Centrifuge tube, method for separating substances in sample and use

Abstract

The embodiment of the invention relates to a centrifugal tube, a method for separating substances in a sample and application. The centrifugal tube comprises a tube body which extends from a tube opening to a tube bottom in a first direction. The centrifugal tube further comprises a filter membrane assembly which is arranged in the tube body and is perpendicular to the first direction. The edge of the filter membrane component is contacted with the inner wall of the pipe body. The density of the filter membrane assembly is greater than that of a first substance in a to-be-separated sample and less than that of a second substance in the sample, and separating the first substance in the sample. The centrifugal tube further comprises a cover body which is arranged on the tube opening of the tube body. In this way, the movable filter membrane assembly can be brought into gradual contact with a second substance dispersed in the sample during movement, thereby avoiding large-area contact or penetration of the second substance through the filter membrane. Therefore, filter membrane blockage and shear fracture of the second substance are avoided.

Description

Technical Field

[0001] The present application relates to the technical field of clinical medical devices, and in particular to a centrifuge tube, a method for separating substances in a sample, and uses thereof. Background Art

[0002] In the biological field, centrifugation is commonly used to separate different substances in a sample. Centrifugal sample separation typically utilizes the centrifugal force generated by a centrifuge to separate components of different densities within a biological sample. When a sample is placed in a centrifugal field, the different components are subjected to varying centrifugal forces due to their varying densities. Denser components experience greater centrifugal forces, causing them to move outward at a faster rate, while less dense components move more slowly or remain in the upper layer, thereby achieving separation of the sample components. Under the influence of centrifugal force, particles of varying sizes, shapes, and densities within the sample have different sedimentation velocities. These differences in sedimentation velocities cause the particles to form layers, arranged in descending order of density, thus achieving separation. Summary of the Invention

[0003] In a first aspect of the present disclosure, a centrifuge tube is provided. The centrifuge tube includes a tube body extending from a tube mouth to a tube bottom along a first direction. The centrifuge tube also includes a filter membrane assembly disposed in the tube body and perpendicular to the first direction. The edge of the filter membrane assembly contacts the inner wall of the tube body. The density of the filter membrane assembly is greater than the density of a first substance in a sample to be separated and less than the density of a second substance in the sample, so as to separate the first substance in the sample. The centrifuge tube also includes a cover body disposed on the tube mouth of the tube body.

[0004] In a second aspect of the present disclosure, a method for separating a substance from a sample is provided. The method comprises placing the sample into the centrifuge tube according to the first aspect of the present disclosure, the sample being placed therein. The method further comprises placing a filter membrane assembly of the centrifuge tube into the tube at an initial position above the sample. The method further comprises centrifuging the centrifuge tube to separate a first substance from the sample. The method further comprises obtaining a supernatant containing the first substance from the centrifuged centrifuge tube.

[0005] In a third aspect of the present disclosure, a use is provided, which is the use of the centrifuge tube according to the first aspect of the present disclosure in the preparation of decellularization, cell debris, and organelle samples.

[0006] It should be understood that the contents described in the Summary of the Invention section are not intended to limit the key or important features of the embodiments of the present disclosure, nor are they intended to limit the scope of the present disclosure. Other features of the present disclosure will become readily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] To better understand the above and other objects, features, advantages, and functions of the present disclosure, reference may be made to the preferred embodiments shown in the accompanying drawings. Like reference numerals in the accompanying drawings refer to like components. Those skilled in the art should understand that the accompanying drawings are intended to schematically illustrate preferred embodiments of the present disclosure and have no limiting effect on the scope of the present disclosure. The components in the drawings are not drawn to scale.

[0008] Figure 1 shows a schematic diagram of an example centrifuge system according to an embodiment of the present disclosure;

[0009] Figure 2 shows a schematic cross-sectional view of an example centrifuge tube according to an embodiment of the present disclosure;

[0010] Figure 3 A flow chart illustrating an example method for separating substances in a sample according to an embodiment of the present disclosure is shown;

[0011] Figure 4 shows a schematic cross-sectional view of an example centrifuge tube during a centrifugation operation according to an embodiment of the present disclosure;

[0012] Figure 5A and 5B A schematic diagram showing example results of comparative experiments according to an embodiment of the present disclosure; and

[0013] Figure 6 A schematic diagram showing example results of a fragment distribution assay according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0014] Various embodiments will now be described with reference to the accompanying drawings, wherein like reference numerals are used to refer to like elements throughout the text. In the following description, for the purpose of explanation, many specific details are set forth to facilitate a thorough understanding of one or more embodiments. However, it may be clear in some or all cases that any of the embodiments described below can be practiced without adopting the specific design details described below. In other examples, well-known structures and devices are shown in block diagram form to facilitate description of one or more embodiments. A simplified overview of one or more embodiments is given below to provide a basic understanding of the embodiments. This overview is not an exhaustive overview of all contemplated embodiments and is not intended to identify the key or important elements of all embodiments, nor is it intended to define the scope of any or all embodiments.

