WO2024109378A1 - Two-dimensional liquid phase-based exosome purity evaluation method - Google Patents

Two-dimensional liquid phase-based exosome purity evaluation method Download PDF

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WO2024109378A1
WO2024109378A1 PCT/CN2023/124403 CN2023124403W WO2024109378A1 WO 2024109378 A1 WO2024109378 A1 WO 2024109378A1 CN 2023124403 W CN2023124403 W CN 2023124403W WO 2024109378 A1 WO2024109378 A1 WO 2024109378A1
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mobile phase
purity
exosomes
dimensional liquid
liquid chromatography
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PCT/CN2023/124403
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French (fr)
Chinese (zh)
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葛啸虎
韩春乐
陆路
何磊
王淼
王娜
高梦雅
孟晓竹
温智钧
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天津外泌体科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of exosome detection and analysis, and in particular relates to a method for evaluating the purity of exosomes based on two-dimensional liquid phase.
  • Exosomes are lipid bilayer vesicles secreted by cells, with a diameter of about 30 to 150 nm. Exosomes are widely present in various body fluids such as saliva, cell culture fluid, milk, blood and urine. Since exosomes exist in a variety of complex biological environments, exosomes from different sources or even from the same source have great differences in morphology and biochemical characteristics. Exosomes have broad application prospects in the field of disease diagnosis and treatment. Obtaining exosomes of high purity and quality is a key factor in scientific research and application. Therefore, it is crucial to establish a more accurate and efficient method for evaluating the purity of exosomes.
  • Liquid chromatographs can be divided into reverse phase chromatography, normal phase chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography according to their separation modes.
  • Size exclusion is a commonly used detection method for liquid chromatographs.
  • the size exclusion chromatography column is filled with fillers with a certain molecular pore size. When the sample flows through the column, the particles with large particle sizes flow out of the column first, and the samples with small particles flow out later. Therefore, the separation effect of particles of different particle sizes can be achieved, but impurities with a particle size close to that of the main component cannot be analyzed.
  • Exosomes are charged, and the negative charge of exosomes is caused by the negative charge of their surface molecules.
  • Ion chromatography is widely used to separate molecules with different charges. These molecules are captured when they bind to the chromatographic column, and are enriched in large quantities on the ion chromatography column. The exosomes are then eluted using the mobile phase, but impurities with the same charge cannot be analyzed. This is the limitation of simple ion chromatography.
  • This method can only determine partially labeled exosomes, such as CD63, CD9, CD81, etc., but the purity of unlabeled exosomes cannot be determined, so there are certain limitations.
  • the protein/particle method needs to detect protein concentration and particle number separately, and needs to combine the results of the two detection methods, and the detection time is long.
  • the q-PCR method also takes a long time to detect, is easy to be contaminated, and requires high professional operation of personnel.
  • Nanoparticle tracking analysis can only determine partially labeled exosomes, but the purity of unlabeled exosomes cannot be determined, so there are certain limitations. Due to the shortcomings of traditional liquid phase (if the main peak and impurity peak have low separation or overlap and cannot be analyzed), if exosomes are detected by liquid phase method, this major problem needs to be overcome.
  • the present invention provides a method for evaluating the purity of exosomes based on two-dimensional liquid phase.
  • the technical solution adopted by the present invention is: a method for evaluating the purity of exosomes based on two-dimensional liquid phase, wherein the purity of exosomes is evaluated by two-dimensional liquid chromatography; the first dimension uses a size exclusion chromatography column and the mobile phase isocratic elution; the second dimension uses an ion chromatography column and the mobile phase is gradient elution.
  • the exosome sample is first analyzed by first-dimension liquid chromatography, and then the six-way valve is switched to perform central cutting of the main peak of the exosomes to enter the second-dimension liquid chromatography.
  • the first-dimension liquid chromatography mobile phase is Tris buffer with a pH value of 7-7.5, preferably a pH value of 7.2;
  • the second-dimension liquid chromatography mobile phase A is Tris buffer, and the second-dimension liquid chromatography mobile phase B is Tris buffer-salt solution;
  • the pH values of mobile phase A and mobile phase B are 7-7.5, preferably a pH value of 7.2.
  • the concentration of Tris buffer is 20 mM
  • the salt solution is NaCl solution
  • the concentration of NaCl salt is 1.0 M.
  • the first dimension mobile phase elution condition is isocratic elution
  • the mobile phase flow rate is 0.3-0.6 ml/min
  • the liquid phase six-way valve switching time is 3-3.8 min.
  • the second dimension mobile phase elution condition is a gradient elution of mobile phases A and B, the mobile phase flow rate is 0.3 ml/min, including more than two fixed value elution stages; including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7.2, 5 5% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  • the size exclusion chromatography column is a Waters BEH SEC, 2.5 ⁇ m, 4.6 ⁇ 150mm; the ion exchange chromatography column is the anion exchange chromatography column is the BIADEAE-0.1Analytical Column, 1.3 ⁇ m.
  • the purity of exosomes in the first-dimensional liquid chromatography and the second-dimensional liquid chromatography are calculated respectively according to the peak area normalization method, and the product of the two is the purity of the detected exosome sample.
  • the purity evaluation method is simple to operate, has a high level of automation, takes up a short time, has high accuracy, is suitable for simultaneous detection of a large number of samples, and is particularly suitable for purity evaluation work for large-scale industrial production of exosomes in the future, is efficient, and has a controllable process.
  • Fig. 1 is a liquid chromatogram of Example 1 of the present invention
  • Fig. 2 is a liquid chromatogram of Example 2 of the present invention.
  • Fig. 3 is a liquid chromatogram of Example 3 of the present invention.
  • Fig. 4 is a liquid chromatogram of Example 4 of the present invention.
  • FIG. 5 is a schematic diagram of a two-dimensional liquid phase six-way valve pipeline of the present invention.
  • Two-dimensional liquid chromatography is a system that connects two chromatographic columns with different separation mechanisms in series. After the sample passes through the first-dimensional chromatographic column and detector, it is switched to the second-dimensional chromatographic column and detector after capture or cutting. Components that cannot be completely separated in a one-dimensional separation system may be better separated in a two-dimensional system. Column switching modes can be divided into full two-dimensional and heart-cutting. The heart-cutting technology can only enter the part that needs secondary analysis into the second dimension for further analysis. Therefore, the development of a two-dimensional liquid phase method for evaluating exosomes can significantly improve the accuracy of exosome purity analysis and provide a more suitable method for quality control of future industrial production of exosomes.
  • the present invention uses a two-dimensional liquid chromatograph to evaluate the purity of exosomes by combining a size exclusion method and an ion exchange chromatography method.
  • the size exclusion method uses different sizes of material particles for analysis. Due to the different sizes of material particles in the sample, the detector will reflect different peak times of different particle sizes. The chromatographic peak that is obviously far away from the main peak is the impurity peak, which is used to distinguish impurities.
  • the ion exchange chromatography method uses a weak anion chromatographic column to specifically capture negatively charged substances, and exosomes are negatively charged under near-neutral mobile phase conditions. After being captured on a weak anion chromatographic column, the exosomes can be eluted by the second-dimensional mobile phase gradient and enter the detector for secondary analysis.
  • FIG. 5 shows the two-dimensional liquid phase six-way valve pipeline.
  • the sample first enters the size exclusion chromatography column of the first dimension of the liquid chromatograph, and isocratically eluted with the mobile phase of the first dimension liquid phase, so that the exosomes enter the detector for the first analysis. Then, by switching the six-way valve of the liquid chromatograph, the main peak of the exosomes is centrally cut and enters the second-dimensional ion chromatography column to capture and collect it, and then enters the detector for the second analysis through the gradient elution of the second-dimensional mobile phase.
