Tracking analysis and detection method for exosome nanoparticles

Abstract

The invention provides an exosome nanoparticle tracking analysis detection method which comprises the following steps: A) treating an exosome sample to obtain a sample with a preset sample loading concentration; and B) detecting the sample; wherein in the step A, the exosome sample is diluted, so that the exosome sample has a preset sample loading concentration, and the preset sample loading concentration is 1.38 E + 08-2.76 E + 09 particle number / ml, preferably 2.00 E + 08-2.00 E + 09 particle number / ml.

Description

Technical Field

[0001] The present invention relates to a method for detecting exosomes, and in particular to a method for detecting exosome nanoparticle tracking analysis. Background Art

[0002] Exosomes are a type of small vesicles secreted by cells, which are wrapped by cell membranes and released outside the cells. Exosomes are mainly divided into two types: exosomes and microvesicles. Exosomes contain biomolecules such as proteins, nucleic acids, and lipids on the cell membrane, and can be transmitted to other cells through body fluids (such as blood, urine, saliva, etc.), and carry out information transmission and regulation between cells. Exosomes play an important role in cell communication. They can bind to receptors on the surface of target cells and release internal biomolecules, thereby regulating the functions and metabolic states of target cells. Exosomes are considered an important intercellular signal transduction medium and can play roles in various physiological and pathological states, such as immune regulation, cell proliferation and apoptosis, and inflammatory responses.

[0003] In recent years, the research on exosomes has gradually attracted attention because they have great potential for disease diagnosis, treatment, and drug delivery. Exosomes can be used as biomarkers for early detection and monitoring of diseases, and can also be used as carriers to deliver drugs or genes to specific cells or tissues. The research on exosomes provides new perspectives and methods for understanding intercellular interactions and disease pathogenesis.

[0004] After the isolation and purification of exosomes, there is an important step - namely, identifying the obtained exosomes.

[0005] Similar to the isolation and purification of exosomes, it is difficult to obtain high-purity exosomes using a single method. Similarly, it is also unrealistic to comprehensively evaluate the quality of exosomes using a single method. According to the recommendations of the International Society for Extracellular Vesicles (abbreviated as ISEV), the identification of exosomes should be carried out from three aspects: size, morphology, and surface markers, which requires a combined evaluation through multiple experiments.

[0006] The sizes of exosomes extracted in experiments are not consistent, so the particle size distribution is commonly used to evaluate the size of exosomes. Techniques such as dynamic light scattering (abbreviated as DLS), nanoparticle tracking analysis (abbreviated as NTA), and nanoflow cytometry (abbreviated as nFCM) can all achieve the detection of exosome particle size distribution.

[0007] The principle of NTA is to irradiate the nanoparticles in the sample with a laser, use a high-speed camera to record the scattered light points of each nanoparticle, obtain the trajectory of its Brownian motion, and then calculate the particle size (fluid dynamics particle size) of the nanoparticles according to the Stokes-Einstein equation. At the same time, count the number of particles in the video window and calculate the concentration. Usually, the NTA test results of exosomes with higher purity show a single peak. It can fully characterize particles with a wide range of particle size distribution in the suspension, and has the advantages of high resolution, fast detection speed, and high accuracy.

[0008] The NAT test process automatically focuses without manual intervention, thus fully ensuring the accuracy of the test results. The NAT test can track each nanoparticle, and the test results are more accurate and more stable. However, how to detect exosomes through NAT to ensure the stability and reliability of the test data is a major problem that needs to be solved in this field. Summary of the invention

[0009] One advantage of the present invention is that it provides an exosome nanoparticle tracking analysis detection method, wherein the exosome nanoparticle tracking analysis detection method is stable and reliable.

[0010] Another advantage of the present invention is that it provides an exosome nanoparticle tracking analysis detection method, wherein the exosome nanoparticle tracking analysis detection method makes the detection result stable and reliable by controlling the appropriate sample concentration range.

