Protein separation and detection device and detection method based on annular non-uniform electric field partition sample introduction

Abstract

The invention relates to the technical field of protein separation and detection, and particularly discloses a protein separation and detection device and detection method based on annular non-uniform electric field partition sample introduction. According to the device, different sample introduction holes matched with an annular non-uniform electric field are formed, different protein separation areas are arranged, the protein separation areas are matched with proteins with different molecular weights, and the characteristic that the annular geometrical characteristics are concentrically converged in the central area of the electric field is combined; therefore, high-resolution separation and high-sensitivity detection of the protein are realized.

Description

Technical Field

[0001] The present application relates to the technical field of protein separation and detection, and in particular to a protein separation and detection device and a detection method based on annular non-uniform electric field partitioned sampling. Background Art

[0002] Protein is the basic unit of life, participates in all major life activities of organisms, and is closely related to the diagnosis and treatment of diseases. Therefore, separating and detecting the structure and function of a large number of protein mixtures is of great significance for understanding the laws of life activities, revealing the mechanisms of various diseases, and diagnosing, treating and preventing them. However, due to the complex biological environment of proteins, their own diverse structures, and low content, how to effectively separate and detect them from a large number of protein samples is a problem that needs to be solved urgently.

[0003] However, existing protein separation and detection devices or methods have technical problems of low resolution and low sensitivity. Summary of the invention

[0004] In view of the above problems, the present application provides a protein separation and detection device and detection method based on annular non-uniform electric field partition injection. The device sets different injection holes that match the annular non-uniform electric field and sets different protein separation zones. These protein separation zones match proteins of different molecular weights, and combine the characteristics of the annular geometric features concentrically converging in the central area of ​​the electric field, thereby achieving high-resolution separation and high-sensitivity detection of proteins.

[0005] The first aspect of the present application is to provide a protein separation and detection device based on annular non-uniform electric field partition injection, the device comprising:

[0006] Electrophoresis structure: including an electrophoresis tank and a gel separation structure arranged in the electrophoresis tank;

[0007] The gel separation structure comprises a gel body and a plurality of injection holes connected to the gel body, a through hole is provided at the center of the gel body, the injection holes are arranged along the circumference of the through hole, and the distances between some injection holes and the through hole are not equal;

[0008] Inner electrode: arranged in the through hole;

[0009] External electrode: arranged along the outer circumference of the gel separation structure;

[0010] And transfer film.

[0011] In some embodiments, the distances between two adjacent injection holes and the through hole are not equal.

[0012] In some embodiments, the sample injection holes include first-class sample injection holes, second-class sample injection holes, and third-class sample injection holes;

[0013] The distance between the first-class sample injection hole and the through hole is d1;

[0014] The distance between the second-class sample injection hole and the through hole is d2;

[0015] The distance between the third-class sample injection hole and the through hole is d3;

[0016] Satisfying: d1 < d2 < d3.

[0017] In some embodiments, the first-class sample injection holes are arranged circumferentially along the through hole.

[0018] In some embodiments, the second-class sample injection holes are arranged circumferentially along the through hole;

[0019] In some embodiments, the third-class sample injection holes are arranged circumferentially along the through hole.

[0020] In some embodiments, the electrophoresis tank is connected to a buffer circulation device;

[0021] In some embodiments, the electrophoresis tank is connected to a temperature control device.

[0022] In some embodiments, a circulating liquid inlet is provided at the central position of the electrophoresis tank, and the circulating liquid inlet is connected to a buffer circulation device.

[0023] In some embodiments, the gel separation structure is connected to the transfer membrane.

[0024] In some embodiments, the inner electrode includes platinum metal.

[0025] In some embodiments, the outer electrode includes a tin foil ring.

[0026] In some embodiments, the gel body includes any one of agarose gel, polyacrylamide gel, or hydrogel.

[0027] In some embodiments, the transfer membrane includes any one of a polyvinylidene fluoride membrane, a nitrocellulose membrane, or a nylon membrane.

[0028] The second aspect of the present application is to provide a method for separating and detecting proteins by the device according to the first aspect, the method comprising:

[0029] Providing a protein sample;

[0030] Injecting the protein sample into the gel separation structure through the respective sample injection holes of the device;

[0031] Apply a voltage to the above electrophoresis structure so that proteins with different molecular weights in the above protein sample are separated in the above gel separation structure and form multiple protein bands;

[0032] Transfer each protein band to the transfer membrane;

[0033] Perform immunoassay on the proteins on the above transfer membrane.