  • the two-dimensional liquid phase six-way valve pipeline is shown in Figure 5.
  • the purity of exosome samples was evaluated by two-dimensional liquid chromatography using a two-dimensional liquid chromatograph; the first dimension used a size exclusion chromatography column with isocratic elution of the mobile phase; the second dimension used an ion chromatography column with gradient elution of the mobile phase.
  • the exosome samples were first analyzed by first-dimensional liquid chromatography, and then the six-way valve was switched to perform central cutting of the main peak of the exosomes to enter the second-dimensional liquid chromatography.
  • the mobile phase of the first-dimensional liquid chromatography was Tris buffer; the mobile phase A of the second-dimensional liquid chromatography was Tris buffer, and the mobile phase B of the second-dimensional liquid chromatography was Tris buffer-salt solution, the Tris buffer concentration was 20mM, the salt solution was NaCl solution, and the NaCl salt concentration was 1.0M; the pH value of mobile phase A and mobile phase B was 7.2.
  • the size exclusion chromatography column is a Waters BEH SEC, 2.5 ⁇ m, 4.6 ⁇ 150mm; the ion exchange chromatography column is the anion exchange chromatography column is the BIA DEAE-0.1Analytical Column, 1.3 ⁇ m.
  • the first-dimension mobile phase elution condition is isocratic elution
  • the mobile phase flow rate is 0.3-0.6 ml/min, preferably 0.5 ml/min
  • the liquid phase six-way valve switching time is 3-3.8 min, wherein the mobile phase flow rate and the six-way valve switching time are inversely proportional.
  • the flow rate is faster, a shorter switching time can be used.
  • the elution time can be longer before switching. The purpose is to ensure that the peak type of the exosomes can be fully reflected.
  • the second dimension mobile phase elution condition was a gradient elution of mobile phases A and B, with a mobile phase flow rate of 0.3 ml/min, including more than two fixed value elution stages; including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7.2, 55% mobile phase A; 7.2-7.3 min, 55-45% mobile phase A; 7.3-8.3 min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  • Liquid phase purity analysis uses the area normalization method.
  • the purity of the main peak of exosomes is the percentage of the main peak area to the total peak area.
  • the purity of exosomes in the first-dimensional liquid chromatography and the second-dimensional liquid chromatography is calculated according to the peak area normalization method.
  • the product of the two is the purity of the detected exosome sample.
  • the main peak purity of exosomes in the first dimension is A
  • the main peak purity of exosomes in the second dimension is B, which is a secondary analysis of the main peak of the first dimension.
  • the final purity of the exosomes is A ⁇ B.
  • the patent "A method for analyzing the charge heterogeneity of exosomes" discloses a method for separating exosome subpopulations based on the charge heterogeneity of exosomes.
  • the present invention is a further study based on this patent.
  • the protective agent is discarded to save costs and simplify the preparation of the mobile phase.
  • ion chromatography is used.
  • the peak time of impurities with the same negative charge in the liquid phase may overlap with the main peak of the exosomes, and the purity of the exosomes cannot be more accurately determined.
  • bovine serum albumin is an impurity that may appear in the process.
  • the present invention adopts a two-dimensional liquid phase method combining size exclusion method with ion chromatography method to make up for this deficiency.
  • the sample first passes through a size exclusion chromatography column, and the main peak of exosomes and impurity peaks can be shown on the liquid chromatogram according to the particle size; for example, according to experimental results, the peak time of bovine serum albumin in the size exclusion method can be clearly distinguished from the time of the main peak of exosomes, and then only the center of the main peak part of the sample is cut into two-dimensional liquid phase and enters the ion chromatography column, and the so-called "pure exosomes" part in the size exclusion method is subjected to secondary analysis.
  • the present invention uses the size exclusion method first, does not use a protective agent, and only analyzes the main peak of exosomes in the size exclusion method by ion chromatography.
  • the mobile phases used in the size exclusion method and the ion chromatography are different mobile phase systems, which allows it to separate five components, which is different from the six components in the previous patent. By developing a liquid phase method, the peaks of different components are farther apart, which facilitates more accurate and complete collection of different fractions.
  • exosomes or exosomes prepared by different processes have different specificities, and a more suitable method needs to be selected according to the type and specificity of exosomes.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the 2D liquid chromatograph was Waters ACQUITY UPLC PLUS Bio.
  • the purity of milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography.
  • the size exclusion column was Waters BEH SEC, 2.5 ⁇ m, 4.6 ⁇ 150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3 ⁇ m; the injection volume was 25 ⁇ l, the column temperature was 25°C, the detection wavelength was 280nm, and the sample chamber was 6°C; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
  • the first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min; the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7 .2min, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the 2D liquid chromatograph was Waters ACQUITY UPLC PLUS Bio.
  • the purity of purified milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography.
  • the size exclusion column was Waters BEH SEC, 2.5 ⁇ m, 4.6 ⁇ 150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3 ⁇ m; the injection volume was 25 ⁇ l, the column temperature was 25°C, the detection wavelength was 280nm, and the sample chamber was 6°C; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
  • the first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min; the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7 .2min, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • the liquid chromatograph was Waters ACQUITY UPLC PLUS Bio, and the purity of milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography.
  • the size exclusion chromatography column was Waters BEH SEC, 2.5 ⁇ m, 4.6 ⁇ 150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3 ⁇ m; the injection volume was 25 ⁇ l, the column temperature was 25°C, the detection wavelength was 280nm, and the sample chamber was 6°C; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
  • the same milk exosomes as in Example 1 were used as samples.
  • the first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min.
  • the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-70% mobile phase A.
  • Phase A 5.0-5.1min, 70-65% mobile phase A; 5.1-6.1min, 65% mobile phase A; 6.1-6.2min, 65-60% mobile phase A; 6.2-7.2min, 60% mobile phase A; 7.2-7.3min, 60-50% mobile phase A; 7.3-8.3min, 50% mobile phase A; 8.3-8.4min, 50-40% mobile phase A; 8.4-9.4min, 40% mobile phase A; 9.4-9.5min, 40-20% mobile phase A; 9.5-10.5min, 20% mobile phase A; 10.5-10.6min, 20-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  • the two-dimensional liquid phase switching valve time is 3.8min.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • the two-dimensional liquid chromatograph was Waters ACQUITY UPLC PLUS Bio, and the purity of the purified milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography as in Example 2.
  • the size exclusion column was Waters BEH SEC, 2.5 ⁇ m, 4.6 ⁇ 150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3 ⁇ m; the injection volume was 25 ⁇ l, the column temperature was 25°C, the detection wavelength was 280nm, and the sample chamber was 6°C; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
  • the first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min; the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7 .2min, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  • the purity of exosomes is evaluated by two-dimensional liquid phase, and the evaluation results are more accurate.
  • the same sample is used for continuous evaluation, which eliminates the sample errors caused by different batches of tests, and is more efficient and faster.
  • the purity detection of milk exosome samples is described in detail in the embodiments of the present invention. This detection scheme can also be used for exosome samples prepared from raw materials such as urine, blood, and saliva.

