Exosome enrichment chip and enrichment method

Abstract

The application relates to the technical field of exosome extraction, and discloses an exosome enrichment chip and an enrichment method.The chip comprises a sample storage box, a sample inlet channel, a vortex reaction enrichment pool, a sample circulation channel and the like.The enrichment method can be used for single-time and high-throughput circulation enrichment of exosomes;exosome-specific enrichment materials are loaded on the filter membrane at the bottom of the vortex reaction enrichment pool, and the asymmetric impinging streams of the bilateral asymmetric sample inlet channels can be matched with the vortex motion, so that the residence time of the exosomes in the enrichment pool and the collision efficiency of the exosomes with the specific enrichment materials are improved, efficient enrichment is realized, then the sample passes through the filter membrane and enters the sample circulation channel, and is returned to the sample storage box, circulation enrichment of the exosomes in the sample is realized, and the exosome recovery rate is improved.The application can realize rapid, high-yield and high-purity enrichment of exosomes in large-volume samples such as urine, thoracic and abdominal fluid and cell culture supernatant.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of exosome extraction, and particularly relates to an exosome enrichment chip and an enrichment method. BACKGROUND

[0002] Exosomes are vesicular structures with a diameter of 30-200 nm secreted by cells. Exosomes are released by various types of cells into body fluids, carrying nucleic acids, proteins, metabolites and other biomolecules derived from the parent cells, and the phospholipid bilayer structure of exosomes can protect the contents from being stable in body fluids. As a carrier of cell communication, exosomes have been proven to play an important role in immune regulation, disease diagnosis, drug delivery, efficacy monitoring, etc.

[0003] Separating exosomes from a complex body fluid environment is the key to exosome research. The ultracentrifugation method is considered the "gold standard" for exosome separation, but it has the disadvantages of long time consumption, dependence on ultracentrifuge, low exosome yield and purity, etc., which is not conducive to clinical application and promotion. The exosomes enriched by the polyethylene glycol precipitation method are easily contaminated by proteins, lipids, etc., and have low purity. Other traditional exosome separation methods such as ultrafiltration and size exclusion have the limitations of tedious operation and long time consumption when separating large-volume samples. In order to improve the selectivity of exosome separation, affinity capture strategies based on molecular recognition have become increasingly attractive. Through the high binding force of specific markers of exosomes and corresponding ligands such as antibodies, peptides, aptamers, etc., the selectivity and specificity of exosome enrichment can be improved. At present, commercial kits based on phosphatidylserine affinity capture and immunomagnetic beads have been widely developed, but the amount of capture reagent increases with the increase of sample volume, which is high in cost and not suitable for exosome separation of large-volume samples. SUMMARY

[0004] In view of the defects and needs of the existing exosome enrichment technology, the application provides an exosome enrichment chip and an enrichment method, which overcome the technical defects of low throughput, long time consumption, high cost, multiple operation steps and low enrichment efficiency of traditional exosome enrichment methods.

[0005] Based on the above technical problems and technical purposes, the application is realized by the following technical solutions:

[0006] On the one hand, the application provides an exosome enrichment chip, which comprises a sample storage tank, a sample inlet channel, a vortex reaction enrichment pool and a sample circulation channel.

[0007] Further, the exosome enrichment chip comprises a total sample inlet arranged at the top of the sample storage tank, a sample outlet arranged at the bottom of the sample storage tank, a sample inlet of the sample inlet channel connected with the sample outlet, and the sample inlet channel connected with the vortex reaction enrichment pool and the sample circulation channel in sequence.

[0008] Preferably, the present application provides a high-throughput exosome circulation enrichment chip, which is composed of a sample storage tank, a double-sided asymmetric sample inlet channel, a vortex reaction enrichment pool and a sample circulation channel; a total sample inlet is arranged at the top of the sample storage tank, and a first sample outlet and a second sample outlet are arranged at the bottom of the sample storage tank; the double-sided asymmetric sample inlet channel is provided with a first sample inlet, a second sample inlet, a first sample input channel, a ring channel, a symmetric radial channel and a second sample input channel; the first sample inlet is connected to the first sample outlet, the first sample input channel is connected to the ring channel, and the ring channel is connected to the vortex reaction enrichment pool through a plurality of symmetric radial channels; the second sample inlet is connected to the second sample outlet, and the second sample input channel is connected to the vortex reaction enrichment pool.

[0009] A circular notch is arranged at the bottom of the vortex reaction enrichment pool for placing a filter membrane, and an exosome-specific enrichment material is loaded on the filter membrane; the sample circulation channel comprises a sample filtration pool, a sample output channel and a total sample outlet.