[0034] In some embodiments, the above sample injection holes include a first type of sample injection hole, a second type of sample injection hole, and a third type of sample injection hole;

[0035] The distance between the above first type of sample injection hole and the above through hole is d1;

[0036] The distance between the above second type of sample injection hole and the above through hole is d2;

[0037] The distance between the above third type of sample injection hole and the above through hole is d3;

[0038] Satisfy: d1 < d2 < d3;

[0039] The above protein sample includes a first type of protein sample, a second type of protein sample, and a third type of protein sample. The molecular weight of the first type of protein sample is M1, the molecular weight of the second type of protein sample is M2, and the molecular weight of the third type of protein sample is M3, satisfying: M1 > M2 > M3;

[0040] And the above first type of protein sample is injected into the above gel separation structure through the above first type of sample injection hole; the above second type of protein sample is injected into the above gel separation structure through the above second type of sample injection hole; the above third type of protein sample is injected into the above gel separation structure through the above third type of sample injection hole.

[0041] In some embodiments, the step of transferring each of the above protein bands to the above transfer membrane includes:

[0042] Stack the electrode, the gel separation structure with protein bands, and the transfer membrane together;

[0043] Place the stacked structure in the transfer buffer and apply a voltage to the above electrode;

[0044] The above protein band is transferred to the above transfer membrane.

[0045] The beneficial technical effects of this application:

[0046] 1. The device provided by this application realizes multi-dimensional optimization of the separation process by setting different sample injection holes that match the annular non-uniform electric field and setting different protein separation regions. These protein separation regions match proteins with different molecular weights. Specifically, they have characteristics such as time difference and diverse movement trajectories, and finally achieve high-resolution separation of proteins with different molecular weights.

[0047] 2. The device provided by this application combines the characteristic of concentric convergence in the central region of the electric field of the annular geometric feature to efficiently concentrate proteins for facilitating further high-sensitivity detection of proteins.

[0048] 3. The separation and detection method provided by this application conveniently shortens the time required to achieve an ideal separation effect by a large margin, and significantly improves the separation efficiency.

[0049] The above description is only an overview of the technical solution of this application. In order to be able to understand the technical means of this application more clearly, it can be implemented in accordance with the content of the specification. And in order to make the above and other purposes, features and advantages of this application more obvious and understandable, the following specifically illustrates the specific implementation manners of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] By reading the detailed description of the preferred embodiments below, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of this application. And throughout the drawings, the same reference numerals are used to represent the same components. In the drawings:

[0051] Figure 1 is a schematic structural diagram of a protein separation and detection device shown in some embodiments of this application;

[0052] Figure 2 is Figure 1 a schematic structural diagram of the electrophoresis tank in ;

[0053] Figure 3 is Figure 1 a schematic structural diagram of the electrode in ;

[0054] Figure 4 is Figure 2 a test diagram of the electric field intensity distribution of the schematic electrophoresis tank;

[0055] Figures 5A to 5E is a schematic structural diagram of a gel separation structure shown in some embodiments of this application;

[0056] Figure 6 is a schematic diagram of the positional relationship of a transfer membrane shown in some embodiments of this application;

[0057] Figure 7Flow chart of the protein separation and detection method shown in some embodiments of the present application;

[0058] Figure 8 Test diagram of the protein separation method shown in some embodiments of the present application;

[0059] Figure 9 Test diagram of the protein separation method shown in some embodiments of the present application;

[0060] Figure 10 Test diagram of the protein detection method shown in some embodiments and comparative examples of the present application;

[0061] Figure 11 Test diagram of the protein detection method shown in some comparative examples of the present application;

[0062] Among them, the numbers in each drawing are as follows:

[0063] 1000, protein separation and detection device;

[0064] 100, electrophoresis structure;

[0065] 110, electrophoresis tank;

[0066] 120, gel separation structure; 121, gel main body; 122, sample injection hole;

[0067] 122a, first type of sample injection hole; 122b, second type of sample injection hole; 122c, third type of sample injection hole;

[0068] 123, through hole;

[0069] 200, internal electrode;

[0070] 300, external electrode;

[0071] 400, transfer membrane;

[0072] 700, filter paper;

[0073] 800, sponge;

[0074] 900a, first electrode; 900b, second electrode. Detailed implementation manners

[0075] Hereinafter, embodiments of the protein separation and detection device and detection method based on annular non-uniform electric field partition injection of the present application will be specifically described in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary details are omitted. For example, there may be cases where detailed descriptions of well-known matters and repeated descriptions of actually identical structures are omitted. This is to prevent the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand the present application, and are not intended to limit the subject matter recited in the claims.

