Centrifugal micro-fluidic chip and method for automatically and rapidly enriching and detecting exosome

Abstract

The invention relates to the technical field of exosome enrichment, in particular to a centrifugal micro-fluidic chip and method for automatically and rapidly enriching and detecting exosomes. Comprising a sample chamber, a sample quantification chamber, a waste sample chamber, a reagent chamber, an efficient mixing chamber, a high-abundance protein removal chamber, an exosome sorting chamber, a carrier generation chamber, an exosome quantification storage chamber, an exosome total storage chamber, a gas generation chamber and a reagent storage chamber, the liquid conveying channel and the gas conveying channel are used for connecting the chambers and controlling the fluid to flow directionally; the rotating speed, direction and time of the chip are controlled through programming of a centrifugal machine, automatic enrichment of exosomes is achieved, and the exosome sorting cavity is configured to capture the exosomes through a bubble carrier and E-Am functional molecules. The exosome sorting device does not depend on a complex liquid pumping system and a mechanical arm, exosome sorting is achieved by programming the centrifugal sample feeding machine, the exosome is not damaged, the sorting speed is high, and the cost is low.

Description

Technical Field

[0001] This invention relates to the field of exosome enrichment technology, specifically to a centrifugal microfluidic chip and method for automated rapid enrichment and detection of exosomes. Background Technology

[0002] Exosomes are extracellular vesicles carrying blastocyst-related proteins and nucleic acids. They enable early detection of tumors in their early stages, providing a diagnostic window earlier than imaging studies and offering new hope for early cancer diagnosis and reduced cancer mortality. However, exosomes are nanoscale vesicles. Due to their small size, gravity, inertia, lift, dielectric force, and acoustic forces have weak effects on them, making it difficult to overcome the resistance of fluid drag and achieve selective enrichment. Efficiently enriching exosomes remains a pressing challenge.

[0003] The commonly used method for extracting exosomes in the laboratory is ultracentrifugation. However, the purity of exosomes obtained by ultracentrifugation is low, the recovery rate is low, and the strong centrifugal force can damage the integrity of some exosomes.

[0004] Exosome separation techniques based on microfluidics and size exclusion methods also suffer from problems such as prolonged separation time, low recovery rate, reduced purity, and high cost. Microfluidic chips based on electric and acoustic fields can damage the integrity of exosomes, and traditional magnetic bead carriers affect downstream analysis. In addition, due to platelet activation and cell lysis, the level of exosomes in plasma or serum changes significantly with the delay in blood processing.

[0005] Therefore, there is an urgent need to develop rapid and effective exosome isolation technologies, especially in remote areas and primary healthcare, so that exosome isolation and detection can be achieved with low cost and portable devices. This is of great significance for realizing the clinical value of exosomes. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a centrifugal microfluidic chip and method for automated and rapid enrichment and detection of exosomes. The entire exosome sorting process can be achieved through a programmed centrifugal sample injector. This sorting method is non-destructive to exosomes, fast in sorting speed, and low in cost. It solves the problem of exosome integrity damage caused by microfluidic chips based on electric and acoustic fields. After exosome enrichment is completed, the bubble carrier ruptures and disappears, avoiding the problem of traditional magnetic bead carriers affecting downstream analysis.

[0007] This invention is achieved through the following technical solution: A centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes is provided, comprising: The substrate has multiple functional chambers, including a sample chamber, a sample quantification chamber, a waste sample chamber, a reagent chamber, a high-efficiency mixing chamber, a high-abundance protein removal chamber, an exosome sorting chamber, a carrier generation chamber, an exosome quantitative storage chamber, a total exosome storage chamber, a gas generation chamber, and a reagent storage chamber. Liquid delivery channels and gas delivery channels are used to connect the various chambers and control the directional flow of fluids; The chip uses centrifuge programming to control the speed, direction and time to achieve automatic enrichment of exosomes, and the exosome sorting chamber is configured to capture exosomes using bubble carriers and E-Am functional molecules.

