Sample elution extraction device and sample elution extraction system

Abstract

The utility model provides a kind of sample elution extraction device and sample elution extraction system, sample elution extraction device includes cavity, cavity includes sample inlet and sample outlet;Cavity includes first cavity and second cavity, first filter layer is arranged between first cavity and second cavity, second filter layer is arranged between second cavity and sample outlet, second cavity is used to exert at least one part of force to first cavity, which prevents liquid in first cavity from flowing to sample outlet through first filter layer.The device of the present application can accommodate liquid reaction system containing treatment for a long time, and is less likely to leak, thereby avoiding incomplete treatment or the need for liquid replacement, reducing reagent use.

Description

[0001] Priority information This application claims priority and benefits to patent application No. 202521245970.8, filed with the China National Intellectual Property Administration on June 17, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This utility model relates to the field of mass spectrometry sample processing technology, and more specifically, to a sample elution and extraction device and a sample elution and extraction system. Background Technology

[0003] Sample elution and extraction is a crucial pretreatment technique widely used in the separation, purification, and enrichment of target substances. Traditional sample elution and extraction methods typically rely on tools such as centrifuge tubes, filter membranes, or adsorbent materials, involving multiple manual operations such as pipetting, centrifugation, or filtration. However, these methods have several drawbacks. Taking the in-gel enzymatic digestion method in gel strip mass spectrometry identification as an example, it involves numerous steps. During sample pretreatment, manual aspiration of waste liquid is required for each sample multiple times, consuming experimental consumables, resulting in long experimental times, and it is difficult to automate. Furthermore, during multiple transfers and processing, for soft protein gels, aspiration of waste liquid may clog the pipette tip or aspirate the gel, leading to sample loss; for brittle protein gels, they are prone to breakage, producing small gel fragments that may also be accidentally aspirated, resulting in loss. Transferring between various consumables increases the risk of non-specific adsorption and reduces the reproducibility of detection. These factors significantly limit the detection efficiency, throughput, and quality of gel strip mass spectrometry identification.

[0004] Therefore, devices for sample elution and extraction still need further research. Utility Model Content

[0005] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application provides a sample elution and extraction device and system, which can complete most elution and extraction reactions within the device without multiple transfers of the processed material, thus avoiding sample loss and contamination risks caused by the transfer process. Simultaneously, the device can hold the liquid reaction system containing the processed material for extended periods without leakage, thereby avoiding incomplete processing or the need for replenishment and reducing reagent usage. Therefore, it can effectively improve the elution and extraction effect and efficiency, and can be widely applied in various sample processing scenarios, demonstrating high application value.

[0006] In one aspect of this application, a sample elution and extraction device is provided, comprising: a cavity including an inlet and an outlet; along the direction from the inlet to the outlet, the cavity includes a first cavity and a second cavity, a first filter layer is disposed between the first cavity and the second cavity, and a second filter layer is disposed between the second cavity and the outlet, the second cavity being used to apply at least a portion of a force to the first cavity to prevent liquid in the first cavity from flowing through the first filter layer to the outlet; the first cavity is connected to the second cavity through a first filter hole on the first filter layer; the second cavity is connected to the outlet through a second filter hole on the second filter layer.

[0007] Furthermore, the average porosity of the first filter layer is greater than or equal to the average porosity of the second filter layer.

[0008] Furthermore, the materials of the first filter layer and the second filter layer are each independently at least one of glass fiber, polyethylene, polypropylene and polytetrafluoroethylene.

[0009] Furthermore, a compaction layer and / or a swelling layer are provided above the first filter layer.

[0010] Furthermore, the swollen layer is sandwiched between the compacted layer and the first filter layer.

[0011] Furthermore, the average inner diameter of the first cavity is greater than the average inner diameter of the second cavity.

[0012] Furthermore, the first cavity and / or the second cavity includes cavity segments of equal diameter.

[0013] Furthermore, for the first cavity and / or the second cavity, along the direction from the inlet to the outlet, the inner diameter of the cavity near the inlet is larger than the inner diameter of the cavity near the outlet.

[0014] Furthermore, the first cavity and / or the second cavity includes concentric tapering cavity segments. Furthermore, a third cavity is provided between the second filter layer and the sample outlet.

[0015] Furthermore, the average inner diameter of the second cavity is greater than the average inner diameter of the third cavity.

[0016] Furthermore, the third cavity comprises cavity segments of equal diameter.

[0017] Furthermore, for the third cavity, along the direction from the inlet to the outlet, the inner diameter of the cavity near the inlet is larger than the inner diameter of the cavity near the outlet.

[0018] Furthermore, the third cavity includes a concentric tapering cavity segment.

[0019] Furthermore, the inlet and / or outlet are each covered with a cap.

[0020] Furthermore, the pore sizes of the first filter pore and the second filter pore are each independently 1~100μm.

[0021] In another aspect of this application, a sample elution and extraction system is proposed, which includes a sample elution and extraction device.

[0022] Furthermore, the sample elution and extraction system further includes a pressure application device for applying pressure toward the sample outlet into the first chamber.

[0023] Furthermore, the sample elution and extraction system further includes: an ultrasonic processing device for applying ultrasonic waves to the first cavity.