[0076] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular range. The range defined in this way can include or exclude the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a specific parameter, ranges of 60 to 110 and 80 to 120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, then the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0 to 5" means that all real numbers between "0 to 5" have been fully listed herein, and "0 to 5" is only an abbreviated representation of these numerical combinations. In addition, when it is stated that a certain parameter is an integer greater than or equal to 2, it is equivalent to disclosing that the parameter is, for example, the integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0077] If there is no special instruction, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0078] If there is no special instruction, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0079] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0080] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0081] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0082] If not otherwise specified, in this application, the terms "first", "second", etc. are only used to distinguish different objects and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity, specific order or primary and secondary relationship of the indicated technical features.

[0083] Unless otherwise specified, in this application, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple sheets" refers to more than two sheets (including two sheets).

[0084] Protein is the basic unit of life, participates in all major life activities of organisms, and is closely related to the diagnosis and treatment of diseases. Therefore, separating and detecting the structure and function of a large number of protein mixtures is of great significance for understanding the laws of life activities, revealing the mechanisms of various diseases, and diagnosing, treating and preventing them. However, due to the complex biological environment of proteins, their own diverse structures, and low content, how to effectively separate and detect them from a large number of protein samples is a problem that needs to be solved urgently.

[0085] Gel electrophoresis is regarded as a common tool for protein separation and detection due to its simple device, versatility, and high throughput. However, ordinary gel electrophoresis is carried out under the drive of a uniform electric field, and has disadvantages such as low resolution, narrow separation range, long separation time, and no concentration effect. Although high-resolution protein separation techniques based on gradient gels and two-dimensional gels have been developed, they still face problems such as cumbersome preparation steps and difficult operation. As the "gold standard" of protein separation and detection technology, the electrophoresis separation part of Western blotting still has the above-mentioned traditional defects, and due to the lack of concentration effect, the content of the separated sample is low, which easily leads to false negatives in subsequent detections.

[0086] Therefore, there is an urgent need for a simple and efficient separation and concentration technology combined with Western blotting to achieve high-resolution separation and high-sensitivity detection of proteins.

[0087] To achieve the above technical objectives, the present application discloses a protein separation and detection device based on circular non-uniform electric field partition injection, as Figure 1 shown. The protein separation and detection device 1000 includes an electrophoresis structure 100, and the electrophoresis structure 100 includes an electrophoresis tank 110 and a gel separation structure 120 disposed in the electrophoresis tank 110; the gel separation structure 120 includes a gel main body 121 and a plurality of injection holes 122 connected to the gel main body 121. A through hole 123 is provided at the central position of the gel main body 121, and each injection hole 122 is arranged along the circumferential direction of the through hole 123, and the distances between some injection holes 122 and the through hole 123 are not equal; the protein separation and detection device 1000 of the present application further includes an inner electrode 200 and an outer electrode 300. Among them, the inner electrode 200 is disposed in the through hole 123; the outer electrode 300 is arranged along the outer circumferential direction of the gel separation structure 120; in addition, the protein separation and detection device 1000 of the present application further includes a transfer membrane 400, which is not shown in Figure 1 but actually exists.

[0088] As Figure 2 shown, the electrophoresis structure 100 of the present application includes an electrophoresis tank 110, which contains a buffer solution, and the working principle of the electrophoresis tank is based on the action of an electric field on charged particles, so that the charged particles move directionally in the electric field.

[0089] In some embodiments, as Figure 1 shown, the electrophoresis tank 110 is connected to a buffer solution circulation device. The buffer solution circulation device is used to supplement the components in the electrophoresis tank 110 to maintain the balance of the components in the electrophoresis tank 110.

[0090] In some embodiments, as Figure 1As shown, the above electrophoresis tank 110 is connected to a temperature control device. As is known to those skilled in the art, with the continuous driving of the electric field in the electrophoresis tank 110, the temperature of the buffer solution in the electrophoresis tank 110 will change. The temperature control device provided in this application is beneficial to maintaining the stability of the system. In some embodiments, the temperature control device includes an ice-water bath.

[0091] In some embodiments, a circulating liquid inlet is provided at the central position of the above electrophoresis tank 110, and the circulating liquid inlet is connected to the above buffer solution circulating device. This design is beneficial to further maintaining the balance of each component in the electrophoresis tank 110.

[0092] The gel separation structure 120 of this application realizes the separation of molecules by utilizing the network structure and molecular sieve effect of the gel. As described above, the gel separation structure 120 of this application includes a sample injection hole 122 to facilitate protein sample injection.

[0093] In some embodiments, the gel main body 121 includes any one of agarose gel, polyacrylamide gel, or hydrogel.

[0094] In some embodiments, the gel separation structure 120 of this application is prepared by a corresponding mold. Specifically, the gel solution is injected into the corresponding mold, and the gel separation structure of this application is obtained after the gel solidifies. In these embodiments of this application, it is disclosed that the shape of the mold matches the shape of the gel separation structure 120, and this application will not elaborate on each mold.

