A microfluidic chip system device and a method for capturing and separating extracellular vesicle subpopulations and applications

Abstract

The application provides a microfluidic chip system device and a method and application of capturing and separating an extracellular vesicle subpopulation. The microfluidic chip system device comprises a capture chip and a magnetic separation chip for separating a captured sample; the capture chip comprises a capture flow channel and a J-shaped stopper arranged in the capture flow channel, an input end of the capture flow channel is adjacent to a sample inlet and a microsphere inlet, and an output end of the capture flow channel is provided with a sample outlet; the arc-shaped free end is used for blocking the sample flow to form a local micro-vortex; and the magnetic separation chip comprises a separation flow channel and a magnetic field device arranged on one side of the separation flow channel, and the separation flow channel is provided with a sample inlet, a buffer inlet and at least two sample outlets. The microfluidic chip can quickly capture and separate extracellular vesicles, and has small sample demand and low cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a microfluidic chip system device and a method for capturing and separating an extracellular vesicle subpopulation and application thereof. BACKGROUND

[0002] Extracellular vesicles (EVs) are a kind of membrane vesicle structure secreted by almost all cells, and the EV membranes carry proteins, lipids, nucleic acids and other substances from the parent cells, thus playing an important role in cell communication, signal transduction, material transport and interaction with the extracellular matrix. Extracellular vesicles also play a role in the occurrence and development of cancer, so detection of extracellular vesicles can also analyze the progression of cancer.

[0003] Although EVs have attracted attention in disease diagnosis and treatment applications, their clinical application has been limited due to the difficulty in isolating EVs. Current isolation methods include exclusion chromatography, ultracentrifugation and precipitation, etc. CN110511902B discloses an extracellular vesicle separation and enrichment method based on exclusion chromatography and ultrafiltration technology, which comprises: mixing a fluorescent dye with a body fluid, adding it to an exclusion chromatography column, collecting and combining fractions according to fluorescence intensity, treating the combined fractions with proteinase K and RNase A in turn, and finally ultrafiltrating and concentrating.

[0004] CN112501112A discloses a separation and enrichment method for rapidly extracting tissue extracellular vesicles, which comprises: A, chopping the target tissue with a mechanical method and adding tissue digestion enzymes to dissociate the tissue, and obtaining a tissue cell suspension by filtering the obtained tissue dissociation solution; B, sequentially performing differential centrifugation, ultra-centrifugation, SEC exclusion and ultrafiltration on the tissue cell suspension to enrich and purify the tissue extracellular vesicles.

[0005] Although the above methods can achieve the separation of extracellular vesicles to some extent, they are time-consuming, require a large amount of sample, have low product recovery rate and purity, and cause damage to EVs during the separation process.

[0006] Therefore, there is an urgent need to develop a device that can quickly separate extracellular vesicles with less sample requirement. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application aims to provide a microfluidic chip system device and a method for capturing and separating an extracellular vesicle subpopulation and an application thereof. In the capture chip, a J-shaped stopper is arranged in the capture flow channel, which can increase the mixing degree of the sample and the microspheres, thereby improving the sample capture efficiency of the microspheres. In the magnetic separation chip, the different characteristics of the magnetic field force received by each microsphere in the magnetic field can be used to realize the rapid separation of the sample. Therefore, the microfluidic chip system device of the present application can quickly capture and separate extracellular vesicles, requires a shorter time, and has the advantages of small size, small sample volume requirement, and high portability.

[0008] To achieve this purpose, the present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides a microfluidic chip system device, which comprises a capture chip and a magnetic separation chip for separating a captured sample.

[0010] The capture chip comprises a capture flow channel and a J-shaped stopper arranged in the capture flow channel. The input end of the capture flow channel is adjacent to a sample inlet and a microsphere inlet, and the output end of the capture flow channel is provided with a sample outlet. The fixed end of the J-shaped stopper is connected with the inner wall of one side of the capture flow channel, and the arc-shaped free end of the J-shaped stopper leaves a gap with the inner wall of the other side of the capture flow channel. The arc-shaped free end is used to block the sample flow to form a local micro-vortex.