[0024] Applying the technical solution of this application, the sample elution and extraction device has two filter layers inside the cavity, dividing the cavity into a first cavity and a second cavity. The first filter layer can support and filter the liquid material contained in the first cavity. When it is necessary to filter the liquid system containing the material, pressure is applied from the inlet to the outlet. Solids are retained on the first filter layer, while liquid flows out through the second filter layer from the outlet. When the liquid system needs to be retained in the first cavity for a period of time, the gas in the second cavity is subjected to an upward force due to the gravity of the liquid system, the first filter layer exerts a supporting force to support the liquid material, and the surface tension between the liquid and the first filter layer, these three forces work together to effectively delay or prevent the downward flow of liquid. This makes it difficult for liquid in the first cavity to enter the second cavity or flow out from the outlet without external pressure, thereby avoiding leakage that could lead to incomplete sample processing or the need for liquid replenishment, and improving the extraction effect, such as the yield. Attached Figure Description

[0025] The accompanying drawings, which form part of this application, are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an undue limitation of the present invention. In the drawings: Figure 1 A schematic diagram of a sample elution and extraction apparatus according to an embodiment of this application is shown; Figure 2 A schematic diagram of a sample elution and extraction apparatus according to another embodiment of this application is shown.

[0026] The above figures include the following reference numerals: 1. Cavity; 10. Inlet; 20. Outlet; 100. First cavity; 200. Second cavity; 300. Third cavity; 400. Auxiliary layer; 410. Compacted layer; 420. Swelling layer; 430. First filter layer; 500. Second filter layer. Detailed Implementation

[0027] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0028] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps described in these embodiments do not limit the scope of this application. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0030] In the description of this application, it should be understood that the orientation or positional relationship indicated by directional terms such as "front, back, up, down, left, right", "horizontal, vertical, horizontal" and "top, bottom" is usually based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this application and simplifying the description. Unless otherwise stated, these directional terms do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the scope of protection of this application; the directional terms "inner" and "outer" refer to the inner and outer contours relative to the outline of each component itself.

[0031] refer to Figure 1This application provides a sample elution and extraction device, comprising: a cavity 1, the cavity 1 including an inlet 10 and an outlet 20; along the direction from the inlet 10 to the outlet 20, the cavity 1 includes a first cavity 100 and a second cavity 200, a first filter layer 430 is disposed between the first cavity 100 and the second cavity 200, and a second filter layer 500 is disposed between the second cavity 200 and the outlet 20, the second cavity 200 being used to apply at least a portion of a force to the first cavity 100 to prevent the liquid in the first cavity 100 from flowing through the first filter layer 430 to the outlet 20; the first filter layer 430 includes a first filter hole, the first filter hole being connected to the first cavity 100 and the second cavity 200 respectively; the second filter layer 500 includes a second filter hole, the second filter hole being connected to the second cavity 200 and the outlet 20 respectively. The sample elution and extraction device of this application has two filter layers inside the cavity, dividing the cavity into a first cavity and a second cavity. The first filter layer, with filter pores, can support and filter the liquid system containing the sample to be processed contained in the first cavity. When it is necessary to filter the liquid system containing the sample to be processed, pressure is applied from the inlet to the outlet. Solids are retained on the first filter layer, while liquid flows out through the second filter layer from the outlet. When the liquid system needs to react in the first cavity for a period of time, the gas in the second cavity is compressed by the gravity of the liquid, forming an upward force. The first filter layer exerts a supporting force to support the liquid, and the surface tension between the liquid and the first filter layer, these three forces work together to effectively delay or prevent the downward flow of the liquid. This makes it difficult for the liquid in the first cavity to enter the second cavity or flow out from the outlet without external pressure, thereby avoiding leakage that could lead to incomplete sample processing or the need for liquid replenishment, and improving the extraction effect, such as the yield.

[0032] In this paper, the term "processing material" refers to the substance to be processed, which can refer to either the sample to be processed or an intermediate product of the sample in the process.

[0033] In this embodiment, a third cavity 300 is provided between the second filter layer 500 and the sample outlet 20.

[0034] Mass spectrometry sample processing includes in-gel digestion, which involves fragmenting the target lanes of the protein gel electrophoresis sample, followed by washing, destaining and dehydration, protein reduction, protein alkylation, protease digestion, and peptide extraction. Currently, in-gel digestion is a cumbersome and time-consuming process. While using in-gel digestion kits reduces processing time, the high cost of these kits increases the overall cost of in-gel digestion.

[0035] Automated pipetting equipment places gel and solvent in centrifuge tubes for reaction. After the gel is immersed in the solvent and undergoes decolorization or intragel peptide extraction, the solution is transferred to the next reaction system using a pipette. Because the gel strip needs to be chopped before decolorization, the chopped gel particles easily stick to the pipette needle, preventing the pipette from reaching the bottom of the centrifuge tube during pipetting. As a result, the amount of solution transferred each time is small. To ensure sufficient solution volume, solvent needs to be added to the centrifuge tube multiple times. Therefore, using automated pipetting equipment for intragel enzymatic hydrolysis is time-consuming and consumes a lot of solvent.

[0036] The sample elution and extraction apparatus of this application can efficiently perform in-gel enzymatic hydrolysis. In some embodiments, when a first liquid is added to the first chamber 100, the first liquid and the upper surface of the first filter layer 430 come into contact. The surface tension of the contact surface and the higher pressure of the second chamber 200 relative to the first chamber 100 balance the hydrostatic pressure of the liquid, thus retaining the first liquid in the first chamber. When external pressure is applied to break this balance, all the first liquid flows through the first filter pores to the sample outlet 20 and is discharged. Optionally, the first liquid can be a decolorizing solution, enzymatic hydrolysis solution, washing solution, or extraction solution, etc.