[0095] In some embodiments, the concentration of the above gel solution can be 3% - 20%.

[0096] In these embodiments of this application, it is disclosed that the concentration of the gel solution includes any one of 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or any one that satisfies the above range values.

[0097] Combined Figure 1 and Figure 3 it can be seen that the protein separation and detection device 1000 provided in this application includes an inner electrode 200 and an outer electrode 300. Among them, the inner electrode 200 is located at the central position of the electrophoresis structure 100, the outer electrode 300 is annular, and is arranged circumferentially along the outer side of the inner electrode 200. And in some embodiments, the inner electrode 200 includes platinum metal, and the outer electrode 300 includes a tin foil ring; therefore, this application establishes an annular non-uniform electric field, as Figure 4 shown, where Figure 4 8 in represents the annular non-uniform electric field, 9 represents the uniform electric field, combined Figure 4It can be known that the characteristics of the annular non-uniform electric field are as follows: the electric field intensity is larger near the center, smaller far from the center, and the closer to the center, the electric field intensity increases exponentially.

[0098] According to the electric field characteristics of the annular non-uniform electric field established above, the present application designs the sample injection holes 122 of the gel separation structure 120. Specifically, each sample injection hole 122 is arranged circumferentially along the through hole 123, and the distances between some sample injection holes 122 and the through hole 123 are not equal. By injecting proteins with different molecular weights into the sample injection holes 122 with unequal distances from the through hole 123, the separation of proteins with different molecular weights is facilitated, and high-resolution separation of proteins is achieved.

[0099] In some embodiments, the distances between two adjacent sample injection holes 122 and the through hole 123 are not equal, that is, as Figure 5A , the arrangement of the sample injection holes 122 is spiral, and the distances between each sample injection hole 122 and the through hole 123 gradually increase.

[0100] In some embodiments, as Figure 5B schematically shown, the sample injection holes 122 include a first type of sample injection hole 122a, a second type of sample injection hole 122b, and a third type of sample injection hole 122c; among them, the distance between the first type of sample injection hole 122a and the through hole 123 is d1; the distance between the second type of sample injection hole 122b and the through hole 123 is d2; the distance between the third type of sample injection hole 122c and the through hole 123 is d3; satisfying: d1 < d2 < d3.

[0101] In the present application, only three types of sample injection holes are schematically shown above Figure 5A and Figure 5B . In fact, the sample injection holes of the present application can also be four types or more, and can be specifically designed according to the molecular weights of the proteins to be separated. The present application only schematically shows in the specification drawings and specific embodiments, and does not make actual limitations.

[0102] In the following of the present application, only three types of sample injection holes are used for explanation, but this is not a limitation on the technical solution of the present application.

[0103] In these embodiments of the present application, in order to cooperate with the above-mentioned three types of injection holes, the protein samples to be separated are further selected to include a first type of protein sample, a second type of protein sample and a third type of protein sample, and the molecular weight of each type of protein sample is different. For example, the molecular weight of the first type of protein sample is M1, the molecular weight of the second type of protein sample is M2, and the molecular weight of the third type of protein sample is M3, satisfying: M1>M2>M3, and the first type of protein sample is injected into the above-mentioned gel separation structure through the above-mentioned first type of injection hole; the second type of protein sample is injected into the above-mentioned gel separation structure through the above-mentioned second type of injection hole; and the third type of protein sample is injected into the above-mentioned gel separation structure through the above-mentioned third type of injection hole.

[0104] In the present application, the first type of protein sample has a high molecular weight. Due to its large mass and volume, it is relatively difficult for the protein to move in the electric field. Loading the sample in the area near the center of the circular non-uniform electric field can provide a sufficiently strong driving force for the high molecular weight protein, overcome its large mass inertia and other obstacles, and promote its efficient separation and concentration; the second type of protein sample has a medium molecular weight, and the protein mass and volume are at a medium level. Compared with small molecular weight proteins, it requires a larger electric field force to drive migration to achieve better separation, but does not require a very strong electric field force like high molecular weight proteins. Loading the sample in the middle electric field area of ​​the ring can just give the medium molecular weight protein a suitable driving force, so that it can be separated and concentrated according to the desired migration behavior. The third type of protein sample has a small molecular weight. The protein itself has a relatively small mass, small volume, and high similarity. Loading it in the outer area of ​​the annular non-uniform electric field with a relatively weak electric field strength not only provides it with a sufficient migration distance, but also allows it to first show different migration behavior differences based on its own characteristics under a relatively mild electric field driving force. Then, with the decrease of the migration radius, the field strength increases exponentially, and the small molecular weight proteins obtain a greater acceleration. With the help of a stronger electric field force, the distance between each other is further increased, thereby accelerating the separation speed. Especially for some proteins with similar properties, this acceleration can more significantly reflect the differences in their motion characteristics, improve the resolution, and finally gradually migrate close to the center to complete the separation and concentration.