[0015] References to "an embodiment" or "one embodiment" in the context of this description are intended to indicate that a particular configuration, structure, or characteristic described with respect to the embodiment is included in at least one embodiment. Therefore, phrases such as "in an embodiment" or "in one embodiment" that may appear in one or more points of this description are not necessarily referring to the same embodiment. Furthermore, in one or more embodiments, the particular configurations, structures, or characteristics may be combined in any appropriate manner.

[0016] In the following disclosure, unless otherwise indicated, when reference is made to absolute position modifiers (such as terms "front", "back", "top", "bottom", "left", "right", etc.) or relative position modifiers (such as terms "above", "below", "higher", "lower", etc.), or when reference is made to directional modifiers (such as "horizontal", "vertical", etc.), reference is made to the orientation shown in the figures.

[0017] As discussed above, centrifugal sample separation is widely used as an important tool in plasma separation to extract circulating cell-free DNA (cfDNA). cfDNA is commonly found in bodily fluids such as plasma and serum. cfDNA testing shows great clinical promise in areas such as early cancer diagnosis, prenatal screening, and transplant rejection monitoring. However, the low content and fragmentation of cfDNA place high demands on sample separation and purification technologies.

[0018] In related technologies, cfDNA extraction generally includes plasma separation and cfDNA extraction. In plasma separation, blood cells in whole blood are usually separated from plasma by centrifugation. However, due to improper operation or delayed post-blood collection processing, blood cells are prone to rupture, releasing a large amount of high-molecular-weight genomic DNA (gDNA), thereby contaminating the target cfDNA. In addition, the operation steps are cumbersome and often require multiple centrifugation steps or sample transfers, which not only increases the complexity of experimental operations but also increases the risk of cross-contamination. In addition, some methods require the use of high-speed centrifugation equipment (such as 12,000-16,000g), which places high demands on equipment performance and laboratory conditions, and increases costs and energy consumption.

[0019] In some related centrifugal separation technologies that use filter membranes for blood separation, cells tend to accumulate on the surface of the filter membrane and clog the filter membrane; or they are sheared and broken when passing through the filter membrane, releasing a large amount of genomic DNA (gDNA), causing cfDNA contamination. In addition, the preparation of samples used to extract cfDNA in related technologies usually requires two or even multiple steps of high-speed centrifugation to obtain relatively pure plasma or supernatant. However, such multi-step centrifugation operations are cumbersome and have high requirements for equipment and technical environment. Furthermore, in related technologies, additional refiltration is often required to remove cell debris and organelles in plasma. This further increases the workload and operational complexity, and easily leads to partial loss or degradation of cfDNA due to repeated operations. It should be understood that although the above-mentioned problems occur in plasma separation, they are common problems in centrifugal separation technology.

[0020] In view of this, the embodiment of the present disclosure proposes a centrifugal separation scheme of centrifugal reverse filtration with simple operation, high efficiency and low cost. In this scheme, a filter membrane assembly is provided in the tube body of a centrifuge tube. The edge of the filter membrane assembly is against the inner wall of the tube body and can move along the inner wall in the direction from the tube mouth toward the bottom of the tube. During the movement, the first substance with a smaller density in the sample can pass through the filter membrane assembly and move between the filter membrane assembly and the tube mouth, while most of the second substance with a larger density in the sample moves to the bottom of the tube under the action of centrifugal force. Since the second substance cannot pass through the filter membrane assembly, during the movement, the filter membrane assembly gradually pushes the remaining part of the second substance to the bottom of the tube. Finally, the first substance is trapped between the filter membrane assembly and the tube mouth, and the second substance is isolated between the filter membrane assembly and the bottom of the tube, thereby achieving separation of the first substance and the second substance.

[0021] According to embodiments of the present disclosure, "reverse filtration" is achieved through a movable filter membrane assembly. Compared to a fixed filter membrane, a movable filter membrane assembly can gradually contact a second substance dispersed in the sample during movement, thereby preventing the second substance from contacting or penetrating the filter membrane over a large area. This prevents filter membrane clogging and shear fracture of the second substance.

