Microfluidic biochip, magnetic beads and kit

Abstract

The application discloses a micro-fluidic biochip, magnetic beads and a kit, and relates to the technical field of biomedical engineering. The micro-fluidic biochip comprises a chip body, and the chip body is provided with a separation channel, a lysis area and a detection area. The micro-fluidic biochip integrates multiple functional areas, has an integrated detection function, and can be used for separation, detection and screening of exosomes and their contents.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, in particular to a microfluidic biochip, magnetic beads and a kit. BACKGROUND

[0002] At present, most of the protein chips on the market rely on large detection equipment (the unit price is more than 1 million) and supporting expensive chip-reagent box consumables (the unit price is more than 5000 yuan), and most of them focus on the detection of a limited number of free proteins with high abundance, and there is no small device for the separation-detection integration of exosomes. The detection field of important pathological monitoring indicators (neuronal proteins and protein post-modification components) in exosomes is even blank. The domestic protein chip industry is mainly concentrated in the low-end field, and mainly relies on the introduction of third-party equipment outsourcing services, and there is no mature domestic protein chip on the market. In addition, the detection of neuronal exosome proteins relies on targeted acquisition of single population of neuronal-derived exosomes, and there is no such product produced at home and abroad. It is still a blank to independently research and develop professional chips for exosome proteomics and post-modification.

[0003] In view of this, the present application is proposed. SUMMARY

[0004] The purpose of the present application is to provide a microfluidic biochip, magnetic beads and a kit, which can be used for the separation and detection of exosomes and their contents (such as proteins, mRNA / miRNA, DNA, lipids, etc.).

[0005] The present application is implemented as follows:

[0006] The present application provides a microfluidic biochip for detecting exosomes or their contents (such as proteins, mRNA / miRNA, DNA, lipids, etc.), which comprises a chip body provided with the following functional zones in sequence: a separation channel for exosome separation, a lysis zone communicating with the separation channel, and a detection zone communicating with the lysis zone.

[0007] One end of the separation channel has a sample input port and a magnetic bead input port, and when the magnetic beads enter from the magnetic bead input port, they can specifically bind to the exosomes entering from the sample input port to form an exosome-magnetic bead complex, which is separated and enriched in the lysis zone under the action of an external force;

[0008] The detection zone is fixed with a capture that can specifically bind to the detected molecules; the detected molecules are derived from the exosomes.

[0009] The microfluidic biochip provided by the application can realize the enrichment and lysis of external vesicles by setting a lysis area. The external vesicle-magnetic bead complex can be enriched by the action of an externally applied magnetic field force. After the lysis solution is added to the area, the external vesicles can be lysed to release the contents (such as proteins, mRNA / miRNA, DNA, lipids, etc.). The contents flow into the detection area, and the corresponding molecules to be detected (i.e., the contents released by the lysis of the external vesicles) are captured by the capture. Then, the corresponding reaction reagent is added to generate a signal reflecting the presence or absence of the molecules to be detected or the content level, so that the detection can be realized.

[0010] The person skilled in the art can reasonably select the type of the capture for the molecules to be detected according to the molecules to be detected.

[0011] For example, when the molecules to be detected are proteins, the capture can be an antibody or a ligand for the proteins; when the molecules to be detected are DNA or RNA fragments, the capture can be a nucleic acid probe complementary to the DNA or RNA fragments.

[0012] Alternatively, in some embodiments, the molecules to be detected are selected from proteins, nucleic acids, or combinations thereof.

[0013] Alternatively, in some embodiments, the nucleic acids are DNA, RNA, or combinations thereof.

[0014] Alternatively, in some embodiments, the capture is selected from an antibody, a ligand, a nucleic acid probe complementary to the nucleic acid, or combinations thereof.

[0015] Alternatively, in some embodiments, the magnetic beads are modified with an antibody or a ligand that can specifically bind to the surface membrane protein of the external vesicles.

[0016] The antibody modified by the magnetic beads described above can be selected according to the specificity of the surface membrane protein of the external vesicles. The surface membrane proteins of external vesicles from different tissues have certain heterogeneity. For example, the specific membrane proteins of external vesicles from neurons include L1CAM, LRRK2, and TREM-2. The specific membrane proteins of external vesicles from cancer tissues include PDL1, FASL, or TRAIL.