[0010] The pore size of the filter membrane is greater than 200 nm.

[0011] The double-sided asymmetric sample inlet channel comprises a plurality of symmetric radial channels and a second sample input channel, the channel diameter of the symmetric radial channels and the channel diameter of the sample input channel are both less than 0.5 mm, when the sample flows through the symmetric radial channels and the second sample input channel at the same time, an asymmetric impinging stream can be formed in the vortex reaction enrichment pool, a stronger oscillation motion can be obtained compared with a symmetric impinging stream, the collision probability of the sample exosomes and the exosome-specific enrichment material is improved, and the exosome enrichment efficiency is improved.

[0012] The sample storage tank can accommodate large-volume samples such as cell supernatant, urine, and pleural effusion, after the sample is added through the total sample inlet, the sample can flow into the double-sided asymmetric sample inlet channel from the first sample outlet and the second sample outlet at the same time, and then enter the vortex reaction enrichment pool through the double-sided asymmetric channel, at this time, the exosomes in the sample combine with the exosome-specific enrichment material in the vortex reaction enrichment pool to realize the enrichment of the exosomes. The sample filtration pool is connected to the vortex reaction enrichment pool, the sample filtered through the filter membrane enters the sample filtration pool, and then reaches the total sample outlet through the sample output channel, and can be reconnected to the sample storage tank to realize the circulation enrichment of the exosomes.

[0013] The exosome-specific enrichment material is one of a microsphere containing an exosome capture probe, a silicon sphere, a metal organic framework material, and the size of the specific enrichment material is greater than the pore size of the filter membrane.

[0014] The exosome capture probe is one or more of a combination of an exosome-specific recognition antibody, an exosome-specific recognition aptamer, an exosome-specific recognition metal ion, arginine, lysine, a polyhydroxy compound, and an exosome-specific recognition membrane protein.

[0015] The metal organic framework material is a MOF-on-MOF material, which specifically binds to the phospholipid bilayer of the exosome through metal-phosphate coordination; the MOF-on-MOF material has a Zr-MOF core and a Fe-MOF grown on the outside of the Zr-MOF.

[0016] The MOF-on-MOF material is synthesized by dispersing ZrCl4 and terephthalic acid in a mixed solution of DMF and glacial acetic acid at a reflux temperature, cooling to 20-25℃, then washing the white precipitate with DMF and anhydrous ethanol by centrifugation, and vacuum drying for 8-12 hours to obtain a white product Zr-MOF; dispersing the Zr-MOF in DMF, adding terephthalic acid powder to obtain a mixture; ultrasonically treating the mixture for 20-30 minutes, then adding a mixed DMF solution containing FeCl3·6H2O and triethylamine to obtain a new mixture; transferring the new mixture to an autoclave and reacting at 100-150℃ for 10-15 hours; cooling to 20-25℃; recovering the orange solid by centrifugation, and washing with DMF and anhydrous methanol to obtain a powder, which is vacuum dried at 80-150℃ for 8-12 hours to obtain the MOF-on-MOF material.

[0017] Preferably, ZrCl4 and terephthalic acid are dissolved in a mixed solution of DMF and glacial acetic acid at a volume ratio of 1-10:1 at a reflux temperature of 100-150℃ for 20-30 hours;

[0018] Preferably, the final concentration of Zr-MOF in the mixture is 4-8 mg / mL, and the final concentration of terephthalic acid is 0.08-0.12 mmol / mL;

[0019] Preferably, an equal volume of a mixed DMF solution containing FeCl3·6H2O and triethylamine with a final concentration of 0.8-1.2 v / v% is added to the above mixture, so that the molar concentration ratio of FeCl3·6H2O to terephthalic acid reaches 1:1 at this time, to obtain a new mixture;

[0020] The DMF (N,N-dimethylformamide, purity 99.5%) and glacial acetic acid are both analytical reagents.

[0021] In a second aspect, the present application also provides an exosome enrichment method, comprising the following steps:

[0022] 1) Load exosome specific enrichment material: after the exosome specific enrichment material is resuspended with buffer, it is injected into the vortex reaction enrichment pool through the second sample inlet;

[0023] 2) Prepare to install the sample storage tank: connect the first sample outlet with the first sample inlet, and connect the second sample outlet with the second sample inlet;

[0024] 3) Sample pretreatment: the sample is centrifuged at 2000g to remove cells and cell fragments, and then filtered through a 0.22 mu m filter to remove large vesicles to obtain the treated sample;

[0025] 4) Add sample: the sample obtained in step 3) is added to the sample storage tank through the total sample inlet;

[0026] 5) Liquid connection: connect the total sample outlet with the inlet of the external suction device, and connect the outlet of the external suction device with the external waste liquid bottle;

[0027] 6) Exosome enrichment: start the external suction device, and through the action of the external suction device (for example: peristaltic pump), the sample in the sample storage tank enters the vortex reaction enrichment pool through the bilateral asymmetric channel, at this time, the exosomes in the sample combine with the exosome specific enrichment material in the vortex reaction enrichment pool, and the exosome enrichment is realized.