[0022] Especially in the separation of plasma samples, blood cells (corresponding to the second substance in the sample) have a greater density than the filter membrane assembly and settle at the bottom of the tube. During the centrifugation process, the weighted filter membrane moves between the blood cells and plasma (corresponding to the first substance in the sample) to separate the blood cells and plasma. In addition, impurities with a density also greater than that of the filter membrane assembly, such as cell debris and organelles, have large particle sizes, are difficult to settle, or are in a floating state and dispersed between the filter membrane assembly and the bottom of the tube. As the filter membrane assembly moves toward the bottom of the tube, these impurities are trapped by the filter membrane between the filter membrane assembly and the bottom of the tube. As a result, the plasma above the filter membrane assembly is filtered "upwards", resulting in a pure supernatant free of cells, cell debris, and organelles. In this process, cells are prevented from contacting or penetrating the filter membrane over a large area, thereby preventing filter membrane clogging and cell shear rupture, significantly reducing gDNA contamination, and ensuring the purity of cfDNA. In addition, the configuration of the filter membrane assembly allows sample separation to be completed in a single centrifugation. Compared with the "two-step method" or multi-step high-speed centrifugation required in the related art, the solution according to the embodiment of the present disclosure can simultaneously complete the separation of plasma and blood cells, as well as the removal of cell fragments and organelles in plasma in a low-speed centrifugation process (such as 2000g~4000g), thereby greatly simplifying the operation process.

[0023] The following will be combined Figures 1 to 6 The centrifuge tube and the method for separating substances in a sample according to the present disclosure are described in detail. Figure 1 Schematic diagram of an example centrifugal system 1 according to an embodiment of the present disclosure is shown. Figure 1 As shown, the centrifugal system 1 includes a centrifuge 10. The centrifuge 10 can use centrifugal force to separate, precipitate and purify the mixed solution. When the sample makes a circular motion in the centrifuge, centrifugal force is generated. Different components in the sample solution settle at different speeds under the action of centrifugal force according to the density difference, thereby achieving separation. The centrifuge 10 includes a drive control system 11, a centrifugal chamber 12 and a rotor 13. The drive control system 11 can accept instructions from the user, and includes a motor and a rotating shaft to provide power for the centrifuge and drive the rotor to rotate at high speed. The centrifugal chamber 12 is a cavity for placing the rotor of the centrifuge. The rotor 13 is a component for placing a centrifuge tube and driving it to rotate. As shown Figure 1 As shown, a centrifuge tube 100 - 1 and a centrifuge tube 100 - 2 are symmetrically placed in the rotor 13 .

[0024] Centrifuge tube 100-1 includes a tube body 110-1. Tube body 110-1 is a hollow tubular structure that extends from the tube mouth to the tube bottom along a first direction. Centrifuge tube 100-1 also includes a filter membrane assembly 120-1. Filter membrane assembly 120-1 is disposed in tube body 110-1 and is perpendicular to the first direction. The edge of filter membrane assembly 120-1 contacts the inner wall of tube body 110-1 so that the edge of filter membrane assembly 120-1 and tube body 110-1 are sealed, so that the liquid in tube body 110-1 can only flow through the filter pores of filter membrane assembly 120-1 and cannot flow between the edge of filter membrane assembly 120-1 and tube body 110-1. In addition, centrifuge tube 100-1 also includes a cover 130-1. Cover 130-1 is disposed on the tube mouth of tube body 110-1 to seal tube body 110-1.

[0025] Accordingly, centrifuge tube 100-2 includes a tube body 110-2. Tube body 110-2 is a hollow tubular structure that extends from the tube mouth to the tube bottom along a first direction. Centrifuge tube 100-2 also includes a filter membrane assembly 120-2. Filter membrane assembly 120-2 is disposed in tube body 110-2 and is perpendicular to the first direction. The edge of filter membrane assembly 120-2 contacts the inner wall of tube body 110-2 so that the edge of filter membrane assembly 120-2 and tube body 110-2 are sealed, so that the liquid in tube body 110-2 can only flow through the filter pores of filter membrane assembly 120-2 and cannot flow between the edge of filter membrane assembly 120-2 and tube body 110-2. In addition, centrifuge tube 100-1 also includes a cover 130-1. Cover 130-1 is disposed on the tube mouth of tube body 110-1 to seal tube body 110-1.