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Abstract

A two-dimensional liquid phase-based exosome purity evaluation method. By using a method combining a size exclusion method with an ion exchange chromatography method, exosome purity is evaluated by means of a two-dimensional liquid chromatograph. A sample firstly enters a first-dimension size exclusion chromatography column of the liquid chromatograph, and is subjected to isocratic elution by using a mobile phase of a first-dimension liquid phase to analyze an exosome for the first time; then, by switching a six-way valve of the liquid chromatograph, the exosome has a main peak portion subjected to heart cutting to enter a second-dimension ion chromatography column so as to capture and collect the exosome, and the exosome is subjected to gradient elution by means of a second-dimension mobile phase to enter a detector for second analysis. The two-dimensional liquid phase-based exosome purity evaluation method is simple to operate, high in automation level, short in occupied time and high in accuracy, suitable for simultaneous detection of large batches of samples, in particular for purity evaluation work of large-scale industrial production of exosomes in the future, and is efficient and controllable in process.

Description

一种基于二维液相评价外泌体纯度的方法A method for evaluating the purity of exosomes based on two-dimensional liquid chromatography 技术领域Technical Field
本发明属于外泌体检测分析领域，尤其是涉及一种基于二维液相评价外泌体纯度的方法。The present invention belongs to the field of exosome detection and analysis, and in particular relates to a method for evaluating the purity of exosomes based on two-dimensional liquid phase.
背景技术Background technique
外泌体是细胞分泌的脂质双层结构囊泡，直径约为30～150nm，外泌体广泛存在于唾液、细胞培养液、乳液、血液及尿液等多种体液中，由于外泌体存在于多种复杂的生物环境中，不同来源甚至是同一来的外泌体在形态和生化特性方面都有着极大差异源。外泌体在疾病诊断和治疗领域有着广阔的应用前景，获得较高纯度和质量的外泌体是科研及应用的关键要素，因此建立更准确而高效的评价外泌体纯度的方法显得至关重要。Exosomes are lipid bilayer vesicles secreted by cells, with a diameter of about 30 to 150 nm. Exosomes are widely present in various body fluids such as saliva, cell culture fluid, milk, blood and urine. Since exosomes exist in a variety of complex biological environments, exosomes from different sources or even from the same source have great differences in morphology and biochemical characteristics. Exosomes have broad application prospects in the field of disease diagnosis and treatment. Obtaining exosomes of high purity and quality is a key factor in scientific research and application. Therefore, it is crucial to establish a more accurate and efficient method for evaluating the purity of exosomes.
目前评价外泌体纯度的方法有多种多样，对于外泌体纯度分析并没有统一的标准，有蛋白/颗粒法、q-PCR法、纳米颗粒跟踪分析法等。蛋白/颗粒法和q-PCR法检测方法耗费时间过长、样品配制方式复杂，不适用于多批次大量检测；而纳米颗粒跟踪分析法虽然检测时间并不长，但是只能测定部分标记的的外泌体，未标记的外泌体纯度无法测定，准确度不高，因此存在一定的局限性。There are many methods for evaluating the purity of exosomes, and there is no unified standard for the analysis of exosome purity, including protein/particle method, q-PCR method, nanoparticle tracking analysis method, etc. The protein/particle method and q-PCR method are time-consuming and complex in sample preparation, and are not suitable for large-scale testing of multiple batches; and although the nanoparticle tracking analysis method does not take a long time to detect, it can only detect partially labeled exosomes, and the purity of unlabeled exosomes cannot be determined, and the accuracy is not high, so there are certain limitations.
液相色谱仪根据其分离模式不同可以分为反相色谱、正相色谱、离子交换色谱、尺寸排阻色谱、亲和色谱。尺寸排阻法是液相色谱仪常用的检测方法，尺寸排阻色谱柱里面填充着一定分子孔径的填料，当样品流经色谱柱时，大粒径的颗粒率先流出色谱柱，小颗粒的样品后流出，因此可以达到不同粒径颗粒的分离效果，但是无法分析出与主成分物质颗粒大小相近的杂质。Liquid chromatographs can be divided into reverse phase chromatography, normal phase chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography according to their separation modes. Size exclusion is a commonly used detection method for liquid chromatographs. The size exclusion chromatography column is filled with fillers with a certain molecular pore size. When the sample flows through the column, the particles with large particle sizes flow out of the column first, and the samples with small particles flow out later. Therefore, the separation effect of particles of different particle sizes can be achieved, but impurities with a particle size close to that of the main component cannot be analyzed.
外泌体是带有电荷的，外泌体的负电荷是由于其表面分子带负电荷导致的，离子色谱法被广泛应用于不同电荷分子的分离，这些分子与色谱柱结合时被捕捉，大量富集在离子色谱柱上然后采用流动相将外泌体洗脱出来，但是同样带电情况的杂质无法分析出来，这是单纯离子色谱法的局限性。Exosomes are charged, and the negative charge of exosomes is caused by the negative charge of their surface molecules. Ion chromatography is widely used to separate molecules with different charges. These molecules are captured when they bind to the chromatographic column, and are enriched in large quantities on the ion chromatography column. The exosomes are then eluted using the mobile phase, but impurities with the same charge cannot be analyzed. This is the limitation of simple ion chromatography.
目前评价外泌体纯度的方法有多种多样，有蛋白/颗粒法、q-PCR法、纳米颗粒跟踪分析法等。例如，专利《一种从动物血浆中分离外泌体并进行纯度检测的方法》采用q-PCR法测定外泌体纯度，q-PCR法对人员操作专业性要求更高， 且实验过程繁琐、耗时长、易污染；专利《从脐带间充质干细胞制备的外泌体制剂及其方法》其中采用纳米颗粒跟踪分析法测定测定纯度，此方法只能测定部分标记的的外泌体，例如CD63、CD9、CD81等，但是未标记的外泌体纯度无法测定，因此存在一定的局限性。蛋白/颗粒法需要分别检测蛋白浓度和颗粒数，需要结合两种检测方法的结果，检测时间较长。q-PCR法同样检测过程时间较长，易污染，且对人员专业操作要求高。纳米颗粒跟踪分析法只能测定部分标记的的外泌体，但是未标记的外泌体纯度无法测定，因此存在一定的局限性。由于传统液相存在的不足(如果主峰和杂质峰分离度较低或者重合无法进行分析)，外泌体如果采用液相方法检测纯度就需要克服这一大难题。There are many methods for evaluating the purity of exosomes, including protein/particle method, q-PCR method, nanoparticle tracking analysis method, etc. For example, the patent "A method for separating exosomes from animal plasma and conducting purity detection" uses q-PCR method to determine the purity of exosomes. The q-PCR method requires higher professionalism of personnel. Moreover, the experimental process is cumbersome, time-consuming, and easy to be contaminated; the patent "Exosome preparation and method prepared from umbilical cord mesenchymal stem cells" uses nanoparticle tracking analysis to determine the purity. This method can only determine partially labeled exosomes, such as CD63, CD9, CD81, etc., but the purity of unlabeled exosomes cannot be determined, so there are certain limitations. The protein/particle method needs to detect protein concentration and particle number separately, and needs to combine the results of the two detection methods, and the detection time is long. The q-PCR method also takes a long time to detect, is easy to be contaminated, and requires high professional operation of personnel. Nanoparticle tracking analysis can only determine partially labeled exosomes, but the purity of unlabeled exosomes cannot be determined, so there are certain limitations. Due to the shortcomings of traditional liquid phase (if the main peak and impurity peak have low separation or overlap and cannot be analyzed), if exosomes are detected by liquid phase method, this major problem needs to be overcome.
发明内容Summary of the invention
为解决上述技术问题，本发明提供一种基于二维液相评价外泌体纯度的方法。In order to solve the above technical problems, the present invention provides a method for evaluating the purity of exosomes based on two-dimensional liquid phase.