[0028] Further, the application also provides a high-throughput exosome circulation enrichment method, based on the above method, after steps 1)-4) are performed, step 5) is connected: the total sample outlet is connected with the inlet of the external suction device, and the outlet of the external suction device is connected with the total sample inlet; after step 6), the sample flows through the filter membrane arranged at the bottom of the vortex reaction enrichment pool and enters the sample filtration pool, and then passes through the sample output channel to reach the total sample outlet, and can flow back to the sample storage tank, the enrichment time is 10-30 minutes, and the circulation enrichment of the exosomes is realized.

[0029] Compared with the prior art, the application has the advantages and beneficial effects that:

[0030] 1) The combination of the exosome specific enrichment material and the asymmetric impinging stream chip can greatly improve the enrichment efficiency of the exosomes. The design of the symmetric radial channel of the chip and the asymmetric fluid channel of the sample input channel can form an asymmetric impinging stream in the vortex reaction enrichment pool, improve the residence time of the sample exosomes in the vortex reaction enrichment pool and the collision probability with the exosome specific enrichment material, and further improve the enrichment efficiency of the exosomes.

[0031] 2) The circulation liquid path design realizes the circulation enrichment of exosomes, and improves the exosome yield in large volume samples such as urine, pleural effusion, cell culture supernatant, etc. The sample in the vortex reaction enrichment pool is filtered into the sample filtration pool through the filter membrane, and then passes through the sample output channel to reach the sample outlet, which can be reconnected to the sample storage tank, realizing the circulation enrichment of exosomes and improving the yield of exosomes in large volume samples.

[0032] 3) The exosome enrichment method based on the high-flux exosome circulation enrichment chip is simple to operate, does not depend on large instruments such as ultracentrifuges, and can realize the rapid and efficient enrichment of exosomes in batch large volume samples within half an hour through a multi-channel peristaltic pump. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a schematic diagram of the layered structure of the high-flux exosome circulation enrichment chip of the application;

[0034] Figure 2 It is a schematic diagram of the overall appearance of the high-flux exosome circulation enrichment chip of the application;

[0035] Figure 3 It is a schematic diagram of the cross-section of the high-flux exosome circulation enrichment chip of the application;

[0036] Figure 4 It is a scanning electron microscope image of the MOF-on-MOF material (Zr-MOF@Fe-MOF material) in the application;

[0037] Figure 5 It is the scanning electron microscope energy spectrum analysis result of the MOF-on-MOF material (Zr-MOF@Fe-MOF material) in the application, wherein the four small images are as follows: the upper left is the electronic image of the Zr-MOF@Fe-MOF material, the upper right is the Zr element distribution map, the lower left is the Fe element distribution map, and the lower right is the combined map of the Zr element and the Fe element;

[0038] Figure 6 It is an infrared spectrum of the MOF-on-MOF material (Zr-MOF@Fe-MOF material) in the application;

[0039] Figure 7 It is a fluorescence image of exosomes enriched by the high-flux exosome circulation enrichment chip with different circulation times, i is a control group, and ii is an experimental group;

[0040] Figure 8 It is a fluorescence image of exosomes enriched by three materials in Example 2;

[0041] Figure 9 It is a scanning electron microscope image of urine exosomes enriched by the MOF-on-MOF material (Zr-MOF@Fe-MOF material) in Example 3, wherein the arrow points to the exosome.

[0042] Figure 10 Transmission electron microscope image of urine exosome release after high-throughput exosome circulation enrichment chip enrichment in Example 3;

[0043] Figure 11 Protein immunoblotting result image of urine exosome enrichment product of three exosome enrichment methods in Example 4;

[0044] Among them, 1-sample storage box, 2-bilateral asymmetric sample inlet channel, 3-vortex reaction enrichment pool, 4-sample circulation channel, 5-total sample inlet, 6-first sample outlet, 7-second sample outlet, 8-first sample inlet, 9-second sample inlet, 10-first sample input channel, 11-ring channel, 12-symmetrical radial channel, 13-second sample input channel, 14-circular notch, 15-filter membrane, 16-sample filtration pool, 17-sample output channel, 18-total sample outlet. DETAILED DESCRIPTION

[0045] The following devices and materials are used in the following examples:

[0046] The external suction device is a peristaltic pump (lubricated); PBS buffer (biosharp, pH = 7.3-7.5); 0.22 μm filter (biosharp); DMF (analytical pure); glacial acetic acid (analytical pure); triethylamine (analytical pure); the synthesis method of Zr-MOF@Fe-MOF material is as follows, Figures 4-6 respectively, are the physical and chemical analysis result images of the material.