[0011] Another advantage of the present invention is that it provides an exosome nanoparticle tracking analysis detection method, wherein the exosome nanoparticle tracking analysis detection method is suitable for Malvern NanoSight NS300 nanoparticle tracking analyzer.

[0012] Another advantage of the present invention is that it provides an exosome nanoparticle tracking analysis detection method to provide an optimal sample concentration range, thereby making the exosome detection results stable and reliable.

[0013] Another advantage of the present invention is that it provides an exosome nanoparticle tracking analysis detection method to provide an optimal sample concentration range, so that the particle size peak diagram obtained by exosome detection is better.

[0014] Another advantage of the present invention is that it provides a method for tracking and analyzing exosome nanoparticles to provide an optimal sample concentration range, thereby stabilizing the particle size obtained by exosome detection.

[0015] Another advantage of the present invention is that it provides an exosome nanoparticle tracking analysis detection method to provide an optimal sample concentration range, so that the reported original concentration obtained by exosome detection is reliable.

[0016] Another advantage of the present invention lies in providing a method for detecting and analyzing exosome nanoparticles to provide a relatively optimal sample loading concentration range, so that the particle size repeatability of exosome detection results is good.

[0017] Another advantage of the present invention lies in providing a method for detecting and analyzing exosome nanoparticles to provide a relatively optimal sample loading concentration range, so that the particle concentration repeatability of exosome detection results is good.

[0018] Other advantages and features of the present invention are fully embodied in the following detailed description and can be achieved by the combination of means and devices specifically pointed out in the appended claims.

[0019] According to one aspect of the present invention, an exosome nanoparticle tracking analysis and detection method of the present invention capable of achieving the foregoing objectives and other advantageous effects includes the following steps:

[0020] A) Processing an exosome sample to obtain a sample with a preset sample loading concentration; and

[0021] B) Detecting the sample;

[0022] Wherein in step A, the exosome sample is diluted to have a preset sample loading concentration, and the preset sample loading concentration is 1.38E+08 - 2.76E+09 particles / ml.

[0023] According to an embodiment of the present invention, the preset sample loading concentration is

[0024] 2.92E+08 - 1.54E+09 particles / ml.

[0025] According to an embodiment of the present invention, the preset sample loading concentration is 1.38E+08 - 2.41E+09 particles / ml.

[0026] According to an embodiment of the present invention, the preset sample loading concentration is 1.38E+08 - 2.76E+09 particles / ml.

[0027] According to an embodiment of the present invention, the preset sample loading concentration is 2.92E+08 - 2.76E+09 particles / ml.

[0028] According to an embodiment of the present invention, the preset sample loading concentration is 2.00E+08 - 2.00E+09 particles / ml.

[0029] According to an embodiment of the present invention, the preset sample loading concentration is 2.92E+08 - 6.60E+08 particles / ml.

[0030] According to an embodiment of the present invention, the nanoparticle tracking analysis detection method detects exosomes through a Malvern NanoSight NS300 nanoparticle tracking analyzer.

[0031] According to an embodiment of the present invention, step A includes the following steps:

[0032] A1) Ultrasonically mix the exosome sample to obtain an ultrasonically mixed sample; and

[0033] A2) Dilute the ultrasonically mixed sample to obtain a diluted sample.

[0034] According to an embodiment of the present invention, step A2 dilutes the ultrasonically mixed sample with a dilution solution, where the dilution solution is PBS.

[0035] According to an embodiment of the present invention, step A2 is diluted by a direct dilution method.

[0036] According to an embodiment of the present invention, the direct dilution method mixes a first preset volume of PBS with a second preset volume of the original sample solution evenly according to a first preset dilution multiple parameter to obtain a directly diluted sample with a preset dilution multiple.

[0037] According to an embodiment of the present invention, step A2 is diluted by direct dilution.

[0038] According to an embodiment of the present invention, the direct dilution method pre-adds a third preset volume of PBS according to a second preset dilution multiple parameter, and then measures a fourth preset volume of the sample dilution solution from the previous dilution multiple solution and mixes them evenly to obtain an indirectly diluted sample with a preset dilution multiple.

