extracellular vesicles (EV) has achieved rapid development in the past decade, with numerous discoveries and reports on its origin, disease relevance, diagnosis, and treatment. In these research fields, comprehensive characterization of EVs is crucial, covering both single particle and overall analysis of EVs, such as particle size, potential, concentration, particle size distribution, as well as detection of characteristic proteins associated with EVs, such as CD9, CD63, CD81, Alix, and TSG1012-5. If a more in-depth analysis of the phenotype of EVs is desired, methods such as Western blotting, ELISA, or single vesicle/whole cell immunolabeling should be used for EV phenotype analysis.
At present, there is no method that can detect proteins on a single vesicle and integrate them with the wholeparticle size distributionCombined with concentration measurement. Nanoparticle tracking analysis (NTA) is a technique applied to characterize the particle size distribution of EVs. NTA is based on the Stokes Einstein equation and tracks the behavior of individual particlesBrownian motionTo determine the size of individual EVs and EV groups. NTA utilizes laser irradiation of particles to theoretically achieve specific excitation of fluorescent dyes and detection of fluorescently labeled EVs (NTA-FL).However, currently only a few studies have rigorously evaluated NTA-FL, and these studies rely on immune labeling of atypical EV protein targets or non-specific markers.
Tufts University, USA（Tufts University）Researchers have reported an optimized algorithm for fluorescent immunostaining of single EVs for NTA-FL, making single vesicle immunostaining and detection based on NTA more accurate for particle size distribution and concentration analysis. This algorithm can effectively improve the credibility of NTA detection results, providing a powerful tool for further research on the functions and mechanisms of EVs.
In the preliminary research, researchers found that traditional利用NTAconductEVFluorescence analysis of subtypes/The scattered light comparison method may result in data deviationIn general, the ratio of the area enclosed by the fluorescence (FM) curve to the area enclosed by the scattered light (LSM) curve can indicate the purity of EVs. However, during the analysis process, these researchers found that in tests 1, 2, and 4 (Table 1, Figure 1), the ratio of FM (blue line) to LSM (red line) was greater than 100%, indicating that the purity of EV was greater than 100%, which is clearly unreasonable. These test results indicate that:During the fluorescence detection process, a large number of false signals may occur.
2. The technical limitations of the NTA system prevent LSM from detecting very small EVs or other particles with low refractive index.Under scattered light mode, EV detection is incomplete, with some EV data not detected, resulting in a smaller LSM area and a ratio greater than 100%.
3. Researchers also found that there are significant signal peaks in the FM mode below 50 nm, while compared to the LSM mode, the signal attenuation below 50 nm is basically zero, which may be one of the reasons for the ratio being greater than 100%.Specifically, this may be due to insufficient removal of free fluorescent molecules, as well as labeling of free proteins in the system with fluorescent molecules, resulting in enhanced fluorescence signals in small-sized partitions.
Table 1 Fluorescence area (FM) to scattered light area (LSM) ratio of different samplesFigure 1 NTA test results of different samples in FM and LSM modes
To address this situation, researchers have proposed two evaluation methods:
The first methodIt is a traditional calculation method that calculates the number of particles larger than 50 nm in the sample and compares the number of particles in FM and LSM to obtain the ratio between FM and LSM. (Figure 2b, c)
the second methodIt is an optimized calculation method that calculates the overlapping area between the particle size distribution curves obtained by the two methods, and compares this area with the total number of particles larger than 50 nanometers in LSM to obtain the proportion of overlapping areas. (Figure 2d)
During the research process, the researchers emphasized their choices regarding particle size selection. In order to analyze EVs more accurately, researchers narrowed down the scope of analysis to50-200 nanometersThe reason for choosing this range is becauseMost EV sizes are within this rangeIn addition,LSM has limited detection capability for particles smaller than 50 nanometersParticles larger than 200 nanometers may not be EVs. Therefore, the analysis scope is narrowed down to50-200Nanoparticles can effectively eliminate other types of particles and improve the accuracy of analysis results.
Figure 2 Schematic diagram of immune labeling algorithm based on NTA
Based on this optimization algorithm, researchers analyzed the data of the six sets of samples mentioned above, and the results showed that the EV purity had all fallen back to less than 100%.
Table 2: The ratio of fluorescence area (FM) to scattered light area (LSM) of different samples after algorithm optimization (SA)
The above algorithm utilizes quantum dot labeling and NTA-FL technology to effectively identify and analyze different subtypes of EVs at the level of individual vesicles by comparing the results of fluorescence mode (FM) and scattered light mode (LSM), and provide more accurate particle size distribution information. However, although NTA-FL technology and optimization algorithms can effectively improve the accuracy of EV subtype analysis, it must also be recognized that NTA technology has some limitations in underlying principles.NTA is detecting very small EVs orWide particle size distributionofSamples (especially EV samples)There is a certain degree of existencedifficultyTherefore, although NTA-FL technology and optimization algorithms can improve the accuracy of particle size distribution and concentration analysisThe technological bottleneck of NTA has not been overcomeCan the subsequent algorithm optimization and fluorescence labeling methods be appliedObtain correct results on the basis of errorsCaution should still be exercised to avoid excessive reliance on the data obtained from this technology.The NanoCoulter nanometer particle size analyzer adoptscoulter principleBreaking the limitations of optical principles, it can accurately analyze each through-hole particle, achieving true single particle detection and detecting particles with wide particle size distributionsDispersed systemThe sample has better measurement performance. And it is not affected by the optical properties such as refractive index and absorbance of the sample, and the data does not fit, which most accurately reflects the state of the sample.