[0047] The following examples use an implementation form of an exosome enrichment chip of the application:

[0048] A high-throughput exosome circulation enrichment chip, consisting of a sample storage box 1, a bilateral asymmetric sample inlet channel 2, a vortex reaction enrichment pool 3, and a sample circulation channel 4. By Figures 1-3 As shown in the figure, the top of the sample storage box 1 is provided with a total sample inlet 5, and the bottom is provided with a first sample outlet 6 and a second sample outlet 7; in the example, four layers of etched thick glass sheets are used to form a bilateral asymmetric sample inlet channel 2, a vortex reaction enrichment pool 3, and a sample circulation channel 4 by cross-etching thin glass sheets;

[0049] Among them, below the sample storage box 1, from top to bottom, a first layer of thick glass sheet with the same length and width, a second layer of thin glass sheet, a third layer of thin glass sheet, and a fourth layer of thick glass sheet are arranged; the first layer and the second layer are fixedly connected, the third layer and the fourth layer are fixedly connected, and after the assembly of the four glass sheets and the filter membrane is completed, a dovetail clamp can be used for fixation, screw fixation, bolt fixation, or any fixation method in the prior art, which will not be described here.

[0050] The first layer thick glass plate is internally etched to form a first sample input channel 10, an annular channel 11, a symmetric radial channel 12, and a second sample input channel 13, thereby forming a double-sided asymmetric sample inlet channel 2; the first layer thick glass plate is internally etched to form a first sample inlet 8 and a second sample inlet 9 on the upper surface; in use, the first sample inlet 8 and the second sample inlet 9 can be connected to the first sample outlet 6 and the second sample outlet 7 respectively to form a connection between the double-sided asymmetric sample inlet channel 2 and the sample storage tank 1.

[0051] The vortex reaction enrichment pool 3 is formed by the cavity internally etched in the first layer thick glass plate, the second layer thin glass plate, and the hollows etched at corresponding positions in the three layer thin glass plates; the circular notch 14 etched in the third layer thin glass plate is used to place the filter membrane 15, and the filter membrane 15 is loaded with exosome-specific enrichment materials.

[0052] The first sample input channel 10 is connected to the annular channel 11, the annular channel 11 is connected to the vortex reaction enrichment pool 3 through at least two symmetric radial channels 12, the second sample inlet 9 is connected to the second sample outlet 7, and the second sample inlet 9 is connected to the vortex reaction enrichment pool 3 through the second sample input channel 13.

[0053] The fourth layer thick glass plate is etched to form a sample filtration pool 16 and a sample output channel 17, another end of the sample output channel 17 and a hollow etched at a corresponding position in the three layer glass plate above the sample output channel 17, and the upper surface of the first layer thick glass plate forms a total sample outlet 18; the sample filtration pool 16 is connected to the vortex reaction enrichment pool 3, the sample enters the sample filtration pool 16 through the filter membrane 15, and then passes through the sample output channel 17 to reach the total sample outlet 18; the total sample outlet 18 is connected to the total sample inlet 5, thereby re-connecting to the sample storage tank 1 and realizing the cyclic enrichment of exosomes.

[0054] The exosome-specific enrichment materials can be one of a microsphere containing an exosome capture probe, a silicon ball, a metal organic framework material, and the like.

[0055] After the sample is added from the sample storage tank 1, the sample enters the vortex reaction enrichment pool 3 through the double-sided asymmetric sample inlet channel 2, at this time, the exosomes in the sample combine with the exosome-specific enrichment materials in the vortex reaction enrichment pool 3, thereby realizing the enrichment of exosomes; the sample flows through the filter membrane 15 at the bottom of the vortex reaction enrichment pool 3, enters the sample filtration pool 16 after being filtered, and then passes through the sample output channel 17 to reach the sample outlet 18, thereby re-connecting to the sample storage tank 1 and realizing the cyclic enrichment of exosomes.

[0056] In order to better explain the present application, the content of the present application is further illustrated in combination with specific examples.