[0039] According to an embodiment of the present invention, step B includes the following steps:

[0040] B1) Turn on the machine;

[0041] B2) Clean;

[0042] B3) Load the sample;

[0043] B4) Set parameters;

[0044] B5) Adjust the field of view;

[0045] B6) Set the save path;

[0046] B7) Detect;

[0047] B8) Analyze the results and generate a report; and

[0048] B9) Unload the sample and turn off the machine after saving.

[0049] Further objects and advantages of the present invention will become fully apparent from the following description and the accompanying drawings.

[0050] These and other objects, features, and advantages of the present invention will become fully apparent from the following detailed description, the accompanying drawings, and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is a flowchart of a method for detecting exosome nanoparticle tracking analysis according to a preferred embodiment of the present invention.

[0052] Figure 2A is a concentration / diameter distribution diagram of sample S1 with a dilution factor of 1 diluted by the direct dilution method detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the concentration / diameter distribution comparison of three repeated experiments.

[0053] Figure 2B is an average concentration / diameter distribution diagram of sample S1 with a dilution factor of 1 diluted by the direct dilution method detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / diameter distribution and its error bars of three repeated experiments.

[0054] Figure 3A is a concentration / diameter distribution diagram of sample S2 with a dilution factor of 2 diluted by the direct dilution method detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the concentration / diameter distribution comparison of three repeated experiments.

[0055] Figure 3B is an average concentration / diameter distribution diagram of sample S2 with a dilution factor of 2 diluted by the direct dilution method detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / diameter distribution and its error bars of three repeated experiments.

[0056] Figure 4A is a concentration / diameter distribution diagram of sample S3 with a dilution factor of 5 diluted by the direct dilution method detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the concentration / diameter distribution comparison of three repeated experiments.

[0057] Figure 4B is an average concentration / diameter distribution diagram of sample S3 with a dilution factor of 5 diluted by the direct dilution method detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / diameter distribution and its error bars of three repeated experiments.

[0058] Figure 5A It is the concentration / diameter distribution diagram of sample S4 diluted by the direct dilution method with a dilution multiple of 10 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0059] Figure 5B It is the average concentration / diameter distribution diagram of sample S4 diluted by the direct dilution method with a dilution multiple of 10 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the average concentration / diameter distribution and its error bars of three repeated experiments.

[0060] Figure 6A It is the concentration / diameter distribution diagram of sample S5 diluted by the direct dilution method with a dilution multiple of 20 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0061] Figure 6B It is the average concentration / diameter distribution diagram of sample S5 diluted by the direct dilution method with a dilution multiple of 20 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the average concentration / diameter distribution and its error bars of three repeated experiments.

[0062] Figure 7A It is the concentration / diameter distribution diagram of sample S6 diluted by the direct dilution method with a dilution multiple of 50 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0063] Figure 7B It is the average concentration / diameter distribution diagram of sample S6 diluted by the direct dilution method with a dilution multiple of 50 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the average concentration / diameter distribution and its error bars of three repeated experiments.

[0064] Figure 8A It is the concentration / diameter distribution diagram of sample S7 diluted by the direct dilution method with a dilution multiple of 100 detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0065] Figure 8BIt is the average concentration / particle size distribution diagram of sample S7 with a dilution factor of 100 diluted by the direct dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / particle size distribution and its error bars of three repeated experiments.

[0066] Figure 9A It is the concentration / particle size distribution diagram of sample S8 with a dilution factor of 200 diluted by the direct dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the comparison of the concentration / particle size distribution of three repeated experiments.

[0067] Figure 9B It is the average concentration / particle size distribution diagram of sample S8 with a dilution factor of 200 diluted by the direct dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / particle size distribution and its error bars of three repeated experiments.

[0068] Figure 10A It is the concentration / particle size distribution diagram of sample S9 with a dilution factor of 500 diluted by the direct dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the comparison of the concentration / particle size distribution of three repeated experiments.