[0008] Furthermore, the gas generating chamber contains sodium bicarbonate powder, and the reagent storage chamber contains acetic acid with a concentration of 20% to 40%. The reaction between the two is controlled by centrifugation to generate bubbles.

[0009] Preferably, the chip includes 2n sets of parallel processing systems symmetrically distributed around the chip center, capable of processing 2n samples simultaneously, where n is a non-zero natural number.

[0010] Furthermore, multiple chips are vertically stacked to form a multi-channel chip.

[0011] As a preferred option, the high-efficiency mixing chamber is a Z-shaped channel.

[0012] Furthermore, it also includes a separate coagulation chamber (13) for receiving whole blood samples and separating serum by centrifugation. After obtaining the serum, it can be transferred to the sample chamber (1) using a pipette.

[0013] Furthermore, the high-efficiency mixing chamber and the high-abundance protein removal chamber are prepared using a high-abundance protein removal reagent (HAPR).

[0014] A method for enriching exosomes using a centrifugal microfluidic chip includes the following steps: S1. Add serum sample to sample chamber, add high-abundance protein reagent to reagent chamber, add acetic acid to reagent storage chamber, add sodium bicarbonate powder to gas generation chamber, and add sorting buffer to exosome sorting chamber. S2. By controlling the speed and direction of the centrifuge through programming, sample quantification, mixing, protein removal, exosome capture and sorting are completed, and the exosomes are transported to the exosome quantitative storage chamber and the exosome total storage chamber. S3. The entire process is completed within 30 minutes.

[0015] Furthermore, in step S2, the steps of sequentially controlling the centrifuge via programming include: a) Run counterclockwise at 1000 rpm for 1 minute to quantitatively transfer the sample from the sample chamber to the sample quantification chamber and the waste sample chamber, thus completing the sample quantification; b) Run clockwise at 2000 rpm for 3 minutes to transfer the sample from the sample quantification chamber and the reagent from the reagent chamber to the high-efficiency mixing chamber and then into the high-abundance protein removal chamber. Let stand for 1 minute. c) Run counterclockwise at 2500 rpm for 3 minutes to complete the precipitation of high-abundance proteins, then increase the speed to 3000 rpm for 1 minute and transfer the supernatant to the sorting chamber; d) Stop rotating and run in an alternating clockwise and counterclockwise oscillation mode for 10 minutes to allow the exosomes to bind to the E-Am functional molecules in the exosome sorting chamber; e) Run counterclockwise at 4000 rpm for 1 minute to deliver acetic acid solution to the gas generating chamber. Reduce the speed to 2000 rpm and run for 10 minutes to complete exosome sorting and deliver to the exosome quantitative storage chamber and the exosome total storage chamber.

[0016] Furthermore, when the sample is whole blood, in the sample addition step, the whole blood sample is added to the coagulation chamber, centrifuged at 500 rpm for 20 min, and then run at 5000 rpm for 10 min to separate the serum and transfer it to the sample chamber.

[0017] The beneficial effects of this invention are: This invention benefits from the fully automated parallel processing capability of the centrifugal microfluidic chip. It completes the steps of quantification, mixing, separation, and enrichment in one go through programmed centrifugation, eliminating the tedious manual transfer and waiting time in traditional methods. This invention can complete the entire process from sample to exosome enrichment within 30 minutes, which is highly efficient and fast, and greatly shortens the detection time.

[0018] This invention employs a specific capture scheme for E-Am functional molecules using bubble carriers. The bubbles rupture and disappear after transport, avoiding the interference that traditional magnetic bead carriers may cause to downstream analyses (such as PCR and sequencing). This further ensures the accuracy and high sensitivity of the analysis, with significant enrichment effect and avoidance of exosome loss. The exosome recovery rate is not less than 80%, and the enrichment ratio is about 80%.

