Recently, the Shanghai Institute of Materia Medica of the Chinese Academy of SciencesInternational Journal of PharmaceuticsPublished an article titled 'Key Parameters of Nanomaterial Size and Concentration for Standardized Nanoparticle Tracking Analysis'. In this study, the authors systematically evaluated the effects of sample composition, sample concentration, instrument setting parameters, camera setting parameters, and liquid flow rate on the measurement accuracy of NTA (NanoSight NS300). During the research process, they simultaneously selected the gold standard for particle size characterization - transmission electron microscopy and resistance pulse based methodsinductionThe principle of RPSNanoCoulterOrthogonal experiments were conducted using a nano Coulter particle size analyzer to cross validate the accuracy of NTA in measuring particle size and concentration.

Compare the accuracy of measurement results under different particle concentrations

Figure 1: The influence of sample concentration on NTA measurement results.

The author used polystyrene (PS) and silicon dioxide (SiO) of different sizes₂）Standard particles were measured at different particle concentrations, and the average size and concentration deviation were compared at different concentrations. The results show thatWhen the particle concentration is10⁸-10⁹ When the concentration is between particles/mL, the measurement results are most accurate and the linear trend is the best.At this time, the corresponding number of particles in the field of view is 10-100 and 400-6500.

Compare the impact of different exposure levels on measurement results

image2. The influence of exposure on NTA measurement results

Unlike DLS, NTA calculates particle size by tracking real-time Brownian motion of particles through a camera. ThereforeOptimizing camera settings parameters can significantly improveThe accuracy of particle size and concentration measured by NTA.The author used a mixture of PS particles of different sizes and characterized them at different camera levels. Exposure is a parameter that indicates the sensitivity of a camera to particle motion. By adjusting the exposure, it can affect the photography results of NTA. The results showed that when the exposure was 6-9, the observation of 100 nm PS particles was poor, while 200 nm PS particles were clearly visible. When the exposure was 10-14, 100 nm PS particles could be clearly observed, while 200 nm PS particles would be overexposed, affecting the results. Subsequently, the author further evaluated the impact of exposure on the particle size peak and distribution of PS particles at 100 nm, 120 nm, 150 nm, 170 nm, and 200 nm. The results showed that changes in exposure did not significantly affect the peak particle size of PS particles, but as exposure increased, the average particle size of PS particles in each group increased. Therefore, the author believes that specificParticles of different sizes will jointly affect the measurement results of particle size with exposure.When the sample dispersion is large,It is difficult for NTA cameras to find the appropriate exposure to ensure that all particles can be captured. When writing relevant reports, it is necessary to submit NTA's exposure and other set parameters together to ensure data transparency.

Comparing the effects of different pump speeds on measurement results

Previous studies have shown that,The viscosity of the solution will affectAccuracy of NTA measurement resultsHowever, the impact of pump speed on measurement results is still unknown. Therefore, the author used PS particles of different sizes to measure at different pump speeds and compared the average size and concentration deviation at different speeds. The results show thatFor less thanParticles with a diameter of 200 nm and a pump speed of 50 AU（arbitrary unit)More accurate results can be obtained within, while for larger particles, lower pump speeds are required to ensure accuracy.

image4. Measurement of Multi dispersed Samples by NTA, DLS, and RPSinfluence

Subsequently, based on these results, the author evaluated the characterization ability of NTA for polydisperse samples according to the standard operating procedure. The author prepared a mixture of 100 nm and 150 nm PS particles with a concentration ratio of 1:1 and 10:1. Based on different exposure levels, NTA was able to successfully measure two particle size peak positions for both samples within the range of 7-10. Subsequently, the author further prepared three mixed and four mixed samples of 1:1:1 at 100 nm, 150 nm, and 200 nm, as well as 1:1:1:1 at 100 nm, 150 nm, 170 nm, and 200 nm. The results showed that NTA could not complete the measurement and could only obtain a broad peak. Subsequently, the author further analyzed the four samples using DLS and RPS,DLS cannot measure the precise peak positions of the four samples mentioned above, only one single peak can be obtained, while NanoCoulter based on RPS theoryBy using single particle detection method, the particle size of the mixture was successfully measured and the accurate peak position was obtainedSubsequently, the author further validated the particle size measurement effect of RPS and prepared mixtures of 80 nm, 120 nm, 160 nm, and 200 nm with concentration ratios of 1:1:1:1. measurement resultsConfirmed againRPS can smoothly measure the corresponding peaks of the mixture, and distinguish between different sample peaks clearly with good resolution.This four distribution sample can more accurately represent the actual sample.

Finally, the author selected two types of extracellular vesicles (derived from HEK293 cells and dried ginger) and performed particle size analysis using NTA, DLS, and RPS techniques, respectively. The results indicate that in terms of particle size analysis, the data obtained by NTA is similar to TEM, while the particle size values obtained by DLS are larger and the particle size results obtained by RPS are smaller. In terms of concentration, RPS exhibits more accurate quantitative analysis capabilities, thanks to its single particle detection technology, which can achieve precise characterization of each particle.

By measuring actual samples, the author summarized the characteristics of four different particle characterization methods (Table 1).The author believesNTA has good resolution and high accuracy in particle size analysis, and can distinguish samples of 2-3 modalities. However, for samples of exosomes, NTA cannot distinguish between exosomes, vesicles, and lipoproteins, and cannot distinguish between exosomes from different sources. For DLS, the author believes that this method is very simple and can be used to measure small amounts of samples. However, the particle size values measured by this method have a large deviation compared to the real situation, and this method cannot measure the particle concentration of the sample. The TEM method can directly achieve visual measurement of sample particles, but this method is costly and time-consuming. For RPS, the author believes that this method can directly measure the true particle size of particles (especially in the case of unknown sample particle characteristics), and has good resolution for multimodal mixed samples.

Table 1Summary of Characteristics of Four Different Particle Size Analysis Methods

Through this work, the authors systematically evaluated the effects of factors including concentration, instrument threshold, exposure, pump flow rate, etc. on NTA performance, and found thatAt a concentration of10⁸~10⁹When the pump flow rate is 50AU, more accurate characterization results can be obtained for particle samplesAt the same time, they also proposedFor the two parameters of instrument threshold and exposure, it is necessary to analyze them specifically based on the particle size of the sampleAt the same time, they also discoveredNTA has poor analytical ability for polydisperse samples, while RPS can successfully distinguish three or four peak samples, indicating that RPS has better discriminative ability for polydisperse samples.At the same time, the author used multiple characterization methods to analyze the two types of extracellular vesicles, and the particle size and concentration obtained by each characterization method were not the same. Therefore, the author believes thatWhen conducting biological particle research, it is necessary to use orthogonal methodology to perform multiple characterizations of particles.

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