[0105] Therefore, the design method provided in this application can add samples to the corresponding separation partitions according to the detection requirements, so that proteins of different molecular weights can be accurately adapted to the electric fields of each region by loading samples in different regions. After the voltage is applied, under the joint action of the electric field in each region and the gel molecular sieve effect, proteins of different molecular weights move toward the inner electrode (positive electrode) at different electrophoretic speeds to achieve high-resolution separation. At the same time, the circular geometric features converge concentrically in the central area to efficiently concentrate the protein, and finally achieve the integration of protein separation and concentration.

[0106] like Figure 5C ,Figure 5D and 5E It can be seen that the present application also separately shows other design manners of the above sample injection holes. In this design manner, the first type of sample injection holes 122a are arranged circumferentially along the above through hole 123, and / or the second type of sample injection holes 122b are arranged circumferentially along the above through hole 123; and / or the third type of sample injection holes 122c are arranged circumferentially along the above through hole 123.

[0107] In some embodiments, the first type of sample injection holes 122a are arranged circumferentially along the above through hole 123, and the second type of sample injection holes 122b are arranged circumferentially along the above through hole 123; and the third type of sample injection holes 122c are arranged circumferentially along the above through hole 123. Along the radial direction of the gel main body 121, the first type of sample injection holes 122a, the second type of sample injection holes 122b and the third type of sample injection holes 122c are not on the same straight line, so as to facilitate protein separation and ensure a certain resolution.

[0108] Combined with Figure 1 It can be seen that the gel separation structure 120 is connected to the above transfer membrane 400. After using the gel separation structure 120 to separate proteins with different molecular weights, as Figure 6 shown in the schematic diagram, the first electrode 900a, the sponge 800, the filter paper 700, the transfer membrane 400, the gel separation structure 120, the filter paper 700, the sponge 800 and the second electrode 900b are stacked together to form a "sandwich" device similar to being immersed in the transfer solution, and then an electric field is applied to make the protein molecules migrate from the gel separation structure to the transfer membrane under the action of the electric field force. The present application controls the protein transfer membrane behavior by adjusting the voltage, current and time. Then, the target protein on the transfer membrane and its expression in the specimen to be detected are detected through the antigen-antibody reaction.

[0109] In some embodiments, the transfer membrane includes any one of a polyvinylidene fluoride membrane, a nitrocellulose membrane or a nylon membrane.

[0110] Therefore, on the basis of achieving the high-resolution separation of the above proteins, the present application can further achieve the detection of proteins.

[0111] The second aspect of the present application is to provide a method for separating and detecting proteins by using the device described in the first aspect, as Figure 7 shown in the schematic diagram, the method includes the following steps:

[0112] S100. Provide a protein sample;

[0113] S200. Inject the above protein sample into the above gel separation structure through the respective sample injection holes of the above device;

[0114] S300. Apply a voltage to the above electrophoresis structure so that proteins with different molecular weights in the above protein sample are separated in the above gel separation structure and form multiple protein bands;

[0115] S400. Transfer each of the above protein bands to the above transfer membrane;

[0116] S500. Perform immunoassay on the proteins on the above transfer membrane.

[0117] In some embodiments, as described above, to cooperate with the above three types of sample injection holes, the protein sample to be separated is further selected to include a first type of protein sample, a second type of protein sample, and a third type of protein sample, and the molecular weights of various protein samples are different. For example, the molecular weight of the first type of protein sample is M1, the molecular weight of the second type of protein sample is M2, and the molecular weight of the third type of protein sample is M3, satisfying: M1 > M2 > M3, and the first type of protein sample is injected into the above gel separation structure through the above first type of sample injection hole; the second type of protein sample is injected into the above gel separation structure through the above second type of sample injection hole; the third type of protein sample is injected into the above gel separation structure through the above third type of sample injection hole.

[0118] In some embodiments, the molecular weight M1 of the first type of protein sample > 70 kDa.

[0119] In some embodiments, the molecular weight M2 of the second type of protein sample is 25 kDa to 70 kDa.

[0120] In some embodiments, the molecular weight M3 of the third type of protein sample < 25 kDa.

[0121] kDa in this application refers to the abbreviation of kilodalton, which is a unit used to represent the molecular weight (or relative molecular mass). The method for measuring the molecular weight of the protein sample in this application includes any conventional measurement method in the art, such as gel permeation chromatography, etc.