[0019] The exosomes enriched by this invention have good integrity and are suitable for various downstream fine analyses. The driving method combines centrifugal force with gentle oscillation, and the gentle transport by bubble carrier avoids the potential damage to the exosome membrane structure caused by strong physical external fields (such as electric fields and sound fields).

[0020] This invention utilizes centrifugal force as the sole driving force and achieves automatic and sequential control of fluids through the ingenious design of the chip structure. The entire process can be completed with only one programmable centrifuge, requiring no complex operations. It is simple to operate, highly automated, and reduces the requirements for personnel and environment. The chip can integrate two or more parallel processing systems, increasing the throughput of a single test and meeting the needs of clinical batch sample testing.

[0021] The chip substrate of this invention uses conventional materials such as acrylic and is processed through CNC and hot-pressing techniques, resulting in low cost. High-abundance protein removal reagents can also be commercially available products, leading to a significant overall cost advantage and facilitating widespread adoption in remote areas and primary care hospitals. Its low cost makes it easy to promote in primary healthcare settings. This invention offers flexible applications, enabling direct processing of whole blood samples. The chip design includes a coagulation chamber, allowing for direct processing of whole blood samples and integrating serum separation and exosome enrichment. By integrating preprocessing functions on the chip, its application scenarios are expanded, making point-of-care testing possible.

[0022] This invention, through the integrated and automated design of centrifugal microfluidic chips and combined with innovative bubble carrier capture technology, successfully achieves rapid, efficient, non-destructive, and low-cost enrichment of exosomes, effectively solving several bottleneck problems in existing technologies. It has significant application value and market prospects in the fields of early diagnosis and basic research of exosome-related tumors. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the basic structure of the chip of the present invention.

[0024] Figure 2 This is a schematic diagram of the high-throughput expansion structure of the chip of the present invention.

[0025] Figure 3 This is an experimental curve showing the relationship between acetic acid concentration and gas generation in this invention.

[0026] Figure 4 This is a comparison chart of NTA test results for exosomes before and after enrichment in the chip of this invention.

[0027] Figure 5 This is the E-Am mass spectrum synthesized in Example 2 of the present invention.

[0028] As shown in the figure: 1-Sample chamber, 2-Sample quantification chamber, 3-Waste sample chamber, 4-Reagent chamber, 5-High-efficiency mixing chamber, 6-High-abundance protein removal chamber, 7-Exosome sorting chamber, 8-Carrier generation chamber, 9-Exosome quantitative storage chamber, 10-Total exosome storage chamber, 11-Gas generation chamber, 12-Reagent storage chamber, 13-Coagulation chamber, 14-Liquid delivery channel, 15-Gas delivery channel. Detailed Implementation

[0029] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0030] Example 1: A centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes, comprising: The substrate has multiple functional chambers, including sample chamber 1, sample quantification chamber 2, waste sample chamber 3, reagent chamber 4, high-efficiency mixing chamber 5, high-abundance protein removal chamber 6, exosome sorting chamber 7, carrier generation chamber 8, exosome quantitative storage chamber 9, total exosome storage chamber 10, gas generation chamber 11, and reagent storage chamber 12.

[0031] Liquid delivery channel 14 and gas delivery channel 15 are used to connect the chambers and control the directional flow of fluid.

[0032] The chip controls the speed, direction and time of the centrifuge through programming to achieve automatic enrichment of exosomes, and the exosome sorting chamber 7 is configured to capture exosomes using bubble carriers and E-Am functional molecules.

[0033] The chip substrate is preferably made of acrylic, and the chambers and microchannels are formed by CNC milling. (See attached image) Figure 1 As shown, the cavity of this chip adopts a radial layout with the chip's center as the core. Based on function and processing order, it can be divided into the following regions: Sample loading and initial processing area (inner core area): This area is located near the center of the chip and mainly includes sample chamber 1, sample quantification chamber 2, and waste sample chamber 3. Placing the sample loading inside allows the liquid to flow naturally from the inside to the outside under centrifugal force, optimizing the efficiency of the initial quantification and dispensing process and facilitating integration with whole blood pretreatment functions.

