The latest advances in exosome charactertation measurement technology.

abstract: exosomes were first found in the in-body
culation
sheep red blood cells on the liquid, is the cell active secretion size is more equal, diameter of 40 to 100 nanometers, density of 1.10 to 1.18 g / ml of the vesicle-like small body.
extracellular progenitor carries a variety of
proteins
, mRNA, miRNA, involved in cell communication, cell migration, angiogenesy and tumor cell growth processes and has the potential to become a natural carrier of drugs for clinical treatment. However, the limitations of measurement techniques limit the progress of exosome research in these areas. This paper summarizes the purification methods of exosomes, compares the existing techniques of exosome measurement, and focuses on a new measurement technique, namely nanoparticle tracking and analysis, in the study of exosome size and characterization.. 1. Exosome extraction and methodological evaluation So far, there is no extraction method that can guarantee the content, purity and biological activity of exosomes at the same time.1.1 centrifugation method
which is currently the most common method of exosome extraction. Simply put, the collection of cell culture fluids is followed by the removal of cell fragments and large molecular proteins at 300 g, 2,000 g, 10,000 g centrifuges, and the last 100,000 g centrifuges are obtained by exosomes. This method obtains a large number of exosomes, but the purity is insufficient, electro-mirror identification found that exosomes gathered into blocks, because micro-bubbles and exosomes do not have a very uniform identification standards, there are some studies that this method is obtained is micro-bubbles are not exosomes.1.2
filtration
centrifugation
filtration centrifugation is the use of different intercepting relative molecular mass (MWCO) ultrafiltration membrane centrifuges to separate exosomes. Intercepting relative molecular mass refers to the relative molecular mass of the largest molecule in a molecule that can pass freely through a perforated material. Exosome is a cystic small body, relative to the molecular mass is greater than the general protein, so the choice of different sizes of MWCO membrane can make the exosome separate from other large molecular substances. This simple operation, time-saving, does not affect the biological activity of exosomes, but there is also a lack of purity.1.3 Density gradient centrifugation method
density gradient centrifugation is the sample and gradient material together over-centrifugal, samples of different parts of the sink to their respective iso-density zones, divided into continuous and seconseconcing gradient centrifugation method. The medium used for density gradient centrifugation requires non-toxicity to cells, low viscosity at high concentrations and easy pH to neutral. In the experiment, the sucrose density gradient centrifugal method was commonly used, and on the basis of centrifugal method, two concentrations of sucrose solution (e.g. 2.5 M and 0.25 M) were pre-arranged into a continuous gradient system in an overspeed centrifuge tube, the sample was laid on the sucrose solution, 16 h, and the exosome was reduced to the equal density zone (1.10 to 1.18/ml). The exosome separated by this method has high purity, but the preparatory work is complicated, time-consuming and the quantity is small.1.4 Immunomagnetic bead method
genesome-related
antigen

antibody
(e.g. CD9, CD63, Alix) and exosomes incubated together, distilled water rinsed, resurfaced in PBS buffer. This method can ensure the integrity of exosome morphology, high specificity, simple operation, no need for expensive equipment, but non-neutral pH and non-physiological salt concentration will affect exosome biological activity, inconvenient to carry out the next experiment.
1.5
Chromatography
method
chromatography is a method of analyzing the separation of solutes based on the relative relationship between the aperture size of the gel pores and the molecular size of the sample. The large molecules in the sample can not enter the gel hole, can only pass through the column along the gap between the porous gel particles, and are first washed out by the flow phase; The separated exosomes are the same size under the electroscope, but require special equipment and are not widely used.. 2. Comparison of various methods of exosome measurement 2.1 Electron
Microscope
Scan Electron Microscope (SEM) works by means of electron beams with an energy of 1-30KV, illuminated to the surface of the sample under analysis by grating scanning, and obtains high resolution of the microscopic structure and shape of the sample surface by means of grating scanning, using secondary electrons and back scattered electron form images generated by the interaction of the subject matter. Due to the development of ultra-high vacuum technology, the application of field-launched electronic guns has been popularized, and the resolution of modern advanced scanning electron mirrors has reached about 1 nanometer, which is sufficient for the measurement of exosome size. In view of the working characteristics of SEM, in the study of exosomes, it is able to directly observe the morphology of chinese and foreign urogenes in samples. And SEM has a high resolution, can identify different sizes of exosomes. However, SEM on the sample pre-treatment and preparation requirements above, the sample preparation stage is more complex, not suitable for a large number of rapid measurement of external urology. And because exosomes go through the pre-treatment and preparation process, it is not possible to accurately measure exosome concentration.