[0026] During centrifugation, samples containing a first substance and a second substance are added to centrifuge tubes 100-1 and 100-2, respectively. The filter membrane assemblies are then installed so that they are positioned above the samples. Centrifuge 10 is then started, causing rotor 13 to rotate centrifuge tubes 100-1 and 100-2. During rotation, centrifugal force causes the majority of the second substance, which has a higher density, in the sample to move to the bottom of centrifuge tube 100-1. Simultaneously, filter membrane assembly 120-1 also moves toward the bottom. The first substance, which has a lower density, in the sample passes through the filter membrane assembly and moves between filter membrane assembly 120-1 and the tube opening. Furthermore, since the second substance cannot pass through the filter membrane assembly, filter membrane assembly 120-1 gradually pushes the remaining portion of the second substance to the bottom of the tube during this rotation. Finally, the first substance is retained between the filter membrane assembly 120-1 and the tube mouth, while the second substance is isolated between the filter membrane assembly 120-1 and the tube bottom, thereby achieving separation of the first substance and the second substance in the centrifuge tube 100-1.

[0027] Similarly, in centrifuge tube 100-2, under the action of centrifugal force, most of the second substance with a higher density in the sample moves to the bottom of the tube. At the same time, filter membrane assembly 120-2 also moves toward the bottom of the tube. The first substance with a lower density in the sample passes through the filter membrane assembly and moves between filter membrane assembly 120-2 and the tube mouth. In addition, since the second substance cannot pass through the filter membrane assembly, during the movement process, filter membrane assembly 120-1 gradually pushes the remaining portion of the second substance to the bottom of the tube. Finally, the first substance is trapped between filter membrane assembly 120-2 and the tube mouth, while the second substance is isolated between filter membrane assembly 120-2 and the tube bottom, thereby achieving the separation of the first substance and the second substance in centrifuge tube 100-2.

[0028] According to embodiments of the present disclosure, "reverse filtration" is achieved through a movable filter membrane assembly. Compared to a fixed filter membrane, a movable filter membrane assembly can gradually contact a second substance dispersed in the sample during movement, thereby preventing the second substance from contacting or penetrating the filter membrane over a large area. This prevents filter membrane clogging and shear fracture of the second substance.

[0029] Figure 2 A schematic cross-sectional view of an exemplary centrifuge tube 100 according to an embodiment of the present disclosure is shown. The centrifuge tube 100 may correspond to Figure 1 The centrifuge tube 100-1 and the centrifuge tube 100-2 are shown. Figure 2 As shown, the centrifuge tube 100 includes a tube body 110. The tube body 110 is a hollow tubular structure. The tube body 110 extends from the tube mouth 111 to the tube bottom 112 along the first direction D. In some embodiments, the tube body 110 is a cylindrical tubular structure that can withstand a certain centrifugal force. In some embodiments, the tube body 110 can be made of a transparent, chemically resistant and mechanically strong material, such as polypropylene (PP), polycarbonate (PC) or other medical grade plastics. Figure 2 As shown, the tube bottom 112 is a round bottom. The tube bottom 112 can also be tapered. In such an embodiment, the tube bottom is arranged to be round or tapered, which is conducive to sample sedimentation and precipitation.

[0030] The centrifuge tube 100 further includes a filter membrane assembly 120. The filter membrane assembly 120-1 is disposed in the tube body 110 and is perpendicular to the first direction D. The edge of the filter membrane assembly 120 contacts the inner wall of the tube body 110 so that the edge of the filter membrane assembly 120 and the tube body 110 are sealed, so that the liquid in the tube body 110 can only flow through the filter pores of the filter membrane assembly 120 and cannot flow between the edge of the filter membrane assembly 120 and the tube body 110. Figure 2As shown, filter membrane assembly 120 includes a filter membrane 121 and a contact ring 122 disposed around the edge of filter membrane 121. Since centrifugal separation is achieved through the different densities of different substances, the density of filter membrane assembly 120 needs to be greater than the density of the first substance and less than the density of the second substance. Furthermore, the density of filter membrane 121 is typically relatively low, so the material of contact ring 122 needs to be selected to achieve a desired overall density of filter membrane assembly 120. In such an embodiment, the material of contact ring 122 is selected such that the density of filter membrane assembly 120 is greater than the density of the first substance and less than the density of the second substance.

[0031] In the embodiment of plasma separation, the first substance may be plasma, and the density of plasma is 1.025 g / cm 3 to 1.030g / cm 3 The second substance may be blood cells, and the density of blood cells is about 1.10 g / cm 3 In such an embodiment, the overall density of the membrane assembly 120 should be greater than 1.030 g / cm 3 And less than 1.10g / cm 3 It should be understood that plasma separation is only an example, and the overall density of the filter membrane assembly 120 can be adjusted according to the type of biological sample to be separated.