[0034] Reaction and mixing zone (intermediate annular zone): Located outside the sample loading zone, this zone includes reagent chamber 4, high-efficiency mixing chamber 5, and high-abundance protein removal chamber 6. Samples and reagents converge, mix, and react in this zone. High-efficiency mixing chamber 5 employs a Z-shaped channel design to enhance mixing efficiency.

[0035] The sorting and final storage area (outer annular region): Located on the outermost layer of the chip, it includes an exosome sorting chamber 7, a carrier generation chamber 8, an exosome quantitative storage chamber 9, and a total exosome storage chamber 10. Simultaneously, a gas generation chamber 11 and a reagent storage chamber 12 are also located in this area, facilitating connection to the exosome sorting chamber 7 via a short channel for rapid bubble generation and delivery. The exosome sorting chamber 7 is the core of this invention, pre-filled with E-Am functional molecules. The E-Am functional molecules in this invention are self-synthesized, and their molecular formula is as follows: R1-R2-R3-COOH; where, R1 is a C15 straight-chain alkyl group; R2 is ; R3 is the following peptide chain: -Cys-Arg-His-Ser-Gln-Met-Thr-Val-Thr-Ser-Arg-Leu-, Wherein: Cys is cysteine, Arg is arginine, His is histidine, Ser is serine, Gln is glutamine, Met is methionine, Thr is threonine, Val is valine, and Leu is leucine.

[0036] R3 is CP05, which is existing technology and can be obtained from the article Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnosis and therapy by Xianjun Gao and Ning Ran et al.

[0037] The principle behind the enrichment of exosomes by E-Am functional molecules used in this invention is the perfect synergy between specific chemical recognition and physical buoyancy transport. At the chemical level: E-Am molecules specifically recognize exosome surface antigens through their peptide chain R3.

[0038] At the physical level: Bubbles use buoyancy to transport bound exosomes rapidly and gently to the collection area, and purify them through differences in foam stability.

[0039] The various areas are interconnected through liquid transport channels 14 and gas transport channels 15, forming a unidirectional, automated processing path from the inside out.

[0040] In addition, the chip may optionally include a coagulation chamber 13 for direct processing of whole blood samples. After chip fabrication, a polypropylene film is bonded to the open surface of the chip using a thermoforming method for encapsulation. Sample application ports, vents, etc., are selectively sealed with sealing films as needed.

[0041] Example 2: like Figure 2 As shown in diagram a, in this embodiment, the chip has four parallel processing systems symmetrically distributed around its center, capable of processing four samples simultaneously. Each system includes... Figure 1 The diagram shows all the functional chambers (sample chamber, quantitative chamber, mixing chamber, sorting chamber, etc.) and the connected liquid delivery channel 14 and gas delivery channel 15. In a... Figure 2The chip designed can process four samples simultaneously. Each sample completes the entire process from quantification to exosome enrichment in its own independent channel without interfering with each other.

[0042] Furthermore, Figure 2 The chip shown in Figure a is formed by multiple vertically stacked layers, as shown in Figure a. Figure 2 The multi-channel chip shown in b. By vertically stacking integrated layers 1, 2, ... and n, the chip's processing capabilities can be further expanded to achieve multi-channel, multi-sample processing.

[0043] Example 3: The method for synthesizing E-Am functional molecules in this invention is as follows: (1) Resin swelling Weigh 0.8 g of 2-chlorotriphenylmethyl chloride resin (Aladdin) with a degree of substitution of 0.3 mmol / g, put the resin into a reaction tube, add 10 mL of dichloromethane with a concentration of 15 mL / g, and shake for 30 min.

