Synchronous separation system and separation method for multiple extracellular vesicle subgroups based on enzyme power probe

Abstract

The invention discloses a system and a method for synchronously separating multiple extracellular vesicle subgroups based on an enzyme power probe. According to the method, a separation reagent containing a plurality of enzyme power probes and a total vesicle sample are co-incubated, then the sample is injected into a sample injection hole shared by a plurality of separation channels, different types of power substrates are arranged in the separation channels, and the power substrates have gradient concentrations; enzyme dynamic probes connected with different extracellular vesicle subgroups are attracted by dynamic substrates in different separation channels, enter different separation channels and are finally captured by a sample settling tank. By utilizing the system, a plurality of target extracellular vesicle subgroups can be respectively separated from a total vesicle sample within 2 minutes, and the efficiency is very high.

Description

Technical Field

[0001] The present invention relates to the technical field of extracellular vesicle subpopulation separation, and in particular to a system and method for synchronously separating multiple extracellular vesicle subpopulations based on enzyme kinetic probes. Background Art

[0002] Extracellular vesicles (EVs) are nanoscale membrane vesicles secreted by cells, which are widely present in various body fluids and tissue gaps of the human body. As an important signal transduction carrier between cells, EVs carry abundant bioactive molecules, such as proteins, nucleic acids and lipids, and play an important role in the physiological and pathological processes of the human body. However, human-derived EVs are composed of secretions from a variety of cells, such as tumor cells, immune cells, endothelial cells, etc. EVs from different cell sources inherit the biological characteristics of different mother cell sources, thus showing their unique biological functions and clinical research significance. Therefore, in order to deeply study the biological characteristics and functions of various EVs subpopulations, the primary task is to achieve the refined separation of different EVs subpopulations.

[0003] At present, the methods commonly used to separate extracellular vesicle subpopulations mainly include ultracentrifugation, exclusion method and ultrafiltration method. Although these separation methods can be used to separate and obtain extracellular vesicles in different application scenarios, none of them can achieve the separation of extracellular vesicle subpopulations. In addition, the immunoaffinity method can separate EVs and their subpopulations based on the specific binding of antibodies to the surface proteins of extracellular vesicle subpopulations, but the separated EVs are often fixed on the interface modified with antibodies, which makes it difficult to further verify the functions of the obtained extracellular vesicle subpopulations. The prior art proposes a strategy for non-destructive separation of extracellular vesicle subpopulations based on the immunoaffinity method, which can achieve non-destructive separation and functional exploration of specific extracellular vesicle subpopulations from complex biological samples. However, these non-destructive separation strategies can only separate and obtain one EV subpopulation at a time. When it is necessary to separate and study multiple extracellular vesicle subpopulations, the current separation methods are not only cumbersome and complex to operate, inefficient, but also cause a large amount of sample loss. Summary of the invention

[0004] The present invention provides an extracellular vesicle subpopulation separation system based on enzyme-powered probe driving, so as to solve the problems of the existing extracellular vesicle subpopulation separation method, such as cumbersome and complicated operation, low efficiency, large sample loss, etc. when separating multiple extracellular vesicle subpopulations.

[0005] The technical solution provided by the present invention is as follows:

[0006] The first aspect of the present invention provides an extracellular vesicle subpopulation separation system, comprising at least:

[0007] A variety of power gels, each of which has a power substrate adsorbed on it;

[0008] A separation device, comprising a plurality of separation channels sharing a common injection hole, wherein a gel placement groove is provided at the other end of each separation channel, a sample sedimentation groove is provided between the gel placement groove and the injection hole, and each gel placement groove is used to place a different type of power gel;

[0009] A diffusion reagent is used to fill the separation channel before injection, and to power the substrate to diffuse along the separation channel toward the injection hole;

[0010] A separation reagent is used for incubation with a total vesicle sample and then injection; the separation reagent contains a variety of enzyme kinetic probes, each of which has an extracellular vesicle subset adapter at one end and a kinetic enzyme at the other end, and the various enzyme kinetic probes have different types of kinetic enzymes; each kinetic enzyme is used to catalyze the decomposition of a kinetic substrate in a different separation channel to guide each enzyme kinetic probe to advance along a different separation channel.

