A replaceable exosome extraction chip

Abstract

This invention relates to the field of exosome extraction technology and discloses a replaceable exosome extraction chip, comprising a detachably connected upper cover and a lower cover. The upper cover has a liquid inlet, and the lower cover has a liquid outlet. The lower cover contains, in sequence, a sealing ring, a buffer pad, a filter membrane, and a lower end liner. The buffer pad has several buffer holes, and the lower end liner has several filter holes. This exosome extraction chip is compact and portable. During extraction, the sample liquid is simply injected into the extraction chip through the liquid inlet, and the exosomes are collected on the filter membrane, achieving exosome extraction. The operation is convenient and effectively improves exosome extraction efficiency.

Description

Technical Field

[0001] This invention relates to the field of exosome extraction technology, and more specifically to a replaceable exosome extraction chip. Background Technology

[0002] Exosomes have been proven useful for the early diagnosis of various diseases, and the most crucial step in their extraction is their isolation and collection. Currently, the commonly used methods for exosome isolation include the following:

[0003] 1. Ultracentrifugation: This method extracts exosomes by increasing centrifugation force or time to separate the precipitate or supernatant. It is suitable for separating exosomes in large-volume samples, but it may cause mechanical damage to some exosomes, destroy the exosome structure, and lead to the aggregation and co-precipitation of exosomes with other amorphous particles in the sample. It may even lead to the rupture of exosomes or their fusion with contaminants and other proteins.

[0004] 2. Density gradient centrifugation: The cell suspension or homogenate is placed on top of a sucrose medium. The cells are separated by centrifugal force, and the exosomes are separated from the mixed substances. Then, the cells are further purified using a filter membrane. The preliminary preparation is cumbersome, the operation is complicated and time-consuming.

[0005] 3. Size exclusion method: The size exclusion SEC principle is used to separate exosomes according to molecular size. The exosomes separated by chromatography are not affected by shear force, which may change the structure of the vesicles.

[0006] 4. Precipitation technology based on PEG polymers: The sample is mixed with a polymer-containing precipitation solution, incubated at 4°C and centrifuged at low speed. However, the extraction purity is insufficient, and the contamination of polymer materials may affect downstream analysis.

[0007] To address the aforementioned issues, this invention proposes a high-efficiency, high-throughput exosome extraction chip that can separate exosomes from sample solutions through simple operation. This not only effectively overcomes the shortcomings of existing technologies but also enables rapid extraction of exosomes, which is of great significance for the early diagnosis of diseases. Summary of the Invention

[0008] The present invention aims to provide a replaceable exosome extraction chip to solve the technical problems of complicated steps and low efficiency in existing exosome extraction methods.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: a replaceable exosome extraction chip, comprising a detachably connected upper cover and a lower cover, wherein the upper cover is provided with a liquid inlet and the lower cover is provided with a liquid outlet; the lower cover is provided with a sealing ring, a buffer pad, a filter membrane and a lower end liner in sequence, wherein the buffer pad is provided with a plurality of buffer holes and the lower end liner is provided with a plurality of filter holes.

[0010] The principle of this scheme is:

[0011] First, the sealing ring, buffer pad, filter membrane, and lower end liner are placed inside the lower cover in top-to-bottom order. Then, the upper and lower covers are rotated and tightened, securing the sealing ring, buffer pad, filter membrane, and lower end liner between the end faces of the upper and lower covers, thus assembling the extraction chip. The buffer pad and lower end liner are positioned on opposite sides of the filter membrane, effectively securing and tightening it. Next, the sample to be extracted is injected into the extraction chip through the inlet. Exosomes in the sample are blocked and collected on the filter membrane, while liquid and other impurities pass through the filter membrane and exit through the outlet. The outlet can be connected to a centrifuge tube or other liquid collection device for collection. Then, the upper and lower covers are unscrewed, the sealing ring and buffer pad are removed, the filter membrane surface is washed with physiological saline, the exosome solution is collected, and the exosomes are extracted and concentrated.

[0012] The advantages of this solution are:

[0013] 1. Compared with existing exosome extraction equipment, which is large and involves complicated steps that affect efficiency, the exosome extraction chip in this solution is small and portable. During extraction, the sample liquid is simply injected into the extraction chip through the inlet, and the exosomes are collected on the filter membrane to achieve exosome extraction. The operation is convenient and effectively improves the exosome extraction efficiency.