[0069] Figure 10B It is the average concentration / particle size distribution diagram of sample S9 with a dilution factor of 500 diluted by the direct dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / particle size distribution and its error bars of three repeated experiments.

[0070] Figure 11A It is the concentration / particle size distribution diagram of the dilution solution PBS (sample S10), detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the comparison of the concentration / particle size distribution of three repeated experiments.

[0071] Figure 11B It is the average concentration / particle size distribution diagram of the dilution solution PBS (sample S10), detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / particle size distribution and its error bars of three repeated experiments.

[0072] Figure 12AIt is the concentration / diameter distribution diagram of sample S11 with a dilution factor of 1 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0073] Figure 12B It is the average concentration / diameter distribution diagram of sample S11 with a dilution factor of 1 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the average concentration / diameter distribution and its error bars of three repeated experiments.

[0074] Figure 13A It is the concentration / diameter distribution diagram of sample S12 with a dilution factor of 2 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0075] Figure 13B It is the average concentration / diameter distribution diagram of sample S12 with a dilution factor of 2 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the average concentration / diameter distribution and its error bars of three repeated experiments.

[0076] Figure 14A It is the concentration / diameter distribution diagram of sample S13 with a dilution factor of 5 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0077] Figure 14B It is the average concentration / diameter distribution diagram of sample S13 with a dilution factor of 5 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the average concentration / diameter distribution and its error bars of three repeated experiments.

[0078] Figure 15A It is the concentration / diameter distribution diagram of sample S14 with a dilution factor of 10 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, which illustrates the concentration / diameter distribution comparison of three repeated experiments.

[0079] Figure 15BIt is the average concentration / particle size distribution map of sample S14 with a dilution factor of 10 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the average concentration / particle size distribution and its error bars of three repeated experiments.

[0080] Figure 16A It is the concentration / particle size distribution map of sample S15 with a dilution factor of 20 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the comparison of the concentration / particle size distribution of three repeated experiments.

[0081] Figure 16B It is the average concentration / particle size distribution map of sample S15 with a dilution factor of 20 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the average concentration / particle size distribution and its error bars of three repeated experiments.

[0082] Figure 17A It is the concentration / particle size distribution map of sample S16 with a dilution factor of 50 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the comparison of the concentration / particle size distribution of three repeated experiments.

[0083] Figure 17B It is the average concentration / particle size distribution map of sample S16 with a dilution factor of 50 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the average concentration / particle size distribution and its error bars of three repeated experiments.

[0084] Figure 18A It is the concentration / particle size distribution map of sample S17 with a dilution factor of 100 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the comparison of the concentration / particle size distribution of three repeated experiments.

[0085] Figure 18B It is the average concentration / particle size distribution map of sample S17 with a dilution factor of 100 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, and illustrates the average concentration / particle size distribution and its error bars of three repeated experiments.

[0086] Figure 19AIt is the concentration / diameter distribution diagram of sample S18 with a dilution factor of 200 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the concentration / diameter distribution comparison of three repeated experiments.

[0087] Figure 19B It is the average concentration / diameter distribution diagram of sample S18 with a dilution factor of 200 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / diameter distribution and its error bars of three repeated experiments.

[0088] Figure 20A It is the concentration / diameter distribution diagram of sample S19 with a dilution factor of 500 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the concentration / diameter distribution comparison of three repeated experiments.

[0089] Figure 20B It is the average concentration / diameter distribution diagram of sample S19 with a dilution factor of 500 diluted by the sequential dilution method, detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / diameter distribution and its error bars of three repeated experiments.

[0090] Figure 21A It is the concentration / diameter distribution diagram of the dilution solution PBS (sample S20), detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the concentration / diameter distribution comparison of three repeated experiments.

[0091] Figure 21B It is the average concentration / diameter distribution diagram of the dilution solution PBS (sample S20), detected by the exosome nanoparticle tracking analysis detection method according to the above preferred embodiment of the present invention, illustrating the average concentration / diameter distribution and its error bars of three repeated experiments.