[0011] In combination with the first aspect of the present invention, in some embodiments, the motive substrate includes one or more of urea, hydrogen peroxide, and glucose; and / or the motive enzyme includes one or more of urease, catalase, and glucose oxidase; and / or the extracellular vesicle subpopulation adapter includes one or more adapters of Sgc8c, epithelial cell adhesion molecule (EpCAM), and platelet endothelial cell adhesion molecule 1 (PECAM-1).

[0012] In combination with the first aspect of the present invention, in some embodiments, the kinetic gel is an agarose gel impregnated with a kinetic substrate; and / or the diffusion reagent is a biological buffer; and / or the extracellular vesicle subpopulation adapter and the kinetic enzyme on the enzyme kinetic probe are connected via biotin.

[0013] In combination with the first aspect of the present invention, in some embodiments, the sample sedimentation tank is disposed adjacent to the gel placement tank, and its bottom surface is lower than the gel placement tank and the separation channel.

[0014] In combination with the first aspect of the present invention, in some embodiments, a gel baffle is provided between the sample sedimentation tank and the gel placement tank to prevent the power gel from moving toward the separation channel.

[0015] In combination with the first aspect of the present invention, in some embodiments, the separation channel is a strip-shaped groove open at the top.

[0016] In combination with the first aspect of the present invention, in some embodiments, the system for synchronously separating multiple extracellular vesicle subpopulations is a kit, and the separation device is a microfluidic chip.

[0017] A second aspect of the present invention provides a method for synchronously isolating multiple extracellular vesicle subpopulations, comprising the following steps:

[0018] Providing a system for synchronously isolating the above-mentioned multiple extracellular vesicle subpopulations;

[0019] Placing power gels with different power substrates in different gel placement tanks, adding diffusion reagents to each separation channel, and then allowing the power substrates to stand until a concentration gradient is formed in each separation channel;

[0020] After the separation reagent is co-incubated with the total vesicle sample, the sample is injected from the injection hole, and the sediment is collected from each sample sedimentation tank.

[0021] In combination with the second aspect of the present invention, in some embodiments, the method for synchronously isolating multiple extracellular vesicle subpopulations further includes: after the separation is completed, taking out the sample that has not entered the separation channel from the injection hole.

[0022] The third aspect of the present invention provides an extracellular vesicle subpopulation separation system, comprising:

[0023] Power gel, a power substrate is adsorbed on the power gel;

[0024] The separation device comprises a separation channel; one end of the separation channel is a sample injection hole, the other end is a gel placement groove, and a sample sedimentation groove is provided between the gel placement groove and the sample injection hole;

[0025] A diffusion reagent is used to fill the separation channel before injection, and to power the substrate to diffuse along the separation channel toward the injection hole;

[0026] A separation reagent is used for incubation with a total vesicle sample and then injection; the separation reagent contains an enzyme kinetic probe, one end of which is an extracellular vesicle subpopulation adapter and the other end is a kinetic enzyme; the kinetic enzyme can catalyze the decomposition of the kinetic substrate and guide the enzyme kinetic probe bound to the target extracellular vesicle subpopulation along the separation channel to the sample sedimentation tank.

[0027] A fourth aspect of the present invention provides a method for isolating an extracellular vesicle subpopulation, comprising:

[0028] Providing the above-mentioned extracellular vesicle subpopulation separation system;

[0029] The power gel is placed in a gel placement tank, and a diffusion reagent is added to the gel placement tank until the diffusion reagent fills the separation channel, and then the separation channel is allowed to stand until the power substrate forms a concentration gradient in the separation channel;

[0030] After the separation reagent is co-incubated with the total vesicle sample, the sample is injected from the injection hole, and the precipitate is collected from the sample sedimentation tank.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] The enzyme kinetic probe in the separation reagent of the present invention can specifically bind to the target extracellular vesicle subpopulation in the total vesicle sample. After the sample is injected, the kinetic enzyme on each enzyme kinetic probe reacts with the gradient concentration kinetic substrate in the corresponding separation channel, and the extracellular vesicle subpopulation can be brought into the corresponding separation channel and captured by the sample sedimentation tank when it is close to the kinetic gel. Using this system, multiple target extracellular vesicle subpopulations can be separated from the total vesicle sample simultaneously within 2 minutes, with very high efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 : A three-dimensional schematic diagram of a system for synchronously isolating multiple extracellular vesicle subpopulations provided by the present invention;