[0014] 2. In this exosome extraction chip, the appropriate size of the filter membrane can be pre-selected and installed according to the type of biological sample and the required size of the exosome, which can effectively realize the extraction of various types of exosomes and effectively meet the exosome extraction needs under different circumstances.

[0015] 3. Compared to existing equipment, which is large and requires multiple samples to be processed in a concentrated manner, thus reducing the timeliness of sample processing, the extraction chip structure of this solution is small and can extract exosomes from small samples and single samples. This effectively improves the timeliness of sample extraction and avoids the accumulation and precipitation of exosomes in the sample, degradation or destruction by enzymes, etc., caused by the failure to extract the samples in time after collection. This effectively improves the extraction yield and integrity of exosomes.

[0016] 4. This solution effectively mitigates the impact on the filter membrane during sample injection by placing a buffer pad above the filter membrane, ensuring the membrane's integrity and effectively filtering impurities smaller than the exosome particle size, thus improving exosome purity. Furthermore, the buffer pad makes the extraction of exosomes by the extraction chip gentler, effectively preventing exosome breakage and improving the integrity of the obtained exosomes. The applicant also discovered through long-term experiments that without the buffer pad, during the assembly of the extraction chip, the sealing ring may rotate with the top cover when tightening it, causing structural damage to the filter membrane surface in direct contact with the sealing ring (such as wrinkles or breaks). Damaged surfaces may lead to increased pore size, pore breakage, or even membrane cracking, resulting in some upstream liquid not being effectively filtered, with exosomes contaminating the filtered liquid and reducing exosome yield. The lower liner not only ensures the stability of the filter membrane during experiments but also guarantees a smooth and orderly flow of the filtered liquid out of the outlet.

[0017] Preferably, as an improvement, the sealing ring, buffer gasket, and filter membrane are all connected to the gap between the lower cover and the casing.

[0018] Technical benefits: The above-mentioned setup facilitates the complete washing away of exosomes on the filter membrane after disassembly and also makes it easy to replace the filter membrane.

[0019] Preferably, as an improvement, the lower cover and the lower end liner are fixedly connected or detachably connected.

[0020] Technical Benefits: This solution, employing the above-described configuration, facilitates the assembly of extraction chips from components made of various materials and using diverse molding processes. The molding process for fixed connections can be integral molding or welding (e.g., with metal materials), while detachable connections are formed separately. If the exosome extraction chip is entirely made of metal, the lower end pad and lower cover can be integrally molded or welded together, or they can be formed separately and then assembled. Conversely, if the exosome extraction chip is entirely made of polymer materials (such as plastics) or composite materials, separate molding can be selected, facilitating replacement even if the lower end pad is damaged, thus extending the overall lifespan of the extraction chip.

[0021] Preferably, as an improvement, when the lower cover and the lower end liner are detachably connected, a rubber gasket is provided between the lower end liner and the lower cover.

[0022] Technical benefits: The above-mentioned design of this solution has the following advantages: First, it ensures that there is more room for positional changes during the design process of the entire chip; second, it provides elastic support for the lower substrate, preventing displacement caused by gaps between the substrate and the lower cover due to their separate molding, which would lead to movement of the filter membrane and affect the extraction effect of the filter membrane on exosomes; third, it facilitates cleaning, disassembly and replacement during replacement.

[0023] Preferably, as an improvement, the bottom of the lower cover can be flat or conical; when the bottom of the lower cover is flat, the rubber pad directly abuts against the flat bottom of the lower cover; when the bottom of the lower cover is conical, the side wall of the lower cover is provided with an annular step for abutting against the rubber pad, thereby supporting the rubber pad.

[0024] The beneficial effect is that the above-mentioned arrangement facilitates the support of the lower cover for the rubber gasket.

[0025] Preferably, as an improvement, the upper cover and the lower cover are threaded together, with the upper cover having an internal thread and the lower cover having an external thread.

[0026] Technical benefits: The above-mentioned design facilitates quick tightening and disassembly of the upper and lower covers, making operation convenient.

[0027] Preferably, as an improvement, both the upper and lower covers are provided with anti-slip railings on their outer walls.

[0028] Technical effect: The above-mentioned design facilitates anti-slip when tightening the top and bottom covers, and improves the tightness of the connection between them.

[0029] Preferably, as an improvement, the diameter of the buffer hole is larger than the diameter of the filter hole.

