Method for detecting the size distribution of extracellular vesicles in plasma and use thereof

Abstract

The application provides a method for detecting the particle size distribution of extracellular vesicles in plasma and application thereof, and belongs to the technical field of extracellular vesicle detection. The method for detecting the particle size distribution of extracellular vesicles in plasma provided by the application comprises the following steps: obtaining an extracellular vesicle sample in plasma by differential centrifugation, and performing dynamic light scattering detection on the filtered extracellular vesicle sample. The application first adopts a scheme of adding a filtering operation on the basis of traditional differential centrifugation, can further remove large-diameter impurity particles often present in the sample, avoids that these large particles seriously interfere with the DLS detection result, significantly improves the stability and reproducibility of the detection result, and makes the sample suitable for DLS detection. Moreover, by selecting a filter combination of a suitable pore size and material, it is finally ensured that the filtering scheme does not have too much influence on the separation recovery rate of EVs.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular vesicle detection, and in particular relates to a method for detecting the particle size distribution of extracellular vesicles in plasma and an application thereof. Background Art

[0002] Extracellular vesicles (EVs) are produced by cells and widely found in various biological fluids, including plasma and urine. They mediate intercellular communication. Their outer lipid bilayer contains a variety of bioactive molecules, including nucleic acids, proteins, lipids, and metabolites. These EVs can deliver biological information to target cells, causing functional or phenotypic changes. Based on their production mechanisms, EVs are primarily classified as exosomes and microvesicles (MVs), with diameters ranging from 50 to 150 nm and 100 to 1000 nm, respectively. In recent years, the important physiological functions of EVs have garnered significant attention in the study of various diseases, including the discovery and utilization of EVs as biomarkers for disease diagnosis and prognosis. As cell-derived products, EVs, in addition to containing traditional bioactive molecules such as nucleic acids, proteins, and lipids, also possess unique biophysical properties, such as number (concentration), size distribution, zeta potential, and mechanical properties. These biophysical properties also hold the potential to serve as biomarkers for disease diagnosis and prognosis. Plasma is the most common, important, and complex biological fluid used in clinical practice. Therefore, accurately and reliably detecting the size distribution of EVs in plasma is of great significance, but also a significant technical challenge.

[0003] Currently, several techniques can directly or indirectly measure the size distribution of EVs, including transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), flow cytometry (FCM), and tunable resistive pulse sensing (TRPS). Each of these methods has its own advantages and limitations, and is suitable for different research purposes. Selection and optimization of the detection process should be tailored to the specific situation. Specifically, TEM and AFM can directly measure the geometric diameter (actual diameter) of EVs and offer high resolution. However, these methods require cumbersome sample preparation and can cause EVs to deform, losing their normal physiological morphology and correspondingly changing their geometric diameter. Furthermore, only a small portion of the entire EV population can be observed at a time, making it difficult to generalize the results to the entire EV population. Approximate size distribution maps require manual counting of individual EVs, which is time-consuming and labor-intensive, hindering their translation to clinical applications. DLS, NTA, and FCM are all based on detecting light scattered by particles in solution and are used to determine the hydrodynamic diameter distribution of EVs in fluid media. The principle of DLS is to illuminate EVs suspended in a solution with a monochromatic laser beam. Since the EV particle size is smaller than the laser wavelength, scattered light is generated. Simultaneously, the EVs in the solution undergo Brownian motion, causing the scattered light intensity to vary over time. This allows the decay of the autocorrelation function to be calculated. Because EVs of different particle sizes undergo varying Brownian motion, the resulting autocorrelation function decays differently. By inferring the decay of the autocorrelation function, the size distribution of the EV population can be determined. DLS offers rapid detection, high sensitivity, minimal operator influence, and direct measurement of the overall EV size distribution. However, DLS is more suitable for monodisperse samples (those containing particles of a specific size), as the presence of small amounts of large particles in the solution can significantly interfere with the particle size distribution measurement. Plasma EV samples prepared using traditional methods are polydisperse and therefore unsuitable for direct DLS analysis. NTA also directly detects the particle size distribution of EVs. The NTA test results reflect the overall particle size distribution of EVs well, but the particle diameters measured by NTA are relatively large and the distribution is more severely broadened. The test results of EV samples isolated from complex biological fluids such as plasma have a large variability. In addition, the instrument's camera and detection parameters need to be set and optimized before each test, and the analysis results also require additional parameter settings. Therefore, they are greatly affected by the operator's subjective influence.FCM can detect, count, and sort different types of cells one by one at a high throughput, and has been very mature and widely used in cytological research. However, it is necessary to first detect standard microspheres to establish a reference before detecting the particle size of EVs, which is an indirect detection. The lower limit of the particle diameter that traditional FCM can detect is 300-500nm, which cannot cover the particle size range of EVs, and the resolution of particles of different sizes is poor. TRPS requires the consumption of a special nanopore membrane adapted to the instrument, and also requires the detection of standard particles as a control. In actual applications, it is found that the TRPS method also has a serious broadening of the particle diameter, and the accuracy of the test results is not high enough. Therefore, there is an urgent need in this field to provide a method for detecting the particle size distribution of EVs in plasma with stable and accurate test results and universal application. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a method for detecting the size distribution of extracellular vesicles in plasma, which has stable, accurate, reproducible, and highly sensitive detection results and is universal.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting the particle size distribution of extracellular vesicles in plasma, comprising the following steps: obtaining an extracellular vesicle sample in plasma by differential centrifugation, filtering the extracellular vesicle sample, and then performing dynamic light scattering detection.