[0035] Figure 2 : A plan view of a system for synchronously isolating multiple extracellular vesicle subpopulations provided by the present invention;

[0036] Figure 3 : A schematic diagram of the process of the method for synchronously isolating multiple extracellular vesicle subpopulations provided by the present invention;

[0037] Figure 4 :The extracellular vesicle subpopulation that specifically binds to the enzyme kinetic probe moves rapidly along the kinetic substrate concentration gradient toward the direction of high concentration in the separation channel within 0 to 1 min;

[0038] Figure 5 :Transmission electron microscopy images of extracellular vesicle subpopulations before and after binding to enzyme dynamics probe;

[0039] Figure 6 : Changes in the concentrations of the extracellular vesicle subpopulations expressing EpCAM and Sgc8c on the surface before and after separation.

[0040] Wherein: 1. gel placement tank; 2. gel block baffle; 3. separation channel; 4. sample sedimentation tank; 5. injection hole; 6. kinetic substrate concentration gradient formation step; 7. enzyme kinetic probe and multiple extracellular vesicle subpopulations co-incubation step; 8. multiple extracellular vesicle subpopulations synchronous separation step; 9. substrate solution for soaking kinetic gel; 10. kinetic gel; 11. diffusion reagent; 12. separation reagent; 13. multiple enzyme kinetic probes; 14. total vesicle sample; 15. multiple extracellular vesicle subpopulations specifically bound to enzyme kinetic probes; 16. injection hole; 17. separation channel; 18. extracellular vesicle subpopulations specifically bound to enzyme kinetic probes move in the separation channel; 19. sample sedimentation tank. DETAILED DESCRIPTION

[0041] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] For simplicity, only some numerical ranges are explicitly disclosed herein. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range. Similarly, any upper limit can be combined with any other upper limit to form an unambiguous range. In addition, although not explicitly stated, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unambiguous range.

[0043] It should be noted that, in the description of this article, unless otherwise specified, "above", "below" are inclusive numbers, and the meaning of "multiple" in "one or more" is two or more. Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment. In the absence of more restrictions, the elements defined by the sentence "including one..." do not exclude the existence of other identical elements in the process, method, article or equipment including the elements.

[0044] In the description of this specification, the description with reference to the terms "any embodiment / method", "one embodiment / method", "some embodiments / methods", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment / method or example are included in at least one embodiment / method or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment / method or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments / methods or examples. In addition, those skilled in the art may combine and combine the different embodiments / methods or examples described in this specification and the features of the different embodiments / methods or examples, unless they are contradictory.

[0045] In the present invention, "extracellular vesicle subpopulation" refers to extracellular vesicles derived from the same cell, and "total vesicle sample" refers to a mixed sample containing multiple "extracellular vesicle subpopulations".

[0046] The above summary of the invention of the present invention is not intended to describe each disclosed embodiment or each implementation in the present invention. The following description more specifically illustrates exemplary embodiments. These embodiments can be used in various combinations. In each embodiment, enumeration is only used as a representative group and should not be interpreted as exhaustive.

[0047] Separation system:

[0048] The present invention provides a system for synchronously separating multiple extracellular vesicle subpopulations, comprising at least:

[0049] A variety of power gels, each of which has a power substrate adsorbed on it;

[0050] A separation device, comprising a plurality of separation channels sharing a common injection hole, wherein a gel placement groove is provided at the other end of each separation channel, a sample sedimentation groove is provided between the gel placement groove and the injection hole, and each gel placement groove is used to place a different type of power gel;

[0051] A diffusion reagent is used to fill the separation channel before injection, and to power the substrate to diffuse along the separation channel toward the injection hole;

[0052] A separation reagent is used for incubation with a total vesicle sample and then injection; the separation reagent contains a variety of enzyme kinetic probes, each of which has an extracellular vesicle subset adapter at one end and a kinetic enzyme at the other end, and the various enzyme kinetic probes have different types of kinetic enzymes; each kinetic enzyme is used to catalyze the decomposition of a kinetic substrate in a different separation channel to guide each enzyme kinetic probe to advance along a different separation channel.