[0017] It should be noted that the selection of the type of the antibody or the ligand that binds to the surface membrane protein of the external vesicles can be reasonably selected according to the protein object to be bound, which is easy for the person skilled in the art to realize. Regardless of the protein or ligand to be modified, it is within the protection scope of the application.

[0018] It should be noted that the antibody or ligand modified by the magnetic beads described above can also be directed to the membrane protein labeled with the membrane protein, such as CD63; it should be noted that the type of antibody or ligand modified by the magnetic beads described above can be reasonably selected by those skilled in the art according to the detection needs in specific application scenarios or practices.

[0019] The selection of the above ligand is similar to the antibody, as long as it can specifically bind to the membrane protein of the extracellular vesicle of interest, and any ligand belongs to the protection scope of the present application.

[0020] Neurodegenerative diseases are a group of diseases caused by chronic and progressive degeneration of central nervous tissue, which are related to aging. Neurodegenerative diseases generally occur through key processes such as protein misfolding and aggregation, neuroinflammation, or changes in cell signal transduction. Brain neurons release extracellular vesicles into the blood circulation, and neuronal extracellular vesicles directly reflect brain cell secretion activity. It is possible to screen high-value biomarkers directly related to Parkinson's disease from neuronal extracellular vesicles. Ultra-high sensitivity and high-throughput protein chips can analyze the content of multiple proteins by collecting a small amount of blood samples, and screen protein biomarkers related to diseases. Thus, it is possible to determine whether the body has a disease or is in which stage of the disease, and to achieve precise health management of the subject. At present, there is no protein chip system for analyzing extracellular vesicle proteomics to study brain secretion activity in Parkinson's disease from the perspective of neuronal extracellular vesicles and to explore high-precision markers. From the perspective of the performance of the biochip, or from the perspective of pathological diagnosis, it is a new technological progress representing precision medicine. However, so far there is no biochip specifically for extracellular vesicle proteomics analysis on the market. The microfluidic biochip of the embodiments of the present application for screening biomarkers from neuronal extracellular vesicles also has great practical significance and urgency for early prevention and intervention of neurodegenerative diseases such as Parkinson's disease, and effectively fills the technical gap in the field of extracellular vesicle proteomics detection.

[0021] Optionally, in some embodiments, the detection area comprises one or more reaction chambers; the capture agent is fixed in the reaction chamber.

[0022] The provision of multiple reaction chambers can realize the synchronous detection of different molecules to be detected, and improve the detection throughput and efficiency.

[0023] Optionally, in some embodiments, a sensor for collecting and transmitting a detection signal is arranged in the reaction chamber.

[0024] Optionally, in some embodiments, the sensor is selected from an electrochemical signal sensor, an electrochemiluminescence signal sensor, and an optical sensor.

[0025] It should be noted that the sensor is not limited to an electrochemical signal sensor and an electrochemiluminescence signal sensor, but can also be any other type of signal sensor, and any sensor falls within the protection scope of the present application.

[0026] Optionally, in some embodiments, a valve is arranged between the detection zone and the lysis zone for controlling the flow of liquid: when the valve is in an open state, the liquid in the lysis zone can flow to the detection zone; when the valve is in a closed state, the liquid in the lysis zone can be blocked from flowing to the detection zone.

[0027] It should be noted that in some embodiments, the valve can be arranged or not arranged, and the selection is reasonable according to the needs of the detection zone.

[0028] Optionally, in some embodiments, the surface of the magnetic beads is further modified with an anti-fouling molecule layer.

[0029] Blood components are complex, and the modification of the anti-fouling molecule layer will greatly shield the non-specific protein adsorption of the outer vesicles in the blood, reduce the false positive signals generated by background noise, and can target and reliably study the fingerprint biological information of the target outer vesicles, thereby effectively improving the proteomic detection depth.

[0030] Through the modification of the anti-fouling molecule layer, the anti-pollution ability of the magnetic beads can be improved, and non-specific protein adsorption can be avoided.

[0031] The components of the anti-fouling molecule layer are hydrophilic polymer materials, zwitterionic polymers, and polysaccharide polymers. Optionally, in some embodiments, the components of the anti-fouling molecule layer are selected from one or a combination of APPC, PEG (Polyethyleneglycol), PMPC (poly(2-methacryloyloxyethyl phosphorylcholine), PSBMA (poly(sulfobetaine methacrylate)), and PCBMA (poly(carboxybetaine methacrylate)).