[0007] Preferably, the filter membrane used for filtration is a polyvinylidene fluoride filter membrane.

[0008] Preferably, the polyvinylidene fluoride filter membrane is a hydrophilic polyvinylidene fluoride filter membrane.

[0009] Preferably, the pore size of the polyvinylidene fluoride filter membrane is 1 μm.

[0010] Preferably, the filter used for filtration is a needle filter.

[0011] Preferably, the filtration speed of the needle filter is 0.1 ml / s.

[0012] Preferably, the instrument used for dynamic light scattering detection is Litesizer TM 500 nm particle size analyzer.

[0013] Preferably, the parameters of the detection setting include selecting a continuous particle size measurement mode, a maximum number of running rounds of 30, a running time of each round of 00:00:10 (hh:mm:ss), a material refractive index of 1.4000, and an absorption coefficient of 0.01001 / m.

[0014] The present invention also provides an application of the above method in preparing disease diagnosis biomarkers or disease prognosis biomarkers.

[0015] Beneficial effects of the present invention:

[0016] Since it is generally believed in the art that the filtration operation itself will also affect the separation and recovery rate of EVs in the blood, and the recovery rate of EVs collected by centrifugation is at a moderate level, there are currently no reports in the art that use filtration solutions when using dynamic light scattering methods to detect the particle size distribution of EVs in plasma. The present invention is the first to adopt a solution that adds a filtration operation on the basis of traditional differential centrifugation, which can further remove large-diameter impurity particles that often appear in the sample, avoiding these large particles from causing serious interference with the DLS detection results, significantly improving the stability and reproducibility of the test results, and making the sample suitable for DLS detection. Moreover, by selecting a filter combination with appropriate pore size and material, it is ultimately ensured that the filtration solution will not have a significant impact on the separation and recovery rate of EVs.

[0017] In addition, the improved method of the present invention can directly detect the overall particle size distribution of plasma EVs conveniently, quickly, stably and reliably, and has the value of being transformed into clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1-Figure 5 These are the plasma EV particle size distribution diagrams of Cases 1 to 5, respectively;

[0019] Figure 6-Figure 8 These are the plasma EV size distribution diagrams of Cases 6-8, respectively;

[0020] Figures 9-11 These are the plasma EV particle size distribution diagrams of Cases 9-11 detected by NTA, Figures 9-11 The left figure is the particle size distribution curve of three consecutive test results, and the right figure is the fusion of the three particle size distribution curves;

[0021] Figure 12-14 The NTA test results are as follows: plasma EV particle size distribution of case 9 (after filtration) - case 11 (after filtration), Figure 12-14 The left figure is the particle size distribution curve of three consecutive test results, and the right figure is the fusion of the three particle size distribution curves;

[0022] Figure 15-17 These are the plasma EV size distribution diagrams for 0.45 μm PES membrane case 1 and 0.45 μm PES membrane case 3, respectively;

[0023] Figures 18-20 These are the plasma EV size distribution diagrams of 0.45 μm hydrophobic PVDF membrane case 1 and 0.45 μm hydrophobic PVDF membrane case 3, respectively;