[0053] The present invention also provides an extracellular vesicle subpopulation separation system for non-simultaneously separating multiple extracellular vesicle subpopulations, comprising:

[0054] Power gel, a power substrate is adsorbed on the power gel;

[0055] The separation device comprises a separation channel; one end of the separation channel is a sample injection hole, the other end is a gel placement groove, and a sample sedimentation groove is provided between the gel placement groove and the sample injection hole;

[0056] A diffusion reagent is used to fill the separation channel before injection, and to power the substrate to diffuse along the separation channel toward the injection hole;

[0057] A separation reagent is used for incubation with a total vesicle sample and then injection; the separation reagent contains an enzyme kinetic probe, one end of which is an extracellular vesicle subpopulation adapter and the other end is a kinetic enzyme; the kinetic enzyme can catalyze the decomposition of the kinetic substrate and guide the enzyme kinetic probe bound to the target extracellular vesicle subpopulation along the separation channel to the sample sedimentation tank.

[0058] Taking the extracellular vesicle subpopulation separation system for non-simultaneous separation of multiple extracellular vesicle subpopulations provided by the present invention as an example, in the separation channel of the separation device, the power gel diffuses the power substrate adsorbed by it into the separation channel to form a concentration gradient, attracting the extracellular vesicle subpopulation connected to the enzyme power probe to move toward the power gel. In this process, the extracellular vesicle subpopulation close to the power gel falls into the sample sedimentation tank and is captured by the sample sedimentation tank. One end of the enzyme power probe is an extracellular vesicle subpopulation aptamer and the other end is a power enzyme; the extracellular vesicle subpopulation aptamer can specifically identify the target extracellular vesicle subpopulation, and the power enzyme can catalyze the rapid degradation of the power substrate in the separation channel, thereby prompting the power enzyme to move rapidly in the direction of higher power substrate concentration, thereby achieving rapid separation of the target extracellular vesicle subpopulation.

[0059] Based on the extracellular vesicle subpopulation separation system for non-simultaneously separating multiple extracellular vesicle subpopulations provided by the present invention, the multiple extracellular vesicle subpopulation synchronous separation system provided by the present invention sets multiple separation channels sharing a sampling hole on a separation device, and incubates the total vesicle sample with a separation reagent containing multiple enzyme dynamic probes, and then injects the sample into the sampling hole shared by multiple separation channels, so that multiple extracellular vesicle subpopulations can enter different separation channels respectively, and then be captured by different sample sedimentation tanks, thereby realizing the simultaneous separation of multiple extracellular vesicle subpopulations from the total vesicle sample.

[0060] In some embodiments of the present invention, the power gel in each gel placement tank adsorbs a power substrate, for example, the power substrate is urea, hydrogen peroxide or glucose; each enzyme power probe in the separation reagent is connected to a power enzyme, for example, the power enzyme is urease, catalase or glucose oxidase. During the separation process, the enzyme power probe connected to urease moves toward the power gel adsorbing urea, the enzyme power probe connected to catalase moves toward the power gel adsorbing hydrogen peroxide, and the enzyme power probe connected to glucose oxidase moves toward the power gel adsorbing glucose.

[0061] In some embodiments of the present invention, each enzyme kinetic probe in the separation reagent is respectively connected to an extracellular vesicle subpopulation aptamer, for example, the extracellular vesicle subpopulation aptamer is an Sgc8c aptamer, an EpCAM aptamer or a PECAM-1 aptamer, and the enzyme kinetic probe connected to the Sgc8c aptamer is used to specifically identify and capture the extracellular vesicle subpopulation expressing Sgc8c, the enzyme kinetic probe connected to the EpCAM aptamer is used to specifically identify and capture the extracellular vesicle subpopulation expressing EpCAM, and the enzyme kinetic probe connected to the PECAM-1 aptamer is used to specifically identify and capture the extracellular vesicle subpopulation expressing PECAM-1. Each enzyme kinetic probe can be connected to different extracellular vesicle subpopulations through the extracellular vesicle subpopulation aptamer, and different extracellular vesicle subpopulations are brought into different separation channels by pairing the enzyme kinetic probe and the kinetic substrate.

[0062] In some embodiments of the present invention, the power gel is an agarose gel impregnated with a power substrate; after the agarose gel adsorbs the power substrate, it can release the power substrate by soaking in a diffusion reagent, thereby forming a gradient concentration of the power substrate in the separation channel. In addition, the pore size of the agarose gel is suitable, which can hinder the extracellular vesicle subpopulation from continuing to move forward or adsorb on the power gel. When preparing the agarose gel of the present invention, high-purity agarose powder is used as a raw material, and its concentration is set to 10 mg / mL, which can ensure the mechanical stability and permeability of the agarose gel.

