CN115541777B - Method for evaluating exosome purity based on two-dimensional liquid phase - Google Patents
Method for evaluating exosome purity based on two-dimensional liquid phase Download PDFInfo
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- 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
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- 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
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- G—PHYSICS
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- 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
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Abstract
The invention discloses a method for evaluating exosome purity based on two-dimensional liquid phase, which evaluates exosome purity by adopting a method of combining a size exclusion method and an ion exchange chromatography through a two-dimensional liquid chromatograph; the method comprises the steps that a sample firstly enters a size exclusion chromatographic column in a first dimension of a liquid chromatograph, and mobile phase isocratic elution of a liquid phase in the first dimension is adopted to make exosomes perform first analysis; and then, through switching a six-way valve of the liquid chromatograph, performing center cutting on the main peak part of the exosome, entering an ion chromatographic column in a second dimension, capturing and collecting the main peak part, and performing secondary analysis by performing gradient elution of a mobile phase in the second dimension, and entering a detector. The purity evaluation method is simple to operate, high in automation level, short in occupied time, high in accuracy, suitable for simultaneous detection of a large number of samples, particularly suitable for purity evaluation work of large-scale industrial production of future exosomes, efficient and controllable in process.
Description
Technical Field
The invention belongs to the field of exosome detection analysis, and particularly relates to a method for evaluating exosome purity based on two-dimensional liquid phase.
Background
The exosomes are lipid bilayer structure vesicles secreted by cells, the diameter is about 30-150 nm, the exosomes are widely existing in saliva, cell culture fluid, emulsion, blood, urine and other various body fluids, and as the exosomes exist in various complex biological environments, different sources and even the same exosomes have extremely different sources in terms of morphology and biochemical characteristics. The exosomes have wide application prospects in the fields of disease diagnosis and treatment, and the exosomes with higher purity and quality are key elements of scientific research and application, so that the establishment of a more accurate and efficient method for evaluating the purity of the exosomes is very important.
The existing methods for evaluating the purity of exosomes are various, and have no unified standard for the purity analysis of exosomes, such as a protein/particle method, a q-PCR method, a nanoparticle tracking analysis method and the like. The protein/particle method and the q-PCR method have the disadvantages of long time consumption, complex sample preparation mode and inapplicability to large-scale detection of multiple batches; however, although the nanoparticle tracking analysis method has a short detection time, only a part of marked exosomes can be detected, and the purity of unmarked exosomes cannot be detected, so that the accuracy is not high, and therefore, certain limitation exists.
Liquid chromatographs can be classified into reverse phase chromatography, normal phase chromatography, ion exchange chromatography, size exclusion chromatography, and affinity chromatography according to their separation modes. The size exclusion chromatography is a common detection method of a liquid chromatograph, a filler with a certain molecular aperture is filled in a size exclusion chromatographic column, when a sample flows through the chromatographic column, large-particle-size particles flow out of the chromatographic column at first, and small-particle samples flow out after flowing out, so that the separation effect of particles with different particle sizes can be achieved, but impurities with the particle size similar to that of main component substances cannot be analyzed.
The exosomes are charged, the negative charge of the exosomes is caused by the fact that the surface molecules of the exosomes are negatively charged, ion chromatography is widely applied to separation of molecules with different charges, the molecules are captured when being combined with a chromatographic column, a large amount of molecules are enriched on the ion chromatographic column, then the exosomes are eluted by adopting a mobile phase, but impurities with the same charged condition cannot be analyzed, and the limitation of the pure ion chromatography is overcome.