[0092] Figure 22A It is the comparison diagram of the single particle size results of three repeated experiments of samples S01 to S20.

[0093] Figure 22B It illustrates the average particle size results of samples S01 to S20.

[0094] Figure 23A It is the comparison diagram of the single original concentration results of three repeated experiments of samples S01 to S20.

[0095] Figure 23BExplains the average results of the original concentrations of the three repeated experiments of samples S01 to S20.

[0096] Figure 24 Explains the calculated loading concentrations of samples S01 to S20.

[0097] Figure 25 Explains the background-subtracted calculated loading concentrations of samples S01 to S20.

[0098] Figure 26 Explains the background-subtracted calculated original concentrations of samples S01 to S20.

[0099] Figure 27 Is a comparison chart of the reported original concentrations and the background-subtracted calculated original concentrations of samples S01 to S20. Detailed implementation mode

[0100] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments in the following description are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other implementation schemes, variation schemes, improvement schemes, equivalent schemes, and other technical schemes that do not deviate from the spirit and scope of the present invention.

[0101] In the accompanying drawings of the specification Figure 1 Explains an exosome nanoparticle tracking analysis (NTA) detection method according to a preferred embodiment of the present invention. The exosome nanoparticle tracking analysis detection method includes the following steps:

[0102] A) Process an exosome sample to obtain a sample with a preset loading concentration;

[0103] B) Detect the sample.

[0104] According to this preferred embodiment of the present invention, the exosome nanoparticle tracking analysis detection method detects an exosome sample through a Malvern NanoSight NS300 nanoparticle tracking analyzer, and step B includes the following steps:

[0105] B1) Turn on the machine;

[0106] B2) Clean;

[0107] B3) Load the sample;

[0108] B4) Set parameters;

[0109] B5) Adjust the field of view;

[0110] B6) Set the save path;

[0111] B7) Detection;

[0112] B8) Analyze the results and issue a report; and

[0113] B9) Save the sample and then shut down the equipment.

[0114] According to this preferred embodiment of the present invention, step A includes the following steps:

[0115] A1) Ultrasonically mix the exosome sample to obtain an ultrasonically mixed sample; and

[0116] A2) Dilute the ultrasonically mixed sample to obtain a diluted sample.

[0117] According to this preferred embodiment of the present invention, the exosome sample is purified milk exosomes with a protein concentration of about 1 μg / μL. To analyze the influence of the loading concentration on the detection results of nanoparticle tracking analysis, after the exosome sample is ultrasonically mixed, it is diluted by multiple different multiples and then detected and analyzed by a Malvern NanoSight NS300 nanoparticle tracking analyzer. To obtain sufficient data support, the exosome sample is directly diluted and serially diluted with a dilution solution respectively to obtain two groups of diluted samples, and each diluted sample is detected three times. According to this preferred embodiment of the present invention, the dilution solution is PBS.

[0118] Specifically, the sample dilution scheme is as follows:

[0119] Samples S1 - S10 are diluted by direct dilution. Specifically, according to a first preset dilution multiple parameter, a first preset volume of PBS is mixed evenly with a second preset volume of the sample stock solution to obtain a directly diluted sample with a preset dilution multiple. According to this preferred embodiment of the present invention, the sample stock solution refers to the ultrasonically mixed sample. According to this preferred embodiment of the present invention, after PBS is pre - added to tubes S1 - S10, the corresponding volumes of the sample stock solution in Table 1 are added respectively for dilution.

[0120] Samples S11 - S19 are diluted by serial dilution. Specifically, according to a second preset dilution multiple parameter, after a third preset volume of PBS is pre - added, a fourth preset volume of the sample dilution is measured from the previous dilution - multiple solution and mixed evenly to obtain an indirectly diluted sample with a preset dilution multiple. According to this preferred embodiment of the present invention, after PBS is pre - added to tubes S11 - S19, the corresponding volumes of the sample dilution in Table 2 are measured from the previous tube number for re - dilution, where the volume of the upper tube of S11 is the volume of the sample stock solution.