[0063] In some embodiments of the present invention, the diffusion reagent is a biological buffer, which can ensure that the extracellular vesicle subpopulation does not break in the diffusion reagent and still has integrity and activity after separation. Preferably, the biological buffer used in the present invention is PBS.

[0064] In some embodiments of the present invention, the extracellular vesicle subpopulation aptamer and the kinase on the enzyme kinetic probe are linked via biotin.

[0065] In some embodiments of the present invention, the sample sedimentation tank is arranged adjacent to the gel placement tank, and its bottom surface is lower than the gel placement tank and the separation channel. This arrangement allows the target extracellular vesicle subpopulation to fall into the sample sedimentation tank due to its own gravity when it is about to reach the power gel. The sample in the sample sedimentation tank can be taken out by a pipette later.

[0066] In some embodiments of the present invention, a gel baffle is provided between the sample sedimentation tank and the gel placement tank to block the power gel from moving toward the separation channel to avoid disturbing the concentration gradient of the power substrate.

[0067] In some embodiments of the present invention, the separation channel is a strip-shaped groove with an open top, which is convenient for sample addition, observation and cleaning.

[0068] The system for synchronously separating multiple extracellular vesicle subpopulations provided by the present invention can be used for industrial purification of extracellular vesicle subpopulations. In some embodiments of the present invention, the system for synchronously separating multiple extracellular vesicle subpopulations is a kit, and the separation device is a microfluidic chip, which can be used for micro-purification of extracellular vesicle subpopulations in the laboratory.

[0069] In some embodiments of the present invention, the separation device comprises three separation channels with an angle of 120° that share a common injection hole. Further, each separation channel has the same length, width and height. Preferably, each separation channel has a length of 36 mm, a width of 9 mm and a height of 3 mm.

[0070] Separation method:

[0071] The method for synchronously isolating multiple extracellular vesicle subpopulations provided by the present invention comprises the following steps:

[0072] Providing a system for synchronously isolating the above-mentioned multiple extracellular vesicle subpopulations;

[0073] Placing power gels with different power substrates in different gel placement tanks, adding diffusion reagents to each separation channel, and then allowing the power substrates to stand until a concentration gradient is formed in each separation channel;

[0074] After the separation reagent is co-incubated with the total vesicle sample, the sample is injected from the injection hole, and the sediment is collected from each sample sedimentation tank.

[0075] The method for synchronously separating multiple extracellular vesicle subpopulations without simultaneously separating multiple extracellular vesicle subpopulations provided by the present invention comprises the following steps:

[0076] Providing the above-mentioned extracellular vesicle subpopulation separation system;

[0077] The power gel is placed in a gel placement tank, and a diffusion reagent is added to the gel placement tank until the diffusion reagent fills the separation channel, and then the separation channel is allowed to stand until the power substrate forms a concentration gradient in the separation channel;

[0078] After the separation reagent is co-incubated with the total vesicle sample, the sample is injected from the injection hole, and the precipitate is collected from the sample sedimentation tank.

[0079] In some embodiments of the present invention, after separation is completed, the sample that has not entered the separation channel is taken out from the injection hole. These mixed vesicles are a mixture of non-target extracellular vesicle subpopulations and can be discarded.

[0080] In some embodiments of the present invention, the precipitate is collected and then ultracentrifugation is used to collect the target extracellular vesicle subpopulation.

[0081] In some embodiments of the present invention, the time for the motive substrate to form a concentration gradient in each separation channel is 20 to 40 minutes.

[0082] In some embodiments of the present invention, the separation reagent and the total vesicle sample are co-incubated for 20 to 30 hours.

[0083] The biological functions of extracellular vesicles are closely related to their donor cells. Therefore, different extracellular vesicle subpopulations have their unique functions. For example, exosomes released by tumor cells may carry factors that promote tumor growth and metastasis, have tumor homing characteristics, and are ideal candidate carriers for the prevention, diagnosis and treatment of cancer, as well as biomarkers for the diagnosis and monitoring of tumors. Exosomes released by immune cells may carry molecules that regulate immune responses and participate in immune regulation. The present invention can separate multiple extracellular vesicle subpopulations simultaneously through the above separation method, and the separation can be completed within 2 minutes, which will provide great help for the purification and research of extracellular vesicle subpopulations.