In the existing method for evaluating the purity of exosomes, for example, the patent 'a method for separating exosomes from animal plasma and detecting the purity' adopts a q-PCR method to determine the purity of the exosomes, and the q-PCR method has higher requirements on the professional operation of personnel, and has the defects of complicated experimental process, long time consumption and easy pollution; the patent "exosome preparation prepared from umbilical cord mesenchymal stem cells and method thereof" uses nanoparticle tracking analysis to determine purity, and this method can only determine partially labeled exosomes, such as CD63, CD9, CD81, etc., but unlabeled exosomes cannot be determined in purity, so there is a limit. The protein/particle method needs to detect the concentration of protein and the number of particles respectively, and needs to combine the results of the two detection methods, so that the detection time is long. The q-PCR method has longer detection process time, is easy to pollute and has high requirements on professional operation of personnel. Nanoparticle tracking assays can only measure partially labeled exosomes, but unlabeled exosomes cannot be measured in purity, and thus have certain limitations. Because of the deficiencies of the traditional liquid phase (if the separation degree of main peaks and impurity peaks is low or coincidence cannot be analyzed), the problem that exosomes need to overcome if the purity is detected by adopting a liquid phase method.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for evaluating exosome purity based on two-dimensional liquid phase.
The technical scheme adopted by the invention is as follows: a method for evaluating exosome purity based on two-dimensional liquid phase, which evaluates exosome purity through two-dimensional liquid chromatography; the first dimension adopts a size exclusion chromatographic column, and the elution is carried out at equal flow degree; and the second dimension adopts an ion chromatographic column, and the mobile phase is eluted in a gradient way.
Preferably, the exosome sample is first analyzed by the first dimension liquid chromatography, and then the main exosome peak is cut by switching the six-way valve to enter the second dimension liquid chromatography.
Preferably, the mobile phase of the first dimension liquid chromatography is Tris buffer with pH 7-7.5, preferably pH 7.2; the second dimension liquid chromatography mobile phase A is Tris buffer solution, and the second dimension liquid chromatography mobile phase B is Tris buffer solution-salt solution; the pH of mobile phase A and mobile phase B is 7-7.5, preferably 7.2.
Preferably, the Tris buffer concentration is 20mM, the salt solution is NaCl solution, and the NaCl salt concentration is 1.0M.
Preferably, the first dimension mobile phase elution condition is isocratic elution, and the mobile phase flow rate is 0.3-0.6 ml/min; the switching valve time of the liquid phase six-way valve is respectively 3-3.8min.
Preferably, the second dimension mobile phase elution condition is mobile phase A and B gradient elution, the mobile phase flow rate is 0.3ml/min, and the second dimension mobile phase elution comprises more than two fixed value elution stages; comprises 0-4.3min,100% mobile phase A;4.3-5.0min,100-75% mobile phase A;5.0-5.1min,75-65% mobile phase A;5.1-6.1min,65% mobile phase A;6.1-6.2min,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.
Preferably, the size exclusion chromatography column is a Waters BEH 450 Ǻ SEC,2.5 μm, 4.6x150 mm; the ion exchange chromatographic column is anion exchange chromatographic column and BIA DEAE-0.1 Analytical Column,1.3 μm.
Preferably, in the obtained spectrum, the purity of the exosome in the first dimension liquid chromatograph and the purity of the exosome in the second dimension liquid chromatograph are respectively calculated according to a peak area normalization method, and the product of the two is the purity of the detected exosome sample.
The invention has the advantages and positive effects that: the purity evaluation method is simple to operate, high in automation level, short in occupied time, high in accuracy, suitable for simultaneous detection of a large number of samples, particularly suitable for purity evaluation work of large-scale industrial production of future exosomes, efficient and controllable in process.
Drawings
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 according to the present invention.
Detailed Description
Two-dimensional liquid chromatography is a system in which two chromatographic columns with different separation mechanisms are connected in series, and a sample passes through a chromatographic column and a detector in a first dimension and then is captured or cut and then is switched into the chromatographic column and the detector in a second dimension. Components that cannot be completely separated in a one-dimensional separation system can be better separated in a two-dimensional system. The column switching pattern can be divided into full two-dimensional and center cut, and the center cut technique can only bring the part requiring secondary analysis into the second dimension for further analysis. Therefore, the development of a two-dimensional liquid phase exosome evaluation method can obviously improve the accuracy of exosome purity analysis, and provides a more suitable method for quality control of future exosome industrial production.