[0030] Technical effect: The above-mentioned settings facilitate the flow of samples from large pores to small pores, increase the liquid flow rate, and improve extraction efficiency.

[0031] Preferably, as an improvement, both the buffer hole and the filter hole are tapered holes, and the diameter of the tapered hole gradually decreases along the direction of liquid flow.

[0032] Technical effects: The above-mentioned setup in this solution allows the conical orifice to facilitate the flow direction of the extracted sample and increases the liquid pressure, accelerating the penetration of the liquid portion of the sample through the filter membrane and further improving the exosome extraction efficiency.

[0033] Preferably, as an improvement, the diameter of the buffer hole region is smaller than the diameter of the filter hole region.

[0034] Technical effects: The above-mentioned settings facilitate the further guidance of sample movement, increase liquid flow pressure, accelerate the penetration of liquid portion of the sample through the filter membrane, and improve exosome extraction efficiency.

[0035] Preferably, as an improvement, the centers of both the buffer pad and the lower end liner are recessed towards the downward cover.

[0036] Technical effect: The above-mentioned configuration of this solution, with the buffer pad and the lower liner recessed in the same direction and with the same curvature, makes the filter membrane located between the two also have the same curvature, which facilitates the diversion of the extracted sample injected to the surrounding area to the center, improves the exosome collection effect, and can further improve the extraction efficiency.

[0037] Preferably, as an improvement, the buffer pad has a raised ring near the edge of the filter membrane, the inner side of the raised ring is gap-connected with the outer side of the lower end liner, and the thickness of the raised ring is less than the thickness of the lower end liner.

[0038] In terms of technical effectiveness, the above-mentioned setup in this solution facilitates the use of buffer pads to fix the edge of the filter membrane onto the lower liner, ensuring the filter membrane remains stable during extraction and improving extraction efficiency and stability.

[0039] Preferably, as an improvement, this solution also provides a method for using a replaceable exosome extraction chip, including the following steps:

[0040] Step 1: Assemble the above exosome extraction chip: Place the sealing ring, buffer pad, filter membrane and lower end liner in the lower cover in the order from top to bottom, then rotate and fasten the upper cover and lower cover together, and press the sealing ring, buffer pad, filter membrane and lower end liner between the end faces of the upper cover and lower cover.

[0041] Step 2: Sample addition: The biological sample enters the extraction chip through the inlet. After being buffered by the buffer pad, the exosomes are blocked on the filter membrane, while the liquid and other impurities pass through the filter membrane and are discharged from the outlet. The outlet is connected to a centrifuge tube or other liquid collection device.

[0042] Step 3: Collect exosomes: Unscrew the top and bottom caps, remove the sealing ring and buffer pad, wash the filter membrane surface with physiological saline, collect the exosome solution, and extract and concentrate to obtain exosomes.

[0043] Technical benefits: The above-mentioned setup facilitates the rapid extraction and separation of exosomes from the sample to be extracted using the extraction chip, and also enables the management and assembly of the extraction chip equipment. Attached Figure Description

[0044] Figure 1 This is a structural diagram of the replaceable exosome extraction chip in Embodiment 1 of the present invention.

[0045] Figure 2 This is an exploded view of the replaceable exosome extraction chip in Embodiment 1 of the present invention.

[0046] Figure 3 This is a partial view of the upper anti-slip fence in the replaceable exosome extraction chip of Embodiment 1 of the present invention.

[0047] Figure 4This is a partial view of the buffer pad in the replaceable exosome extraction chip in Embodiment 1 of the present invention (top view, bottom side view).

[0048] Figure 5 This is a top-axis side view of the buffer pad in the replaceable exosome extraction chip in Embodiment 1 of the present invention.

[0049] Figure 6 This is a partial view of the lower end substrate of the replaceable exosome extraction chip in Embodiment 1 of the present invention (top view, bottom side view).

[0050] Figure 7 This is a cross-sectional view of the lower cover of the replaceable exosome extraction chip in Embodiment 1 of the present invention.

[0051] Figure 8 This is a particle size distribution diagram of plasma exosomes in Experimental Example 1 of the present invention.

[0052] Figure 9 The number of plasma exosome concentrated particles in Experimental Example 1 of this invention.

[0053] Figure 10 This is an electron micrograph of plasma exosomes (double-membrane teacup structure) from Experimental Example 1 of this invention.