[0037] Compared to the first chamber, the second chamber has stronger "resistance" (i.e., the second chamber provides greater flow resistance, resulting in higher pressure or a larger pressure drop in this area). When there is no external pressure, i.e., under natural reaction conditions, the decolorizing solution, enzymatic hydrolysate, washing solution, or extraction solution above the first filter layer 430 will come into full contact with the granular material (gel), allowing the gel to be immersed in the liquid system and complete decolorization, enzymatic hydrolysis, washing, or extraction, thereby completing the extraction of peptides within the gel. The decolorizing solution, enzymatic hydrolysate, washing solution, or extraction solution is less likely to flow out from the sample outlet 20. When decolorization... When the enzymatic hydrolysis or extraction is completed, the pressure in the first chamber 100 or the second chamber 200 is adjusted until the reacted liquid flows smoothly out through the first filter hole, the second filter hole and the sample outlet 20. There is no longer a need for manual removal of the reacted liquid, which reduces the reaction steps, shortens the reaction time and reduces the manpower required. This solves the problem that the existing in-gel enzymatic hydrolysis process cannot simultaneously achieve the advantages of saving time and labor as well as saving solvent. At the same time, it avoids sample loss during the operation, ensures the sample recovery rate and improves the reproducibility of the detection.

[0038] Specifically, because the pore size of the first filter hole is small enough, the liquid contained in the first cavity can form a surface tension with the first filter layer 430. The surface tension and the pressure of the second cavity 200 together overcome the gravity of the liquid and the pressure of the first cavity 100, preventing the liquid from flowing out of the first filter hole. This ensures that when the pressure in the first cavity 100 is less than the pressure in the second cavity 200, the liquid and particulate matter above the first filter layer 430 are in full contact.

[0039] Specifically, when the pressure in the first chamber 100 is greater than the pressure in the second chamber 200 and the third chamber 300, the liquid in the first chamber can flow smoothly out from the first filter hole, the second filter hole, and the sample outlet. When the pressure in the first chamber 100 is less than the pressure in the second chamber 200, the gas in the second chamber 200 is under the pressure of the third chamber 300, and the liquid will not flow out from the second filter hole. This ensures that the pressure in the second chamber 200 is stable and prevents the pressure in the second chamber 200 from decreasing to less than the pressure in the first chamber 100. This ensures that the pressure in the second chamber 200 and the surface tension of the first filter layer 430 together overcome the gravity of the liquid and the pressure in the first chamber 100, preventing the liquid from flowing out from the first filter hole.

[0040] In practice, high-pressure gas is introduced into the first cavity 100 through a pressurizing component, so that the pressure in the first cavity 100, the second cavity 200 and the third cavity 300 decreases in sequence, ensuring that the liquid flows out smoothly from the first filter hole, the second filter hole and the sample outlet.

[0041] To minimize or prevent liquid from flowing out of the sample outlet through the first filter layer 430 in the first chamber 100, the sample elution and extraction device provided in this application ensures that the pressure in the second chamber is greater than the pressure in the first chamber when liquid is present in the first chamber. This pressure arises from the resistance difference between the first and second filter layers. Therefore, the resistance of each filter layer is first derived from the Carman-Kozeny equation: First filter layer resistance R1:

[0042] Second filter layer resistance R2:

[0043] in, : Filter layer porosity (dimensionless, 0 < <1) A m Area of ​​the second filter layer A u Area of ​​the first filter layer d p : Sieve aperture or particle characteristic size (m) μ Dynamic viscosity of fluid (Pa·s) L : Thickness of the filter layer (m) To enhance the "resistance" of the second chamber, i.e., to increase the pressure in the second chamber, it is necessary to reduce the pressure drop of the first filter layer (i.e., reduce the resistance of the first filter layer) and at the same time increase the pressure drop of the second filter layer (i.e., increase the resistance of the second filter layer), so that R2 > R1. In this way, the pressure drops significantly at the second filter layer, causing the pressure in the second chamber to accumulate and increase.

[0044] When all filter layers are made of the same material This can be considered a constant, and at this point, the porosity and area of ​​each filter layer must meet the following relationship:

[0045] Therefore, the porosity is selected. 1 / 2≥1, and the area of ​​the filter layer A u / A m When ≥1, and not all are equal signs, it helps to satisfy R 2> R 1. This allows the second cavity to withstand high resistance forces generated by gravity compression.

[0046] In this embodiment, there are multiple first filter pores spaced apart on the first filter layer 430, and multiple second filter pores spaced apart on the second filter layer 500. The average porosity of the first filter layer 430 is greater than or equal to the average porosity of the second filter layer 500. This more effectively prevents liquid from flowing from the first cavity through the second filter layer to the sample outlet without external pressure, thus preventing accidental leakage of liquid during the reaction process and ensuring the stability of the reaction system.

[0047] In this embodiment, the porosity of the first and second filter pores is independently 50%-70%, for example, 60%. This not only effectively delays or prevents liquid leakage when no external pressure is applied to the first chamber, ensuring the stability of the reaction system and avoiding sample loss and contamination; but also allows the liquid to flow rapidly through the first and second filter pores to the sample outlet when external pressure is applied to the first chamber. Furthermore, it reduces the occurrence of material clogging the filter pores.

[0048] In this embodiment, the pore sizes of the first filter pore and the second filter pore are each independently 1~100 μm, for example, they can be 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, or 100μm; preferably, the pore size of the first filter pore is greater than or equal to the pore size of the second filter pore. Therefore, the first filter layer has multiple small-diameter filter holes, which makes the surface tension of the liquid sufficient to prevent the liquid from flowing out of the first filter layer towards the sample outlet. When no external pressure is applied to the first chamber, it can effectively slow down or prevent liquid leakage, ensure the stability of the reaction system, and avoid sample loss and contamination. When external pressure is applied to the first chamber, it can make the liquid flow out of the sample outlet quickly through the first filter hole and the second filter hole.