The method evaluates the purity of the exosome by adopting a method combining a size exclusion method and an ion exchange chromatography through a two-dimensional liquid chromatograph. The size exclusion method utilizes different particle sizes of substances to analyze, and due to different particle sizes of substances in a sample, different peak-out times of different particle sizes can be reflected on a detector, and chromatographic peaks which are obviously far away from a main peak are impurity peaks, so that impurities are distinguished. The ion exchange chromatography can specifically trap negatively charged substances by using a weak anion chromatographic column, and exosomes are negatively charged under the condition of a near neutral mobile phase, and the exosomes can be eluted by a second dimension mobile phase gradient after being trapped on the weak anion chromatographic column and enter a detector for secondary analysis.
The conventional liquid phase can only be analyzed by adopting a certain chromatographic method, the impurities with the particle size similar to that of the exosomes cannot be analyzed by adopting a size exclusion chromatography method alone, and the impurities such as main peaks, bovine serum albumin and the like cannot be analyzed by adopting an ion exchange chromatography method alone, so that the exosomes purity can be measured by creatively adopting a two-dimensional liquid phase to combine the size exclusion method with the ion exchange chromatography method and adopting a central cutting method, and the exosome purity can be measured more accurately by adopting a two-dimensional liquid phase six-way valve pipeline as shown in fig. 5 and performing analysis and evaluation through the two-dimensional liquid phase. The purity calculation in the first dimension is preliminary purity calculation, the pure exosome of the main peak part in the first dimension is analyzed in the second dimension, and the more accurate judgment of the sample is realized through the comprehensive analysis in the two dimensions.
The sample firstly enters a size exclusion chromatographic column in a first dimension of a liquid chromatograph, and the mobile phase of the liquid phase in the first dimension is adopted for equal elution, so that the exosome enters a detector for first analysis. And then, through switching a six-way valve of the liquid chromatograph, performing center cutting on the main peak part of the exosome, entering an ion chromatographic column in a second dimension to capture and collect the main peak part, and then, performing secondary analysis by performing gradient elution of a mobile phase in the second dimension, entering a detector. The two-dimensional liquid phase six-way valve pipeline is shown in fig. 5.
In the implementation, the exosome sample is subjected to two-dimensional liquid chromatography to evaluate the purity of the exosome, and a two-dimensional liquid chromatograph is used; the first dimension adopts a size exclusion chromatographic column, and the elution is carried out at equal flow degree; and the second dimension adopts an ion chromatographic column, and the mobile phase is eluted in a gradient way. The exosome sample is firstly analyzed by the first dimension liquid chromatography, and then the main exosome peak is cut by switching the six-way valve to enter the second dimension liquid chromatography. The mobile phase of the first dimension liquid chromatograph is Tris buffer solution; the second dimension liquid chromatography mobile phase A is Tris buffer solution, the second dimension liquid chromatography mobile phase B is Tris buffer solution-salt solution, the concentration of the Tris buffer solution is 20mM, the salt solution is NaCl solution, and the concentration of NaCl salt is 1.0M; the pH of mobile phase A and mobile phase B was 7.2.
In certain embodiments of the invention, the size exclusion chromatography column is Waters BEH 450 Ǻ SEC,2.5 μm, 4.6x150 mm; the ion exchange chromatographic column is anion exchange chromatographic column and BIA DEAE-0.1 Analytical Column,1.3 μm.
The first dimension mobile phase elution condition is isocratic elution, the flow rate of the mobile phase is 0.3-0.6 ml/min, preferably 0.5ml/min, the liquid phase six-way valve switching valve time is 3-3.8min respectively, wherein the flow rate of the mobile phase and the six-way valve switching time are inversely proportional, when the flow rate is higher, the shorter switching time can be adopted, and when the flow rate is lower, the mobile phase can be eluted for a longer time and then is switched, and the aim is to ensure that the peak where an exosome is located can be completely reflected. The second dimension mobile phase elution condition is mobile phase A and B gradient elution, the mobile phase flow rate is 0.3ml/min, and the second dimension mobile phase elution comprises more than two fixed value elution stages; comprises 0-4.3min,100% mobile phase A;4.3-5.0min,100-75% mobile phase A;5.0-5.1min,75-65% mobile phase A;5.1-6.1min,65% mobile phase A;6.1-6.2min,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.