本发明采用的技术方案是：一种基于二维液相评价外泌体纯度的方法，通过二维液相色谱评价外泌体纯度；第一维采用尺寸排阻色谱柱，流动相等度洗脱；第二维采用离子色谱柱，流动相梯度洗脱。The technical solution adopted by the present invention is: a method for evaluating the purity of exosomes based on two-dimensional liquid phase, wherein the purity of exosomes is evaluated by two-dimensional liquid chromatography; the first dimension uses a size exclusion chromatography column and the mobile phase isocratic elution; the second dimension uses an ion chromatography column and the mobile phase is gradient elution.
优选地，先通过第一维液相色谱对外泌体样本首次分析，再切换六通阀对外泌体主峰进行中心切割进入第二维液相色谱。Preferably, the exosome sample is first analyzed by first-dimension liquid chromatography, and then the six-way valve is switched to perform central cutting of the main peak of the exosomes to enter the second-dimension liquid chromatography.
优选地，第一维液相色谱流动相为Tris缓冲液，pH值为7-7.5，优选pH值为7.2；第二维液相色谱流动相A为Tris缓冲液，第二维液相色谱流动相B为Tris缓冲液-盐溶液；流动相A和流动相B的pH值为7-7.5，优选pH值为7.2。Preferably, the first-dimension liquid chromatography mobile phase is Tris buffer with a pH value of 7-7.5, preferably a pH value of 7.2; the second-dimension liquid chromatography mobile phase A is Tris buffer, and the second-dimension liquid chromatography mobile phase B is Tris buffer-salt solution; the pH values of mobile phase A and mobile phase B are 7-7.5, preferably a pH value of 7.2.
优选地，Tris缓冲液浓度为20mM，盐溶液为NaCl溶液，NaCl盐浓度为1.0M。Preferably, the concentration of Tris buffer is 20 mM, the salt solution is NaCl solution, and the concentration of NaCl salt is 1.0 M.
优选地，第一维流动相洗脱条件为等度洗脱，流动相流速为0.3-0.6ml/min；液相六通阀切换阀时间分别为3-3.8min。Preferably, the first dimension mobile phase elution condition is isocratic elution, the mobile phase flow rate is 0.3-0.6 ml/min; the liquid phase six-way valve switching time is 3-3.8 min.
优选地，第二维流动相洗脱条件为流动相A和B梯度洗脱，流动相流速为0.3ml/min，包括多余两个的固定值洗脱阶段；包括0-4.3min，100％流动相A；4.3-5.0min，100-75％流动相A；5.0-5.1min，75-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-55％流动相A；6.2-7.2，55％流动相A；7.2-7.3min，55-45％流动相A；7.3-8.3min，45％流动相A；8.3-8.4min，45-35％流动相A；8.4-9.4min，35％流动相A；9.4-9.5min，35-15％流动相A；9.5-10.5min，15％流动相A；10.5-10.6min，15-0％流动相A；10.6-12min，0％流动相A。 Preferably, the second dimension mobile phase elution condition is a gradient elution of mobile phases A and B, the mobile phase flow rate is 0.3 ml/min, including more than two fixed value elution stages; including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7.2, 5 5% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
优选地，尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为阴离子交换色谱柱为BIADEAE-0.1Analytical Column，1.3μm。Preferably, the size exclusion chromatography column is a Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column is the anion exchange chromatography column is the BIADEAE-0.1Analytical Column, 1.3μm.
优选地，在所得图谱中，根据峰面积归一化法分别计算第一维液相色谱和第二维液相色谱中外泌体纯度，二者的乘积即为所检测外泌体样本的纯度。Preferably, in the obtained spectrum, the purity of exosomes in the first-dimensional liquid chromatography and the second-dimensional liquid chromatography are calculated respectively according to the peak area normalization method, and the product of the two is the purity of the detected exosome sample.
本发明具有的优点和积极效果是：本纯度评价方法操作简单，自动化水平高，占用时间短，准确度高，适合大批样品同时检测，尤其适合未来外泌体大规模工业化生产的纯度评价工作，高效且过程可控。The advantages and positive effects of the present invention are: the purity evaluation method is simple to operate, has a high level of automation, takes up a short time, has high accuracy, is suitable for simultaneous detection of a large number of samples, and is particularly suitable for purity evaluation work for large-scale industrial production of exosomes in the future, is efficient, and has a controllable process.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是本发明实施例1的液相色谱图；Fig. 1 is a liquid chromatogram of Example 1 of the present invention;
图2是本发明实施例2的液相色谱图；Fig. 2 is a liquid chromatogram of Example 2 of the present invention;
图3是本发明实施例3的液相色谱图；Fig. 3 is a liquid chromatogram of Example 3 of the present invention;
图4是本发明实施例4的液相色谱图；Fig. 4 is a liquid chromatogram of Example 4 of the present invention;
图5是本发明二维液相六通阀管路示意图。FIG. 5 is a schematic diagram of a two-dimensional liquid phase six-way valve pipeline of the present invention.
具体实施方式Detailed ways
二维液相色谱是将分离机理不同的两支色谱柱串联起来的系统，样品经过第一维的色谱柱和检测器后，通过捕集或切割后切换进入第二维色谱柱和检测器中。在一维分离系统中不能完全分离的组分，在二维系统中可能可以更好地分离。柱切换模式可以分为全二维和中心切割，中心切割技术可以只将需要二次分析的部分进入到第二维中进一步分析。因此，开发一种二维液相评价外泌体的方法可以明显提高针对外泌体纯度分析的准确度，对于未来外泌体工业化生产的质量控制提供一种更适宜的方法。Two-dimensional liquid chromatography is a system that connects two chromatographic columns with different separation mechanisms in series. After the sample passes through the first-dimensional chromatographic column and detector, it is switched to the second-dimensional chromatographic column and detector after capture or cutting. Components that cannot be completely separated in a one-dimensional separation system may be better separated in a two-dimensional system. Column switching modes can be divided into full two-dimensional and heart-cutting. The heart-cutting technology can only enter the part that needs secondary analysis into the second dimension for further analysis. Therefore, the development of a two-dimensional liquid phase method for evaluating exosomes can significantly improve the accuracy of exosome purity analysis and provide a more suitable method for quality control of future industrial production of exosomes.
本发明通过二维液相色谱仪采用尺寸排阻法和离子交换色谱法结合的方法来评价外泌体纯度。尺寸排阻法利用的物质颗粒大小不同进行分析，由于样品内物质颗粒大小不同，在检测器会反映出不同颗粒大小的出峰时间不同，明显远离主峰的色谱峰为杂质峰，以此来区分杂质。离子交换色谱法利用弱阴离子色谱柱可以特异性捕集带负电荷的物质，而外泌体在近中性流动相条件下带负电荷，外泌体可以在弱阴离子色谱柱上捕集后通过第二维流动相梯度洗脱，进入检测器进行二次分析。The present invention uses a two-dimensional liquid chromatograph to evaluate the purity of exosomes by combining a size exclusion method and an ion exchange chromatography method. The size exclusion method uses different sizes of material particles for analysis. Due to the different sizes of material particles in the sample, the detector will reflect different peak times of different particle sizes. The chromatographic peak that is obviously far away from the main peak is the impurity peak, which is used to distinguish impurities. The ion exchange chromatography method uses a weak anion chromatographic column to specifically capture negatively charged substances, and exosomes are negatively charged under near-neutral mobile phase conditions. After being captured on a weak anion chromatographic column, the exosomes can be eluted by the second-dimensional mobile phase gradient and enter the detector for secondary analysis.