[0084] Example

[0085] The technical scheme of the present invention is described in detail by way of examples below. If the manufacturer's brand or preparation method is not specified, the raw materials, equipment or solvents used are all common raw materials, equipment or solvents on the market. Unless otherwise specified, the raw materials of the same name used in the following examples and comparative examples are all the same raw materials.

[0086] The sources of the reagents used in the examples of the present invention are as follows:

[0087] Urea: purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10023218;

[0088] Hydrogen Peroxide: The item number is 88597;

[0089] glucose: The item number is BS099;

[0090] Agarose: The item number is BS081;

[0091] Urease: The item number is U1500;

[0092] Catalase: The item number is C40;

[0093] Glucose oxidase: The item number is G7141;

[0094] Sulfo-NHS-biotin: The item number is B2100;

[0095] SA (streptavidin): S103034;

[0096] Biotinylated Sgc8c aptamer, biotinylated EpCAM aptamer, biotinylated PECAM-1 aptamer:

[0097] Example 1

[0098] 1. Preparation of power gel:

[0099] (1) Preparation of substrate solution: Weigh appropriate amounts of urea, hydrogen peroxide, and glucose respectively, and use ddH2O to accurately prepare 1 mM urea solution, hydrogen peroxide solution, and glucose solution, and ensure that the solutions are uniform and free of precipitation.

[0100] (2) Slowly add high-purity agarose powder to double distilled water (ddH2O), stirring while adding, and heat until completely dissolved (usually above the boiling point, the specific temperature is adjusted according to the type of agarose) to obtain a 10 mg / mL agarose hot solution. Then, pour the hot agarose solution into a pre-cleaned and dried mold to avoid bubbles. Let it stand at room temperature to cool naturally until a transparent, solid gel is formed. Cut the gel into 1 cm*1 cm*1 cm gel blocks for later use.

[0101] (3) The cut gel blocks were carefully immersed in urea solution, hydrogen peroxide solution, and glucose solution, respectively, and allowed to stand at room temperature in a dark environment for 12 hours; then the gel blocks were taken out and the surface of the gel blocks was gently rinsed with freshly prepared ddH2O to remove the residual substrate solution that had not penetrated into the gel blocks, thereby obtaining urea-powered gel, hydrogen peroxide-powered gel, and glucose-powered gel, respectively.

[0102] (4) Place the processed gel block in a sealed container and store it at room temperature and away from light to maintain its biological activity and stability.

[0103] 2. Preparation of enzyme dynamics probe:

[0104] Urease, catalase, and glucose oxidase were incubated with Sulfo-NHS-biotin in PBS at 4°C overnight (1 mg / mL enzyme solution corresponds to 16.6 μM biotin), and then 20 mg / mL streptavidin (SA) solution was added (ensuring that the SA concentration was 1 / 4 of that of biotin) and incubated at 37°C for 1 hour to form stable urease-biotin-SA solution, catalase-biotin-SA solution, and glucose oxidase-biotin-SA solution, respectively.

[0105] The biotinylated Sgc8c aptamer was added to the above urease-biotin-SA solution at 10 μM, and the unbound biotin, SA and aptamer were removed and purified using ultracentrifugation technology (120,000 g, 70 min, 8° C.) to obtain the urease-Sgc8c probe.

[0106] The biotinylated EpCAM aptamer was added to the above catalase-biotin-SA solution at 10 μM, and the unbound biotin, SA and aptamer were removed and purified using ultracentrifugation technology (120,000 g, 70 min, 8° C.) to obtain the catalase-EpCAM probe.

[0107] The biotinylated PECAM-1 aptamer was added to the glucose oxidase-biotin-SA solution at 10 μM, and the unbound biotin, SA and aptamer were removed and purified using ultracentrifugation technology (120,000 g, 70 min, 8° C.) to obtain the glucose oxidase-PECAM-1 probe.

[0108] Finally, the purified enzyme dynamics probe was packaged and stored in a 4°C refrigerator to ensure its stability and activity.