[0122] In some embodiments, the molecular weight M1 of the first type of protein sample includes any one or more of 71 kDa, 75 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, etc.

[0123] In some embodiments, the molecular weight M2 of the second type of protein sample includes any one or more of 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, etc.

[0124] In some embodiments, the molecular weight M3 of the third type of protein sample includes any one or more of 24 kDa, 23 kDa, 22 kDa, 21 kDa, 20 kDa, 19 kDa, 15 kDa, etc.

[0125] In some embodiments, the step of transferring each of the above protein bands to the above transfer membrane includes:

[0126] Stack the electrodes, the gel separation structure with the protein band, and the transfer membrane together;

[0127] Place the stacked structure in the transfer buffer and apply a voltage to the above electrodes;

[0128] The above protein band is transferred to the transfer membrane.

[0129] In some embodiments, the transfer conditions of the present application include: controlling the voltage to be 1V to 300V, the current to be 10mA to 400mA, and the time to be 5min to 180min. The present application can specifically adjust the above transfer conditions according to the protein transfer effect.

[0130] After the above protein of the present application is transferred to the above transfer membrane, the transfer membrane is incubated in the first antibody solution, the protein on the transfer membrane binds to the first antibody, and then it is incubated in the second antibody solution, the second antibody binds to the first antibody, and after binding, immunoassay is performed to complete the qualitative detection of the protein. Among them, the first antibody of the present application includes but is not limited to antibody CD9, antibody TSG101, etc.

[0131] The second antibody of the present application includes but is not limited to HRP-linked goat anti-rabbit IgG (H+L) antibody. And after the HRP-labeled second antibody binds to the first antibody, a luminescence signal is generated through a chemiluminescent substrate (such as ECL), which is convenient for detection.

[0132] Example 1

[0133] A method for separating a protein sample by a protein separation and detection device based on annular non-uniform electric field partition injection is provided, and the method includes the following steps:

[0134] (1) Construct a gel separation structure: Prepare 35 mL of a 10% sodium dodecyl sulfate polyacrylamide gel solution and pour the gel into a mold. After solidification, a gel separation structure 120 is formed. The radius of the gel separation structure 120 is 37 mm, and its structure is as Figure 5A shown;

[0135] (2) Provide a transfer membrane: PVDF transfer membrane;

[0136] (3) Construct the electrophoresis structure: Transfer the gel separation structure 120 with the sample injection hole 122 into the electrophoresis tank 110 filled with the buffer solution. The drain hole at the bottom of the electrophoresis tank 110 is connected to the drain pipe and the pump for draining the buffer solution. Use tin foil to construct the annular outer electrode 300 and adhere it to the inner side wall of the electrophoresis tank 110. Use platinum metal to construct the cylindrical inner electrode 200, insert one end of the inner electrode 200 into the bottom of the through hole 123, and fix the other end on the iron stand to establish an annular non-uniform electric field system. Fix the liquid inlet pipe above the through hole 123. The liquid inlet pipe and the inner electrode 200 are fixed on the iron stand together. The liquid inlet pipe is connected to the pump and the buffer solution storage bottle, and the buffer solution storage bottle is placed in the ice-water mixture for cooling. Drain the liquid through the drain pipe and inject the liquid through the liquid inlet pipe to realize the circulating condensation of the buffer solution in the through hole 123 and maintain the temperature and pH stability of the buffer solution in the through hole 123;

[0137] (4) Prepare the protein sample: Taking the preparation of the exosome protein sample as an example in this embodiment, specifically collect the urine samples within 24 hours, centrifuge at 300 g and 2000 g for 10 minutes respectively and then centrifuge at 10000 g for 30 minutes to remove cells, dead cells and cell debris. Finally, centrifuge at 100000 g for 70 minutes, resuspend the exosome sample with PBS solution for collection, and store it at -80 °C. Use the BCA method to quantify the protein concentration of the exosomes. Mix the exosome sample with the loading buffer (loading buffer, conventional in the art) at a ratio of 4:1, and boil it in a water bath at 95 °C for 15 min to obtain the exosome protein sample.

[0138] (5) Electrophoresis: After injecting the protein sample and the protein Marker prepared in step (4) into the above-mentioned sample injection hole 122 respectively, apply a voltage (80 V) to start electrophoresis. The protein sample migrates to the central region in turn according to the molecular weight, forming a target distribution, where the protein marker (containing 9 bands: 150 kDa, 100 kDa, 70 kDa, 50 kDa, 40 kDa, 35 kDa, 25 kDa, 20 kDa, 15 kDa) is used as an indicator.

[0139] Obtain Figure 8 the schematic test chart, combined with Figure 8 it can be seen that proteins with different molecular weights are well separated.