传统液相只能单独采用某一种色谱方法进行分析，单独使用尺寸排阻色谱法 无法分析出和外泌体颗粒大小相近的杂质，而单独使用离子交换色谱法又无法将主峰和牛血清白蛋白等杂质分析出来，因此，我们创造性地采用二维液相将尺寸排阻法和离子交换色谱法结合起来采用中心切割测定外泌体纯度，如图5所示为二维液相六通阀管路，通过二维液相进行分析评价，可以更准确地测定外泌体纯度。第一维的纯度计算是初步纯度计算，第二维将第一维主峰部分的“纯外泌体”再进行第二次分析，通过两个维度的综合分析，实现对样本更为准确的判断。Traditional liquid chromatography can only be analyzed using a single chromatographic method, and size exclusion chromatography alone Impurities with similar sizes to exosome particles cannot be analyzed, and ion exchange chromatography alone cannot analyze the main peak and impurities such as bovine serum albumin. Therefore, we creatively use two-dimensional liquid phase to combine size exclusion method and ion exchange chromatography to determine the purity of exosomes by central cutting. Figure 5 shows the two-dimensional liquid phase six-way valve pipeline. Through two-dimensional liquid phase analysis and evaluation, the purity of exosomes can be determined more accurately. The purity calculation of the first dimension is a preliminary purity calculation, and the second dimension performs a second analysis on the "pure exosomes" of the main peak of the first dimension. Through the comprehensive analysis of the two dimensions, a more accurate judgment of the sample can be achieved.
样品首先进入液相色谱仪第一维的尺寸排阻色谱柱，采用第一维液相的流动相等度洗脱，使外泌体进入检测器进行首次分析。再通过切换液相色谱仪的六通阀，对外泌体主峰部分进行中心切割进入第二维的离子色谱柱对其进行捕捉收集，之后通过第二维的流动相梯度洗脱进入检测器进行第二次分析。二维液相六通阀管路如图5所示。The sample first enters the size exclusion chromatography column of the first dimension of the liquid chromatograph, and isocratically eluted with the mobile phase of the first dimension liquid phase, so that the exosomes enter the detector for the first analysis. Then, by switching the six-way valve of the liquid chromatograph, the main peak of the exosomes is centrally cut and enters the second-dimensional ion chromatography column to capture and collect it, and then enters the detector for the second analysis through the gradient elution of the second-dimensional mobile phase. The two-dimensional liquid phase six-way valve pipeline is shown in Figure 5.
实施时，将外泌体样本通过二维液相色谱评价外泌体纯度，使用二维液相色谱仪；第一维采用尺寸排阻色谱柱，流动相等度洗脱；第二维采用离子色谱柱，流动相梯度洗脱。先通过第一维液相色谱对外泌体样本首次分析，再切换六通阀对外泌体主峰进行中心切割进入第二维液相色谱。第一维液相色谱流动相为Tris缓冲液；第二维液相色谱流动相A为Tris缓冲液，第二维液相色谱流动相B为Tris缓冲液-盐溶液，Tris缓冲液浓度为20mM，盐溶液为NaCl溶液，NaCl盐浓度为1.0M；流动相A和流动相B的pH值为7.2。During implementation, the purity of exosome samples was evaluated by two-dimensional liquid chromatography using a two-dimensional liquid chromatograph; the first dimension used a size exclusion chromatography column with isocratic elution of the mobile phase; the second dimension used an ion chromatography column with gradient elution of the mobile phase. The exosome samples were first analyzed by first-dimensional liquid chromatography, and then the six-way valve was switched to perform central cutting of the main peak of the exosomes to enter the second-dimensional liquid chromatography. The mobile phase of the first-dimensional liquid chromatography was Tris buffer; the mobile phase A of the second-dimensional liquid chromatography was Tris buffer, and the mobile phase B of the second-dimensional liquid chromatography was Tris buffer-salt solution, the Tris buffer concentration was 20mM, the salt solution was NaCl solution, and the NaCl salt concentration was 1.0M; the pH value of mobile phase A and mobile phase B was 7.2.
本发明某些实施例中，尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为阴离子交换色谱柱为BIA DEAE-0.1Analytical Column，1.3μm。In certain embodiments of the present invention, the size exclusion chromatography column is a Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column is the anion exchange chromatography column is the BIA DEAE-0.1Analytical Column, 1.3μm.
第一维流动相洗脱条件为等度洗脱，流动相流速为0.3-0.6ml/min，优选为0.5ml/min，液相六通阀切换阀时间分别为3-3.8min，其中流动相流速和六通阀切换时间呈反比，流速较快时，可采用较短的切换时间，流速较慢时，可洗脱较长时间后再切换，其目的在于，保证外泌体所在峰型能够完整体现。第二维流动相洗脱条件为流动相A和B梯度洗脱，流动相流速为0.3ml/min，包括多余两个的固定值洗脱阶段；包括0-4.3min，100％流动相A；4.3-5.0min，100-75％流动相A；5.0-5.1min，75-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-55％流动相A；6.2-7.2，55％流动相A；7.2-7.3min，55-45％流动相A；7.3-8.3min， 45％流动相A；8.3-8.4min，45-35％流动相A；8.4-9.4min，35％流动相A；9.4-9.5min，35-15％流动相A；9.5-10.5min，15％流动相A；10.5-10.6min，15-0％流动相A；10.6-12min，0％流动相A。The first-dimension mobile phase elution condition is isocratic elution, the mobile phase flow rate is 0.3-0.6 ml/min, preferably 0.5 ml/min, and the liquid phase six-way valve switching time is 3-3.8 min, wherein the mobile phase flow rate and the six-way valve switching time are inversely proportional. When the flow rate is faster, a shorter switching time can be used. When the flow rate is slower, the elution time can be longer before switching. The purpose is to ensure that the peak type of the exosomes can be fully reflected. The second dimension mobile phase elution condition was a gradient elution of mobile phases A and B, with a mobile phase flow rate of 0.3 ml/min, including more than two fixed value elution stages; including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7.2, 55% mobile phase A; 7.2-7.3 min, 55-45% mobile phase A; 7.3-8.3 min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
液相纯度分析采用面积归一化法，外泌体主峰纯度为主峰峰面积占全部峰面积的百分比，在所得图谱中，根据峰面积归一化法分别计算第一维液相色谱和第二维液相色谱中外泌体纯度，二者的乘积即为所检测外泌体样本的纯度。如，第一维中外泌体主峰纯度为A，第二维中外泌体主峰纯度B是对第一维主峰部分的二次分析，其外泌体最终纯度为A×B。Liquid phase purity analysis uses the area normalization method. The purity of the main peak of exosomes is the percentage of the main peak area to the total peak area. In the obtained spectrum, the purity of exosomes in the first-dimensional liquid chromatography and the second-dimensional liquid chromatography is calculated according to the peak area normalization method. The product of the two is the purity of the detected exosome sample. For example, the main peak purity of exosomes in the first dimension is A, and the main peak purity of exosomes in the second dimension is B, which is a secondary analysis of the main peak of the first dimension. The final purity of the exosomes is A×B.