[0109] 3. Synchronous Isolation of Multiple Extracellular Vesicle Subpopulations

[0110] like Figure 1 As shown, the separation device provided in this embodiment is provided with three separation channels sharing one injection hole and having an angle of 120°, and each separation channel is provided with a gel placement groove at the other end, and a sample sedimentation groove is provided between the gel placement groove and the injection hole, and the sample sedimentation groove is arranged adjacent to the gel placement groove. Among them, each separation channel has a length of 36 mm, a width of 9 mm, and a height of 3 mm.

[0111] The power gels were placed in the gel placement tanks of the separation device. Then, an appropriate amount of PBS reagent was added to the separation channel to fill the separation channel with PBS solution, and the separation channel was left to stand for half an hour until the power substrate formed a stable concentration gradient.

[0112] The enzyme dynamics probes (urease-Sgc8c probe, catalase-EpCAM probe, glucose oxidase-PECAM-1 probe) and the mixture of three extracellular vesicle subpopulations expressing Sgc8c, EpCAM, and PECAM-1 on the surface were added to the test tube and incubated at room temperature for 24 hours. The incubated sample was added to the injection hole of the separation device, and after standing for 30 minutes, the solution was aspirated from the three sample sedimentation tanks into the centrifuge tube with a pipette to collect the extracellular vesicle subpopulations enriched in each of the three sample sedimentation tanks.

[0113] 4. Verification

[0114] Taking the separation of the extracellular vesicle subpopulation expressing Sgc8c on the surface and the extracellular vesicle subpopulation expressing EpCAM as an example, the extracellular vesicle subpopulation expressing EpCAM on the surface was first mixed with 1000 times of hydroxyfluorescein diacetate succinimidyl ester (CFSE), 1 μL of CFSE was added to each 1000 μL sample, and incubated in the dark for 15 minutes. The extracellular vesicle subpopulation expressing Sgc8c on the surface was mixed with 1000 times of exosome red fluorescent marker dye (PKH26), 1 μL of exosome red fluorescent marker dye was added to each 1000 μL sample, and incubated in the dark for 15 minutes. Both were washed by ultracentrifugation (120000g, 70min) to obtain the extracellular vesicle subpopulation expressing Sgc8c on the surface stained with CFSE and the extracellular vesicle subpopulation expressing EpCAM on the surface stained with PKH26.

[0115] The two stained extracellular vesicle subpopulations were mixed in equal amounts to prepare a mixed sample, and the mixed sample was injected into the injection hole. The separation device was quickly placed in a fluorescent microplate reader to detect the concentrations of the extracellular vesicle subpopulations expressing EpCAM on the surface and the extracellular vesicle subpopulations expressing Sgc8c on the surface before and after separation in the sample sedimentation tank of the separation channel where the hydrogen peroxide power gel was placed. The results are as follows:

[0116] like Figure 6 As shown in the figure, after synchronous separation, the concentration of the extracellular vesicle subpopulation expressing EpCAM on the surface (T-extracellular vesicle subpopulation, marked with CFSE green fluorescence) in the sample sedimentation tank increased significantly, while the concentration of the extracellular vesicle subpopulation expressing Sgc8c on the surface (I-extracellular vesicle subpopulation, marked with PKH26 red fluorescence) remained relatively stable with almost no change. This result intuitively proves that the separation method of the present invention is not only effective, but also can highly purify the target extracellular vesicle subpopulation, and realizes the precise separation of the two extracellular vesicle subpopulations expressing Sgc8c and EpCAM on the surface.

[0117] like Figure 4As shown, the extracellular vesicle subpopulation specifically bound to the enzyme kinetic probe moved rapidly along the kinetic substrate concentration gradient toward the direction of high concentration in the separation channel within 0 to 1 min, confirming that the extracellular vesicle subpopulation connected to the enzyme kinetic probe can move rapidly toward the direction of high kinetic substrate concentration, thereby achieving rapid and efficient separation.

[0118] like Figure 5 As shown, there is no obvious change in the morphology of the extracellular vesicle subpopulation before and after binding to the enzyme dynamics probe, indicating that the binding to the enzyme dynamics probe does not affect the morphology of the extracellular vesicle subpopulation.