[0024] Figure 21-23 These are the plasma EV size distribution diagrams for 0.8 μm MCE membrane case 1 and 0.8 μm MCE membrane case 3, respectively;

[0025] Figure 24-26 These are the plasma EV particle size distribution diagrams for 0.8 μm PA6 membrane case 1 and 0.8 μm PA6 membrane case 3, respectively;

[0026] Figure 27-Figure 29 These are the plasma EV size distribution diagrams for 1.2 μm PES membrane case 1 and 1.2 μm PES membrane case 3, respectively;

[0027] Figure 30-Figure 32 These are the plasma EV particle size distribution diagrams of 1μm hydrophobic PVDF membrane example 1-1μm hydrophobic PVDF membrane example 3 respectively. DETAILED DESCRIPTION

[0028] The present invention provides a method for detecting the particle size distribution of extracellular vesicles in plasma, comprising the following steps: obtaining an extracellular vesicle sample in plasma by differential centrifugation, filtering the extracellular vesicle sample, and then performing dynamic light scattering detection.

[0029] In the present invention, the filtration membrane is preferably a polyvinylidene fluoride membrane, more preferably a hydrophilic polyvinylidene fluoride membrane. The specific source of the polyvinylidene fluoride membrane is not particularly limited in the present invention; any commercially available product in the art can be used. In the present invention, the pore size of the polyvinylidene fluoride membrane is preferably 1 μm.

[0030] In the present invention, the filter used for filtration is preferably a needle filter. The present invention does not specifically limit the specific source of the needle filter, and any commercially available product in the art can be used. When using a needle filter for filtration, the filtration speed is preferably 0.1 ml / s.

[0031] In the present invention, when dynamic light scattering is used for detection, the instrument used is preferably Litesizer TM 500 nanometer particle size analyzer, the parameters of the detection setting are preferably set as: continuous particle size measurement mode, the maximum number of running rounds is 30, the running time of each round is 00:00:10 (hh:mm:ss), the material refractive index is 1.4000, and the absorption coefficient is 0.01001 / m. In the present invention, the parameters of the detection setting preferably also include the solvent being PBS, the solvent refractive index is 1.3318, and the viscosity is 0.9041mPa.s.

[0032] The present invention also provides an application of the above method in preparing disease diagnosis biomarkers or disease prognosis biomarkers.

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] In the following examples, unless otherwise specified, all methods are conventional.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] Blood collection and processing:

[0038] In the morning, on an empty stomach (no smoking or drinking for one day, no eating or drinking for ≥8 hours), 2 ml of peripheral venous blood was drawn from the subject and placed into a BD vacuum anticoagulant tube containing sodium citrate. The drawn venous blood was centrifuged in a low-speed centrifuge (4°C, 2000 g, 20 minutes) within 2 hours. The upper plasma was transferred to a 2 ml sterile EP tube using a pipette, taking care not to aspirate the precipitated blood cells in the lower layer. The plasma was centrifuged in a high-speed centrifuge (4°C, 11000 g, 2 minutes) to obtain the supernatant, platelet-poor plasma (PPP). 400 μl of the upper PPP was gently aspirated using a pipette and transferred to a 1.5 ml sterile EP tube.

[0039] EV isolation from plasma:

[0040] Filter the room temperature phosphate buffered saline (PBS) through a sterile needle filter with a pore size of 0.22 μm and set aside. Add 1 ml of filtered PBS to each EP tube containing 400 μl PPP, cover the tube tightly, and turn it upside down several times to mix and dilute the plasma. Centrifuge the diluted plasma in a high-speed centrifuge (4°C, 13000g, 50min). Gently remove the centrifuged plasma sample, open the tube cap, slowly tilt the tube mouth to pour out the supernatant, and turn the EP tube upside down on absorbent paper for 2 minutes. Add 1 ml of filtered PBS to each EP tube, cover the tube cap tightly, turn it upside down several times to resuspend the precipitate, and centrifuge the sample in a high-speed centrifuge (4°C, 13000g, 50min). Gently remove the sample, open the tube cap, slowly pour off the supernatant, turn the EP tube upside down on absorbent paper for 2 minutes, add 1 ml of filtered PBS to each EP tube, close the tube cap tightly, and oscillate on a vortex oscillator for 10 seconds to obtain preliminary separated EVs in plasma.