[0044] (2) Connect the first amino acid The solvent was removed by filter-through of a sand filter. Three molar amounts of excess leucine (Leu) contained in 9-fluorenylmethoxycarbonyl (Fmoc) were added to the resin, followed by ten molar amounts of excess N,N-diisopropylethylamine (DIEA). Finally, 3 mL of N,N-dimethylformamide (DMF) was added to dissolve the solvent, and the mixture was shaken for 1 hour. The solution was then washed six times alternately with N,N-dimethylformamide and dichloromethane.

[0045] (3) Deprotection Add 15 ml of 20% piperidine DMF solution, react for 5 min, discard the supernatant, add another 15 ml of 20% piperidine DMF solution, and react for 15 min.

[0046] (4) Detection Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add 3 drops of Kaiser Test detection solution (Solution A: 20% anhydrous ethanol + 80% phenol, Solution B: pyridine, Solution C: 5g ninhydrin + 100ml anhydrous ethanol, add one drop of each solution during testing, there is no order), heat at 105-110℃ for 5 minutes, a deep blue color indicates a positive reaction.

[0047] (5) Wash Wash twice with DMF solution (10 ml / g), twice with methanol solution (10 ml / g), and twice with DMF solution (10 ml / g).

[0048] (6) Condensation Fmoc-protected arginine in excess (3 times) and O-benzotriazole-tetramethylurea hexafluorophosphate in excess (3 times) were dissolved in DMF, added to a reaction tube, and immediately 10 times the amount of DIEA was added. The reaction was carried out for 40 minutes.

[0049] (7) Wash Wash once with DMF solution (10 ml / g), twice with methanol solution (10 ml / g), and twice with DMF solution (10 ml / g).

[0050] (8) Repeat steps (2) to (7) to connect the amino acids in the E-Am molecular peptide chain sequence from right to left.

[0051] (9) Detection Wash twice with DMF solution (10 ml / g), twice with DCM solution (10 ml / g), and twice with DMF solution (10 ml / g). Dry under vacuum for 10 min. Ninhydrin test negative.

[0052] (10) Wash Wash three times with methanol solution (10 ml / g).

[0053] (11) Cutting peptides from resin Prepare 10g of cutting fluid: 94% trifluoroacetic anhydride; 2.5% water; 2.5% ethylenediaminetetraacetic acid; 1% triisopropylsilane.

[0054] The prepared resin was placed in a flask, and cutting fluid was added so that the resin ratio in the cutting fluid was 10 ml / g. The mixture was shaken at 40°C for 120 min to obtain the pyrolysis solution.

[0055] (12) Drying and washing The lysis buffer was dried with nitrogen gas for at least 1 hour, then extracted with 10 ml of diethyl ether. After washing with diethyl ether six times, the buffer was allowed to stand at room temperature for at least 2 hours to obtain the crude peptide sequence.

[0056] (13) Purify polypeptides by HPLC Detailed operation steps: Take 200 mg of crude peptide and place it in a container. Add 5 ml of 50% acetonitrile aqueous solution and sonicate for 2 min.

[0057] Filtered using a 0.45μm filter membrane.

[0058] Analysis: Take 3 μl of the crude product and analyze it using analytical grade HPLC. The mobile phase was water and acetonitrile, the time was 30 min, and gradient elution was used. The HPLC was first equilibrated with the initial gradient for 5 min before injection. The initial gradient was 95% water and 5% acetonitrile, and the final gradient was 5% water and 95% acetonitrile.

[0059] Preparation: Prepare the dissolved sample for injection. Equilibrate with preparative HPLC for 10 min, starting with a gradient of 95% water and 5% acetonitrile, and ending with a gradient of 25% water and 75% acetonitrile over a gradient time of 40 min. Collect the sample exiting the detector.

[0060] Identification: The collected fraction was freeze-dried to obtain a pure white powder. Samples were then analyzed by mass spectrometry (MS), and the results are as follows: Figure 5 As shown, the molecular weight is 1890.6 Da, consistent with the target molecular weight, and the purity, as determined by HPLC, is 98.6%. The resulting E-Am molecular powder was sealed and stored at -20°C for later use. The E-Am molecule synthesized in this embodiment has a well-defined structure and high purity, and can be used in the exosome enrichment process described in this invention.