专利《一种分析外泌体电荷异质性的方法》公开一种根据外泌体电荷异质性分离得到外泌体亚群的方法，本发明为在此专利基础上的更深入研究。舍去了保护剂以节省成本并简化流动相配制。该发明方案中采用离子色谱法，带有同样负电荷的杂质在液相的出峰时间可能会与外泌体主峰重合，无法更准确测定外泌体纯度；例如，根据我们后续实验发现，牛血清白蛋白作为工艺中可能出现的杂质，在离子交换色谱法中液相色谱图中，其牛血清白蛋白会与外泌体其中一个峰的保留时间相同，因此单独使用离子交换色谱法对于更准确地测定外泌体纯度的专属性不适用。而本发明采用尺寸排阻法结合离子色谱法的二维液相方法可以弥补这种不足，本发明方法中样品首先通过尺寸排阻色谱柱，可以根据颗粒大小在液相色谱图上表现出外泌体主峰及杂质峰；例如，根据实验结果表明，牛血清白蛋白在尺寸排阻法中出峰时间与外泌体主峰的时间可以明显区分开，之后再通过二维液相只将主峰部分样品中心切割进入离子色谱柱，将在尺寸排阻法中所谓的“纯外泌体”部分进行二次分析，通过带电量不同可以分析出尺寸排阻法无法分析出的杂质，使纯度测定更为准确，更准确地确定图谱中各组分的相对含量。而且本发明相对于之前的专利，由于变更了先使用尺寸排阻法、不使用保护剂且只将尺寸排阻法中外泌体主峰部分再通过离子色谱法分析，尺寸排阻法与离子色谱法使用的流动相为不同流动相系统方式，这使得其分出了五个组分，不同于之前专利的六个组分，且通过开发液相方法使其不同组分的峰离得更远，便于更准确、更完整地收集不同馏分。 The patent "A method for analyzing the charge heterogeneity of exosomes" discloses a method for separating exosome subpopulations based on the charge heterogeneity of exosomes. The present invention is a further study based on this patent. The protective agent is discarded to save costs and simplify the preparation of the mobile phase. In this invention scheme, ion chromatography is used. The peak time of impurities with the same negative charge in the liquid phase may overlap with the main peak of the exosomes, and the purity of the exosomes cannot be more accurately determined. For example, according to our subsequent experimental findings, bovine serum albumin is an impurity that may appear in the process. In the liquid chromatogram of ion exchange chromatography, its bovine serum albumin will have the same retention time as one of the peaks of the exosomes. Therefore, the use of ion exchange chromatography alone is not suitable for more accurate determination of the purity of exosomes. The present invention adopts a two-dimensional liquid phase method combining size exclusion method with ion chromatography method to make up for this deficiency. In the method of the present invention, the sample first passes through a size exclusion chromatography column, and the main peak of exosomes and impurity peaks can be shown on the liquid chromatogram according to the particle size; for example, according to experimental results, the peak time of bovine serum albumin in the size exclusion method can be clearly distinguished from the time of the main peak of exosomes, and then only the center of the main peak part of the sample is cut into two-dimensional liquid phase and enters the ion chromatography column, and the so-called "pure exosomes" part in the size exclusion method is subjected to secondary analysis. The impurities that cannot be analyzed by the size exclusion method can be analyzed by the different charges, so that the purity determination is more accurate and the relative content of each component in the spectrum can be determined more accurately. Moreover, compared with the previous patent, the present invention uses the size exclusion method first, does not use a protective agent, and only analyzes the main peak of exosomes in the size exclusion method by ion chromatography. The mobile phases used in the size exclusion method and the ion chromatography are different mobile phase systems, which allows it to separate five components, which is different from the six components in the previous patent. By developing a liquid phase method, the peaks of different components are farther apart, which facilitates more accurate and complete collection of different fractions.
不同种类的外泌体或者其不同制备工艺的外泌体有其不同的专属性，需要根据外泌体种类和特异性进行选择更为适合的方法。Different types of exosomes or exosomes prepared by different processes have different specificities, and a more suitable method needs to be selected according to the type and specificity of exosomes.
下面结合附图对本发明方案做出说明，其中，未具体说明操作步骤的实验方法，均按照相应商品说明书进行，实施例中所用到的仪器、试剂、耗材如无特殊说明，均可从商业公司购买得到。The scheme of the present invention is described below in conjunction with the accompanying drawings, wherein the experimental methods without specific operating steps are all carried out in accordance with the corresponding product instructions, and the instruments, reagents, and consumables used in the examples can all be purchased from commercial companies unless otherwise specified.
实施例1：Embodiment 1:
二维液相色谱仪为Waters ACQUITY UPLC PLUS Bio，采用尺寸排阻法结合离子交换色谱法分析牛奶外泌体纯度，尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为弱阴离子色谱柱，BIA DEAE-0.1 Analytical Column，1.3μm；进样量为25μl，柱温25℃，检测波长280nm，样品仓6℃；第一维流动相和第二维流动相A均使用20mM的Tris缓冲液(pH 7.2)，第二维流动相B为20mM的Tris，1M NaCl溶液(pH 7.2)。The 2D liquid chromatograph was Waters ACQUITY UPLC PLUS Bio. The purity of milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography. The size exclusion column was Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3μm; the injection volume was 25μl, the column temperature was 25℃, the detection wavelength was 280nm, and the sample chamber was 6℃; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
第一维流动相洗脱条件为流动相A等度洗脱，流速为0.5ml/min；第二维流动相洗脱条件为流动相A和B梯度洗脱，流速为0.3ml/min，包括0-4.3min，100％流动相A；4.3-5.0min，100-75％流动相A；5.0-5.1min，75-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-55％流动相A；6.2-7.2min，55％流动相A；7.2-7.3min，55-45％流动相A；7.3-8.3min，45％流动相A；8.3-8.4min，45-35％流动相A；8.4-9.4min，35％流动相A；9.4-9.5min，35-15％流动相A；9.5-10.5min，15％流动相A；10.5-10.6min，15-0％流动相A；10.6-12min，0％流动相A。二维液相切换阀时间为3.5min。The first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min; the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7 .2min, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A. The 2D liquid phase switching valve time is 3.5min.
结果如图1所示，0-3.5min为第一维尺寸排阻法色谱图，3.5-12min为第二维离子交换色谱法色谱图，根据峰面积归一化法，在第一维中外泌体纯度为74.15％，在第二维中外泌体纯度为99.22％，外泌体最终纯度则为74.15％×99.22％＝73.57％。对于第一维和第二维都检测出杂质，色谱图峰型和每个峰之间的分离度良好，纯度检测仅需要12分钟，高效快捷。从图1结果也能够看出，第二维离子交换色谱中能够体现第一维离子交换色谱无法分辨的杂质。 The results are shown in Figure 1. 0-3.5min is the first-dimension size exclusion chromatogram, and 3.5-12min is the second-dimension ion exchange chromatography chromatogram. According to the peak area normalization method, the purity of exosomes in the first dimension is 74.15%, and the purity of exosomes in the second dimension is 99.22%. The final purity of exosomes is 74.15% × 99.22% = 73.57%. Impurities were detected in both the first and second dimensions, and the separation between the chromatogram peak shape and each peak was good. The purity detection only took 12 minutes, which was efficient and fast. It can also be seen from the results in Figure 1 that the second-dimensional ion exchange chromatography can reflect the impurities that cannot be distinguished by the first-dimensional ion exchange chromatography.