[0032] It should be noted that any polymer that can play an anti-fouling role to reduce non-specific binding can be applied to the present application, and all fall within the protection scope of the present application.

[0033] Optionally, in some embodiments, the anti-fouling molecule layer is formed by a polymerization reaction of a compound selected from the following monomers: PSBMA and PMPC.

[0034] Through the use of the anti-fouling modification of the two-component zwitterionic molecules, stable targeted extraction and outer vesicle purification in a complex sample matrix (such as serum) are achieved.

[0035] Optionally, in some embodiments, the chip body comprises a substrate layer at the bottom and a chip layer attached to the substrate layer, the separation channel and the lysis zone are configured on the side of the chip away from the substrate layer, and the detection zone is configured on the side of the chip layer close to the substrate layer.

[0036] Optionally, in some embodiments, the sensor can be arranged on the chip layer or on the substrate layer, as long as it can collect and detect the reaction signal of the detection zone. Optionally, in some embodiments, the material of the substrate layer is selected from paper, glass, PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), COC (cyclic olefin copolymer), PC (polycarbonate), and PS (polystyrene).

[0037] Optionally, in some embodiments, the material of the chip layer is selected from paper, glass, PDMS, PMMA, COC, PC, and PS.

[0038] It should be noted that using other high polymer materials to make the chip of the present application also falls within the scope of the present application.

[0039] In another aspect, the present application provides a magnetic bead, the surface of which is modified with an antibody or a ligand that specifically binds to a membrane protein of an exovesicle, and an anti-fouling molecule layer.

[0040] The components of the anti-fouling molecule layer are hydrophilic polymer materials, zwitterionic polymers, and polysaccharide polymers. Optionally, in some embodiments, the anti-fouling polymer is selected from one or a combination of several of APPC, PEG, PMPC, PSBMA, and PCBMA.

[0041] In another aspect, the present application provides a kit for detecting an exovesicle or its contents, which comprises the microfluidic biochip of any one of the above and / or the magnetic bead of any one of the above. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0043] Figure 1 Structure diagram of the microfluidic biochip for detecting or screening exovesicle proteins in Example 1.

[0044] Figure 2The schematic diagram of the chip layer structure of the microfluidic biochip in Example 1 (the size of each functional area in the figure is only exemplary, and can be set as needed in other examples).

[0045] Figure 3 The schematic diagram of the structure of the microfluidic biochip in Example 2.

[0046] Figure 4 The schematic diagram of the detection process of the microfluidic biochip in Example 2.

[0047] Figure 5 The photograph of the physical diagram of the microfluidic biochip in Example 2.

[0048] Figure 6 The SDS polyacrylamide gel electrophoresis result of the anti-fouling magnetic bead conjugate in the example.

[0049] Figure 7 The schematic diagram of the principle of the anti-fouling modification of the magnetic beads in the example.

[0050] Figure 8 The detection result of the anti-fouling ability of the magnetic beads modified by different concentrations of APPC in Example 4.

[0051] Figure 9 The Western blot analysis result of the expression of proteins (CD9 protein, Syntenin protein, L1 cell adhesion molecule protein (L1CAM)) in extracellular vesicles in the example.

[0052] Figure 10 The TEM image of the extracellular vesicles in the example.

[0053] Figure 11 The nanotracking analysis (NTA) of the size distribution of the extracellular vesicles in the example.

[0054] Figure 12 The sensitivity result of the microfluidic biochip in detecting L1CAM protein in the example.

[0055] Figure 13 The result determination of the electrochemical sensor of the microfluidic biochip in the example.

[0056] Figure 14 The specificity detection result of the microfluidic biochip in the example.

[0057] Figures 1-3 The labels in the middle are respectively: 100, 200-chip body, 110-separation channel, 111-sample input port, 112-immunomagnetic bead input port, 120-lysis area, 130-detection area, 131-reaction chamber, 140-chip layer, 150-substrate layer, 160-valve, 170-electrochemical signal sensor. DETAILED DESCRIPTION

[0058] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below clearly and completely. If specific conditions are not indicated in the embodiments, the conventional conditions or the conditions suggested by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not indicated, the conventional products that can be purchased in the market are adopted.