[0032] In some of these embodiments, the contact ring 122 may comprise an elastic material, and the elastic material may be pressed against the inner wall of the tube body 110. In such embodiments, the contact ring 122 can fit tightly against the inner wall of the tube body 110, achieving an effective seal, ensuring that liquid can only pass through the microporous filter membrane and does not leak out from around the filter membrane assembly 120. In some alternative embodiments, the contact ring 122 and the inner wall of the tube body 110 may also have a certain matching tolerance to achieve a seal between the contact ring 122 and the inner wall of the tube body 110.

[0033] In some embodiments, the elastic material includes a plastic or rubber material, and the contact ring 122 further includes a metal support ring enclosed in the plastic or rubber material. In addition, the support ring can also be integrally formed with high-density plastic or other materials that can increase mass (specific gravity) so as to act as a counterweight during the centrifugation process.

[0034] The filter membrane 121 is used to filter substances with lower density in the sample. In the embodiment of plasma separation, the filter membrane 121 can be used to filter cell debris, organelles and other suspended impurities. In such an embodiment, the pore size of the filter can be in the range of 0.1 μm to 1 μm. Alternatively, the pore size of the filter can be in the range of 0.2 μm to 0.8 μm to effectively remove cells and organelles. In some embodiments, the microporous filter membrane can be composed of one of the following materials: polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES) or mixed cellulose ester (MCE). In such an embodiment, the filter membrane 121 supported by the above materials is not easy to break during centrifugation and has good chemical compatibility with plasma, cells and cfDNA.

[0035] In some embodiments, the filter membrane 121 and the contact element 122 can be connected by heat pressing, ultrasonic welding, or other feasible sealing methods. This ensures the stability of the filtration function while preventing liquid leakage. In some embodiments, due to the small pore size of the filter pores of the filter membrane 121, a grid support structure is provided on the side of the filter membrane 121 near the tube orifice 111, on the side of the filter membrane 121 near the tube bottom 112, or on both sides to reduce the risk of damage to the filter membrane itself.

[0036] In some embodiments, the tube body 110 may include at least one limiting strip extending along the first direction D. Figure 2 As shown, a limit strip 113 is provided on the inner wall of the tube body 110. Correspondingly, the filter membrane assembly 120 may include at least one limit groove opened on the edge and matched with the limit strip. Figure 2 As shown, a contact ring 122 at the edge of the filter membrane assembly 120 is provided with a retaining groove 123 extending in a first direction D from the side of the filter membrane assembly 120 near the tube opening 111 to the side of the filter membrane assembly near the tube bottom 112. The inner wall of the retaining groove 123 abuts against the retaining bar 113, thereby tightly abutting the retaining groove 123 and the retaining bar 113. In some embodiments, the edge of at least one retaining groove 123 can be chamfered or rounded to ensure smooth cooperation between the retaining groove and the retaining bar, thereby facilitating assembly of the filter membrane assembly before centrifugation and disassembly after centrifugation.

[0037] In such an embodiment, the cooperation between the limiting strips on the tube body and the corresponding limiting grooves on the filter membrane assembly can guide the filter membrane assembly to move only in the axial direction during centrifugation, preventing the filter membrane assembly from tilting, rotating, or shifting under the action of centrifugal force. In addition, the filter membrane can be prevented from leaking, rupturing, or shearing. In some alternative embodiments, the contact ring 122 may include a flange extending in the first direction D. The flange is tightly coupled to the inner wall of the tube body, thereby preventing the filter membrane assembly from deflecting during centrifugation.

[0038] In addition, the centrifuge tube 100 further includes a cover 130. The cover 130 is disposed on the tube opening of the tube body 110 to seal the tube body 110, thereby preventing the sample from leaking or volatilizing during the centrifugation process. Figure 2 As shown, the cover 130 can be screwed or snap-fitted onto the nozzle 111. In the embodiment using the screw-fitting method, a leak-proof gasket can be provided at the position where the nozzle 111 and the cover 130 meet to enhance airtightness. In the embodiment using the snap-fitting method, the cover can be ensured not to loosen or fall off under centrifugal force.

[0039] Figure 3 FIG. 3 is a flow chart illustrating an exemplary method 300 for separating substances in a sample according to an embodiment of the present disclosure. Figure 4 To describe the method 300. Figure 3 As shown, at 302, method 300 includes placing a sample in a centrifuge tube. At 304, method 300 includes placing a filter membrane assembly of the centrifuge tube into the tube at an initial position above the sample. Figure 4 A schematic cross-sectional view of an example centrifuge tube during a centrifugation operation according to an embodiment of the present disclosure is shown.