[0041] EV sample filtration:

[0042] Prepare a syringe filter with a 1 μm pore size and a 13 mm diameter, using a hydrophilic polyvinylidene fluoride (PVDF) membrane. Rinse the filter with 1 ml of filtered PBS at a PBS flow rate of 0.1 ml / s. Use a 1 ml syringe to aspirate the EV sample from the EP tube. Connect the syringe to the rinsed syringe filter. Place the syringe on a syringe pump, secure it securely, set the syringe pump flow rate to 0.1 ml / s, and begin constant-rate filtration. Transfer the filtered EV sample to a new, sterile 1.5 ml EP tube.

[0043] EV samples were tested on the machine for particle size distribution:

[0044] Using the Litesizer from Anton-Paar, Austria TM The 500 nm particle size analyzer was used for detection. The instrument operating software was opened, and “continuous particle size measurement” was selected. The detection parameters were set as follows: sample cell: disposable sample cell; measurement angle: automatic; target temperature: 25.0 °C; equilibrium time: 00:00:20 (hh:mm:ss); analysis model: conventional; accumulation model: advanced; quality mode: fast; maximum number of running rounds: 30; running time per round: 00:00:10 (hh:mm:ss); light intensity filter mode: automatic; focus point mode: automatic; material refractive index: 1.4000; absorption coefficient: 0.01001 / m; solvent name: PBS; solvent refractive index: 1.3318; viscosity: 0.9041 mPa.s; series type: repeat; keep focus point: √; keep filtering: √; generation parameters: repeat 3 times.

[0045] Use a pipette to transfer 1 ml of EV sample into a fluorescent cuvette imported from Sarstedt, Germany. Keep the cuvette clean to avoid the entry of dust and other foreign particles, and be careful not to create bubbles. Insert the cuvette into the cuvette slot and push it to the bottom of the cuvette slot. Close the sample pool module cover and click the "Start" button on the software interface to begin the test.

[0046] Example 2

[0047] The dynamic light scattering method of Example 1 was used to detect the particle size distribution of EVs in venous blood samples PPP of 5 different individuals (humans), respectively recorded as Example 1 to Example 5. The particle size distribution diagrams (light intensity mode) obtained by the detection are as follows: Figure 1-Figure 5 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 1 to 5 respectively.

[0048] Table 1 Plasma EV particle size distribution of case 1

[0049]

[0050] Table 2 Plasma EV particle size distribution of case 2

[0051]

[0052] Table 3 Plasma EV particle size distribution of case 3

[0053]

[0054] Table 4 Plasma EV particle size distribution of case 4

[0055]

[0056] Table 5 Plasma EV particle size distribution of case 5

[0057]

[0058] The test results from five different individuals, including Examples 1-5, demonstrate that the method of the present invention demonstrates excellent stability and reproducibility in the particle size distribution of plasma EVs, with high sensitivity, capable of detecting even small EVs with diameters less than 100 nm. Furthermore, the instrument automatically selects the measurement angle and adjusts the optimal focus point, minimizing the influence of subjective factors on the tester. The entire testing process is highly automated, convenient, and rapid. The method of the present invention can directly measure the particle size distribution of the entire EV sample, eliminating the need to extrapolate test results.

[0059] Comparative Example 1

[0060] The particle size distribution of EVs in venous blood samples PPP from three different individuals (humans) was detected. The specific detection method was the same as Example 1, except that the EV samples were not filtered. The remaining steps were recorded as Examples 6 to 8, respectively. The particle size distribution diagrams (light intensity mode) obtained by the detection were as follows: Figure 6-Figure 8 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 6 to 8 respectively.

[0061] Table 6 Plasma EV particle size distribution of case 6

[0062]

[0063] Table 7 Plasma EV particle size distribution of case 7

[0064]

[0065] Table 8 Plasma EV particle size distribution of case 8

[0066]

[0067] From the test results of three different people, it can be seen that the results of simply separating plasma EVs by differential centrifugation without filtering and directly detecting the EV particle size distribution by DLS show great instability and poor reproducibility.