[0011] The magnetic separation chip comprises a separation flow channel and a magnetic field device arranged on one side of the separation flow channel. The separation flow channel is provided with a sample inlet, a buffer inlet, and at least 2 sample outlets, for example, 2 sample outlets, 3 sample outlets, 4 sample outlets, 5 sample outlets, 6 sample outlets, 7 sample outlets, 8 sample outlets, or 10 sample outlets, etc.

[0012] The present application provides a microfluidic chip system device. In the capture chip, a J-shaped stopper is arranged in the capture flow channel. The arc-shaped free end of the J-shaped stopper can block the sample flow to form a local micro-vortex, increase the mixing degree of the sample and the microspheres, thereby improving the sample capture efficiency of the microspheres, and the arc-shaped structure can reduce the generation of bubbles. In the magnetic separation chip, the different characteristics of the magnetic field force received by each microsphere in the magnetic field can be used to realize the rapid separation of the sample. Therefore, the microfluidic chip system device of the present application can quickly capture and separate extracellular vesicles, requires a shorter time, and has the advantages of small size, small sample volume requirement, and high portability.

[0013] In the present application, after the J-shaped stopper is arranged in the capture flow channel, a new curved flow channel is formed for the sample flow.

[0014] Preferably, the material of the capture chip comprises polydimethylsiloxane (PDMS).

[0015] Preferably, the capture chip is bonded on a glass substrate, and the inside of the chip is blocked with 1-5% (w / v) BSA, for example, 1% (w / v) BSA, 2% (w / v) BSA, 3% (w / v) BSA, 4% (w / v) BSA, or 5% (w / v) BSA, etc.

[0016] Preferably, the material of the magnetic separation chip includes polydimethylsiloxane (PDMS).

[0017] Preferably, the magnetic separation chip is bonded on a glass substrate, and the inside of the chip is blocked with 1-5% (w / v) BSA, for example, 1% (w / v) BSA, 2% (w / v) BSA, 3% (w / v) BSA, 4% (w / v) BSA, or 5% (w / v) BSA, etc.

[0018] Preferably, the sample inlet of the capture flow channel is connected with a sample inlet channel, and the magnetic bead inlet is connected with a magnetic bead inlet channel, and the sample inlet channel is arranged vertically to the magnetic bead inlet channel.

[0019] Preferably, at least 2 J-shaped baffles are arranged in the capture flow channel, for example, 2, 5, 10, 15, 20, 25, or 30, etc., preferably 5-20.

[0020] Preferably, the at least 2 J-shaped baffles are arranged in parallel and at intervals.

[0021] Preferably, the adjacent 2 J-shaped baffles are staggered in the capture flow channel.

[0022] In the present application, a plurality of J-shaped baffles are arranged in parallel and at intervals in the capture flow channel, and a gap is left between the arc-shaped free end of each J-shaped baffle and the inner wall of one side of the capture flow channel, so as to form a serpentine curved flow channel, and the sample can flow along the serpentine curved flow channel, and the arc-shaped free end can also block the sample flow to form a local micro-vortex, thereby enhancing the mixing.

[0023] Preferably, the distance between the adjacent 2 J-shaped baffles is 0.2-4mm, for example, 0.2mm, 0.3mm, 0.5mm, 0.7mm, 1mm, 1.5mm, 2mm, 3mm, or 4mm, etc.

[0024] Preferably, the gap between the arc-shaped free end of the J-shaped baffle and the inner wall of the capture flow channel is 0.8-3mm, for example, 0.8mm, 1mm, 1.2mm, 1.5mm, 2mm, 2.5mm, or 3mm, etc.