实施例2：Embodiment 2:
二维液相色谱仪为Waters ACQUITY UPLC PLUS Bio，采用尺寸排阻法结合离子交换色谱法分析已进行纯化后的牛奶外泌体纯度，尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为弱阴离子色谱柱，BIA DEAE-0.1 Analytical Column，1.3μm；进样量为25μl，柱温25℃，检测波长280nm，样品仓6℃；第一维流动相和第二维流动相A均使用20mM的Tris缓冲液(pH 7.2)，第二维流动相B为20mM的Tris，1M NaCl溶液(pH 7.2)。The 2D liquid chromatograph was Waters ACQUITY UPLC PLUS Bio. The purity of purified milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography. The size exclusion column was Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3μm; the injection volume was 25μl, the column temperature was 25℃, the detection wavelength was 280nm, and the sample chamber was 6℃; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
第一维流动相洗脱条件为流动相A等度洗脱，流速为0.5ml/min；第二维流动相洗脱条件为流动相A和B梯度洗脱，流速为0.3ml/min，包括0-4.3min，100％流动相A；4.3-5.0min，100-75％流动相A；5.0-5.1min，75-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-55％流动相A；6.2-7.2min，55％流动相A；7.2-7.3min，55-45％流动相A；7.3-8.3min，45％流动相A；8.3-8.4min，45-35％流动相A；8.4-9.4min，35％流动相A；9.4-9.5min，35-15％流动相A；9.5-10.5min，15％流动相A；10.5-10.6min，15-0％流动相A；10.6-12min，0％流动相A。二维液相切换阀时间为3.5min。The first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min; the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7 .2min, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A. The 2D liquid phase switching valve time is 3.5min.
结果如图2所示，第一维尺寸排阻法色谱图和第二维离子交换色谱法色谱图中均无杂质峰出现；本实施例为对纯化后的外泌体样本进行评价，样品纯度为100％，可作为“标准品”色谱图，能够直观对比其他实施例中杂质出现的位置。The results are shown in Figure 2. No impurity peaks appeared in the first-dimensional size exclusion chromatography chromatogram and the second-dimensional ion exchange chromatography chromatogram. This example evaluates the purified exosome samples, and the sample purity is 100%, which can be used as a "standard" chromatogram to intuitively compare the locations of impurities in other examples.
实施例3：Embodiment 3:
维液相色谱仪为Waters ACQUITY UPLC PLUS Bio，采用尺寸排阻法结合离子交换色谱法分析牛奶外泌体纯度，尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为弱阴离子色谱柱，BIA DEAE-0.1 Analytical Column，1.3μm；进样量为25μl，柱温25℃，检测波长280nm，样品仓6℃；第一维流动相和第二维流动相A均使用20mM的Tris缓冲液(pH 7.2)，第二维流动相B为20mM的Tris，1M NaCl溶液(pH 7.2)。The liquid chromatograph was Waters ACQUITY UPLC PLUS Bio, and the purity of milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography. The size exclusion chromatography column was Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3μm; the injection volume was 25μl, the column temperature was 25℃, the detection wavelength was 280nm, and the sample chamber was 6℃; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
采用与实施例一相同的牛奶外泌体作为样品，第一维流动相洗脱条件为流动相A等度洗脱，流速为0.5ml/min；第二维流动相洗脱条件为流动相A和B梯度洗脱，流速为0.3ml/min，包括0-4.3min，100％流动相A；4.3-5.0min，100-70％流动 相A；5.0-5.1min，70-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-60％流动相A；6.2-7.2min，60％流动相A；7.2-7.3min，60-50％流动相A；7.3-8.3min，50％流动相A；8.3-8.4min，50-40％流动相A；8.4-9.4min，40％流动相A；9.4-9.5min，40-20％流动相A；9.5-10.5min，20％流动相A；10.5-10.6min，20-0％流动相A；10.6-12min，0％流动相A。二维液相切换阀时间为3.8min。The same milk exosomes as in Example 1 were used as samples. The first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min. The second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-70% mobile phase A. Phase A; 5.0-5.1min, 70-65% mobile phase A; 5.1-6.1min, 65% mobile phase A; 6.1-6.2min, 65-60% mobile phase A; 6.2-7.2min, 60% mobile phase A; 7.2-7.3min, 60-50% mobile phase A; 7.3-8.3min, 50% mobile phase A; 8.3-8.4min, 50-40% mobile phase A; 8.4-9.4min, 40% mobile phase A; 9.4-9.5min, 40-20% mobile phase A; 9.5-10.5min, 20% mobile phase A; 10.5-10.6min, 20-0% mobile phase A; 10.6-12min, 0% mobile phase A. The two-dimensional liquid phase switching valve time is 3.8min.
结果如图3所示，0-3.8min为第一维尺寸排阻法色谱图，3.8-12min为第二维离子交换色谱法色谱图，根据峰面积归一化法，在第一维中外泌体纯度为73.97％，在第二维中外泌体纯度为100.00％，外泌体最终纯度则为74.15％×100.00％＝73.97％；第二维未检测到杂质。与实施例1比较，本实施例第二维流动相采用了不同的梯度洗脱条件，能够看出，流动相盐浓度对于液相色谱仪的检测有较大影响。The results are shown in Figure 3. 0-3.8min is the first dimension size exclusion chromatogram, 3.8-12min is the second dimension ion exchange chromatography chromatogram, according to the peak area normalization method, the purity of exosomes in the first dimension is 73.97%, the purity of exosomes in the second dimension is 100.00%, and the final purity of exosomes is 74.15% × 100.00% = 73.97%; no impurities were detected in the second dimension. Compared with Example 1, the second dimension mobile phase of this embodiment adopts different gradient elution conditions, and it can be seen that the salt concentration of the mobile phase has a great influence on the detection of the liquid chromatograph.
实施例4：Embodiment 4:
二维液相色谱仪为Waters ACQUITY UPLC PLUS Bio，采用尺寸排阻法结合离子交换色谱法分析与实施例2相同的已进行纯化后的牛奶外泌体纯度，尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为弱阴离子色谱柱，BIA DEAE-0.1 Analytical Column，1.3μm；进样量为25μl，柱温25℃，检测波长280nm，样品仓6℃；第一维流动相和第二维流动相A均使用20mM的Tris缓冲液(pH 7.2)，第二维流动相B为20mM的Tris，1M NaCl溶液(pH 7.2)。The two-dimensional liquid chromatograph was Waters ACQUITY UPLC PLUS Bio, and the purity of the purified milk exosomes was analyzed by size exclusion method combined with ion exchange chromatography as in Example 2. The size exclusion column was Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column was a weak anion chromatography column, BIA DEAE-0.1 Analytical Column, 1.3μm; the injection volume was 25μl, the column temperature was 25℃, the detection wavelength was 280nm, and the sample chamber was 6℃; the first-dimension mobile phase and the second-dimension mobile phase A both used 20mM Tris buffer (pH 7.2), and the second-dimension mobile phase B was 20mM Tris, 1M NaCl solution (pH 7.2).
第一维流动相洗脱条件为流动相A等度洗脱，流速为0.5ml/min；第二维流动相洗脱条件为流动相A和B梯度洗脱，流速为0.3ml/min，包括0-4.3min，100％流动相A；4.3-5.0min，100-75％流动相A；5.0-5.1min，75-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-55％流动相A；6.2-7.2min，55％流动相A；7.2-7.3min，55-45％流动相A；7.3-8.3min，45％流动相A；8.3-8.4min，45-35％流动相A；8.4-9.4min，35％流动相A；9.4-9.5min，35-15％流动相A；9.5-10.5min，15％流动相A；10.5-10.6min，15-0％流动相A；10.6-12min，0％流动相A。二维液相切换阀时间为2.5min。The first dimension mobile phase elution condition was isocratic elution of mobile phase A at a flow rate of 0.5 ml/min; the second dimension mobile phase elution condition was gradient elution of mobile phases A and B at a flow rate of 0.3 ml/min, including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55% mobile phase A; 6.2-7 .2min, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A. The 2D liquid phase switching valve time is 2.5min.