[0061] Example 4: Taking serum samples as an example, a method for exosome enrichment using a centrifugal microfluidic chip includes the following steps: Serum is added to sample chamber 1, high-abundance protein removal reagent (HAPR) is added to chamber 4, acetic acid (preferably 30%) is added to reagent storage chamber 12, sodium bicarbonate powder is added to gas generation chamber 11, and sorting buffer is added to chamber 7. The high-abundance protein removal reagent is a commercially available kit; HAPR can be purchased from Dolemy Biotechnology (Wuhan) Co., Ltd.

[0062] Sample quantification: Centrifuge at 1000 rpm counterclockwise for 1 min. The sample flows from sample chamber 1 into quantification chamber 2 through channel 14. Excess sample enters waste sample chamber 3.

[0063] Mixing and protein removal: Centrifuge at 2000 rpm clockwise for 3 min. The sample from chamber 2 and the reagent from chamber 4 are transferred to the high-efficiency mixing chamber 5 for mixing, and then enter the high-abundance protein removal chamber 6.

[0064] Protein precipitation and supernatant transfer: Centrifuge at 2500 rpm counterclockwise for 3 min to allow the protein to precipitate fully; then increase the speed to 3000 rpm and centrifuge for 1 min, and transfer the supernatant to sorting chamber 7.

[0065] Exosome capture: Stop the rotation and start the clockwise / counterclockwise alternating oscillation mode (e.g., 500-1000 rpm) for 10 minutes to allow the exosomes to fully and gently bind to the E-Am molecules in chamber 7.

[0066] Bubble transport and sorting: Centrifuge counterclockwise at 4000 rpm for 1 min to rapidly transport acetic acid from reagent storage chamber 12 to gas generation chamber 11, where it reacts with sodium bicarbonate to generate a large number of bubbles; then reduce the speed to 2000 rpm and centrifuge for 10 min. The bubbles act as carriers, transporting E-Am molecules bound to exosomes to quantitative exosome storage chamber 9 and total exosome storage chamber 10. The bubbles burst and disappear during transport or upon reaching the storage chambers, avoiding interference with downstream analysis.

[0067] If whole blood samples are used, pretreatment is required: add whole blood to coagulation chamber 13, centrifuge at 500 rpm for 20 min, then run at 5000 rpm for 10 min to separate the serum and transfer it to sample chamber 1. Subsequent steps are the same as for serum processing.

[0068] To optimize bubble generation conditions, the optimal acetic acid concentration was determined experimentally. With a fixed mass of sodium bicarbonate (850 mg), the acetic acid concentration was varied, and the results are as follows: Figure 3 As shown, the amount of gas generated peaks at an acetic acid concentration of 30%. Therefore, an acetic acid concentration range of 20% to 40% is preferred.

[0069] The exosome enrichment effect after processing with the chip of this invention was verified by NTA, and the results are as follows: Figure 4 As shown, the concentration of exosomes increased significantly after enrichment, and the particle size distribution was concentrated in the typical exosome size range (about 100 nm), indicating that the enrichment effect was good and did not cause exosome aggregation or damage.

[0070] Calculations show that the enrichment ratio of this method is about 80, the recovery rate is not less than 80%, the entire process can be completed within 30 minutes, which is much faster than ultracentrifugation (2-5 hours), and it can well maintain the integrity of exosomes.

[0071] Of course, the above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments and drawings are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. This invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes, characterized in that: include: The substrate has multiple functional chambers, including a sample chamber (1), a sample quantification chamber (2), a waste sample chamber (3), a reagent chamber (4), a high-efficiency mixing chamber (5), a high-abundance protein removal chamber (6), an exosome sorting chamber (7), a carrier generation chamber (8), an exosome quantitative storage chamber (9), a total exosome storage chamber (10), a gas generation chamber (11), and a reagent storage chamber (12). Liquid delivery channel (14) and gas delivery channel (15) are used to connect the chambers and control the directional flow of fluid; The chip controls the speed, direction and time of the centrifuge through programming to achieve automatic enrichment of exosomes, and the exosome sorting chamber (7) is configured to capture exosomes using bubble carriers and E-Am functional molecules.