[0054] Figure 11 This is a particle size distribution diagram of urinary exosomes in Experiment Example 2 of the present invention.

[0055] Figure 12 This refers to the number of concentrated exosome particles in urine in Experimental Example 2 of this invention.

[0056] Figure 13 This is an electron micrograph of urinary exosomes (double-layered membrane saucer structure) from Experimental Example 2 of the present invention.

[0057] Figure 14 This is a particle size distribution diagram of avocado exosomes in Experimental Example 3 of the present invention.

[0058] Figure 15 The number of avocado exosome particles in Experiment Example 3 of this invention.

[0059] Figure 16 The exosomes in Comparative Example 1 of this invention are damaged (a double phospholipid bilayer is visible, but the outline is irregular and the structure is not closed).

[0060] Figure 17 This is a particle size distribution diagram of exosomes in Comparative Example 2 of the present invention.

[0061] Figure 18 The exosomes and other impurities in Comparative Example 2 of this invention are damaged (intact exosomes are shown in the red circle, and other impurities are shown in the yellow circle). Detailed Implementation

[0062] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0063] Explanation of reference numerals in the attached drawings: 1. Top cover, 11. Liquid inlet, 12. Upper anti-slip rail, 2. Sealing ring, 3. Buffer pad, 31. Filter hole, 4. Filter membrane, 5. Lower end liner, 51. Filter hole, 6. Rubber pad, 7. Bottom cover, 71. Lower thread, 72. Liquid outlet, 73. Lower anti-slip rail, 74. Annular step.

[0064] Example 1

[0065] This solution provides a replaceable exosome extraction chip, such as Figures 1-6 As shown: It includes a detachable upper cover 1 and a lower cover 7. For reference, the upper cover 1 and the lower cover 71 are connected by a thread. The upper cover 1 has an internal thread (also called an upper thread), and the lower cover 7 has an external thread (also called a lower thread 71). The outer walls of the upper cover 1 and the lower cover 7 are provided with anti-slip rails. Specifically, the outer wall of the upper cover 1 is provided with an upper anti-slip rail 12, and the outer wall of the lower cover 7 is provided with a lower anti-slip rail 73, which facilitates the quick tightening and disassembly of the upper cover 1 and the lower cover 7.

[0066] The upper cover 1 is provided with a liquid inlet 11, and the lower cover 7 is provided with a liquid outlet 72; the lower cover 7 is provided with a sealing ring 2, a buffer pad 3, a filter membrane 4 and a lower end liner 5 in sequence, and the sealing ring 2, the buffer pad 3 and the filter membrane 4 are all connected to the lower cover 7 with gaps.

[0067] In another embodiment, such as Figure 5 As shown, the buffer pad 3 has a raised ring on its edge near the filter membrane 4. The inner side of the raised ring is gapped to the outer side of the lower liner 5, and the thickness of the raised ring is less than the thickness of the lower liner. This design allows the lower liner 5 to firmly hold the edge of the filter membrane 4 against the raised ring of the buffer pad 3, improving the fixation effect of the filter membrane 4 and ensuring its stability during extraction, thereby increasing extraction efficiency and stability. Simultaneously, the buffer pad 3 also prevents damage to the filter membrane 4 due to high internal pressure during exosome extraction when the sealing ring 2 is in direct contact with it, effectively buffering and reducing shock and protecting the integrity of the filter membrane 4.

[0068] The buffer pad 3 is provided with several buffer holes 31, and the lower liner 5 is provided with several filter holes 51. The diameter of the buffer hole 31 is larger than that of the filter hole 51. As an improvement, both the buffer hole 31 and the filter hole 51 are conical holes. The diameter of the conical holes gradually decreases along the direction of liquid flow, which facilitates flow guidance and increases the liquid flow rate, thereby improving the efficiency of exosome extraction.

[0069] In another embodiment, as an improvement, the diameter of the buffer hole 31 region is smaller than the diameter of the filter hole 51 region, and the center of the buffer pad 3 and the lower end liner 5 are both recessed towards the lower cover 7, which facilitates the flow of the sample injected into the edge region to the middle, avoids sample waste, and can further guide the sample liquid.

[0070] For reference, the lower cover 7 and the lower end liner 5 are either fixedly connected or detachably connected. When the lower cover 7 and the lower end liner 5 are detachably connected, a rubber gasket 6 is provided between the lower end liner 5 and the lower cover 7.