[0057] Example 1: Effect of sample circulation time on exosome enrichment performance

[0058] The cycle time will affect the final enrichment efficiency of the exosome. In order to evaluate the influence of cycle time on the enrichment abundance of exosomes, the exosomes are enriched from the cell culture supernatant by ultracentrifugation, and the exosomes are stained with red exosome membrane dye (Dil), and resuspended with pure water. The experimental group uses the aforementioned high-flux exosome circulation enrichment chip based on Zr-MOF@Fe-MOF material to enrich exosomes in the cell supernatant, and the cycle time is set to 5 min, 10 min, 20 min, 30 min; The control group uses the aforementioned high-flux exosome circulation enrichment chip without loading Zr-MOF@Fe-MOF material to enrich exosomes in the cell supernatant. The specific operation steps are as follows:

[0059] 1) Dil-stained cell exosome preparation: cell culture was performed, when the cell density reached 70%-80%, the culture medium was removed, washed with PBS buffer for 2 times, and FBS-free fetal bovine serum was added to the culture medium for starvation culture. After 36 hours of starvation treatment, the cell supernatant was collected. The cell supernatant was centrifuged at 300g for 10 minutes to remove dead cells, and the supernatant was collected. The supernatant was further centrifuged at 2000g for 10 minutes to remove cell debris, and the supernatant was collected. The supernatant was centrifuged at 10000g for 30 minutes to remove cell debris and large vesicles, and the supernatant was collected. The obtained supernatant was transferred to an ultracentrifuge tube, centrifuged at 120000g for 70 minutes, and the supernatant was removed and the exosome precipitate was collected. Add PBS buffer to resuspend the precipitate, add the prepared Dil working dye to the resuspension, incubate at 37°C for 15 minutes in the dark, and then centrifuge at 120000g for 70 minutes. Remove the unbound dye to obtain Dil-stained cell exosomes; add an appropriate amount of pure water to resuspend to obtain an exosome sample.

[0060] 2) Synthesis of MOF-on-MOF material, exemplary, MOF-on-MOF material is Zr-MOF@Fe-MOF material. The specific synthesis method is: 2.0 mmol ZrCl4 and 3.0 mmol terephthalic acid are dispersed and dissolved in a mixture of 30 mL DMF and 5 mL glacial acetic acid; then the mixture is refluxed at 100°C for 24 hours. After cooling to room temperature after the reaction is completed, the white precipitate is collected by centrifugal washing with DMF, anhydrous ethanol in turn, and then vacuum dried at 80°C for 10 hours to obtain white product Zr-MOF. 16.8 mg Zr-MOF is dispersed in 3 mL DMF, and 0.281 mmol terephthalic acid is added. The mixture is ultrasonically treated for 30 minutes, and then 3 mL DMF solution containing 0.281 mmol FeCl3·6H2O and 32 μL triethylamine is added to the mixture to obtain a new mixture. The new mixture is transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 100°C for 12 hours. After cooling to room temperature (20°C), the obtained orange solid is recovered by centrifugation, washed with DMF twice, then washed with anhydrous methanol twice, and finally the powder is vacuum dried at 80°C overnight to obtain Zr-MOF@Fe-MOF material.

[0061] 3) Load Zr-MOF@Fe-MOF material: inject the Zr-MOF@Fe-MOF material obtained in step 2) into the vortex reaction enrichment pool 3 through the second sample inlet 9 after being prepared into a 1 mg / mL suspension with pure water, and the control group is not loaded with Zr-MOF@Fe-MOF material;

[0062] 4) Install sample storage tank: connect the first sample outlet 6 and the second sample outlet 7 of the sample storage tank 1 with the first sample inlet 8 and the second sample inlet 9, respectively;

[0063] 5) Add sample: add the 10 mL exosome sample obtained in step 1) into the sample storage tank 1 through the total sample inlet 5;

[0064] 6) Connect the liquid circuit: connect the total sample outlet 18 with the inlet of the external suction device, and connect the outlet of the suction device with the total sample inlet 5;

[0065] 7) Exosome enrichment: Set the flow rate of the external suction device to 4 mL / min, start the suction device, and under the action of the external suction device (peristaltic pump), the exosome sample in the sample storage tank 1 enters the chip through the first sample inlet 8 and the second sample inlet 9; the exosome sample in the second sample inlet 9 enters the vortex reaction enrichment pool 3 through the second sample input channel 13, at the same time, the exosome sample in the first sample inlet 8 enters the annular channel 11 through the first sample input channel 10, and then enters the vortex reaction enrichment pool 3 through the symmetric radial channel 12; at this time, the exosome sample forms an asymmetric impinging stream in the vortex reaction enrichment pool 3, and is vortexed with the Zr-MOF@Fe-MOF material loaded in the vortex reaction enrichment pool 3, which is specifically affinity with the exosome phospholipid bilayer through metal-phosphate coordination, to achieve the capture of exosomes; after the sample flows through the filter membrane 15 arranged at the bottom of the vortex reaction enrichment pool 3, it enters the sample filtration pool 16 and reaches the total sample outlet 18 through the sample output channel 17, which can be reconnected to the sample storage tank 1, to realize the cyclic enrichment of exosomes.