[0059] The features and performances of the present application are further described in detail below in combination with the embodiments.

[0060] Embodiment 1

[0061] The present embodiment provides a microfluidic biochip, the structure of which is shown in Figure 1 and Figure 2 .

[0062] The microfluidic biochip comprises a chip body 100, which comprises a substrate layer 150 at the bottom and a chip layer 140 attached to the substrate layer 150. The materials of the substrate layer 150 and the chip layer 140 can be reasonably selected according to the materials well known to those skilled in the art.

[0063] The chip layer 140 has the following functional zones on the surface: a separation channel 110 for separation of extracellular vesicles, a lysis zone 120 communicating with the extracellular separation channel 110, and a detection zone 130 communicating with the lysis zone 120. In the present embodiment, the detection zone 130 has one reaction chamber, and in other embodiments, it can have multiple reaction chambers. A valve 160 is arranged between the detection zone 130 and the lysis zone 120, for controlling the flow of liquid: when the valve is in an open state, the liquid in the lysis zone can flow to the detection zone; when the valve is in a closed state, the liquid in the lysis zone can be blocked from flowing to the detection zone.

[0064] The separation channel 110 has a sample input port 111 and an immunomagnetic bead input port 112. The magnetic beads are modified with capture antibodies (for example, the capture antibodies can specifically bind to the membrane proteins of extracellular vesicles of neurons), which can specifically bind to the membrane proteins of extracellular vesicles of specific sources. The shape of the separation channel 120 can be set according to the actual situation, and in the present embodiment, it is set to be serpentine (or in the shape of a paperclip). The sample input port 111 and the immunomagnetic bead input port 112 are in a Y-shaped structure.

[0065] The detection area 130 is fixed with a fixed antibody (i.e. a capture, the specific antibody type can be reasonably selected according to the target protein to be detected, in other embodiments, the capture can also be a ligand specifically combined with the detected molecule, or when the detection molecule is a nucleic acid, the capture can be a probe complementary thereto) that can specifically bind to the target protein (i.e. the detected molecule, the protein released after the outer vesicle is lysed). An electrochemical signal sensor 170 is arranged in the detection area 130. The electrochemical signal sensor can be connected and recognized by the interface of the detection system, realizing the collection and reading of the target signal in the detection area, and further realizing the detection of the detected molecule.

[0066] The separation channel 120 and the lysis area 120 are arranged on the side of the chip layer 140 away from the substrate layer 10, the detection area 130 is arranged on the side of the chip layer 140 close to the substrate layer 10, and the sensor is arranged on the substrate layer and can detect and collect the detection signal of the detected molecule in the reaction chamber.

[0067] In the embodiment, the above functional areas can be arranged as a groove structure.

[0068] In the embodiment, the material of the chip layer 140 is PDMS, and the material of the substrate layer 150 can be glass. In other embodiments, the materials can be reasonably selected according to actual needs.

[0069] Embodiment 2

[0070] The embodiment provides a microfluidic biochip, the structure of which is shown in Figure 3 The structure of the microfluidic biochip is basically the same as that of the microfluidic biochip of embodiment 1, and the difference is that in the embodiment, the detection area 130 has a plurality of reaction chambers 131; each reaction chamber 131 is in communication with the lysis area 120. Each reaction chamber 131 is modified with a fixed antibody. The fixed antibody modified in each reaction chamber 131 can be the same or different, and each reaction chamber is provided with a sensor.

[0071] By arranging the structure of a plurality of reaction chambers, synchronous detection of a plurality of different target markers can be realized.

[0072] The above-mentioned chip screening method of neuron outer vesicle protein is exemplary to introduce the use method and principle (see Figure 4 ) of the above-mentioned embodiment:

[0073] The serum sample (or other liquid sample) is pushed by an external precision syringe pump from the sample input port into the separation channel. The immunomagnetic beads are pushed by an external precision syringe pump from the immunomagnetic bead input port, and contact and bind with the outer vesicles in the separation channel (the antibody on the immunomagnetic beads specifically binds to the membrane protein on the outer vesicles). After passing through the separation channel, the outer vesicles are separated from the sample (e.g., serum). The magnetic beads bound with the outer vesicles (outer vesicle-magnetic bead complex) flow to the lysis region, where the outer vesicle-magnetic bead complex is enriched under the action of the magnetic field, and the outer vesicles are also enriched in this region. The remaining liquid components flow out and are collected for removal. After the magnetic bead enrichment step is completed, a lysis solution such as 1% Triton X-100 is added to the lysis region, and the internal proteins of the outer vesicles are released after lysis. The proteins released from the outer vesicles flow into the detection region through the valve opening under the pressure applied by the pump, and are specifically bound and fixed by the corresponding fixed antibody. Then, an enzyme-linked detection antibody (e.g., horseradish oxidase-detection antibody) is added to form a sandwich structure of fixed antibody-target protein-enzyme-linked detection antibody. The addition of substrate lumiol and hydrogen peroxide can be catalyzed by horseradish oxidase, and the product of the enzymatic reaction can emit light (i.e., a detectable label, the light emission intensity is collected by an electrochemical signal sensor and can be transmitted to a detector for detection), and the intensity of the light emission is directly proportional to the content of the target outer vesicle protein. By comparing the expression levels (i.e., light emission intensity) of these proteins in the disease group and the healthy group, the detection region can screen for highly correlated markers of diseases and construct a composite biomarker (a-Synuclein+X) to improve the accuracy of diagnosis.

[0074] The chip provided in the above embodiment can further screen for markers highly correlated with diseases and construct a composite biomarker (a-Synuclein+X) to improve the accuracy of diagnosis. For example, the biomarker protein can be screened from the outer vesicles of neurons, and then used for early prevention and intervention of neurodegenerative diseases (e.g., Parkinson's disease).

[0075] Example 3

[0076] The reference preparation method of the microfluidic biochip provided in this embodiment includes the following steps:

[0077] Mold preparation:

[0078] The first step is the preparation of the chip substrate, which requires cleaning and drying. First, take a piece of silicon wafer and put it into a cleaning solution (concentrated sulfuric acid: hydrogen peroxide = 3:1), shake the dish gently every 2-3 min to ensure the silicon wafer is fully cleaned; then, quickly clean the silicon wafer with ultrapure water, making sure not to let the surface dry during the cleaning process; then, treat the silicon wafer with ethanol and then acetone for 1 min each to clean the organic matter on the surface of the silicon wafer; again, clean the silicon wafer with ultrapure water and place it above the electric hot plate. When the water on the surface of the silicon wafer is completely evaporated, place the silicon wafer in the center of the electric hot plate for heating and dehydration (200°C, 10 min). During this process, you can rotate the silicon wafer to ensure even heating. After heating, wait for the silicon wafer to cool down to room temperature, and then expose the silicon wafer to hexamethyldisilazane vapor for 5 min. This step can effectively enhance the adhesion of the photoresist on the surface of the silicon wafer.

[0079] The second step is to spin-coat the photoresist on the surface of the silicon wafer. First, spread the photoresist evenly by spinning at low speed (500 rpm) for 15 s; then, spin at high speed (4000 rpm) for 75 s to obtain a smooth photoresist layer.

[0080] The third step is pre-baking to remove excess organic solvents in the photoresist and release some of the stress in the photoresist. The parameter settings and baking sequence are as follows: 65°C for 1 min; 95°C for 5 min; 65°C for 1 min.

[0081] The fourth step is to use a high-power ultraviolet light source and a mask containing the chip design pattern to expose the photoresist to ultraviolet light for 90 s.

[0082] The fifth step requires post-baking the exposed silicon wafer to ensure good adhesion between the photoresist and the silicon wafer during development. The parameters are as follows: 65°C for 1 min; 95°C for 5 min; 65°C for 1 min. After post-baking, the design pattern can be observed slightly on the surface of the silicon wafer.

[0083] The sixth step is the development process. After the post-baking is completed, place the silicon wafer in a dish and let it cool to room temperature. Slowly add the developing solution until it completely covers the silicon wafer. Develop by alternating between shaking and standing. During this process, you can clearly see the unhardened photoresist on the silicon wafer being washed away by the developing solution, leaving behind the hardened structure. Develop for about 1-3 min to ensure that the unhardened photoresist is completely removed. Then, use a low-pressure air gun to blow dry the developing solution on the surface of the silicon wafer.

[0084] The seventh step is hard baking (also known as hard mask), which is used to remove residual solvents and reinforce the structure. The parameters are as follows: 150°C for 5 min.