[0049] In this embodiment, the thicknesses of the first and second filter layers are each independently 0.1–5 mm, for example, 0.1 mm, 0.2 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, 4 mm, and 5 mm. The thickness of the first filter layer satisfies these conditions, providing suitable barrier properties to ensure no liquid leakage under no external pressure, thereby maintaining the stability and sealing of the reaction system and preventing sample loss and contamination. The thickness of the second filter layer satisfies these conditions, allowing liquid to pass through smoothly under applied external pressure, improving the efficiency of elution and extraction. Furthermore, the layered structure with the aforementioned thicknesses maintains suitable mechanical strength of the filter layer and is easy to prepare.

[0050] In this paper, the term "thickness" refers to the length along the direction from the inlet to the outlet.

[0051] In this embodiment, the first filter layer 430 and the second filter layer 500 are materials known in the art. They can be hydrophobic materials, or the materials can be surface-modified to suit the characteristics of the liquid to be retained, thereby enhancing the capillary blocking effect of the filter pores. When capillary blocking is enhanced, the liquid is more difficult to pass through the filter pores without external force, effectively preventing spontaneous leakage of the liquid and ensuring that the liquid only flows through the filter pores when an external force (such as pressure or centrifugal force) is applied. In some embodiments, the materials of the first filter layer and the second filter layer can each independently be, for example, at least one of glass fiber, polyethylene, polypropylene, and polytetrafluoroethylene. The above materials are hydrophobic, do not easily react with samples and reagents, and have a stable structure that is not easily damaged, which helps to improve the elution and extraction efficiency. The compaction layer material can be polyethylene, polypropylene, etc.; the swelling layer material can be glass fiber, polyethylene, polypropylene, polytetrafluoroethylene, etc., with a flocculent structure.

[0052] In some embodiments, the edges of the first filter layer 430 and the second filter layer 500 abut against the respective cavity sidewalls to remain stable from top to bottom.

[0053] In this embodiment, an auxiliary layer 400 is further included above the first filter layer 430. The auxiliary layer comprises at least one selected from the compacted layer 410 or the swollen layer 420. Preferably, the auxiliary layer comprises the compacted layer 410 and the swollen layer 420; when both a compacted layer and a swollen layer are present, preferably, the swollen layer is sandwiched between the compacted layer and the first filter layer; preferably, the auxiliary layer is configured as a single layer that functions as both the compacted layer 410 and the swollen layer 420.

[0054] The auxiliary layer serves three design purposes: First, it prevents liquid splashing during sample addition, specifically avoiding splashing during liquid addition and reducing sample loss. Second, through the swelling layer, it prevents liquid loss. Specifically, when the liquid flows through the auxiliary layer, its volume expands slightly, allowing it to fully contact the inner wall of the cavity, preventing liquid from flowing out from the gap between the sieve plate and the cavity wall, which would lead to incomplete sample processing or the need for liquid replenishment, resulting in liquid waste and poor elution and extraction effects. Third, through the compaction layer, it prevents the first filter layer 430 from rebounding and detaching after external pressure is released. Specifically, the auxiliary layer, by its own weight and the frictional force generated with the inner wall of the tube, forms a stable and uniform pressure constraint on the lower first filter layer, eliminating the risk that the sieve plate will be sucked up or pushed up due to pressure difference during pressure relief.

[0055] Since all materials inevitably adsorb samples to varying degrees, the thicker the liquid flows through each layer of material, the larger the flow area and the greater the degree of sample adsorption. Therefore, it is necessary to control the thickness of the filter layer and auxiliary layer to minimize sample adsorption and achieve high sample recovery rate and good repeatability.

[0056] In some embodiments, the total thickness of the auxiliary layer 400 is 0.1 to 4 mm, for example, 0.1 mm, 0.2 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.8 mm, 1 mm, 2 mm, 3 mm, or 4 mm; wherein, the thickness of the compacted layer 410 is less than 1 mm and greater than or equal to 0 mm, and the thickness of the swelling layer 420 is less than 3 mm and greater than or equal to 0 mm, preferably less than 2 mm; the thicknesses of the first filter layer 430 and the second filter layer 500 are each independently less than 2 mm and greater than or equal to 0 mm, for example, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, or 1 mm.

[0057] In some embodiments, the pore sizes of the compacted layer 410 and the swollen layer 420 are larger than the pore size of the first filter pore. Specifically, the compacted layer 410 and the swollen layer 420 can be 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, or 500 μm. This helps to reduce sample loss.

[0058] In some embodiments, the compaction layer material may be polyethylene, polypropylene, etc.; the swelling layer material may be glass fiber, polyethylene, polypropylene, polytetrafluoroethylene, etc., with a flocculent structure.

[0059] In some embodiments, the pore size of the second filter layer 500 is less than or equal to that of the first filter layer 430. In some embodiments, the pore sizes of the first filter layer 430 and the second filter layer 500 are each independently less than 30 μm, for example, 30 μm, 20 μm, 19 μm, 18 μm, 17 μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, and 1 μm. This facilitates the prevention of liquid leakage from the first filter layer when no external force is applied to the first cavity, and allows the liquid to flow out smoothly when an external force is applied to the first cavity.

[0060] In some embodiments, the diameter of the auxiliary layer is 0-0.5 mm larger than the inner diameter of the first cavity. This ensures sufficient contact between the auxiliary layer and the inner wall of the cavity, increasing friction and effectively preventing the sieve plate from rebounding and detaching during pressure release.