[0040] For example, Figure 4 In the illustrated embodiment, a predetermined amount of blood sample is first added to the tube body 110. The filter membrane assembly 120 is then inserted into the tube body 110, aligning and engaging the retaining groove 123 with the retaining bar 113 within the tube body 110. During insertion, the filter membrane should be kept parallel to the tube base 112 to prevent jamming or damage caused by tilting. After confirming that the filter membrane assembly 120 can slide freely in the first direction within the tube body 110 without leakage or excessive looseness, the cover 130 is installed.

[0041] At 306, method 300 includes performing a centrifugal operation on the centrifuge tube, causing the filter membrane assembly to move in a first direction, so that a first substance in the sample passes through the filter membrane assembly and is trapped between the tube opening and the filter membrane assembly, and a second substance in the sample is isolated between the filter membrane assembly and the bottom of the tube. Figure 4 In the embodiment shown, the centrifuge tube 100 is placed vertically in the centrifuge, ensuring that the filter membrane assembly 120 is facing upward and the sample is facing downward, and is not tilted. The centrifuge is started and the centrifugal force is set to, for example, 2000 g to 4000 g, and a centrifugal operation is performed for 5 to 10 minutes.

[0042] Under the action of centrifugal force, higher-density components such as blood cells 411 in the blood sample 410 are pushed toward the tube bottom 113. Since the density of the filter membrane assembly 120 is between that of the blood cells 411 and the plasma, it moves in the plasma layer along the first direction D, thereby intercepting or retaining larger particles 412, such as cell debris or organelles, thereby purifying the plasma 413 above the filter membrane.

[0043] At 308, method 300 includes obtaining a supernatant containing the first substance from the centrifuge tube after the centrifugation operation. Figure 4 In the illustrated embodiment, after centrifugation is complete, the centrifuge is opened and the centrifuge tube 100 is removed and placed in a vertical position. After opening the lid, the purified plasma (i.e., the target supernatant containing cfDNA) can be removed from the space above the filter membrane assembly 120 using a pipette or other sampling tool.

[0044] according to Figure 3 In the illustrated embodiment, through the "reverse filtration" principle and the specialized design of the filter assembly, blood cells sink to the bottom of the tube under centrifugal force. The filter assembly moves between the plasma and cells, preventing large-scale cell contact or penetration of the filter membrane. This prevents filter clogging and cell shearing, significantly reducing gDNA contamination and ensuring the purity of cfDNA. Furthermore, sample separation and removal of cell debris and organelles can be completed in a single centrifugation, eliminating the need for multiple high-speed centrifugation steps, thus simplifying the operational process.

[0045] Example

[0046] The following will describe the technical effects of the centrifugal separation scheme based on reverse filtration according to the present disclosure in conjunction with comparative experiments. This embodiment aims to verify the separation efficiency and purity of the centrifugal reverse filtration device according to the embodiment of the present disclosure in plasma separation, removal of cells and cell debris to obtain cfDNA, and compare it with the commercially available Streck blood collection tubes. Specific objectives include: 1. Evaluate the cfDNA yield and purity of the centrifuge tube according to the embodiment of the present disclosure, and verify whether the effect of the traditional two-step centrifugation method can be achieved in a single low-speed centrifugation; 2. Compare the ease of operation, total centrifugation time, and equipment requirements, and evaluate whether the centrifuge tube according to the embodiment of the present disclosure simplifies the operating process; 3. Analyze the integrity and contamination of the final cfDNA, and determine whether the centrifuge tube according to the embodiment of the present disclosure can reduce gDNA contamination and cell residues, and improve separation quality.

[0047] The comparative experimental materials include blood samples from the subjects. The blood samples from the subjects were selected from approximately 20 mL of peripheral blood from each of 10 healthy volunteers (without hemolysis, coagulation abnormalities, etc.). The consumables for the comparative experiment include the centrifuge tube according to the embodiment of the present disclosure and the Streck cfDNA blood collection tube (commercially available standard product) as a comparison. The instruments used in the comparative experiment include two centrifuges. The centrifuge for the centrifuge tube of the embodiment of the present disclosure has a centrifugal force of 3000 g, and the centrifuge for the Streck blood collection tube has a centrifugal force option of 16000 g.

[0048] In addition, cfDNA measurement instruments and reagents are also required, including: Qubit fluorometer and dsDNA HS kit (for cfDNA quantification); LabChip for cfDNA fragment distribution and purity assessment; cfDNA extraction kit (QIAamp Circulating Nucleic Acid Kit).