[0068] Comparative Example 2

[0069] The NTA detection method was used to detect the particle size distribution of EVs in venous blood samples PPP from three different individuals (humans). The specific detection method was as follows: blood collection, processing, and specific steps for separating EVs from plasma were the same as in Example 1. After centrifugation to obtain plasma EVs, the particle size distribution of EVs was directly detected by NTA (the same sample was tested three times). The NTA detection parameters were set as follows: NTA Version: NTA 3.3 Dev Build 3.3.301, Script Used: SOP Standard Measurement, Diluent: 1, Camera Type: sCMOS, Laser Type: Blue488, Camera Level: 14, Slider Shutter: 1259, Slider Gain: 366, FPS: 25.0, Number of Frames: 1498, Temperature: 25.0℃, Viscosity: (Water) 0.9 cP, Detect Threshold: 3, Blur Size: Auto, Max Jump Distance: Auto. The particle size distribution diagrams (quantity patterns) obtained from the tests are respectively as follows: Figures 9-11 As shown ( Figures 9-11 The left figure is the particle size distribution curve of each of the three consecutive test results, and the right figure is the fusion of these three particle size distribution curves. The solid line represents the mean and the bandwidth represents the standard error. The particle size distribution tables (quantity mode) obtained from the tests are shown in Tables 9 to 11 respectively.

[0070] Table 9 NTA test example 9 plasma EV particle size distribution

[0071]

[0072] Table 10. Plasma EV particle size distribution of NTA test example 10

[0073]

[0074]

[0075] Table 11. Plasma EV particle size distribution in NTA test example 11

[0076]

[0077] The test results from three different individuals, from Examples 9 to 11, indicate that direct NTA analysis of the particle size distribution of plasma EV samples separated by differential centrifugation without needle filtration yielded significant variability, manifested by poor overlap in the distribution curves for the three tests on the same sample, a wide error band, and unstable test results. Furthermore, because NTA testing requires the examiner to manually set key parameters such as camera brightness and detection threshold, it is susceptible to subjective influences. NTA requires thorough flushing and cleaning of the tubing before and after testing different samples to ensure accurate test results, making sample testing cumbersome and time-consuming. Furthermore, the adequacy of tubing cleanliness requires subjective judgment by the examiner. NTA is unable to effectively detect particles with a diameter less than 100 nm.

[0078] Comparative Example 3

[0079] The steps of blood collection and treatment and EV separation in plasma in Example 1 were adopted. After centrifugal separation to obtain plasma EVs, the plasma EVs were filtered through a needle filter with a hydrophilic PVDF filter membrane with a diameter of 13 mm and a pore size of 1 μm (the specific operation details of the filtration were the same as those in Example 1). The particle size distribution of EVs was detected by NTA method. The NTA detection parameter settings were the same as those in Comparative Example 2 (each sample was tested three times). They are respectively recorded as Example 9 (after filtration) to Example 11 (after filtration). The particle size distribution diagrams (quantity patterns) obtained by detection are as follows: Figure 12-14 As shown ( Figure 12-14 The left figure is the particle size distribution curve of each of the three consecutive test results, and the right figure is the fusion of these three particle size distribution curves. The solid line represents the mean and the bandwidth represents the standard error. The particle size distribution tables (quantity mode) obtained from the tests are shown in Tables 12 to 14 respectively.

[0080] Table 12: Plasma EV particle size distribution in NTA test example 9 (after filtration)

[0081]

[0082]

[0083] Table 13. Plasma EV particle size distribution in NTA test example 10 (after filtration)

[0084]

[0085] Table 14. Plasma EV particle size distribution in NTA test example 11 (after filtration)

[0086]

[0087] Compared with the results of Comparative Example 2, it can be seen that filter filtration can reduce the variability of NTA test results to a certain extent, as shown by the improved overlap of the particle size distribution curves of the three tests for the same sample and the narrowing of the error band, but the degree of improvement is not significant. In addition, the degree of improvement in the stability of the test results after filtration varies from sample to sample. For example, the stability of the results of Example 10 was significantly improved after filtration, but there was no significant improvement in Examples 9 and 11. Even Example 10, which showed a significant improvement in result stability, failed to meet the sufficiently high detection reliability requirements. Therefore, NTA is not suitable for detecting the particle size distribution of plasma EV samples.

[0088] Comparative Example 4

[0089] The particle size distribution of EVs in PPP venous blood samples from three different individuals (humans) was detected. The specific detection method was the same as Example 1, except that the filter membrane used in the filtration step was a polyethersulfone (PES) membrane with a pore size of 0.45 μm. The remaining steps were recorded as PES membrane example 1 to PES membrane example 3, respectively. The particle size distribution diagrams obtained by detection (light intensity mode) are shown as follows: Figure 15-17 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 15 to 17 respectively.