[0061] In this embodiment, along the direction from the inlet 10 to the outlet 20, the average inner diameter of the first chamber 100 is larger than the average inner diameter of the second chamber 200, and the average inner diameter of the second chamber 200 is larger than the average inner diameter of the third chamber 300. This helps increase flow resistance, effectively preventing spontaneous liquid flow without external force, thus avoiding liquid leakage and ensuring the sealing and stability of the reaction system. Furthermore, it helps reduce liquid residue within the chambers, minimizing losses and improving elution and extraction efficiency.

[0062] In this embodiment, the average inner diameter of the first cavity 100 is greater than the average inner diameter of the second cavity 200, and the average inner diameter of the second cavity 200 is greater than the average inner diameter of the third cavity 300.

[0063] For example, the first cavity 100, the second cavity 200 and / or the third cavity 300 include cavity segments of equal diameter.

[0064] For example, for one or more of the first cavity 100, the second cavity 200, and the third cavity 300, along the direction from the inlet 10 to the outlet 20, the inner diameter of the cavity near the inlet 10 is larger than the inner diameter of the cavity near the outlet 20. This helps to increase the liquid flow rate, reduce liquid loss during flow, and allow liquid to flow out quickly and in large quantities from the first, second, and third cavities. Furthermore, it helps to reduce liquid residue within the cavities, thus reducing losses and improving elution and extraction efficiency. Specifically, the cavity tube diameter can be a reduced-diameter tube, and the tube diameter change method can be a stepped reduction, that is, the change of tube diameter is achieved through a series of discrete steps, at each step the tube diameter suddenly decreases, forming a clear stepped structure; it can also be a gradual reduction, that is, the change of tube diameter is smooth and continuous, gradually transitioning from a larger tube diameter to a smaller tube diameter; it can also be a segmented reduction, that is, the tube diameter is reduced in multiple regions; it can also be a composite reduction, that is, the above two or more reduction methods are combined, and the specific choice can be made flexibly according to actual needs.

[0065] In some embodiments, the first cavity 100, the second cavity 200, and the third cavity 300 include concentric tapering cavity sections, the inner diameter of which gradually decreases along the direction from the sample inlet 10 to the sample outlet 20. Further, the tapering is a linear tapering or a curved tapering.

[0066] In this embodiment, the first cavity 100, the second cavity 200, and the third cavity 300 each independently comprise a cavity segment of equal diameter and / or a concentrically tapering cavity segment. The inner diameter of the concentrically tapering cavity segment gradually decreases along the direction from the inlet 10 to the outlet 20. Further, the tapering is linear or curvilinear. Thus, the inner diameter of each cavity remains constant, continuously decreases, or simultaneously comprises a cavity segment of equal diameter and a concentrically tapering cavity segment. Furthermore, along the direction from the inlet to the outlet, the inner diameter between adjacent cavities can decrease gradually or continuously. This helps increase the liquid flow rate, reduces liquid loss during flow, and allows liquid to flow out quickly and in large quantities from the first, second, and third cavities. It also helps reduce liquid residue within the cavities, thus reducing losses and improving elution and extraction efficiency.

[0067] In some embodiments, the first cavity 100 and the second cavity 200 and / or the second cavity 200 and the third cavity 300 have a stepped diameter reduction in the sample elution and extraction apparatus.

[0068] In some embodiments, the first cavity 100 and the second cavity 200 and / or the second cavity 200 and the third cavity 300 form a continuous tapering diameter in the sample elution and extraction apparatus.

[0069] In some specific embodiments, the first cavity 100, the second cavity 200 and the third cavity 300 are each independently a cavity of equal diameter, and the cavities are in a stepped diameter reduction manner in the device.

[0070] In some specific embodiments, the first cavity 100 includes a concentric tapering cavity section, the second cavity 200 and the third cavity 300 are concentric tapering cavities, and the adjacent cavities have a stepped or continuous tapering diameter in the device.

[0071] In some embodiments, the inner diameter of the end of the second cavity 200 facing the inlet 10 is smaller than the average inner diameter of the first filter layer 430. Specifically, the inner diameter of the end of the second cavity 200 facing the inlet 10 is less than 50% of the average inner diameter of the first filter layer 430, for example, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%.

[0072] In some embodiments, the inlet 10 and / or outlet 20 are respectively covered with a cap. This prevents sample contamination.

[0073] This application also provides a sample elution and extraction system, including the above-described sample elution and extraction device. The sample elution and extraction system further includes a pressure application device for applying pressure toward the sample outlet 20 into the first chamber 100. Specifically, the pressurizing device includes a pressure transformer, a controller, and at least one liquid injection device. The pressure transformer and at least two liquid injection devices are all communicatively connected to the controller. The pressure transformer is used to control the pressure at various locations within the first chamber 100, and the at least two liquid injection devices are used to inject liquid into the first chamber 100. When the controller controls the liquid injection devices to inject liquid into the first chamber 100, it controls the pressure transformer to adjust the pressure of the first chamber 100 to be less than the pressure of the second chamber 200. After a first predetermined time, the pressure of the first chamber 100 is adjusted to be greater than the pressure of the second chamber 200 so that the reacted liquid flows out from the sample outlet 20. Subsequently, the controller controls another liquid injection device to inject another liquid into the first chamber 100, and controls the pressure transformer to adjust the pressure of the first chamber 100 to be less than the pressure of the second chamber 200. After a second predetermined time, the pressure of the first chamber 100 is adjusted to be greater than the pressure of the second chamber 200 so that the reacted liquid flows out from the sample outlet 20.