[0140] This embodiment further obtains Figure 8 the partition and band peak chart of the schematic 45-min protein Marker spiral separation chart, as shown in Figure 9 (a), Figure 9 (b), Figure 9 (c) and Figure 9 (d). And Figure 9(a) is the preliminary regional division of the spiral separation diagram of the 45min protein Marker, Figure 9 (b) is the peak diagram of 10 protein bands, Figure 9 (c) is the peak diagram of 11 protein bands, Figure 9 (d) is the peak diagram of 12 protein bands, where 10, 11, and 12 are Figure 9 (a) The protein separation bands at different injection hole positions shown, combined with Figure 9 (b) to Figure 9 (d) It can be seen that the gray scale values of the peak diagrams of protein bands with different molecular weights are obvious, indicating that proteins with different molecular weights are Figure 5A well separated in the gel separation structure shown.

[0141] Example 2

[0142] A method for separating and detecting a protein sample by using a protein separation and detection device based on annular non-uniform electric field partition injection is provided. The difference between this method and Example 1 lies in that in step (1), the gel separation structure 120 is as Figure 5B shown: The gel separation structure 120 includes a first type of injection hole 122a, a second type of injection hole 122b, and a third type of injection hole 122c;

[0143] Among them, the radius of the through hole 123 is 2.5 mm, the distance d1 between the first type of injection hole 122a and the through hole 123 is 13.5 mm, the distance d2 between the second type of injection hole 122b and the through hole 123 is 17.5 mm, and the distance d3 between the third type of injection hole 122c and the through hole 123 is 25.5 mm, satisfying d1 < d2 < d3;

[0144] The construction of the electrophoresis structure, the preparation of the protein sample, and the electrophoresis process in the separation method of this example are the same as those in Example 1; at the same time, protein detection is further carried out in this example, specifically as follows:

[0145] (6) Transfer and immunodetection: After the above electrophoresis is completed, the gel separation structure 120 and the transfer membrane 400 are tightly clamped between the sponge 800 and the filter paper 700 to form a sandwich structure, specifically as Figure 6It is shown that wet transfer is carried out in the electrotransfer buffer (such as 300 mA for 20 min). After the transfer is completed, take out the transfer membrane printed with the concentrated protein sample and block it with 5% skim milk for 1 h. The blocked transfer membrane is cut as needed and soaked separately in the prepared primary antibody (CD9 and TSG101, diluted with TBST to a ratio of 1:1000) solution, and incubated overnight at 4 °C. Then recover the primary antibody, wash the membrane with TBST at room temperature for 10 min, and repeat three times. Next, soak the transfer membrane in the secondary antibody (HRP-linked goat anti-rabbit IgG (H+L) antibody, diluted with TBST to a ratio of 1:10000) solution and incubate at room temperature for 1 h. After the incubation is completed, recover the secondary antibody, wash the membrane with TBST at room temperature for 10 min, and repeat three times. Take out the transfer membrane and lay it flat in the imager with the protein side up, drop the developing solution onto the transfer membrane, start exposure after setting the parameters, adjust the brightness and contrast of the imaging diagram and save it to obtain Figure 10 , combined with Figure 10 It can be seen that compared with the traditional Western blotting method of the comparative example, the detection method of this example has significantly better separation and detection effects on proteins, and the detection sensitivity is significantly improved.

[0146] Comparative Example 1

[0147] A traditional method for separating and detecting protein samples is provided. This method uses a conventional gel separation structure (vertical slab gel electrophoresis) and includes the following specific processes:

[0148] (1) Electrophoresis: The processed urine exosome protein sample and protein Marker are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Obtain Figure 11 The schematic protein Marker band separation diagram and band peak diagram shown, where Figure 11 (a) is the protein Marker band separation diagram (45 min) of the gel electrophoresis of this comparative example, and Figure 11 (b) is Figure 11 The peak diagram of the band in (a).

[0149] (2) Transfer: After electrophoresis, take out the electrophoresis gel, trim the size of the gel, cut off the edges and excess parts, and cut a PVDF membrane of the corresponding size according to the size of the trimmed gel, and activate it in an appropriate amount of methanol. Soak 8 filter papers in the pre-cooled transfer buffer, and make a transfer "sandwich" in the order of negative electrode - sponge - filter paper - electrophoresis gel - membrane - filter paper - sponge - positive electrode.