[0121] As shown below, Table 1 and Table 2 respectively illustrate the parameters of the directly diluted samples and the serially diluted samples.

[0122] Table 1 Direct Dilution Sample Parameter Table

[0123]

[0124] Table 2 Indirect Dilution Sample Parameter Table

[0125]

[0126] Tables 3 to 6 show the original data obtained by analyzing the above samples S01 to S20 with a nanoparticle tracking analyzer.

[0127] Table 3 Original Data Table of NTA Detection for Samples S01 to S05

[0128]

[0129] Table 4 Original Data Table of NTA Detection for Samples S06 to S10

[0130]

[0131] Table 5 Original Data Table of NTA Detection for Samples S11 to S15

[0132]

[0133] Table 6 Original Data Table of NTA Detection for Samples S16 to S20

[0134]

[0135] The concentration fold difference described in the table refers to the ratio of the original concentration reported by the machine to the original sample concentration calculated after subtracting the number of PBS background particles.

[0136] According to this preferred embodiment of the present invention, each of the dilution solutions S01 to S20 is tested three times, that is, each dilution solution is tested three times by a Malvern NanoSight NS300 nanoparticle tracking analyzer.

[0137] In the accompanying drawings of the specification Figures 2A to 21B respectively illustrate the NTA test result graphs of samples S01 to S20. Among them, in the accompanying drawings of the specification Figure 2A 、 Figure 3A 、 Figure 4A 、 Figure 5A 、 Figure 6A 、 Figure 7A 、 Figure 8A 、 Figure 9A 、 Figure 10A 、 Figure 11A 、 Figure 12A 、 Figure 13A 、 Figure 14A 、 Figure 15A 、 Figure 16A 、Figure 17A , Figure 18A , Figure 19A , Figure 20A and Figure 21A are the concentration / diameter distribution diagrams of three repeated detections of samples S01 to S20, respectively.

[0138] In the accompanying drawings of the specification Figure 2B , Figure 3B , Figure 4B , Figure 5B , Figure 6B , Figure 7B , Figure 8B , Figure 9B , Figure 10B , Figure 11B , Figure 12B , Figure 13B , Figure 14B , Figure 15B , Figure 16B , Figure 17B , Figure 18B , Figure 19B , Figure 20B and Figure 21B are the average concentration / diameter distribution diagrams of three repeated detections of samples S01 to S20, respectively.

[0139] Refer to Figures 2A to 21B in the accompanying drawings of the specification, and particularly refer to Figures 4A to 7A , Figures 4B to 7B , Figures 14A to 17A as well as Figures 14B to 17B . The results are better when the dilution factor is 5× to 50×, where the number of peaks is relatively single and relatively stable. Since the concentration of exosomes at different dilution factors is affected by the concentration of the original experimental sample itself. This experiment reflects the influence of the loading concentration on the NTA detection result. According to this preferred embodiment of the present invention, the loading concentration range of the directly diluted 5× to 50× sample is 1.36E+09 to 3.38E+08, and the loading concentration range of the successively diluted 5× to 50× sample is 1.54E+09 to 2.92E+08. Therefore, preferably, in the exosome NTA detection method, the loading concentration is 1.36E+09 to 3.38E+08. More preferably, in the exosome NTA detection method, the loading concentration is 1.54E+09 to 2.92E+08.

[0140] In the accompanying drawings of the specification Figure 22A is the comparison diagram of the single particle size results of three repeated experiments of samples S01 to S20. Figure 22B in the accompanying drawings of the specification Figure 22A and Figure 22B, within the dilution factor range of 2× to 100×, the particle size is relatively stable. Among them, the loading concentration range of the directly diluted samples with a dilution factor of 2× to 100× is 1.63E+08 to 2.41E+09, and the loading concentration range of the sequentially diluted samples with a dilution factor of 2× to 100× is 1.38E+08 to 2.14E+09. In other words, based on the particle size detection results, preferably, in the exosome NTA detection method, the loading concentration is 1.38E+08 to 2.41E+09. More preferably, in the exosome NTA detection method, the loading concentration is 1.63E+08 to 2.14E+09.

