cytometry is 1-1.5 hours, while the current Malvern NanoSight, the average time of each sample is about 10 minutes, and the analysis results are comprehensive and reliable. For many customers, this not only saves a lot of time, accelerates the entire research process, but also saves them valuable labor costs.

Summary and outlook

As a leader in materials and biophysical characterization, Malvern continues to bring top-notch technology to China, allowing customers to buy more than just a single piece of hardware. In addition to technological innovation, Malvern has always focused on customer service. The technical team is the company's main force, covering academic experts, application experts and service engineers, and always participates in customer research, in-depth understanding of customer needs and enhance product value.

Today, with the rapid development of biomedicine, many research fields are still in their infancy in China, and Malvern Instruments continues to promote applications and scientific research with new products. With the disclosure of more and more scientific research institutions and research projects, the new generation of NTA technology represented by Malvern NanoSight will make greater contributions to the promotion of exosome research and even human health. I hope this article can be used to encourage more customers and researchers to explore the unknowns in the field of scientific research with Malvern.

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