[0150] (3) Immunoassay: Add transfer buffer into the tank, assemble the transfer device, add floating ice, bury the device in an ice bath, turn on the power supply, and transfer the membrane at 300 mA for 20 min. After the transfer is completed, take out the PVDF membrane, soak the membrane with the protein side up in 5% skim milk in TBST, and block for 1 h. Then, incubate with TSG101 (1:1000), CANX (1:1000), and CD9 (1:1000) overnight at 4 °C. After washing the membrane, incubate with the enzyme-labeled secondary antibody (1:10000) at room temperature for 1 h. Finally, use a gel imaging system to display the obtained immunoreactive bands. Adjust the brightness and contrast of the imaging diagram and save it. Obtain Figure 10 。

[0151] Combined Figure 10 it can be seen that the design method provided by this application has higher detection sensitivity compared with the comparative example.

[0152] In summary, the design method provided by this application realizes high-resolution separation and high-sensitivity detection of proteins.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and the description of this application. In particular, as long as there is no structural conflict, the technical features mentioned in each embodiment can be combined in any way. This application is not limited to the specific embodiments disclosed in the text, but includes all technical solutions falling within the scope of the claims.

Claims

1. A protein separation and detection device based on annular non-uniform electric field partition injection, characterized in that: Comprising: Electrophoresis structure: including an electrophoresis tank and a gel separation structure disposed in the electrophoresis tank; The gel separation structure includes a gel main body and a plurality of sample injection holes connecting the gel main body. A through hole is provided at the central position of the gel main body. Each of the sample injection holes is arranged circumferentially along the through hole, and the distances between some of the sample injection holes and the through hole are not equal; Inner electrode: disposed in the through hole; Outer electrode: arranged circumferentially along the outer side of the gel separation structure; And a transfer membrane.

2. The protein separation and detection device according to claim 1, wherein: The distances between two adjacent sample injection holes and the through hole are not equal.

3. The protein separation and detection device according to claim 1, characterized in that: The sample injection holes include first-class sample injection holes, second-class sample injection holes and third-class sample injection holes; The distance between the first-class sample injection hole and the through hole is d1; The distance between the second-class sample injection hole and the through hole is d2; The distance between the third-class sample injection hole and the through hole is d3; Satisfying: d1 < d2 < d3.

4. The protein separation and detection device according to claim 3, wherein: The first-class sample injection holes are arranged circumferentially along the through hole; And / or; The second-class sample injection holes are arranged circumferentially along the through hole; And / or; The third-class sample injection holes are arranged circumferentially along the through hole.

5. The protein separation and detection device according to any one of claims 1 to 4, characterized in that: The electrophoresis tank is connected to a buffer circulation device; And / or; The electrophoresis tank is connected to a temperature control device; And / or; The gel separation structure is connected to the transfer membrane.

6. The protein separation and detection device according to claim 5, characterized in that: A circulation liquid inlet is provided at the central position of the electrophoresis tank, and the circulation liquid inlet is connected to a buffer circulation device.

7. The protein separation and detection device according to any one of claims 1 to 3, characterized in that: The inner electrode includes platinum metal; And / or; The outer electrode includes a tin foil ring; And / or; The gel main body includes any one of agarose gel, polyacrylamide gel or hydrogel; And / or; The transfer membrane includes any one of polyvinylidene fluoride membrane, nitrocellulose membrane or nylon membrane.

8. A method for separating and detecting proteins by the device according to claim 1, characterized in that: Providing a protein sample; Injecting the protein sample into the gel separation structure through each of the sample injection holes of the device; Applying a voltage to the electrophoresis structure so that proteins with different molecular weights in the protein sample are separated in the gel separation structure and form a plurality of protein bands; Transferring each of the protein bands to the transfer membrane; Performing immunoassay on the proteins on the transfer membrane.

9. The method according to claim 8, wherein: The sample injection holes include first-class sample injection holes, second-class sample injection holes and third-class sample injection holes; The distance between the first-class sample injection hole and the through hole is d1; The distance between the second-class sample injection hole and the through hole is d2; The distance between the third-class sample injection hole and the through hole is d3; Satisfying: d1 < d2 < d3; The protein sample includes a first-class protein sample, a second-class protein sample and a third-class protein sample. The molecular weight of the first-class protein sample is M1, the molecular weight of the second-class protein sample is M2, and the molecular weight of the third-class protein sample is M3, satisfying: M1 > M2 > M3; And the first-class protein sample is injected into the gel separation structure through the first-class sample injection hole; the second-class protein sample is injected into the gel separation structure through the second-class sample injection hole; the third-class protein sample is injected into the gel separation structure through the third-class sample injection hole.

10. The method according to any one of claims 8 to 9, characterized in that: The step of transferring each of the protein bands to the transfer membrane includes: Stacking an electrode, a gel separation structure having protein bands, and a transfer membrane together; Placing the stacked structure in a transfer solution and applying a voltage to the electrode; Transferring the protein bands to the transfer membrane.

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