[0085] Finally, place the silicon wafer under a microscope for inspection, marking, evaluation, and storage.

[0086] Chip preparation:

[0087] The first step is the pretreatment of the mold. In order to effectively increase the adhesion between PDMS and the mold, a high-fidelity microfluidic chip is formed. First, the previously prepared silicon wafer mold is pasted to the center of the petri dish (110 mm) with double-sided tape, and then it is moved into a vacuum dryer. Add 3-5 drops of trimethylchlorosilane to the dish on the side, and fumigate for 5 min.

[0088] The second step is the weighing and curing of the PDMS prepolymer. Weigh 25 g (the number of grams can be adjusted according to the desired height of the chip) of PDMS prepolymer (mass ratio A:B = 10:1); manually stir to mix the prepolymer thoroughly, then slowly pour the prepolymer into the dish containing the mold; after pouring, use a tool to scrape the wall of the cup, and place the dish in a vacuum dryer to extract air bubbles under negative pressure; after the air bubbles are completely extracted, place the dish in an oven, and the chip curing conditions are 80°C for 30 min.

[0089] The third step is to separate and assemble the chip. After the cured PDMS chip is taken out of the oven, it is cooled to room temperature, then the PDMS layer is cut along the edge of the silicon wafer with a special knife, and the PDMS layer is slowly separated from the silicon wafer; then, using a special cutting knife, the PDMS chip is separated from the whole according to the planned blank area, and the chips are classified according to different types and placed, and relevant labels are made. The preparation of the microfluidic chip is completed, and the physical photo of the prepared chip is shown in Figure 5 .

[0090] Example 4

[0091] Detection of the capture ability of anti-pollution magnetic beads

[0092] The specific steps are as follows:

[0093] 1. Take 1-micron-diameter NHS-modified magnetic beads

[0094] 2. Prepare an antibody (such as an anti-CD63 antibody) solution (concentration 0.1-2 mg / mL);

[0095] 3. Wash the NHS-magnetic beads once to activate the carboxyl group;

[0096] 4. Discard the supernatant, and add the antibody solution. Incubate at room temperature for 1-2 h under vertical suspension;

[0097] 5. Keep the flow-through liquid. Wash the magnetic beads 4 times with M-Water;

[0098] 6. Discard the supernatant, and add APPC (4-aminophenylphosphorylcholine) at the same concentration as the antibody solution. Incubate at room temperature for 1-2 h under vertical suspension in the dark;

[0099] 7. Discard the supernatant, wash the M-Water once, and wash the PBS twice;

[0100] 8. Add the preservative solution (0.1% proclin-300 PBS) and store at 4°C; obtain the anti-CD63 antibody and anti-fouling polymer modified magnetic beads (CD63-MB).

[0101] Detect the ability of magnetic beads to capture the outer vesicles:

[0102] (1) Take the CD63-MBs, incubate with serum (from patient blood, obtained by gradient centrifugation) overnight at room temperature; discard the supernatant, collect the magnetic beads, add the lysis solution (0.1% Triton-X100), and lyse the outer vesicles; collect the supernatant, measure the protein concentration with BSA, aliquot, and store at -20°C for later use.

[0103] (2) Take the CD63-MBs, incubate with serum. Remove the unabsorbed material, and elute the bound material with 0.1% Triton-X 100 solution. Resuspend all samples in loading buffer, and separate by SDS polyacrylamide gel electrophoresis. Perform Western blot analysis with horseradish peroxidase secondary antibody. The positions of the molecular weight markers (in kilodaltons) are shown on the right. Lanes 1, 2, and 3, 4 are from different patients. Determine whether the material captured by the CD63-MB is an outer vesicle sample by detecting the marker protein CD63 of the outer vesicle. The molecular weight of the CD63 protein is 26 kDa, Figure 6 The Western blot is consistent with the expected molecular weight of the CD63 protein. Therefore, it can be concluded that the captured material contains the target protein CD63.

[0104] In other embodiments, with reference to the method of the present embodiment, the magnetic beads can be modified with the corresponding antibody of any target protein of interest for specific binding to the outer vesicles, which is easily achieved by those skilled in the art.