[0119] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A system for synchronously separating multiple extracellular vesicle subpopulations, comprising at least: A variety of power gels, each of which has a power substrate adsorbed on it; A separation device, comprising a plurality of separation channels sharing a common injection hole, wherein a gel placement groove is provided at the other end of each separation channel, a sample sedimentation groove is provided between the gel placement groove and the injection hole, and each gel placement groove is used to place a different type of power gel; A diffusion reagent is used to fill the separation channel before injection, and to power the substrate to diffuse along the separation channel toward the injection hole; A separation reagent is used for incubation with a total vesicle sample and then injection; the separation reagent contains a variety of enzyme kinetic probes, each of which has an extracellular vesicle subset adapter at one end and a kinetic enzyme at the other end, and the various enzyme kinetic probes have different types of kinetic enzymes; each kinetic enzyme is used to catalyze the decomposition of a kinetic substrate in a different separation channel to guide each enzyme kinetic probe to advance along a different separation channel.

2. The system for synchronously separating multiple extracellular vesicle subpopulations according to claim 1, characterized in that: The power substrate includes one or more of urea, hydrogen peroxide, and glucose; and / or, The kinetic enzyme comprises one or more of urease, catalase and glucose oxidase; and / or, The extracellular vesicle subpopulation adapter includes one or more adapters of Sgc8c, EpCAM, and PECAM-1.

3. The system for synchronously separating multiple extracellular vesicle subpopulations according to claim 1, characterized in that: The power gel is an agarose gel impregnated with a power substrate; and / or, The diffusion reagent is a biological buffer; and / or, The extracellular vesicle subpopulation adapter and the kinetic enzyme on the enzyme kinetic probe are connected via biotin.

4. The system for synchronously separating multiple extracellular vesicle subpopulations according to claim 1, characterized in that: The sample sedimentation tank is arranged adjacent to the gel placement tank, and its bottom surface is lower than the gel placement tank and the separation channel.

5. The system for synchronously separating multiple extracellular vesicle subpopulations according to claim 1, characterized in that: Therefore, a gel baffle is provided between the sample sedimentation tank and the gel placement tank to prevent the power gel from moving toward the separation channel.

6. The system for synchronously isolating multiple extracellular vesicle subpopulations according to claim 1, characterized in that: The separation channel is a strip-shaped groove open at the top.

7. The system for synchronously isolating multiple extracellular vesicle subpopulations according to claim 1, characterized in that: The system for synchronously separating multiple extracellular vesicle subpopulations is a kit, and the separation device is a microfluidic chip.

8. A method for synchronously isolating multiple extracellular vesicle subpopulations, characterized in that: The following steps are involved: Providing a system for synchronously isolating multiple extracellular vesicle subpopulations as claimed in claim 1; Placing power gels with different power substrates in different gel placement tanks, adding diffusion reagents to each separation channel, and then allowing the power substrates to stand until a concentration gradient is formed in each separation channel; After the separation reagent is co-incubated with the total vesicle sample, the sample is injected from the injection hole, and the sediment is collected from each sample sedimentation tank.

9. An extracellular vesicle subpopulation separation system, characterized in that: include: A kinetic gel having a kinetic substrate adsorbed thereon; a separation device comprising at least one separation channel; One end of the separation channel is a sample injection hole, and the other end is a gel placement tank, and a sample sedimentation tank is provided between the gel placement tank and the sample injection hole; A diffusion reagent is used to fill the separation channel before injection, and to power the substrate to diffuse along the separation channel toward the injection hole; A separation reagent is used for incubation with a total vesicle sample and then injection; the separation reagent contains an enzyme kinetic probe, one end of the enzyme kinetic probe is an extracellular vesicle subpopulation adapter, and the other end is a kinetic enzyme; the kinetic enzyme can catalyze the decomposition of the kinetic substrate and guide the enzyme kinetic probe bound to the target extracellular vesicle subpopulation to move along the separation channel toward the sample sedimentation tank.

10. A method for isolating extracellular vesicle subpopulations, characterized in that: include: Providing the extracellular vesicle subpopulation separation system according to claim 9; The power gel is placed in a gel placement tank, and a diffusion reagent is added to the gel placement tank until the diffusion reagent fills the separation channel, and then the separation channel is allowed to stand until the power substrate forms a concentration gradient in the separation channel; After the separation reagent is co-incubated with the total vesicle sample, the sample is injected from the injection hole, and the precipitate is collected from the sample sedimentation tank.

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