And the liquid phase purity analysis adopts an area normalization method, the purity of the exosome main peak is the percentage of the main peak area to the total peak area, and in the obtained atlas, the exosome purity in the first dimension liquid chromatography and the exosome purity in the second dimension liquid chromatography are respectively calculated according to the peak area normalization method, and the product of the two is the purity of the detected exosome sample. For example, the purity of the main peak of the exosome in the first dimension is A, the purity of the main peak of the exosome in the second dimension is B, and the final purity of the exosome is A×B.
The patent 'a method for analyzing exosome charge heterogeneity' discloses a method for separating exosome subgroup according to exosome charge heterogeneity, and the invention is more intensive research based on the patent. The protectant is omitted to save costs and simplify mobile phase formulation. In the scheme of the invention, an ion chromatography method is adopted, the peak-out time of impurities with the same negative charge in a liquid phase can be overlapped with the main peak of an exosome, and the purity of the exosome can not be measured more accurately; for example, according to our subsequent experiments, bovine serum albumin was found to be an impurity that may occur in the process, in the liquid chromatogram of ion exchange chromatography, its retention time is the same as that of one of the peaks of exosomes, so ion exchange chromatography alone is not suitable for more accurately determining the specificity of exosome purity. The two-dimensional liquid phase method combining the size exclusion method with the ion chromatography can make up for the defect, and the sample in the method can show the main peak and the impurity peak of exosomes on the liquid phase chromatogram according to the particle size by passing through the size exclusion chromatographic column; for example, according to experimental results, the peak time of bovine serum albumin in the size exclusion method can be obviously distinguished from the time of the main peak of exosome, then only the center of the main peak part sample is cut into an ion chromatographic column through a two-dimensional liquid phase, the so-called 'pure exosome' part in the size exclusion method is subjected to secondary analysis, and impurities which cannot be analyzed by the size exclusion method can be analyzed through different charge amounts, so that the purity measurement is more accurate, and the relative content of each component in the spectrum is more accurately determined. Compared with the prior patent, the method changes the mode that the size exclusion method is firstly used, the protective agent is not used, only the main peak part of an exosome in the size exclusion method is analyzed by the ion chromatography, and the mobile phases used by the size exclusion method and the ion chromatography are different mobile phase systems, so that five components are separated, which is different from the six components of the prior patent, and the peaks of different components are separated farther by developing a liquid phase method, thereby being convenient for collecting different fractions more accurately and more completely.
Different exosomes or exosomes prepared by different processes have different specificities, and a more suitable method is needed to be selected according to the types and the specificities of the exosomes.
The following description of the present invention is made with reference to the accompanying drawings, wherein the experimental methods without specific description of the operation steps are performed according to the corresponding commodity specifications, and the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies without specific description.
Example 1:
the two-dimensional liquid chromatograph is Waters ACQUITY UPLC PLUS Bio, the purity of the milk exosome is analyzed by adopting a size exclusion method and combining an ion exchange chromatography, and a size exclusion chromatographic column is Waters BEH 450 Ǻ SEC,2.5 mu m and 4.6X106 mm; the ion exchange chromatographic column is weak anion chromatographic column, BIA DEAE-0.1 Analytical Column,1.3 μm; the sample injection amount is 25 μl, the column temperature is 25 ℃, the detection wavelength is 280nm, and the sample cabin is 6 ℃; both the first mobile phase and the second mobile phase A were used with 20mM Tris buffer (pH 7.2) and the second mobile phase B was 20mM Tris,1M NaCl solution (pH 7.2).
The first dimension mobile phase elution condition is mobile phase A isocratic elution, the flow rate is 0.5ml/min; the second dimension of mobile phase elution condition is mobile phase A and mobile phase B gradient elution, the flow rate is 0.3ml/min, including 0-4.3min,100% mobile phase A;4.3-5.0min,100-75% mobile phase A;5.0-5.1min,75-65% mobile phase A;5.1-6.1min,65% mobile phase A;6.1-6.2min,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 two-dimensional liquid phase switching valve time is 3.5min.