[0090] Table 15: Plasma EV particle size distribution of Example 1 with 0.45 μm PES membrane

[0091]

[0092] Table 16: Plasma EV particle size distribution of 0.45 μm PES membrane Example 2

[0093]

[0094] Table 17: Plasma EV particle size distribution of 0.45 μm PES membrane example 3

[0095]

[0096] It can be seen from the above results that when using a filter with a pore size of 0.45μm and a PES filter membrane for testing, the average light intensity count that the instrument can detect is too low (less than 20kcps), the detection time is significantly extended, and the data quality of the test results is affected; Peak 1 shifts significantly to the left, and the deviation is large compared with the unfiltered sample; the proportion of light intensity distribution in the small particle size range below 100nm increases significantly, especially the distribution peak area of ​​about 10nm increases significantly; the stability and reproducibility of the particle size distribution curve of the test results are not good enough.

[0097] Comparative Example 5

[0098] The particle size distribution of EVs in venous blood samples (PPP) from three different individuals (humans) was detected. The specific detection method was the same as Example 1, except that the filter membrane used in the filtration step had a pore size of 0.45 μm and was made of hydrophobic polyvinylidene fluoride (PVDF). The remaining steps were recorded as Hydrophobic PVDF Membrane Example 1 to Hydrophobic PVDF Membrane Example 3, respectively. The particle size distribution diagrams (light intensity mode) obtained by detection are shown as follows: Figures 18-20 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 18 to 20 respectively.

[0099] Table 18: Plasma EV particle size distribution of 0.45 μm hydrophobic PVDF membrane Example 1

[0100]

[0101] Table 19: Plasma EV particle size distribution of Example 2 with 0.45 μm hydrophobic PVDF membrane

[0102]

[0103] Table 20. Plasma EV particle size distribution of Example 3 with 0.45 μm hydrophobic PVDF membrane

[0104]

[0105] The above results show that when using a filter with a 0.45 μm pore size and a hydrophobic polyvinylidene fluoride membrane, Peak 1 exhibits a significant leftward shift, and the particle size distribution curve lacks reproducibility and stability. Compared to the filter with a 0.45 μm pore size and a PES membrane in Comparative Example 4, the filter type in Comparative Example 5 does not exhibit excessively low average light intensity counts, and is overall superior to the former, but it is not the optimal filter choice.

[0106] Comparative Example 6

[0107] The particle size distribution of EVs in PPP venous blood samples from three different individuals (humans) was detected. The specific detection method was the same as Example 1, except that the filter membrane used in the filtration step had a pore size of 0.8 μm and was a mixed cellulose ester (MCE) filter membrane. The remaining steps were recorded as MCE Membrane Example 1 to MCE Membrane Example 3, respectively. The particle size distribution diagrams (light intensity mode) obtained by detection are shown as follows: Figure 21-23 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 21 to 23 respectively.

[0108] Table 21. Plasma EV particle size distribution of 0.8 μm MCE membrane example 1

[0109]

[0110] Table 22. Plasma EV particle size distribution of 0.8 μm MCE membrane Example 2

[0111]

[0112] Table 23. Plasma EV particle size distribution of 0.8 μm MCE membrane example 3

[0113]

[0114] The above results show that when using a filter with a pore size of 0.8 μm and an MCE filter membrane for testing, the average light intensity count that the instrument can detect is too low (<20 kcps), and even one detection failure occurs; the particle size distribution curve results are unstable and have poor reproducibility.

[0115] Comparative Example 7

[0116] The particle size distribution of EVs in PPP venous blood samples from three different individuals (humans) was detected. The specific detection method was the same as Example 1, except that the filter membrane used in the filtration step was nylon 6 (polycaprolactam, PA6) with a pore size of 0.8 μm. The remaining steps were recorded as PA6 membrane example 1 to PA6 membrane example 3, respectively. The particle size distribution diagrams (light intensity mode) obtained by detection are shown as follows: Figure 24-26 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 24 to 26 respectively.

[0117] Table 24 0.8μm PA6 membrane example 1 plasma EV particle size distribution

[0118]

[0119]

[0120] Table 25 0.8μm PA6 membrane example 2 plasma EV particle size distribution

[0121]

[0122] Table 26 0.8μm PA6 membrane example 3 plasma EV particle size distribution

[0123]

[0124] The above results show that the particle size distribution curve stability and reproducibility are relatively good when tested using a 0.8μm pore size PA6 filter. However, the height stability and reproducibility of Peak 1 are poor, indicating significant deviations in the Peak 1 Distribution (%) between three test results for the same sample. The average light intensity counts after filtration were within a relatively suitable range, consistently exceeding 20 kcps. However, compared to the 1μm pore size, hydrophilic PVDF filter used in Example 2, the 0.8μm pore size PA6 filter performed inferiorly.