[0066] 8) Waste liquid discharge: After the end of the cyclic enrichment at different times according to the foregoing, connect the total sample outlet 18 and the external waste liquid bottle, and under the action of the external suction device, discharge the waste liquid in the chip.

[0067] 9) Observe the enrichment abundance of exosomes in the control group and each experimental group under a fluorescence microscope. For example Figure 7 , the red fluorescence of the control group is basically not observed, and the fluorescence intensity of the exosomes in the experimental group increases with the increase of the cyclic enrichment time, and the fluorescence intensity of the exosomes is basically unchanged after 20 min.

[0068] Example 2: Comparison test of exosome enrichment with different materials

[0069] In order to illustrate that the Zr-MOF@Fe-MOF material of the present application has more excellent performance in enriching exosomes, the exosome enrichment experiments are carried out by using Zr-MOF material, Fe-MOF material and Zr-MOF@Fe-MOF material.

[0070] 1) Preparation of Dil-stained exosomes: the method is the same as that in Example 1

[0071] 2) Synthesis of materials:

[0072] ① Synthesis of Zr-MOF material: 2.0 mmol of ZrCl4 and 3.0 mmol of terephthalic acid are dispersed and dissolved in a mixture of 30 mL of DMF and 5 mL of glacial acetic acid. Then the mixture is refluxed at 100°C for 24 hours. After the reaction is completed and cooled to room temperature, it is washed by centrifugation with DMF, anhydrous ethanol in turn, and then the white precipitate is collected, and then dried at 80°C under vacuum for 10 hours to obtain the white product Zr-MOF.

[0073] ii. Fe-MOF material synthesis: 1.6 mmol of terephthalic acid was dissolved in 6 mL of DMF, and then 180 μL of triethylamine was added; then, 2 mL of DMF solution containing 1.6 mmol of FeCl3·6H2O was slowly added, and the vial was subjected to simple ultrasonic treatment. Heating was performed at 100 °C for 12 hours. After cooling to room temperature, the obtained orange solid was recovered by centrifugation, and was washed with DMF and anhydrous methanol in sequence for 2 times, and the powder was finally dried at 80 °C under vacuum for 10 hours to obtain an orange product Fe-MOF.

[0074] iii. Zr-MOF@Fe-MOF material synthesis: same as Example 1.

[0075] 3) Exosome enrichment: 1 mg of each of the Zr-MOF, Fe-MOF and Zr-MOF@Fe-MOF materials prepared in step 2) of the example was added into a suspension of 1 mL of an exosome sample (prepared in the same manner as in Example 1), and incubation was performed at room temperature for 1 hour.

[0076] 4) Exosome separation: after the incubation at room temperature was completed, centrifugation was performed at 12000 g for 5 minutes to obtain a MOF precipitate containing exosomes.

[0077] 5) Observation of the exosome enrichment abundance of the control group and each experimental group under a fluorescence microscope. As shown in FIG. 2, the exosomes enriched by the Zr-MOF@Fe-MOF had the strongest fluorescence intensity, indicating that the exosome enrichment effect of the Zr-MOF@Fe-MOF was better than that of the Zr-MOF or the Fe-MOF. Figure 8

[0078] Example 3: Exosome enrichment and release of Zr-MOF@Fe-MOF material

[0079] In order to illustrate the enrichment and release effects of the exosome circulation enrichment chip of the application, an exosome enrichment and release experiment was performed using a urine sample, and the Zr-MOF@Fe-MOF material was prepared in the same manner as in Example 1.

[0080] 1. Urine sample pretreatment: the urine was centrifuged at 2000 g for 10 minutes to remove urine sediments, and the supernatant was filtered through a 0.22 μm filter to remove large vesicular structures.

[0081] 2. Exosome circulation enrichment based on the aforementioned high-throughput exosome circulation enrichment chip of Zr-MOF@Fe-MOF material: 10 mL of the pretreated urine was added and circulated for 20 minutes, as described in Example 1.

[0082] 3. Waste liquid removal: as described in Example 1.

[0083] ​4, enrichment experiment group characterization: disassemble the chip and take off the filter membrane, dry and then take SEM photos, the results are shown in Figure 9 Exosomes are adsorbed on the surface of Zr-MOF@Fe-MOF material.