2.2 Dynamic light scattering technology
Dynamic light scattering is the collection solution to do Brown motion particle scattering light intensity fluctuations, through the correlation instrument? the velocity of the particles is calculated by converting the fluctuations of the light force into the relevant curves, thus obtaining the speed of the fluctuations of the light force and calculating the diffusion velocity information of the particles and the particle size. Brown of small particle samples has fast motion, rapid fluctuations in light strength, faster attenuation of related curves, and the opposite of large particles.
in exosome studies, dynamic light scattering measurement sensitivity is high, with a lower measurement limit of 10 nanometers. Compared with SEM technology, sample preparation is simple, only simple filtration is required, and measurement speed is fast. However, because dynamic light scattering technology is to measure the fluctuation data of light strength, so the light wave signal of large particles will mask the light wave signal of smaller particles, so dynamic light scattering is not suitable for the measurement of complex exosome samples of different sizes, only suitable for measuring the size of exosomes of the same size through chromatography, and can not measure the concentration of the sample exosomes.2.3 nanoparticle tracking analysis
nanoparticle tracking analysis (NTA) is a relatively novel method of studying nanoparticles, can directly and in real-time observation of nanoparticles. NTA collects scattered light signals from nanoparticles through optical microscopes, captures images of nanoparticles doing Brown motion in the solution, and tracks and analyzes the Brown motion of each particle to calculate the fluid mechanics radius and concentration of nanoparticles.
NTA system works by irradiated the sample (the solution of suspended particles) through a glass prism with an energy-concentrated laser
the laser beam is introduced into the sample solution from a smaller angle, illuminating the particles in the solution. Optical microscopes equipped with cameras are placed in specific positions to collect light scattering signals emitted by illuminated nanoparticles in the field of view. The sample pool has a depth of about 500 microns, and the sampling point laser illuminates at a width of 20 microns, which matches the focus depth of the optical microscope. The camera will take 60 seconds of image shooting, with 30 samples per second. The motion of the particles is analyzed by the NTA software. The NTA software identifies and tracks nanoparticles that do Brown's motion in each recorded image.
the particle's fluid mechanics radius is calculated using the two-dimensional Stokes-Einstein equation, depending on the particle's . In equation 2 is the verb displacement, KB is the Boltzmann constant; T is the temperature of the solution in Kelvin;ts is the sampling time, for example, 1/30 fpsec is 33 msec; η is the viscosity of the solution; dh is the fluid mechanics diameter. The range of particle sizes detected by the NTA is related to the releass index of the particles themselves. The lower limit of the measurement depends on the signal-to-noise ratio between the particle under study and the background, i.e. the scattering light intensity of the particle and the light intensity difference in the background. The scattering light intensity of the particles is influenced by rayleigh scattering equations where d is the diameter of the particles, the wavelength of the incoming light, and n is the reculsion coefficient ratio of the particles and solutions. Generally speaking, biological samples, such as exosomes, have a low recourse coefficient, so the lower measurement limit is 30-40 nanometers.NTA technology has a very high resolution for complex samples because of the direct tracking of every nanoparticle in the sample. To demonstrate NTA's ability to distinguish complex samples, we mixed two different sizes of polystyrene particles, 100nm and 300nm, in 5:1 quantities and measured them using NTA. Although there is some overlap in its distribution graphs, the peaks of two nanoparticles of different sizes are clearly distinguished. This ability to distinguish complex samples is very important for study subjects such as exosomes.
NTA can also directly measure sample concentrations. A series of 100 nanometer single dispersion samples with concentrations of 1×108 - 8×108 can be measured and a good linear correlation between the NTA measurement concentration results and the actual concentration can be seen. For multi-dispersion systems, the accuracy of the measurement results depends on the setting of the instrument parameters (camera shutter speed and aperture), the appropriate parameter settings can ensure that particles of different sizes can be tracked and calculated by NTA software.
NTA also has the ability to analyze fluorescent samples. NTA has four different wavelengths of lasers to choose from, including 405nm, 488nm, 532nm and 635nm lasers, with the appropriate filters for measuring fluorescent samples. The 100 nm fluorescently labeled particles and 200 nm non-fluorescent particles are mixed with the same solvent and measured using NTA. The blue line shows the light scattering mode of the NTA, which clearly distinguishes between the peaks of 100 nanometers and 200 nanometers, despite overlapping distribution maps of 100 nanometers and 200 nm nanoparticles. Then the fluorescent filter was used for analysis, only 100 nanometers of fluorescently labeled nanoparticles (red lines) were observed
due to the presence of cross-membrane molecules such as markers CD9 and CD63 on the surface of the exosome, in complex background environments (such as
serum
), the exosome can be labeled with fluorescent antibodies, and then the fluorescence measurement function of NTA can be used to achieve the measurement of exosomes in complex backgrounds. Compared with the fluorescence function of flow cytometer, NTA resolution is high, and the lower limit of measuring fluorescent particles can reach 30-40 nanometers, while the lower limit of measuring flow cytometer is 400 nanometers. Even for the latest generation of digital streaming cytometers, the measurement floor has reached 100 nanometers, but because it is still based on monitoring light signals, measurements and accuracy and resolution are still unreliable. Therefore, NTA has a unique advantage in the measurement of exosome fluorescence function. . 3. Summary The as biomarkers of external urogenes is still in its infancy, but it has shown good prospects in clinical applications. In clinical diagnosis, the size and characterization of exosomes are required to measure exosome concentrations quickly and easily in complex biological contexts (e.g. plasma, urine). But the current methods are not perfect to solve this problem. As a relatively new measurement technology, NTA has a high resolution, providing real-time observations as well as accurate concentration and fluorescence measurements, and providing new ideas for the study of exosome size and concentration.
Zhang Shuai, a bioscience expert at Malvern Instruments Co., Ltd.,
.