[0074] Specifically, the first predetermined time refers to the time required for the granular gel in the first cavity 100 to decolorize once.

[0075] Optionally, the transformer is a high-pressure air pump, which can be used to introduce high-pressure gas into the first chamber 100 to increase the pressure inside the first chamber 100, so that the pressure inside the first chamber 100 is greater than the pressure inside the second chamber 200. Optionally, the transformer is a movable piston, which can be pulled to adjust the pressure inside the first chamber.

[0076] In this embodiment, the sample elution and extraction system further includes an ultrasonic processing device for applying ultrasonic waves to the first cavity. This helps the system within the first cavity to react fully.

[0077] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0078] Example 1 1. Test Case 1 See sample elution and extraction device. Figure 2The first cavity 100 is a cavity of uniform diameter with a height of 41.4 mm. The inner diameter of the port closest to the second cavity 200 is 5.55 mm. The second cavity 200 is a concentrically tapering cavity with a height of 11.2 mm. The inner diameter of the port closest to the first cavity 100 is 3.10 mm, and the inner diameter of the port furthest from the first cavity 100 is 2.55 mm. The third cavity 300 has a height of 9.90 mm. The inner diameter of the port closest to the second cavity is 1.55 mm, and the inner diameter of the port furthest from the second cavity is 0.9 mm. The first, second, and third cavities are stepped together. The auxiliary layer (combining the compaction layer and the swelling layer into one layer) is made of polypropylene, with a diameter of 5.7 mm and a thickness of 1 mm. The first filter layer is made of polytetrafluoroethylene, with a diameter of 5.5 mm, a thickness of 1.6 mm, a pore size of 10 μm, and a porosity of 65%. The second filter layer is also made of polytetrafluoroethylene, with a diameter of 2.5 mm, a pore size of 10 μm, a thickness of 1.6 mm, and a porosity of 65%.

[0079] The sample elution and extraction devices were tested in groups as follows: a) Inject acetonitrile into the first chamber of the sample elution and extraction device, then apply a pressure of 5 psi into the first chamber and record the time interval between the first drop and the next drop.

[0080] b) Inject 100 μL of acetonitrile into the first chamber of the sample elution extraction device, then apply a pressure of 5 psi into the first chamber and record the time required for the liquid to flow out.

[0081] 2. Comparative Example 1 The difference from Test Example 1 in this embodiment is that the sample elution and extraction device does not contain a second filter layer.

[0082] Comparative Example 2 The difference from Test Example 1 in this embodiment is that the sample elution and extraction device does not contain the first filter layer and its auxiliary layer.

[0083] Test results: As shown in Table 1, in Comparative Example 1, the interval between two drops of acetonitrile was 3.8 s, and the time required for 100 μL of acetonitrile to flow out was 27.7 s; in Comparative Example 2, the interval between two drops of acetonitrile was 31.8 s, and the time required for 100 μL of acetonitrile to flow out was 133.1 s; in Test Example 1, the interval between two drops of acetonitrile was 41.2 s, and the time required for 100 μL of acetonitrile to flow out was greater than 1200 s.

[0084] As can be seen from the above experiments, compared with the single-layer devices of Comparative Examples 1 and 2, the sample elution and extraction device of Test Example 1 significantly prolonged the interval between two drops of acetonitrile and the eluent time under a pressure of 5 psi, indicating that the sample elution and extraction device of Test Example 1 can more effectively maintain the retention time and amount of acetonitrile in the chamber.

[0085] Table 1. Detection of Sample Elution and Extraction Device

[0086] Example 2 1. Test Case 1 See sample elution and extraction device. Figure 2 Add adhesive granules to the first cavity. The adhesive granules are composed of small pieces of approximately 0.1cm×0.1cm×0.1cm cut from a 1cm×0.5cm×0.1cm adhesive strip.

[0087] The sample elution and extraction device was tested in four groups as follows: a) Inject water into the first chamber of the sample elution and extraction device, then apply a pressure of 100 psi into the first chamber and record the time interval between the first drop of liquid falling and the next drop of liquid falling.

[0088] b) Inject 100 μL of water into the first chamber of the sample elution extraction device, then apply a pressure of 100 psi into the first chamber and record the time required for the liquid to flow out.

[0089] c) Inject acetonitrile into the first chamber of the sample elution and extraction device, then apply a pressure of 10 psi into the first chamber and record the time interval between the first drop and the next drop.

[0090] d) Inject 100 μL of acetonitrile into the first chamber of the sample elution extraction device, then apply a pressure of 10 psi into the first chamber and record the time required for the liquid to flow out.

[0091] 2. Comparative Example 1 The difference from Test Example 1 in this embodiment is that the sample elution and extraction device includes a second filter layer but does not include the first filter layer and its auxiliary layer.

[0092] The test results are shown in Table 2.

[0093] Table 2. Detection of Sample Elution and Extraction Device

[0094] The test example contains the first filter layer, its auxiliary layer, and the second filter layer of the sample elution and extraction device. The colloidal particles contact the first filter layer. Since the comparative example only contains the second filter layer and not the first, the colloidal particles directly contact the second filter layer. Because acetonitrile causes the gel to shrink and water causes it to swell, after placing the colloidal particles in the comparative example, although the acetonitrile solution could be drained normally at 10 psi pressure, the particles swelled upon contact with water. Furthermore, in the comparative example, the particles directly contacted the second filter layer, which had a small pore size and was prone to clogging. Therefore, even at 100 psi pressure, effective drainage was impossible, preventing the gel decolorization process from completing. This demonstrates that while the sample elution and extraction device retains only the second filter layer, with its smaller diameter and lower porosity, it can effectively retain acetonitrile solution, but it also suffers from sample clogging of the filter pores, preventing effective liquid expulsion and hindering the elution function. This fully illustrates the necessity of this sample elution and extraction device using two filter layers to synergistically achieve effective retention and rapid elution.