[0084] 5, release experiment group characterization: take off the sample storage box, add 200 μL PBS buffer to the vortex reaction enrichment pool through the sample inlet, stand for 5 minutes, and then suck out the eluent through the sample outlet to obtain exosomes. Take TEM photos of the obtained exosomes, the results are shown in Figure 10 Exosomes after release present tea tray shape and complete morphology.

[0085] Example 4: Comparison test of different methods of enriching urine exosomes based on Zr-MOF@Fe-MOF material

[0086] In order to illustrate the enrichment effect of the exosome circulation enrichment chip of the application, three groups of comparison experiments were carried out using the same urine sample in this embodiment, including Zr-MOF@Fe-MOF material shock incubation enrichment of exosomes, one-off enrichment of exosomes based on Zr-MOF@Fe-MOF material chip, and the aforementioned high-throughput exosome circulation enrichment chip based on Zr-MOF@Fe-MOF material for circulation enrichment of urine exosomes, and the preparation method of Zr-MOF@Fe-MOF material is the same as that of Example 1.

[0087] 1, urine sample pretreatment: same as Example 3.

[0088] 2, Zr-MOF@Fe-MOF material shock incubation enrichment of exosomes: take 10 mL of pretreated urine in a centrifugal tube, add Zr-MOF@Fe-MOF material and mix well by blowing, and then place it on a shaking bed for shock incubation for half an hour. After incubation, centrifuge at 12000g for 5 minutes, discard the supernatant, add 200 μL PBS buffer to the precipitate and mix well by blowing, stand for 5 minutes, release the exosomes on the material, centrifuge at 12000g for 5 minutes, and collect the supernatant containing exosomes.

[0089] 3, one-off enrichment of exosomes based on Zr-MOF@Fe-MOF material chip: load Zr-MOF@Fe-MOF material, install sample storage box, add 10 mL of pretreated urine, directly connect the sample outlet with the waste liquid bottle, and the urine sample only flows through the vortex reaction enrichment pool once. After all the urine samples are enriched, take off the sample storage box, add 200 μL PBS buffer to the vortex reaction enrichment pool through the sample inlet, stand for 5 minutes, and then suck out the eluent through the sample outlet to obtain exosomes.

[0090] 4. Circulating enrichment of exosomes based on the aforementioned high-flux exosome circulating enrichment chip of Zr-MOF@Fe-MOF material: After 20 minutes of circulating enrichment after adding 10 mL of pretreated urine, the waste liquid was discharged, the sample storage box was removed, 200 μL of PBS buffer was added to the vortex reaction enrichment pool through the sample inlet, and the eluent was sucked out through the sample outlet after standing for 5 minutes, and the exosomes were obtained.

[0091] 5. Western blotting was performed on the exosome samples obtained in the above three groups of experiments to detect the exosome negative marker (calnexin), positive markers (CD9, TSG101 and Alix), and compare the exosome enrichment efficiency. The results are shown in Figure 11 Based on the Zr-MOF@Fe-MOF material, the chip can obtain high-purity and high-abundance urine exosomes.

Claims

1. An exosome enrichment chip, characterized in that, The chip includes: a sample storage box (1), a sample inlet channel, a vortex reaction enrichment cell (3), and a sample circulation channel (4); the top of the sample storage box (1) is provided with a main inlet (5), the sample storage box (1) is connected to the sample inlet channel, and the sample inlet channel is connected in sequence to the vortex reaction enrichment cell (3) and the sample circulation channel (4); one end of the sample circulation channel (4) is provided with a main outlet (18); the sample inlet channel is a double-sided asymmetric sample inlet channel (2), and the bottom of the sample storage box (1) is provided with a first outlet (6) and a second outlet (7); the first outlet (6) and the second outlet (7) are injected into the vortex reaction enrichment cell (3) through the double-sided asymmetric sample inlet channel (2); the double-sided asymmetric sample inlet channel (2) is provided with a first inlet (8) and a second inlet. (9) First sample input channel (10), annular channel (11), symmetrical radial channel (12), and second sample input channel (13); the first sample outlet (6) is connected to the annular channel (11) and the symmetrical radial channel (12) through the first sample inlet (8) and the first sample input channel (10), and the second sample outlet (7) is connected to the second sample input channel (13) through the second sample inlet (9); the symmetrical radial channel (12) and the second sample input channel (13) are respectively connected to the vortex reaction enrichment cell (3); the sample circulation channel (4) includes a sample filter cell (16) and a sample output channel (17); the vortex reaction enrichment cell (3) is connected to the total sample outlet (18) through the sample filter cell (16) and the sample output channel (17).

2. The exosome enrichment chip according to claim 1, characterized in that, The annular channel (11) is connected to the vortex reaction enrichment tank (3) by at least two symmetrical radial channels (12).