[0105] Example 5

[0106] Effect of different concentrations of APPC modification on the anti-fouling ability of magnetic beads

[0107] 1. Prepare anti-fouling magnetic beads, and set different experimental groups:

[0108] 1) Wash the magnetic beads three times with the washing solution;

[0109] 2) Add the protein (1 mg / mL, BSA protein modified with fluorescent molecules) solution and incubate at room temperature for 3 h;

[0110] 3) Wash three times with PBS solution;

[0111] 4) Add APPC solution (concentration: 0, 0.167, 0.33, 0.66, 1.32, 2.64 mg / mL) under light-proof condition, incubate at room temperature for 3 h;

[0112] 5) Set up positive control group: ethanolamine (3M, PH = 9) as blocking solution.

[0113] 6) Wash with PBS for three times;

[0114] 7) Resuspend to appropriate concentration for standby use.

[0115] 2. Characterization of anti-fouling magnetic beads:

[0116] 1) Incubate magnetic beads with BSA-FITC at room temperature for 1 hour under light-proof condition. BSA-FITC concentration: 1 mg / mL;

[0117] 2) Wash with PBS for three times, keep the concentration of magnetic beads: 10 mg / mL, pay attention to light-proof;

[0118] 3) Dilute the concentration of magnetic beads to 1 mg / mL, add to non-transparent 384-well plate, cover with tin foil to avoid light;

[0119] 4) Immediately test the fluorescence value at 495-525 mm.

[0120] The results are shown in Figure 7 and Figure 8 :

[0121] Figure 7 The principle of anti-fouling modification of magnetic beads is shown, which avoids the adsorption of specific proteins through the modification of anti-fouling polymers, Figure 8 It is shown that the fluorescence value of BSA-FITC increases with the increase of the concentration of APPC, until the concentration reaches 2.63 mg / mL. The anti-fouling rate of APPC for magnetic beads gradually increases until 2.63 mg / mL, and then decreases, which may be related to the long-chain structure of APPC. And APPC as an anti-fouling agent, the effect is better than that of the positive control ethanolamine.

[0122] Example 6

[0123] The present embodiment provides a kit for detecting or screening exovesicular proteins, which comprises a microfluidic biochip and magnetic beads;

[0124] Wherein, the structure of the microfluidic biochip is basically the same as that of Example 1, and the magnetic beads are basically the same as those of Example 5, except that the magnetic beads are modified with anti-L1CAM, LRRK2, or TREM-2 antibodies, and 1.32 mg / ml APCC is used for modification.

[0125] Example 7

[0126] Application of microfluidic biochip: microfluidic chip successfully separates extracellular vesicles.

[0127] Using the microfluidic chip of Example 1 to separate neuronal extracellular vesicles, then Western blotting of extracellular vesicles, further confirmed the expression of four transmembrane proteins CD9, syntenin and neuronal extracellular vesicle specific membrane protein L1CAM, which were positively expressed in extracellular vesicles compared with healthy controls and blank controls Figure 9 ). The captured extracellular vesicles have a typical cup-shaped morphology, which can be seen in TEM images Figure 10 . NTA shows that the average size of the extracellular vesicles separated by the chip of Example 1 is 148.5 ± 4.3 nm, and the concentration is 2.25 x 10 8 ± 1.51 x 10 7 particles / ml Figure 11 .

[0128] From the perspective of biomarkers, morphology, particle size and distribution, the microchip of Example 1 can be specifically used to separate neuronal extracellular vesicles for downstream detection.

[0129] Example 8

[0130] L1CAM protein sensitivity

[0131] Using the microfluidic chip of Example 1, different concentrations of L1CAM standard protein were added to the detection area, respectively 300, 250, 150, 75, 25, 5, 1, 0.5, 0 pg / mL, and incubated with the sensor for detection, as shown in Figure 12 , with the decrease of standard antigen concentration, the output electrochemical signal value gradually decreased, until the concentration of 0.5 pg / mL, the electrochemical signal value tended to be consistent with the background signal value. The chip sensor of Example 1 has a sensitivity of 1 pg / mL for detecting L1CAM antigen.

[0132] Example 9

[0133] Specific detection

[0134] Using the microfluidic chip of Example 1, adding related extracellular vesicle proteins (L1CAM, CD81), as well as other protein components in blood (IgG, BSA, IL-6) respectively for electrochemical reaction verification, to confirm the specificity of the chip. As shown in Figure 13 and Figure 14 , by comparing the results, the chip has no electrochemical reaction with the proteins that may form infection, and has good specificity. Therefore, the chip can specifically detect the specific target protein L1CAM of neuronal-derived extracellular vesicles.