[0071] For reference, the bottom of the lower cover 7 can be flat or conical. When the bottom of the lower cover 7 is flat, the rubber pad 6 directly abuts against the flat bottom of the lower cover 7, thus supporting the rubber pad 6. When the bottom of the lower cover 7 is conical, the side wall of the lower cover 7 is provided with an annular step 74 for abutting against the rubber pad 6 (e.g., Figure 7 As shown in the figure, this provides support for the rubber pad 6.

[0072] In actual production, this solution can replace the exosome extraction chip, where all parts except the sealing ring and rubber gasket are made of hard materials, including metal materials, polymer materials or composite materials, etc. The manufacturing process includes molding, 3D printing and other feasible processes. This solution does not impose any special limitations.

[0073] Specifically, the molding process for fixed connections can be integral molding or welding (e.g., metal materials), while detachable connections are formed separately. For reference, in actual production, if the exosome extraction chip is made of metal, the lower end pad 5 and the lower cover 7 can be integrally molded or welded together, or they can be separately molded and then assembled. However, if the exosome extraction chip is entirely made of polymer materials (plastics, etc.) or composite materials, separate molding can be chosen, facilitating timely replacement of the lower end pad 5 after damage and extending the overall lifespan of the extraction chip.

[0074] In addition, this solution can replace the size of the exosome extraction chip and can be customized according to the sample volume of the application scenario, including but not limited to various sizes such as outer ring diameter of 13mm, 15mm, 30mm, 45mm, etc. This solution does not have any special limitations.

[0075] This solution also provides a method for using a replaceable exosome extraction chip, including the following steps:

[0076] Step 1: Assemble the above exosome extraction chip: Place the sealing ring 2, buffer pad 3, filter membrane 4 and lower end liner 5 in the lower cover 7 in the order from top to bottom, and then rotate and fasten the upper cover 1 and the lower cover 7 to press the sealing ring 2, buffer pad 3, filter membrane 4 and lower end liner 5 against the bottom end face of the upper cover 1 and the lower cover 7.

[0077] Step 2: Sample addition: The supernatant obtained after pretreatment of the biological sample is injected into the extraction chip through the inlet 11. After being buffered by the buffer pad 3, the exosomes are blocked on the filter membrane 4. The liquid and other impurities pass through the filter membrane 4 and are discharged from the outlet 72. The outlet 72 is connected to a centrifuge tube or other liquid collection device.

[0078] Step 3: Collect exosomes: Unscrew the top cap 1 and the bottom cap 7, remove the sealing ring 2 and the buffer pad, wash the surface of the filter membrane 4 with physiological saline, collect the exosome solution, and extract and concentrate to obtain exosomes.

[0079] The replaceable exosome extraction chip in this solution has the following advantages:

[0080] 1. Exosomes can be directly extracted after conventional instrument pretreatment;

[0081] 2. It employs a single physical filtration extraction method, requiring no additional biological reagents;

[0082] 3. Samples can be added using a syringe to achieve pressure extraction, without the need for any external pressure pump;

[0083] 4. The extraction process is simple, the operation is safer, no specific operating environment is required, and the sealing is good;

[0084] 5. Personalized exosome extraction is possible, allowing for stepwise extraction within a specific particle size range based on the required exosome particle size.

[0085] 6. The pretreatment and exosome extraction process takes less time and costs less, has high extraction efficiency, and produces high-quality exosomes.

[0086] Experimental Example 1: Extraction of plasma exosomes

[0087] Operating steps:

[0088] 4.1 ml of blood was collected using an EDTA anticoagulant tube and centrifuged at 4°C for 15 min at 1500 g to remove red blood cells. The supernatant was then collected. The collected supernatant was then centrifuged again at 4°C for 3000 g for 15 min, and the supernatant was collected once more.

[0089] The collected supernatant is extracted and concentrated using the exosome extraction chip of this scheme. In this embodiment, the exosome extraction chip has a diameter of 25 mm, and the filter membrane material is PES with a diameter of 25 mm and pore sizes of 0.45 μm, 0.22 μm, and 0.05 μm. The collected supernatant is then passed through exosome extraction chips with pore sizes of 0.45 μm, 0.22 μm, and 0.05 μm, respectively. After passing through the exosome extraction chip with a pore size of 0.05 μm, the desired exosome extract is the membrane-bound substance.