2. The centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes according to claim 1, characterized in that: The gas generating chamber (11) contains sodium bicarbonate powder, and the reagent storage chamber (12) contains acetic acid with a concentration of 20%~40%. The reaction between the two is controlled by centrifugation to generate bubbles.

3. The centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes according to claim 1, characterized in that: The chip includes 2n sets of parallel processing systems symmetrically distributed around the chip's center, capable of processing 2n samples simultaneously, where n is a non-zero natural number.

4. The centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes according to claim 3, characterized in that: Multiple chips are stacked vertically to form a multi-channel chip.

5. The centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes according to claim 1, characterized in that: The high-efficiency mixing chamber (5) is a Z-shaped channel.

6. The centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes according to claim 1, characterized in that: It also includes a separate coagulation chamber (13), which is used to receive whole blood samples and separate serum by centrifugation. After obtaining the serum, it can be transferred to the sample chamber (1) using a pipette.

7. The centrifugal microfluidic chip for automated rapid enrichment and detection of exosomes according to claim 1, characterized in that: The high-efficiency mixing chamber (5) and the high-abundance protein removal chamber (6) are prepared using the high-abundance protein removal reagent (HAPR).

8. A method for enriching exosomes using a centrifugal microfluidic chip according to any one of claims 1 to 7, characterized in that: Includes the following steps: S1. Add serum sample to sample chamber (1), add high abundance protein reagent to reagent chamber (4), add acetic acid to reagent storage chamber (12), add sodium bicarbonate powder to gas generation chamber (11), and add sorting buffer to exosome sorting chamber (7). S2. By controlling the speed and direction of the centrifuge through programming, sample quantification, mixing, protein removal, exosome capture and sorting are completed, and the exosomes are transported to the exosome quantitative storage chamber (9) and the exosome total storage chamber (10). S3. The entire process is completed within 30 minutes.

9. The method for enriching exosomes in a centrifugal microfluidic chip according to claim 8, characterized in that: In step S2, the steps of sequential control of the centrifuge via programming include: a) Run counterclockwise at 1000 rpm for 1 minute to quantitatively transfer the sample from the sample chamber (1) to the sample quantification chamber (2) and the waste sample chamber (3) to complete the sample quantification; b) Run clockwise at 2000 rpm for 3 min, transfer the sample from the sample quantification chamber (2) and the reagent from the reagent chamber (4) to the high-efficiency mixing chamber (5) and then into the high-abundance protein removal chamber (6), and let stand for 1 min; c) Run counterclockwise at 2500 rpm for 3 minutes to complete the precipitation of high-abundance proteins, increase the speed to 3000 rpm for 1 minute, and transfer the supernatant to the sorting chamber (7). d) Stop rotating and run in an alternating clockwise and counterclockwise oscillation mode for 10 minutes to allow the exosomes to bind to the E-Am functional molecules in the exosome sorting chamber (7); e) Run counterclockwise at 4000 rpm for 1 min to deliver acetic acid solution to the gas generating chamber (11), reduce the speed to 2000 rpm for 10 min to complete exosome sorting and deliver to the exosome quantitative storage chamber (9) and the exosome total storage chamber (10).

10. The method for enriching exosomes in a centrifugal microfluidic chip according to claim 8, characterized in that: When the sample is whole blood, in the sample addition step, the whole blood sample is added to the coagulation chamber (13), centrifuged at 500 rpm for 20 min, and then run at 5000 rpm for 10 min to separate the serum and transfer it to the sample chamber (1).

Images