[0141] Of the accompanying drawings of the specification Figure 23A It is a comparison chart of the single - result of the original concentration of three repeated experiments of samples S01 to S20. Figure 23B Illustrates the average result of the original concentration of the report of three repeated experiments of samples S01 to S20. Refer to Figure 23A and Figure 23B , as the dilution factor increases, the concentration becomes larger and larger. Above 100×, the concentration is unstable. Generally speaking, the sequentially dilution method has a smaller increase amplitude than the direct dilution method and is relatively stable. In the range of 20× to 100×, the concentration is relatively reliable. Among them, the loading concentration range of the directly diluted samples with a dilution factor of 20× to 100× is 1.63E+08 to 6.60E+08, and the loading concentration range of the sequentially diluted samples with a dilution factor of 20× to 100× is 1.38E+08 to 5.50E+08. In other words, based on the parameter result of the original report concentration, preferably, in the exosome NTA detection method, the loading concentration is 1.38E+08 to 6.60E+08. More preferably, in the exosome NTA detection method, the loading concentration is 1.63E+08 to 5.50E+08. It is worth mentioning that the exosome NTA detection method according to this preferred embodiment of the present invention is related to the result of the parameter of the original report concentration using a Malvern NanoSight NS300 nanoparticle tracking analyzer. According to other embodiments of the present invention, when using other instruments to perform NTA detection on exosomes, the influence of different dilution factors on the original report concentration is relatively small.

[0142] Figure 24 Illustrates the calculated loading concentration of samples S01 to S20. Figure 25 Illustrates the calculated loading concentration after background subtraction of samples S01 to S20. Figure 26 Illustrates the calculated original concentration after background subtraction of samples S01 to S20. Figure 27 It is a comparison chart of the original concentration of the report and the calculated original concentration after background subtraction of samples S01 to S20. Refer to Figures 24 to 27, the gradient from 20× (loading concentration 6.60E+08) diluted to 100× (loading concentration 1.38E+08) is closer to the truth. From the results of calculating the loading concentration after background subtraction and calculating the original concentration after background subtraction, the difference from before subtraction increases with the increase of the dilution factor. The difference from 20× (loading concentration 6.60E+08) diluted to 100× (loading concentration 1.38E+08) increases from 2% to about 10%. Therefore, when the dilution factor is large, it is necessary to consider the influence of the background particle concentration of the diluent.

[0143] From the perspective of the stability of multiple detection data, referring to FIGS. Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A , Figure 8A , Figure 9A , Figure 10A , Figure 11A , Figure 12A , Figure 13A , Figure 14A , Figure 15A , Figure 16A , Figure 17A , Figure 18A , Figure 19A , Figure 20A and Figure 21A , from the single - repetition results of particle size, the data from 1× to 50× are relatively stable. Starting from 100×, the data fluctuates greatly. It is speculated that the lower limit of the instrument particle size detection stability is the loading concentration of 2.00E+08. (Loading concentration 2.76E+09~2.92E+08)(Loading concentration 1.38E+08)

[0144] Referring to FIGS. Figure 23A , from the single - repetition results of reporting the original concentration, relatively speaking, the data from 1× to 100× (loading concentration 2.76E+09~1.38E+08) are relatively stable. Starting from 200× (loading concentration 5.10E+07), the data fluctuates greatly. Thus, it is speculated that the lower limit of the instrument particle size detection stability is the loading concentration of 1.00E+08.