The results are shown in fig. 1, wherein 0-3.5min is a first dimension size exclusion chromatography chromatogram, 3.5-12min is a second dimension ion exchange chromatography chromatogram, the exosome purity is 74.15% in the first dimension, 99.22% in the second dimension, and 74.15% x 99.22% = 73.57% in the final exosome purity according to peak area normalization. For the impurities detected in the first dimension and the second dimension, the separation degree between the chromatogram peak type and each peak is good, the purity detection only needs 12 minutes, and the method is efficient and quick. As can also be seen from the results of fig. 1, impurities that cannot be resolved by the first dimension ion exchange chromatography can be represented in the second dimension ion exchange chromatography.
Example 2:
the two-dimensional liquid chromatograph is Waters ACQUITY UPLC PLUS Bio, the purity of the purified milk exosome is analyzed by adopting a size exclusion method and combining an ion exchange chromatography, and the size exclusion chromatographic column is Waters BEH 450 Ǻ SEC,2.5 mu m and 4.6X106 mm; the ion exchange chromatographic column is weak anion chromatographic column, BIA DEAE-0.1 Analytical Column,1.3 μm; the sample injection amount is 25 μl, the column temperature is 25 ℃, the detection wavelength is 280nm, and the sample cabin is 6 ℃; both the first mobile phase and the second mobile phase A were used with 20mM Tris buffer (pH 7.2) and the second mobile phase B was 20mM Tris,1M NaCl solution (pH 7.2).
The first dimension mobile phase elution condition is mobile phase A isocratic elution, the flow rate is 0.5ml/min; the second dimension of mobile phase elution condition is mobile phase A and mobile phase B gradient elution, the flow rate is 0.3ml/min, including 0-4.3min,100% mobile phase A;4.3-5.0min,100-75% mobile phase A;5.0-5.1min,75-65% mobile phase A;5.1-6.1min,65% mobile phase A;6.1-6.2min,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 two-dimensional liquid phase switching valve time is 3.5min.
The results are shown in fig. 2, and no impurity peak appears in the first dimension size exclusion chromatography chromatogram and the second dimension ion exchange chromatography chromatogram; in this example, the purified exosome sample was evaluated, and the purity of the sample was 100%, which was used as a "standard" chromatogram, and the positions where impurities appeared in other examples were visually compared.
Example 3:
the two-dimensional liquid chromatograph is Waters ACQUITY UPLC PLUS Bio, the purity of the milk exosome is analyzed by adopting a size exclusion method and combining an ion exchange chromatography, and a size exclusion chromatographic column is Waters BEH 450 Ǻ SEC,2.5 mu m and 4.6X106 mm; the ion exchange chromatographic column is weak anion chromatographic column, BIA DEAE-0.1 Analytical Column,1.3 μm; the sample injection amount is 25 μl, the column temperature is 25 ℃, the detection wavelength is 280nm, and the sample cabin is 6 ℃; both the first mobile phase and the second mobile phase A were used with 20mM Tris buffer (pH 7.2) and the second mobile phase B was 20mM Tris,1M NaCl solution (pH 7.2).
Adopting the milk exosomes as the sample in the first embodiment, wherein the first dimension mobile phase elution condition is that the mobile phase A is eluted at equal degree, and the flow rate is 0.5ml/min; the second dimension of mobile phase elution condition is mobile phase A and mobile phase B gradient elution, the flow rate is 0.3ml/min, including 0-4.3min,100% mobile phase A;4.3-5.0min,100-70% mobile 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.
As shown in fig. 3, 0-3.8min is a first dimension exclusion chromatography chromatogram, 3.8-12min is a second dimension ion exchange chromatography chromatogram, according to peak area normalization method, the exosome purity is 73.97% in the first dimension, 100.00% in the second dimension, and 74.15% x 100.00% = 73.97% in the final exosome purity; no impurity was detected in the second dimension. Compared with example 1, the second mobile phase of this example uses different gradient elution conditions, and it can be seen that the mobile phase salt concentration has a large effect on the detection by the liquid chromatograph.