[0025] Preferably, the height of the J-shaped stopper and the capture flow channel is independently 20-80 μm, for example, it can be 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm or 80 μm, etc.

[0026] Preferably, the height of the J-shaped stopper and the capture flow channel is equal.

[0027] Preferably, the output end of the separation flow channel is provided with at least 2 U-shaped cavities, for example, it can be 2, 5, 10, 15, 20, 25 or 30, etc., and each of the U-shaped cavities is provided with a sample outlet.

[0028] In the present application, by using the characteristics that the microspheres with magnetism will displace in the magnetic field, different microspheres can be used to make them receive different magnetic forces in the magnetic field, thereby producing different displacements, and then the microspheres can be separated, and the separated microspheres enter different U-shaped cavities and are discharged through the sample outlet.

[0029] Preferably, the at least 2 U-shaped cavities are arranged side by side.

[0030] Preferably, the input end of the separation flow channel is provided with the sample inlet, and the sample inlet is directly opposite the U-shaped cavity located at the edge of the separation flow channel.

[0031] Preferably, the sample inlet of the separation flow channel is connected with a sample channel, and the width of the sample channel is 0.08-0.3 mm, for example, it can be 0.08 mm, 0.1 mm, 0.12 mm, 0.15 mm, 0.18 mm, 0.2 mm, 0.25 mm or 0.3 mm, etc.

[0032] Preferably, the input end of the separation flow channel is further provided with the buffer inlet.

[0033] Preferably, the height of the separation flow channel is 20-80 μm, for example, it can be 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm or 80 μm, etc.

[0034] Preferably, the magnetic field device is arranged on the side of the separation flow channel away from the sample inlet and closely arranged with the separation flow channel.

[0035] Preferably, the magnetic field strength of the magnetic field device is 100-300 mT, for example, it can be 100 mT, 120 mT, 140 mT, 160 mT, 180 mT, 200 mT, 220 mT, 240 mT, 260 mT, 280 mT or 300 mT, etc., preferably 180-203 mT.

[0036] In the present application, the magnetic field strength is the field strength at the sample inlet.

[0037] In a second aspect, the present application provides a method for capturing and separating a subpopulation of extracellular vesicles, which is performed by using the microfluidic chip system device of the first aspect, and comprises:

[0038] (1) introducing an extracellular vesicle sample and at least two kinds of microspheres, each having a different antibody, into the capture flow channel through the sample inlet and the microsphere inlet, respectively, for capturing, and then obtaining a captured sample through the sample outlet; wherein at least one of the microspheres has magnetism;

[0039] (2) injecting a buffer into the separation flow channel through the buffer inlet, and introducing the captured sample into the separation flow channel through the sample inlet, and the captured sample is separated under the action of a magnetic field.

[0040] In the present application, step (1) uses microspheres with different antibodies to capture extracellular vesicles, and each microsphere captures one type of extracellular vesicle; step (2) uses the feature that each microsphere experiences different magnetic forces in a magnetic field to cause different lateral displacements of each microsphere, thereby achieving the separation of a subpopulation of extracellular vesicles. The method of the present application has simple steps and low cost.

[0041] In the present application, the at least two kinds of microspheres can include one kind of microsphere without magnetism.

[0042] Alternatively, the extracellular vesicle sample includes MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231 cell lines. The culture supernatant containing MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231 cell lines can be obtained by first centrifuging at 500-1000g for 3-8min, and then centrifuging at 1500-2200g for 15-25min, as the extracellular vesicle sample.

[0043] Preferably, the antibodies include EGFR and / or CD133.

[0044] Preferably, the sample flow rate of the extracellular vesicle sample is 10-90μL / min, for example, it can be 10μL / min, 20μL / min, 30μL / min, 40μL / min, 50μL / min, 60μL / min, 70μL / min, 80μL / min or 90μL / min, etc.

[0045] Preferably, in the capture flow channel, the time for capturing is 1-10min, for example, it can be 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min or 10min, etc.