[0090] Add 180 μL of physiological saline and rinse the filter membrane repeatedly 15-20 times. Finally, collect 120 μL of the washed liquid and perform NTA and TEM experiments.

[0091] The total time for pretreatment and exosome extraction was 45 minutes, and the results are as follows: Figures 8-10 As shown.

[0092] Figures 8-10 The images show the particle size distribution of the extracted material, the number of particles in the extracted material, and an electron microscope image of the extracted material. Figure 8 The extracted material showed that the particle size was mainly 100 nm. Figure 9 The particle count is displayed as 9.8 × 10⁻⁶. 9 pcs / ml Figure 10 The extracted substance exhibits a typical double-membrane cup structure. The particle size and apparent structure of the extracted substance confirm that it is an exosome. Furthermore, the high particle count indicates a high exosome concentration, confirming that the exosome extraction chip in this scheme can extract and concentrate exosomes while ensuring the stability of the exosome structure.

[0093] Experimental Example 2: Extraction of Urinary Exosomes

[0094] Operating steps:

[0095] 75 ml of urine was collected in a centrifuge tube and centrifuged at 4°C with the following parameters: 2000 g and 20 min. The supernatant was then collected.

[0096] The collected supernatant was extracted and concentrated using an exosome extraction chip. In this embodiment, the inner diameter of the exosome extraction chip is 25 mm and the outer diameter is 30 mm. The filter membrane material in this embodiment is PES with a diameter of 25 mm and pore sizes of 0.45 μm, 0.22 μm, and 0.05 μm. The final extracted supernatant was passed through exosome extraction chips with pore sizes of 0.45 μm, 0.22 μm, and 0.05 μm, respectively. After passing through the exosome extraction chip with a pore size of 0.05 μm, the desired exosome extract was obtained from the membrane. 400 μL of physiological saline was added, and the filter membrane was repeatedly rinsed 15-20 times. Finally, 350 μL of the washed liquid was collected and subjected to NTA and TEM experiments.

[0097] The total time for pretreatment and exosome extraction was 35 minutes, and the results are as follows: Figures 11-13 As shown.

[0098] Figures 11-13 The images show the particle size distribution of the extracted material, the number of particles in the extracted material, and an electron microscope image of the extracted material. Figure 11 The extracted material showed that its particle size was mainly 100–200 nm. Figure 12 The particle count is displayed as 4.1 × 10⁻⁶. 8 pcs / ml Figure 13 The extracted substance exhibits a typical double-membrane cup structure. The particle size and apparent structure of the extracted substance confirm that it is an exosome. Furthermore, the high particle count indicates a high concentration of exosomes, demonstrating that this exosome chip can extract and concentrate exosomes while maintaining the stability of the exosome structure.

[0099] Experimental Example 3: Extraction of exosomes from avocados

[0100] Operating steps:

[0101] Peel 130g of fresh avocado and place it in a juicer, adding physiological saline solution. Juice for 10 minutes. After juicing, filter the pulp through sterile gauze and collect the lower layer of juice. Centrifuge the filtered juice at 4°C for the first time at 2000g for 10 minutes, collecting the supernatant. Centrifuge the supernatant again at 10000g for 30 minutes, collecting the supernatant from the second centrifugation. Then, extract and concentrate the supernatant using an exosome extraction chip.

[0102] In this embodiment, the inner diameter of the exosome extraction chip is 25 mm and the outer diameter is 30 mm. The filter membrane material is PES, with a diameter of 25 mm and pore sizes of 0.8 μm, 0.45 μm, 0.22 μm, and 0.05 μm. The final extracted supernatant is passed through exosome extraction chips with pore sizes of 0.8 μm, 0.45 μm, 0.22 μm, and 0.05 μm, respectively. After passing through the exosome extraction chip with a pore size of 0.05 μm, the desired extract is the substance on the membrane. 400 μL of physiological saline is added, and the filter membrane is repeatedly rinsed 15-20 times. Finally, 350 μL of the washed liquid is collected and used for NTA experiments.

[0103] The total time for pretreatment and exosome extraction was 70 minutes, and the results are as follows: Figures 14-15 As shown.

[0104] Figures 14-15 These are the particle size distribution diagram of the extracted substance and the number of particles of the extracted substance, respectively. Figure 14 The extracted material showed that the particle size was mainly 90-150 nm. Figure 15 The particle count is displayed as 1.7 × 10⁻⁶. 12 per ml.