[0135] In summary:

[0136] (1) The new anti-pollution magnetic beads provided by the embodiment of the present application; realize the high-purity outer vesicle subpopulation purification system with good stability and good anti-pollution effect. The method is simpler than the method reported in the literature, and is more widely applicable.

[0137] (2) The microfluidic chip for detecting or screening outer vesicle protein provided by the embodiment of the present application is a microfluidic chip integrating magnetic bead extraction-cracking-detection three modules. In the separation-detection integrated microfluidic chip, the outer vesicles extracted by the magnetic beads release the inner protein components after cracking and are recognized and combined by the antibodies on the chip, and then the enzyme-labeled detection antibodies mixed in advance are used, so as to form a sandwich three-layer structure of one antibody-antigen-two antibodies. Finally, the chemiluminescence reagent (such as lumino+H2O2) is added, the chip is connected to the detector, and the detection of the target protein can be completed. The content of the protein marker is directly reflected by the luminescence intensity, so as to screen out the proteins with large expression difference in different disease groups or disease groups and healthy control groups. The detection area of the chip can be customized according to the user's needs, and is not limited to a few concentrated target detection.

[0138] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A microfluidic biochip for exovesicle or its content detection, characterized by, It comprises: a chip body, which is provided with the following functional areas in sequence: a separation channel for external vesicle separation, a lysis area communicating with the separation channel, and a detection area communicating with the lysis area; one end of the separation channel is provided with a sample input port and a magnetic bead input port, when the magnetic beads enter from the magnetic bead input port, they can specifically bind to the external vesicles entering from the sample input port to form an external vesicle-magnetic bead complex, which is separated and enriched in the lysis area under the action of external force; the surface of the magnetic beads is further modified with an anti-fouling molecule layer; the components of the anti-fouling molecule layer are hydrophilic polymer materials, zwitterionic polymers, and polysaccharide polymers; the components of the anti-fouling molecule layer are one or a combination of several selected from the group consisting of APPC, PEG, PMPC, PSBMA, and PCBMA; the detection area is fixed with a capture capable of specifically binding to a to-be-detected molecule; the to-be-detected molecule is derived from the external vesicle.

2. The microfluidic biochip of claim 1, wherein the surface of the magnetic beads is modified with an antibody or a ligand capable of specifically binding to the surface membrane protein of the external vesicle; the to-be-detected molecule is selected from a protein, a nucleic acid, or a combination thereof; the nucleic acid is DNA, RNA, or a combination thereof; the capture is selected from an antibody, a ligand, a nucleic acid probe complementary to the nucleic acid, or a combination thereof.

3. The microfluidic biochip of claim 2, wherein, the detection area comprises one or more reaction chambers; the reaction chamber is fixed with the capture; the reaction chamber is provided with a sensor for collecting and transmitting a detection signal; the sensor is selected from an electrochemical signal sensor, an electrochemiluminescence signal sensor, and an optical sensor.

4. The microfluidic biochip of claim 1, wherein The chip body comprises a substrate layer at the bottom and a chip layer attached to the substrate layer, the separation channel, the lysis area, and the detection area are arranged on the side of the chip away from the substrate layer, and the detection area is arranged on the side of the chip layer close to the substrate layer.

5. The microfluidic biochip of claim 4, wherein, The material of the substrate layer is selected from paper, glass, PDMS, PMMA, COC, PC, and PS.

6. The microfluidic biochip of claim 5, wherein, The material of the chip layer is selected from paper, glass, PDMS, PMMA, COC, PC, and PS.

7. A magnetic bead, characterized in that, The surface is modified with an antibody or a ligand capable of specifically binding to the membrane protein of the external vesicle, and an anti-fouling molecule layer; the components of the anti-fouling molecule layer are hydrophilic polymer materials, zwitterionic polymers, and polysaccharide polymers; wherein the anti-fouling molecule layer is a combination of one or several selected from the group consisting of APPC, PEG, PMPC, PSBMA, and PCBMA.

8. A kit for exovesicle or its content detection, characterized in that, It comprises the microfluidic biochip of any one of claims 1-6 and / or the magnetic beads of claim 7.

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