[0125] Comparative Example 8

[0126] The particle size distribution of EVs in PPP venous blood samples from three different individuals (humans) was detected. The specific detection method was the same as in Example 1, except that the filter membrane used in the filtration step was a polyethersulfone (PES) membrane with a pore size of 1.2 μm. The remaining steps are respectively recorded as 1.2 μm PES membrane example 1 to 1.2 μm PES membrane example 3. The particle size distribution diagrams (light intensity mode) obtained by detection are shown as follows: Figure 27-Figure 29 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 27 to 29 respectively.

[0127] Table 27. Plasma EV particle size distribution of 1.2 μm PES membrane example 1

[0128]

[0129]

[0130] Table 28: Plasma EV particle size distribution in Example 2 using 1.2 μm PES membrane

[0131]

[0132] Table 29: Plasma EV particle size distribution of Example 3 using 1.2 μm PES membrane

[0133]

[0134] The above results show that when using a PES filter with a pore size of 1.2μm for testing, the particle size distribution curve is not stable enough and the reproducibility is poor; the removal effect of large-particle impurity particles is not ideal and the interference of large-particle particles cannot be effectively eliminated; the average light intensity count detected after filtration is still within the appropriate range.

[0135] Comparative Example 9

[0136] The particle size distribution of EVs in venous blood samples PPP from three different individuals (humans) was detected. The specific detection method was the same as Example 1, except that the filter membrane used in the filtration step had a pore size of 1 μm and was a hydrophobic polyvinylidene fluoride (PVDF) filter membrane. The remaining steps were recorded as 1 μm hydrophobic PVDF membrane example 1 to 1 μm hydrophobic PVDF membrane example 3, respectively. The particle size distribution diagrams (light intensity mode) obtained by detection are shown as follows: Figure 30-Figure 32 As shown, the particle size distribution tables (light intensity mode) obtained by detection are shown in Tables 30 to 32 respectively.

[0137] Table 30 1μm hydrophobic PVDF membrane example 1 plasma EV particle size distribution

[0138]

[0139] Table 31 1μm hydrophobic PVDF membrane example 2 plasma EV particle size distribution

[0140]

[0141]

[0142] Table 32: Plasma EV particle size distribution of Example 3 with 1 μm hydrophobic PVDF membrane

[0143]

[0144] The above results show that when a hydrophobic PVDF filter with a pore size of 1 μm is used for testing, the stability and reproducibility of the particle size distribution curve are improved, but not ideal, and the results vary greatly between different samples. The average light intensity count after filtration is often too low (<20 kcps). Compared with the results of Example 2, it can be seen that the filter with a pore size of 1 μm and a hydrophobic PVDF membrane is significantly inferior to the filter with a pore size of 1 μm and a hydrophilic PVDF membrane.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting the particle size distribution of extracellular vesicles in plasma, characterized in that: The method comprises the following steps: obtaining an extracellular vesicle sample in plasma by differential centrifugation, filtering the extracellular vesicle sample and then performing dynamic light scattering detection; The filter membrane used for filtration is a polyvinylidene fluoride filter membrane; The polyvinylidene fluoride filter membrane is a hydrophilic polyvinylidene fluoride filter membrane; The pore size of the polyvinylidene fluoride filter membrane is 1 μm.

2. The method according to claim 1, characterized in that The filter used for filtration is a needle filter.

3. The method according to claim 2, characterized in that The filtration speed of the needle filter is 0.1 ml / s.

4. The method according to claim 1, wherein The instrument used for dynamic light scattering detection is Litesizer TM 500 nm particle size analyzer.

5. The method according to claim 4, characterized in that The test settings included selecting the continuous particle size measurement mode, a maximum number of runs of 30, a run time of 00hh:00mm:10ss per run, a material refractive index of 1.4000, and an absorption coefficient of 0.0100 l / m.

6. Use of the method according to any one of claims 1 to 5 in preparing disease diagnostic biomarkers.

7. Use of the method according to any one of claims 1 to 5 in preparing a disease prognosis biomarker.

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