结果如图4所示，由于切换阀时间过早，造成外泌体主峰不能完全显示进入第二维，并且第二维出现的峰型与图3标准品的峰型不一致；由此可见，切换阀 时间对外泌体分析有较大影响。The results are shown in Figure 4. Due to the early switching time of the valve, the main peak of the exosomes cannot be fully displayed in the second dimension, and the peak shape in the second dimension is inconsistent with the peak shape of the standard in Figure 3. It can be seen that the switching valve Time has a significant impact on exosome analysis.
通过二维液相来评价外泌体纯度，评价结果更为精准，采用相同的样本连续测评，杜绝了不同批次检测时带来的样本误差，并且效率更高，检测速度更快。本发明实施例中详细描述了对牛奶外泌体样本的纯度检测，本检测方案还能够用于尿液、血液、唾液等原料制备得到的外泌体样本。The purity of exosomes is evaluated by two-dimensional liquid phase, and the evaluation results are more accurate. The same sample is used for continuous evaluation, which eliminates the sample errors caused by different batches of tests, and is more efficient and faster. The purity detection of milk exosome samples is described in detail in the embodiments of the present invention. This detection scheme can also be used for exosome samples prepared from raw materials such as urine, blood, and saliva.
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。 The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of application of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims (8)

  1. 一种基于二维液相评价外泌体纯度的方法，其特征在于：通过二维液相色谱评价外泌体纯度；第一维采用尺寸排阻色谱柱，流动相等度洗脱；第二维采用离子色谱柱，流动相梯度洗脱。A method for evaluating the purity of exosomes based on two-dimensional liquid chromatography, characterized in that: the purity of exosomes is evaluated by two-dimensional liquid chromatography; the first dimension uses a size exclusion chromatography column and the mobile phase is isocratic elution; the second dimension uses an ion chromatography column and the mobile phase is gradient elution.
  2. 根据权利要求1所述的基于二维液相评价外泌体纯度的方法，其特征在于：先通过第一维液相色谱对外泌体样本首次分析，再切换六通阀对外泌体主峰进行中心切割进入第二维液相色谱。The method for evaluating the purity of exosomes based on two-dimensional liquid chromatography according to claim 1 is characterized in that: the exosome sample is first analyzed by first-dimensional liquid chromatography, and then the six-way valve is switched to perform central cutting of the main peak of the exosomes to enter the second-dimensional liquid chromatography.
  3. 根据权利要求2所述的基于二维液相评价外泌体纯度的方法，其特征在于：第一维液相色谱流动相为Tris缓冲液，pH值为7-7.5；第二维液相色谱流动相A为Tris缓冲液，第二维液相色谱流动相B为Tris缓冲液-盐溶液；流动相A和流动相B的pH值为7-7.5。The method for evaluating the purity of exosomes based on two-dimensional liquid chromatography according to claim 2, characterized in that: the first-dimensional liquid chromatography mobile phase is Tris buffer with a pH value of 7-7.5; the second-dimensional liquid chromatography mobile phase A is Tris buffer, and the second-dimensional liquid chromatography mobile phase B is Tris buffer-salt solution; the pH values of mobile phase A and mobile phase B are 7-7.5.
  4. 根据权利要求3所述的基于二维液相评价外泌体纯度的方法，其特征在于：Tris缓冲液浓度为20mM，盐溶液为NaCl溶液，NaCl盐浓度为1.0M。The method for evaluating the purity of exosomes based on two-dimensional liquid phase according to claim 3, characterized in that the concentration of Tris buffer is 20 mM, the salt solution is NaCl solution, and the NaCl salt concentration is 1.0 M.
  5. 根据权利要求1-4中任一所述的基于二维液相评价外泌体纯度的方法，其特征在于：第一维流动相洗脱条件为等度洗脱，流动相流速为0.3-0.6ml/min，液相六通阀切换阀时间分别为3min-3.8min。The method for evaluating the purity of exosomes based on two-dimensional liquid phase according to any one of claims 1 to 4, characterized in that: the elution condition of the first-dimensional mobile phase is isocratic elution, the mobile phase flow rate is 0.3-0.6 ml/min, and the liquid phase six-way valve switching valve time is 3 min-3.8 min respectively.
  6. 根据权利要求5所述的基于二维液相评价外泌体纯度的方法，其特征在于：第二维流动相洗脱条件为流动相A和B梯度洗脱，流动相流速为0.3ml/min，包括多余两个的固定值洗脱阶段；包括0-4.3min，100％流动相A；4.3-5.0min，100-75％流动相A；5.0-5.1min，75-65％流动相A；5.1-6.1min，65％流动相A；6.1-6.2min，65-55％流动相A；6.2-7.2，55％流动相A；7.2-7.3min，55-45％流动相A；7.3-8.3min，45％流动相A；8.3-8.4min，45-35％流动相A；8.4-9.4min，35％流动相A；9.4-9.5min，35-15％流动相A；9.5-10.5min，15％流动相A；10.5-10.6min，15-0％流动相A；10.6-12min，0％流动相A。The method for evaluating the purity of exosomes based on two-dimensional liquid phase according to claim 5, characterized in that: the second-dimensional mobile phase elution condition is a gradient elution of mobile phases A and B, the mobile phase flow rate is 0.3 ml/min, including more than two fixed value elution stages; including 0-4.3 min, 100% mobile phase A; 4.3-5.0 min, 100-75% mobile phase A; 5.0-5.1 min, 75-65% mobile phase A; 5.1-6.1 min, 65% mobile phase A; 6.1-6.2 min, 65-55 % mobile phase A; 6.2-7.2, 55% mobile phase A; 7.2-7.3min, 55-45% mobile phase A; 7.3-8.3min, 45% mobile phase A; 8.3-8.4min, 45-35% mobile phase A; 8.4-9.4min, 35% mobile phase A; 9.4-9.5min, 35-15% mobile phase A; 9.5-10.5min, 15% mobile phase A; 10.5-10.6min, 15-0% mobile phase A; 10.6-12min, 0% mobile phase A.
  7. 根据权利要求1-4和6中任一所述的基于二维液相评价外泌体纯度的方法，其特征在于：尺寸排阻色谱柱为Waters BEHSEC，2.5μm，4.6×150mm；离子交换色谱柱为阴离子交换色谱柱为BIA DEAE-0.1 Analytical Column，1.3μm。The method for evaluating the purity of exosomes based on two-dimensional liquid chromatography according to any one of claims 1 to 4 and 6, characterized in that: the size exclusion chromatography column is a Waters BEH SEC, 2.5μm, 4.6×150mm; the ion exchange chromatography column is the anion exchange chromatography column is the BIA DEAE-0.1 Analytical Column, 1.3μm.
  8. 根据权利要求1-4和6中任一所述的基于二维液相评价外泌体纯度的方法，其特征在于：在所得图谱中，根据峰面积归一化法分别计算第一维液相色谱和第二维液相色谱中外泌体纯度，二者的乘积即为所检测外泌体样本的纯度。 The method for evaluating the purity of exosomes based on two-dimensional liquid chromatography according to any one of claims 1 to 4 and 6, characterized in that: in the obtained spectrum, the purity of the exosomes in the first-dimensional liquid chromatography and the second-dimensional liquid chromatography are calculated respectively according to the peak area normalization method, and the product of the two is the purity of the detected exosome sample.
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