[0145] Since the dilution factor is related to the sample, based on the conclusions of this preferred embodiment of the present invention, the loading concentration is as follows:

[0146] For the loading concentration of 2.92E+08~1.54E+09, the particle size peak pattern is good;

[0147] For the loading concentration of 1.38E+08~2.41E+09, the particle size is relatively stable;

[0148] The sample loading concentration is 1.38E+08 to 6.60E+08, and the reported original concentration is relatively reliable;

[0149] Among them, the greater the dilution factor, the greater the influence of the solvent background. Preferably, the sample loading is not diluted more than 50 times.

[0150] The sample loading concentration is 2.92E+08 to 2.76E+09, and the particle size repeatability is good;

[0151] The sample loading concentration is 1.38E+08 to 2.76E+09, and the particle concentration repeatability is good.

[0152] In summary, preferably, the sample loading concentration is 1.38E+08 to 2.76E+09; preferably, the sample loading concentration is 2.00E+08 to 2.00E+09; more preferably, the sample loading concentration is

[0153] 2.92E+08 to 6.60E+08, where the sample loading concentration = reported original concentration / dilution factor.

[0154] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the drawings are only examples and do not limit the present invention. The object of the present invention has been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments. Without departing from the said principles, the embodiments of the present invention can have any deformation or modification.

Claims

1. An exosome nanoparticle tracking analysis detection method, characterized in that, It includes the following steps: A) Processing an exosome sample to obtain a sample with a preset loading concentration; and B) Detecting the sample; In step A, the exosome sample is diluted to have a preset loading concentration, where the preset loading concentration is 1.38E+08 to 2.76E+09 particles / ml.

2. The exosome nanoparticle tracking analysis detection method according to claim 1, wherein the preset loading concentration is 2.92E+08 to 1.54E+09 particles / ml.

3. The exosome nanoparticle tracking analysis detection method according to claim 1, wherein the preset loading concentration is 1.38E+08 to 2.41E+09 particles / ml.

4. The exosome nanoparticle tracking analysis detection method according to claim 1, wherein the preset loading concentration is 1.38E+08 to 2.76E+09 particles / ml.

5. The exosome nanoparticle tracking analysis detection method according to claim 1, wherein the preset loading concentration is 2.00E+08 to 2.00E+09 particles / ml.

6. The exosome nanoparticle tracking analysis detection method according to claim 1, wherein the preset loading concentration is 2.92E+08 to 2.76E+09 particles / ml.

7. The exosome nanoparticle tracking analysis detection method according to claim 1, wherein the preset loading concentration is 2.92E+08 to 6.60E+08 particles / ml.

8. The exosome nanoparticle tracking analysis detection method according to any one of claims 1 to 7, wherein the exosome nanoparticle tracking analysis detection method detects exosomes through a Malvern NanoSight NS300 nanoparticle tracking analyzer.

9. The exosome nanoparticle tracking analysis detection method according to any one of claims 1 to 7, wherein step A includes the following steps: A1) Ultrasonically mixing the exosome sample to obtain an ultrasonically mixed sample; and A2) Diluting the ultrasonically mixed sample to obtain a diluted sample.

10. The exosome nanoparticle tracking analysis detection method according to claim 9, wherein in step A2, the ultrasonically mixed sample is diluted with a dilution solution, and the dilution solution is PBS.

11. The exosome nanoparticle tracking analysis detection method according to claim 10, wherein step A2 is diluted by a direct dilution method.

12. The exosome nanoparticle tracking analysis detection method according to claim 11, wherein in the direct dilution method, a first preset volume of PBS is mixed evenly with a second preset volume of the sample stock solution according to a first preset dilution multiple parameter to obtain a directly diluted sample with a preset dilution multiple.

13. The exosome nanoparticle tracking analysis detection method according to claim 10, wherein step A2 is diluted by direct dilution.

14. The method for detecting exosome nanoparticle tracking analysis according to claim 13, wherein in the direct dilution method, after adding a third preset volume of PBS in advance according to a second preset dilution multiple parameter, a fourth preset volume of sample diluent is respectively measured from the solution of the previous dilution multiple and mixed evenly to obtain an indirectly diluted sample with a preset dilution multiple.

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