3. The exosome enrichment chip according to claim 2, characterized in that, The diameter of the symmetrical radial channel (12) and the diameter of the second sample input channel (13) are both less than 0.5 mm, which can form an asymmetric impact flow in the vortex reaction enrichment cell (3).

4. The exosome enrichment chip according to any one of claims 1-3, characterized in that, The bottom of the vortex reaction enrichment cell (3) is provided with a circular notch (14) for placing a filter membrane (15), and the filter membrane (15) is loaded with exosome-specific enrichment material.

5. The exosome enrichment chip according to claim 4, characterized in that, The specific enrichment material is any one of microspheres, silica spheres, or metal-organic framework materials containing exosome capture probes, and the size of the specific enrichment material is larger than the pore size of the filter membrane (15); the exosome capture probe is one or more of the following combinations: exosome-specific recognition antibody, exosome-specific recognition aptamer, exosome-specific recognition metal ion, arginine, lysine, polyhydroxy compounds, and exosome-specific recognition membrane protein.

6. The exosome enrichment chip according to claim 5, characterized in that, The metal-organic framework material is a MOF-on-MOF material, which includes a core Zr-MOF and an Fe-MOF grown on the outside of the Zr-MOF.

7. The exosome enrichment chip according to claim 6, characterized in that, The method for synthesizing the MOF-on-MOF material includes the following steps: ZrCl4 and terephthalic acid were dispersed in a mixture of DMF and glacial acetic acid and refluxed. After the reflux reaction was completed, the mixture was cooled to 20-25°C and then washed with DMF and anhydrous ethanol by centrifugation to collect the white precipitate. The precipitate was then dried under vacuum for 8-12 hours to obtain the white product Zr-MOF. The Zr-MOF was dispersed in DMF and then terephthalic acid powder was added and mixed evenly to obtain a mixture. After sonicating the mixture for 20-30 minutes, a mixed DMF solution containing FeCl3·6H2O and triethylamine was added to obtain a new mixture; the volume percentage concentration of triethylamine in the mixed DMF solution was 0.8-1.2%. The new mixture was transferred to an autoclave and reacted at 100-150°C for 10-15 hours; then cooled to 20-25°C, and the resulting orange solid was recovered by centrifugation. The solid was then washed with DMF and anhydrous methanol in sequence to obtain a powder. The powder was then vacuum dried at 80-150°C for 8-12 hours to obtain the MOF-on-MOF material.

8. The exosome enrichment chip according to claim 7, characterized in that, In the method for synthesizing MOF-on-MOF materials, ZrCl4 and terephthalic acid were dispersed and dissolved in a mixture of DMF and glacial acetic acid with a volume ratio of 1-10:1 at a molar ratio of 1:1-1.5, and refluxed at 100-150℃ for 20-30 hours. The final concentration of Zr-MOF in the mixture is 4-8 mg / mL, and the final concentration of terephthalic acid is 0.08-0.12 mmol / mL. The mixed DMF solution containing FeCl3·6H2O and triethylamine is of equal volume to the mixture, such that the molar ratio of FeCl3·6H2O to terephthalic acid is 1:1, thus obtaining a new mixture.

9. A method for enriching exosomes, characterized in that, Includes the following steps: 1) Loading exosome-specific enrichment material: After resuspending the exosome-specific enrichment material in buffer solution, inject it into the vortex reaction enrichment cell (3) through the second injection port (9); 2) Assemble the exosome enrichment chip as described in any one of claims 1-8; 3) Sample pretreatment: The sample was centrifuged at 2000g to remove cells and cell debris, and then filtered with a 0.22μm filter membrane to remove large vesicles to obtain the pretreated sample; 4) Add sample: Add the sample obtained in step 3) into the sample storage box (1) through the main injection port (5); 5) Liquid circuit connection: Connect the main sample outlet (18) to the inlet of the external suction device, and connect the outlet of the suction device to the external waste liquid bottle; 6) Exosome enrichment: Start the suction device. Under the action of the suction device, the sample enters the vortex reaction enrichment cell (3) through the double asymmetric injection channel (2). The exosomes in the sample combine with the exosome-specific enrichment material in the vortex reaction enrichment cell (3) to complete the exosome enrichment.

10. The method for enriching exosomes according to claim 9, characterized in that, In step 5), the outlet of the suction device is connected to the main inlet (5) to form a circulation path; after step 6) is completed, the sample flows through the filter membrane (15) set at the bottom of the vortex reaction enrichment cell (3) and enters the sample filtration cell (16), and reaches the main outlet (18) through the sample output channel (17), and flows back to the sample storage box (1). The enrichment time is 10-30 minutes, and the high-throughput circulation enrichment of exosomes is completed.

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