[0049] The experimental steps included sample collection and grouping, centrifugation, supernatant collection, and cfDNA extraction. During the sample collection and grouping phase, each subject collected 20 mL of peripheral blood using two Streck blood collection tubes and was divided into two groups: 10 mL of whole blood collected using one Streck blood collection tube was transferred to a centrifuge tube according to an embodiment of the present disclosure; 10 mL of whole blood collected using the other Streck blood collection tube was used directly. All blood samples were centrifuged within 30 minutes of collection to prevent factors such as transportation and storage from affecting cfDNA stability.

[0050] During the centrifugation operation, 10 mL of whole blood collected from the Streck blood collection tube was transferred to the tube body 110 of the centrifuge tube according to the embodiment of the present disclosure, and then the filter membrane assembly 120 (average density 1.08 g / cm 3 ) Insert the tube body 110 correctly, ensuring that the limit groove is aligned with the limit bar to avoid tilting or loosening. Finally, close the lid to ensure a complete seal. When placing the sealed centrifuge tube in the centrifuge, ensure that the tube body 110 is placed vertically, with the filter membrane facing up and the blood facing down, and place them symmetrically to ensure balance. Perform the centrifugation operation under the conditions of relative centrifugal force of 3000g, centrifugation time of 10 minutes, and temperature of 4°C.

[0051] The centrifugal result is that the blood cells settle to the bottom of the tube; the filter membrane assembly 120 remains between the plasma and the blood cells, forming a stable interface; cell debris and organelles are intercepted by the microporous filter membrane, ensuring that the upper plasma is purer.

[0052] For the Streck blood collection tubes used as the control group, the manufacturer's instructions were followed: first centrifugation: 1600g, 10 minutes, 4°C; collect the supernatant and transfer it to a new centrifuge tube; second centrifugation: 16000g, 10 minutes, 4°C; collect the supernatant for cfDNA extraction.

[0053] During supernatant collection and cfDNA extraction, for centrifuge tubes according to embodiments of the present disclosure, after centrifugation, remove the device vertically to avoid disturbing the sedimentation layer. Open the lid and use a pipette to aspirate purified plasma from above the membrane assembly 120, avoiding contact with the cell pellet below. For Streck blood collection tubes, plasma is collected using a two-step centrifugation method.

[0054] After plasma collection, all plasma samples were analyzed using a Nanodrop analyzer at 414 nm to determine hemoglobin concentration, ensuring the same experimental process to reduce variability. Concurrently, all plasma samples were extracted using the same kit and eluted with 30 μL of elution buffer.

[0055] By comparing the steps of the two sets of experiments, it can be seen that the centrifuge tube according to the embodiment of the present disclosure only needs one centrifugation, eliminating the 16000g high-speed centrifugation and additional tube changing operations, which is simpler; the Streck blood collection tube requires two centrifugations, which is not only complicated to operate, but also requires a high-speed centrifuge, increasing equipment costs and experimental complexity.

[0056] also, Figure 5A The intensity statistics 520 of the hemoglobin absorption peak of the plasma obtained by using the centrifuge tube according to the embodiment of the present disclosure and the intensity statistics 510 of the hemoglobin absorption peak of the plasma obtained by using the Streck blood collection tube are shown. Figure 5A As shown, the absorption peak intensity of hemoglobin at 414 nm in plasma obtained using the centrifuge tube according to the embodiment of the present disclosure is lower than that of the Streck blood collection tube, indicating that reverse filtration effectively reduces cell residue and hemoglobin contamination. Furthermore, the absorption peak intensity of hemoglobin at 414 nm obtained using the solution according to the embodiment of the present disclosure exhibits less fluctuation, indicating that the solution according to the embodiment of the present disclosure is less likely to cause red blood cells to rupture and release hemoglobin, leading to hemolysis.

[0057] Figure 5B The total amount of cfDNA extracted from plasma obtained using a centrifuge tube according to an embodiment of the present disclosure 540 and the total amount of cfDNA extracted from plasma obtained using a Streck blood collection tube 530 are shown. Here, the extracted cfDNA was quantified using Qubit dsDNA quantification reagent, and the yield of cfDNA was as follows: Figure 5BAs shown, the cfDNA recovery amount of the centrifuge tube according to the embodiment of the present disclosure is comparable to that of the Streck blood collection tube, but the CV is lower, indicating that the scheme according to the embodiment of the present disclosure is stable and the experimental repeatability is good.