Example 4:
the purity of the purified milk exosome was analyzed by size exclusion chromatography in combination with ion exchange chromatography as in example 2 using a two-dimensional liquid chromatograph of Waters ACQUITY UPLC PLUS Bio, a size exclusion column of Waters BEH 450 Ǻ SEC,2.5 μm, 4.6x150 mm; the ion exchange chromatographic column is weak anion chromatographic column, BIA DEAE-0.1 Analytical Column,1.3 μm; the sample injection amount is 25 μl, the column temperature is 25 ℃, the detection wavelength is 280nm, and the sample cabin is 6 ℃; both the first mobile phase and the second mobile phase A were used with 20mM Tris buffer (pH 7.2) and the second mobile phase B was 20mM Tris,1M NaCl solution (pH 7.2).
The first dimension mobile phase elution condition is mobile phase A isocratic elution, the flow rate is 0.5ml/min; the second dimension of mobile phase elution condition is mobile phase A and mobile phase B gradient elution, the flow rate is 0.3ml/min, including 0-4.3min,100% mobile phase A;4.3-5.0min,100-75% mobile phase A;5.0-5.1min,75-65% mobile phase A;5.1-6.1min,65% mobile phase A;6.1-6.2min,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 two-dimensional liquid phase switching valve time is 2.5min.
As a result, as shown in fig. 4, the main peak of the exosome cannot be completely displayed into the second dimension due to the early switching valve time, and the peak pattern appearing in the second dimension is inconsistent with the peak pattern of the standard in fig. 2; it can be seen that the switching valve time has a large impact on the exosome analysis.
The exosome purity is evaluated through the two-dimensional liquid phase, the evaluation result is more accurate, the same sample is adopted for continuous evaluation, sample errors caused by different batches of detection are avoided, the efficiency is higher, and the detection speed is higher. The embodiment of the invention describes the purity detection of the milk exosome sample in detail, and the detection scheme can also be used for exosome samples prepared from raw materials such as urine, blood, saliva and the like.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (2)
1. A method for evaluating exosome purity based on two-dimensional liquid phase is characterized in that: evaluating exosome purity by two-dimensional liquid chromatography; the first dimension adopts a size exclusion chromatographic column, and the elution is carried out at equal flow degree; the second dimension adopts an ion chromatographic column, and mobile phase gradient elution is carried out; firstly analyzing an exosome sample through a first dimension liquid chromatograph, and then switching a six-way valve to perform center cutting on a main exosome peak to enter a second dimension liquid chromatograph;
the first dimension mobile phase elution condition is isocratic elution, the flow rate of the mobile phase is 0.3-0.6 ml/min, the mobile phase of the first dimension liquid chromatography is Tris buffer, and the pH value is 7-7.5;
the second dimension mobile phase elution condition is gradient elution, and the liquid phase six-way valve switching valve time is 3min-3.8min; the second-dimension mobile phase elution condition is that mobile phases A and B are subjected to gradient elution, the second-dimension liquid chromatography mobile phase A is Tris buffer solution, and the second-dimension liquid chromatography mobile phase B is Tris buffer solution-salt solution; the pH value of the mobile phase A and the mobile phase B is 7-7.5; the flow rate of the mobile phase is 0.3ml/min, comprising more than two fixed-value elution stages; comprises 0-4.3min,100% mobile phase A;4.3-5.0min,100-75% mobile phase A;5.0-5.1min,75-65% mobile phase A;5.1-6.1min,65% mobile phase A;6.1-6.2min,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;
the concentration of Tris buffer solution is 20mM, the salt solution is NaCl solution, and the concentration of NaCl salt is 1.0M;
in the obtained spectrum, the purity of the exosome in the first dimension liquid chromatogram and the purity of the exosome in the second dimension liquid chromatogram are calculated according to a peak area normalization method, and the product of the two is the purity of the exosome sample to be detected.
2. The method for evaluating exosome purity based on two-dimensional liquid phase according to claim 1, wherein: the size exclusion column was a Waters BEH 450 Ǻ SEC,2.5 μm, 4.6x150 mm; the ion exchange chromatographic column is anion exchange chromatographic column and BIA DEAE-0.1 Analytical Column,1.3 μm.
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