[0105] In summary, the components and substances contained in different samples vary considerably, and the proportion of the same substance also varies significantly. This patented technology, during the extraction process, not only ensures the number and concentration of exosomes but also preserves the integrity of the exosome structure, preventing damage.

[0106] Comparative Example 1

[0107] The difference between this comparative example and Example 1 is that exosomes were extracted using ultracentrifugation.

[0108] Figure 16 Electron micrographs of the fragmented exosomes extracted in this comparative example are shown. Fragmentation of exosomes reduces the number of effective exosomes and decreases their purity, affecting their physical properties and downstream analysis.

[0109] Comparative Example 2

[0110] The difference between this comparative example and Example 1 is that the replaceable exosome extraction chip in this comparative example does not have a buffer pad.

[0111] In this comparative example, exosomes can also be extracted, but during the tightening of the device, wrinkles or damage easily appear on the surface of filter membrane 4, leading to unstable exosome extraction results. See [link to relevant documentation]. Figures 17-18 .

[0112] Experimental results show that the damaged filter membrane particles range in size from 50 nm to 250 nm, exceeding the theoretical particle size of exosomes. Electron microscopy images show that, under the same field of view, the sample liquid collected from the outlet contains both exosomes and impurities with particle sizes larger than or equal to those of exosomes. This indicates that without a buffer pad, filter membrane 4 is prone to surface structural damage, affecting its filtration efficiency. Undamaged filter membrane 4 successfully filters exosomes, while damaged surfaces may lead to increased pore size, pore rupture, or membrane cracking. Consequently, some upstream liquid fails to pass through effective filtration, resulting in exosome contamination in the filtered liquid and significantly reducing the yield of extracted exosomes.

[0113] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for using a replaceable exosome extraction chip, characterized in that: The extraction chip includes a detachably connected upper cover and a lower cover. The upper cover has a liquid inlet, and the lower cover has a liquid outlet. The lower cover contains, in sequence, a sealing ring, a buffer pad, a filter membrane, and a lower end liner. The buffer pad has several buffer holes, and the lower end liner has several filter holes. A raised ring is located near the edge of the filter membrane on the buffer pad. The inner surface of the raised ring is gap-connected to the outer surface of the lower end liner, and the thickness of the raised ring is less than the thickness of the lower end liner. Both the buffer holes and the filter holes are tapered, with the diameter of the tapered holes gradually decreasing along the liquid flow direction. The diameter of the buffer holes is larger than the diameter of the filter holes, and the diameter of the buffer hole region is smaller than the diameter of the filter hole region. The method of using the extraction chip includes the following steps: Step 1: Assemble the above exosome extraction chip: Place the sealing ring, buffer pad, filter membrane and lower end liner in the lower cover in the order from top to bottom, then rotate and fasten the upper cover and lower cover together, and press the sealing ring, buffer pad, filter membrane and lower end liner between the end faces of the upper cover and lower cover. Step 2: Sample addition: The supernatant obtained after pretreatment of the biological sample enters the extraction chip through the inlet. After being buffered by the buffer pad, the exosomes are blocked on the filter membrane, while the liquid and other impurities pass through the filter membrane and are discharged from the outlet. The outlet is connected to a centrifuge tube or other liquid collection device. Step 3: Collect exosomes: Unscrew the top and bottom caps, remove the sealing ring and buffer pad, wash the filter membrane surface with physiological saline, collect the exosome solution, and extract and concentrate to obtain exosomes.

2. The method of using a replaceable exosome extraction chip according to claim 1, characterized in that: The sealing ring, buffer gasket, and filter membrane are all connected to the gap between the bottom cover and the bottom cover.

3. The method of using a replaceable exosome extraction chip according to claim 1, characterized in that: The lower cover and the lower end liner are either fixedly connected or detachably connected. When the lower cover and the lower end liner are detachably connected, a rubber gasket is provided between the lower end liner and the lower cover.

4. The method of using a replaceable exosome extraction chip according to claim 1, characterized in that: The upper cover and the lower cover are threaded together, with the upper cover having internal threads and the lower cover having external threads.

5. The method of using a replaceable exosome extraction chip according to claim 4, characterized in that: Both the upper and lower covers are equipped with anti-slip railings on their outer walls.

6. The method of using a replaceable exosome extraction chip according to claim 5, characterized in that: The center of both the buffer pad and the lower end liner is recessed towards the downward cover.

Images