[0058] Figure 6 The results 610 of measuring the fragment distribution of plasma obtained by using a centrifuge tube according to an embodiment of the present disclosure using a labchip and the results 620 of measuring the fragment distribution of plasma obtained by using a Streck blood collection tube using a labchip are shown. Figure 6 As shown, the main peak of cfDNA obtained by the centrifuge tube according to the embodiment of the present disclosure is at 170-180bp, which is consistent with the cfDNA fragment distribution obtained by two-step centrifugation in Streck blood collection tubes, and is consistent with the characteristics of cfDNA. In addition, the results obtained by the solution according to the embodiment of the present disclosure show that there is essentially no gDNA contamination at positions greater than 1000bp, indicating that the solution according to the embodiment of the present disclosure can reduce the release of gDNA caused by cell rupture.

[0059] The embodiments of the present disclosure have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the various implementations disclosed herein.

Claims

1. A centrifuge tube (100), comprising: A tube body (110) extending from a tube opening (111) to a tube bottom (112) along a first direction; a filter membrane assembly (120) disposed in the tube body (110) and perpendicular to the first direction, wherein an edge of the filter membrane assembly (120) contacts an inner wall of the tube body (110), and a density of the filter membrane assembly (120) is greater than a density of a first substance in a sample to be separated and less than a density of a second substance in the sample, so as to separate the first substance in the sample; as well as The cover body (130) is arranged on the tube mouth (111) of the tube body (110).

2. The centrifuge tube (100) according to claim 1, wherein the filter membrane assembly (120) is configured to move along the inner wall in the first direction when a centrifugal operation is performed on the sample, thereby allowing the first substance to pass through the filter membrane assembly (120) and be retained between the tube mouth (111) and the filter membrane assembly (120), and allowing the second substance to be isolated between the tube bottom (112) and the filter membrane assembly (120).

3. The centrifuge tube (100) according to claim 1, wherein the filter membrane assembly (120) comprises a filter membrane (121) and a contact ring (122) arranged around the edge of the filter membrane (121), and The material of the contact ring (122) is selected so that the density of the filter membrane assembly (120) is greater than the density of the first substance and less than the density of the second substance.

4. The centrifuge tube (100) according to claim 3, wherein the first substance is plasma, and the density of plasma is 1.025 g / cm 3 to 1.030g / cm 3 The second substance is a blood cell, and the density of the blood cell is about 1.10 g / cm 3 .

5. The centrifuge tube (100) according to claim 3, wherein the contact ring (122) comprises an elastic material, and the elastic material is pressed against the inner wall of the tube body (110).

6. The centrifuge tube (100) according to claim 5, wherein the elastic material comprises a plastic or rubber material, and the contact ring (122) comprises a metal support ring enclosed in the plastic or rubber material.

7. The centrifuge tube (100) according to claim 3, wherein the filter membrane (121) comprises filter pores, and the pore size of the filter pores is in the range of 0.1 μm to 1 μm, preferably in the range of 0.2 μm to 0.8 μm.

8. The centrifuge tube (100) according to claim 3, wherein the filter membrane (121) is made of one of the following: polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES) or mixed cellulose ester (MCE).

9. The centrifuge tube (100) according to claim 3, wherein the filter membrane assembly (120) further comprises a grid support structure for the filter membrane (121).

10. The centrifuge tube (100) according to claim 1, wherein the tube body (110) includes at least one limiting strip (113) extending along the first direction, and the filter membrane assembly (120) includes at least one limiting groove (123) opened at the edge, wherein the inner wall of the at least one limiting groove (123) abuts against the at least one limiting strip (113) so that the filter membrane assembly (120) moves along the at least one limiting strip (113) during centrifugal operation.

11. The centrifuge tube (100) according to claim 10, wherein the edge of the at least one limiting groove (123) is chamfered or rounded.

12. A method (300) for separating substances in a sample, comprising: Placing a sample into the tube body (110) of the centrifuge tube (100) according to any one of claims 1 to 11; placing the filter membrane assembly (120) of the centrifuge tube (100) into the tube body (110) at an initial position above the sample; Performing a centrifugal operation on the centrifuge tube (100) to separate the first substance in the sample; as well as The supernatant containing the first substance is obtained from the centrifuge tube (100) that has undergone the centrifugal operation.

13. The method according to claim 12, wherein the centrifugal force of the centrifugation operation is in the range of 2000 g to 4000 g, and the centrifugation time is in the range of 5 minutes to 10 minutes.

14. Use of the centrifuge tube (100) according to any one of claims 1 to 11 in the preparation of samples for removing cells, cell debris and organelles.

Images