[0095] Example 3 1. Test Case The following operations were performed using the same sample elution and extraction apparatus as in Example 1: 1) Cutting the gel: Place the gel after SDS-PAGE electrophoresis in a glass dish, cut off the strip, then cut the strip into small pieces, rinse twice with distilled water, and then put the granular gel into the first chamber. 2) Decolorization: The transformer is controlled to not apply additional pressure. At this time, the pressure in the first chamber is less than the pressure in the second chamber. The controller controls the first injection device to inject decolorizing liquid (30% acetonitrile aqueous solution) into the first chamber, and the granular gel begins to decolorize. 3) Drainage: After 30 minutes, adjust the pressure in the first chamber to be greater than the pressure in the second chamber using the pressure transformer, so that the reaction solution flows out from the sample outlet; 4) Repeat decolorization: Repeat steps 2-3 several times until the granules are colorless or very light in color, at which point the decolorization is complete; 5) Drying: Control the transformer (high-pressure air pump) to not apply additional pressure. The controller controls the second injection device to inject acetonitrile into the first chamber to rinse the granular gel. After standing for 10 minutes, control the transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the reaction liquid flows out from the sample outlet. Use high-pressure gas to blow air to dry the granular gel. 6) Control the transformer to not apply additional pressure, and control the third injection device to inject 20mM tris(2-carboxyethyl)phosphine (TECP) solution into the first chamber. Incubate at room temperature for 30 minutes, and then control the transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the TECP solution flows out from the sample outlet. 7) Control the transformer to not apply additional pressure, and control the fourth injection device to inject 20mM iodoacetamide (IAA) solution into the first chamber. Place the sample elution and extraction device in a light-proof environment for 30 minutes. Then control the transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the IAA solution flows out from the sample outlet. 8) Control the transformer to not apply additional pressure. The controller controls the second injection device to inject an appropriate amount of 50 mM ammonium bicarbonate (ABC) solution into the first chamber to soak the granular gel for 30 minutes. Then, control the transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the ABC solution flows out from the outlet and dries naturally at room temperature. 9) Control the transformer to not apply additional pressure, and control the second injection device to inject an appropriate amount of acetonitrile into the first chamber to soak the granular gel for 10 minutes. Then control the transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the acetonitrile flows out from the outlet. Use high-pressure gas to blow air to dry the granular gel. 10) Control the pressure transformer to not apply additional pressure. The controller controls the fifth injection device to inject trypsin solution into the first chamber to cover the granular gel. Place the sample elution and extraction device in a 37°C constant temperature incubator. After the second predetermined time (12-16 hours), the controller controls the sixth injection device to inject 1 μL of 10% trifluoroacetic acid solution into the first chamber to terminate the enzymatic hydrolysis reaction. Then, the controller controls the pressure transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the trifluoroacetic acid solution and trypsin solution flow out from the sample outlet into the first new centrifuge tube. 11) Control the transformer to not apply additional pressure, and the controller controls the seventh injection device to inject the extraction solution (50% acetonitrile, 0.5% trifluoroacetic acid) into the first chamber. Place the sample elution and extraction device in the ultrasonic instrument. After 30 minutes, the controller controls the transformer to adjust the pressure of the first chamber to be greater than the pressure of the second chamber so that the extraction solution flows out from the sample outlet into the first new centrifuge tube. 12) Repeat step 11). 13) Place the first new centrifuge tube into a vacuum centrifuge concentrator to concentrate, add 10 μL of 0.1% formic acid and 2% acetonitrile solution to reconstitute, sonicate to mix, centrifuge, and take 1 μL of supernatant for mass spectrometry analysis.

[0096] 2. Comparative Example The difference from Test Example 1 in this embodiment is that the following operations are performed entirely manually: 1) Cutting the gel: Place the gel after SDS-PAGE electrophoresis in a glass dish, cut off the strip, then cut the strip into small pieces, rinse twice with distilled water, and then put the granular gel into the first chamber. 2) Decolorization: Manually add decolorizing solution (30% acetonitrile aqueous solution), place the sample tube in a vortex mixer for vortexing, and the granular gel will begin to decolorize; 3) Remove the decolorizing solution: After decolorizing for 30 minutes, manually remove the dyeing solution to avoid adsorption of colloidal particles; 4) Repeat decolorization: Repeat steps 2-3 several times until the granules are colorless or very light in color, at which point the decolorization is complete; 5) Drying: Manually add acetonitrile to rinse the granular gel, let it stand for 10 minutes, then aspirate the solution to dehydrate and dry the granular gel; 6) Reduction: Manually add 20 mM tris(2-carboxyethyl)phosphine (TECP) solution, incubate at room temperature for 30 minutes, and manually remove the TECP solution; 7) Alkylation: Manually add 20 mM iodoacetamide (IAA) solution, place the sample elution and extraction device in a light-protected environment for 30 minutes, and manually remove the IAA solution; 8) Manually add an appropriate amount of 50 mM ammonium bicarbonate (ABC) solution to soak the granular gel for 30 minutes, then manually remove the ABC solution. 9) Manually add an appropriate amount of acetonitrile to soak the granular gel for 10 minutes, manually remove the acetonitrile, and use a vacuum centrifuge to dehydrate and dry the granular gel. 10) Enzymatic hydrolysis: Manually add trypsin solution to cover the granular gel, place the sample elution and extraction device in a 37℃ constant temperature incubator, and after the second predetermined time (12-16 hours), add 1 μL of 10% trifluoroacetic acid solution to terminate the enzymatic hydrolysis reaction. 11) Peptide extraction: Add extraction solution (50% acetonitrile, 0.1% formic acid), place the sample tube in an ultrasonic instrument, and after 3 minutes, transfer the supernatant to a new centrifuge tube; 12) Repeat step 11). 13) Place the first new centrifuge tube into a vacuum centrifuge concentrator to concentrate, add 10 μL of 0.1% formic acid and 2% acetonitrile solution to reconstitute, sonicate to mix, centrifuge, and take 1 μL of supernatant for mass spectrometry analysis.

[0097] The test results are shown in Tables 3 and 4. As the experiments show, for the identification of single proteins within the gel, with sample loading amounts ranging from 5 μg to 50 ng, the sequence coverage of the sample elution and extraction device in the test examples was not significantly different from that of the manual extraction method. For the analysis of complex proteomes within the gel, with sample loading amounts ranging from 5 μg to 50 ng, the number of proteins identified by the manual extraction method was slightly higher with 5 μg of gel protein, and slightly lower with 0.5 μg and 0.05 μg of gel protein, but the differences were not significant. Using the sample elution and extraction device of this application for automatic sample processing, the sequence identification coverage can be maintained close to that of the manual method with sample amounts ranging from 5 μg to 50 ng, without affecting the identification results.

[0098] Table 3. Comparison of the effects of Coomassie Brilliant Blue staining of BSA protein gels using a sample elution extraction device (A) and manual extraction (M).

[0099] Table 4. Comparison of the effects of Coomassie Brilliant Blue staining of 293F total protein gel using sample elution and extraction device (A) and manual extraction method (M).

[0100] For ease of description, spatial relative terms such as "above," "on top of," "on the upper surface of," "above," etc., are used herein to describe the spatial positional relationship of a device or feature as shown in the figures to other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations in use or operation beyond the orientation of the device as described in the figures. For example, if the device in the figures were inverted, a device described as "above" or "on top of" other devices or structures would subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0101] Furthermore, it should be noted that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore cannot be construed as limiting the scope of protection of this application.

[0102] The above description is merely a preferred embodiment of this utility model and is not intended to limit the utility model. Various modifications and variations can be made to this utility model by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this utility model should be included within the protection scope of this utility model.

Claims

1. A sample elution and extraction device, characterized in that, include: A cavity, the cavity including an inlet and an outlet; Along the direction from the inlet to the outlet, the cavity includes a first cavity and a second cavity. A first filter layer is disposed between the first cavity and the second cavity, and a second filter layer is disposed between the second cavity and the outlet. The second cavity is used to apply at least a portion of the force to the first cavity to prevent the liquid in the first cavity from flowing through the first filter layer to the outlet. The first cavity is connected to the second cavity through the first filter pore of the first filter layer; the second cavity is connected to the sample outlet through the second filter pore of the second filter layer.

2. The sample elution and extraction apparatus according to claim 1, characterized in that, The average porosity of the first filter layer is greater than or equal to the average porosity of the second filter layer; The materials of the first filter layer and the second filter layer are each independently at least one of glass fiber, polyethylene, polypropylene and polytetrafluoroethylene.

3. The sample elution and extraction apparatus according to claim 1, characterized in that, An auxiliary layer is provided above the first filter layer, the auxiliary layer including a compaction layer and / or a swelling layer.

4. The sample elution and extraction apparatus according to claim 3, characterized in that, The swelling layer is sandwiched between the compacted layer and the first filter layer.

5. The sample elution and extraction apparatus according to claim 1, characterized in that, The sample elution and extraction device also satisfies at least one of the following characteristics: The average inner diameter of the first cavity is greater than the average inner diameter of the second cavity; The first cavity and / or the second cavity includes cavity segments of equal diameter; For the first cavity and / or the second cavity, along the direction from the inlet to the outlet, the inner diameter of the cavity near the inlet is larger than the inner diameter of the cavity near the outlet. The first cavity and / or the second cavity includes concentric tapering cavity segments.

6. The sample elution and extraction apparatus according to claim 1, characterized in that, A third cavity is provided between the second filter layer and the sample outlet.

7. The sample elution and extraction apparatus according to claim 6, characterized in that, The sample elution and extraction device also satisfies at least one of the following characteristics: The average inner diameter of the second cavity is greater than the average inner diameter of the third cavity; The third cavity comprises cavity segments of equal diameter; For the third cavity, along the direction from the inlet to the outlet, the inner diameter of the cavity near the inlet is larger than the inner diameter of the cavity near the outlet. The third cavity includes a concentric tapering cavity segment.

8. The sample elution and extraction apparatus according to claim 1, characterized in that, The inlet and / or outlet are respectively covered with a cover; The pore sizes of the first filter pore and the second filter pore are each independently 1~100μm.

9. A sample elution and extraction system, characterized in that, include: The sample elution and extraction apparatus according to any one of claims 1 to 8.

10. The sample elution and extraction system according to claim 9, characterized in that, The sample elution and extraction system further includes: a pressure application device for applying pressure toward the sample outlet into the first chamber; and / or The sample elution and extraction system further includes: an ultrasonic processing device for applying ultrasonic waves to the first cavity.

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