[0046] Preferably, the flow rate ratio of the captured sample to the buffer solution is 1:(1-4), for example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 or 1:4, etc.

[0047] Thirdly, the present invention provides an application of the microfluidic chip system device described in the first aspect, wherein the microfluidic chip system device is used to capture and separate extracellular vesicles of breast cancer cells.

[0048] The system refers to an equipment system, device system, or production device.

[0049] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0050] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0051] This invention provides a microfluidic chip system device. In the capture chip, a J-shaped baffle is arranged within the capture channel. Its arc-shaped free end can impede sample flow to form localized micro-vortices, increasing the mixing degree between the sample and microspheres, thereby improving the sample capture efficiency of the microspheres. Furthermore, its arc-shaped structure reduces bubble generation. In the magnetic separation chip, the different magnetic forces experienced by each type of microsphere in a magnetic field are utilized to achieve rapid sample separation. Therefore, the microfluidic chip system device of this invention can rapidly capture and separate extracellular vesicles in a short time, and has the advantages of small size, small sample volume requirement, and high portability. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the capture chip provided in Embodiments 1-3 of the present invention;

[0053] Figures 2-3 This is a schematic diagram of the capture channel structure in the capture chip provided in Embodiments 1-3 of the present invention;

[0054] Figure 4 The above are schematic diagrams of the magnetic separation chip provided in Embodiments 1-3 of the present invention.

[0055] Figures 5-6 This is a schematic diagram of the separation channel structure in the magnetic separation chip provided in Embodiments 1-3 of the present invention;

[0056] Figures 7-9 This is a partially enlarged schematic diagram of the separation channel in the magnetic separation chip provided in Embodiments 1-3 of the present invention;

[0057] Figure 10 The capture efficiency performance diagram provided in Test Example 1 of this invention;

[0058] Figure 11 A capture efficiency performance graph provided for the present application comparative test example 1;

[0059] Figure 12 A morphology graph of magnetic beads provided for the present application test example 2 (left graph), and a morphology graph after the magnetic beads capture extracellular vesicles (right graph);

[0060] Figure 13 A separation effect graph provided for the present application test example 2;

[0061] Figure 14 A separation effect graph provided for the present application comparative test example 2;

[0062] Figure 15 A protein expression quantity distribution graph of extracellular vesicles from four cell lines provided for the present application application example 1.

[0063] Wherein, 1-capture flow channel; 2-J-shaped stopper; 3-magnetic bead inlet channel; 4-sample inlet channel; 5-sample outlet; 6-separation flow channel; 7-U-shaped cavity; 8-sample inlet channel; 9-buffer inlet; 10-magnetic field device. DETAILED DESCRIPTION

[0064] It should be understood that, in the description of the present application, the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements indicated must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second" and the like are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" and the like can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0065] It should be noted that, in the description of the present application, unless otherwise specified and limited, the terms "provided", "connected", "connected" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be connected inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood through specific circumstances.

[0066] The technical solutions of the present application are further illustrated by specific embodiments.

[0067] Embodiment 1

[0068] The present embodiment provides a microfluidic chip system device, which comprises a capture chip and a magnetic separation chip for separating a captured sample.

[0069] The capture chip is made of PDMS and bonded to a glass substrate, as shown in Figures 1-3 The capture chip comprises a capture flow channel 1, the input end of which is adjacent to a sample inlet and a microsphere inlet, the sample inlet of the capture flow channel 1 is connected with a sample inlet channel 4, the width of the sample inlet channel 4 is 0.2mm, the microsphere inlet is connected with a magnetic bead inlet channel 3, the sample inlet channel 4 and the magnetic bead inlet channel 3 are vertically arranged, and the output end of the capture flow channel 1 is provided with a sample outlet 5.

[0070] The capture flow channel 1 is provided with 10 J-shaped baffles 2, the 10 J-shaped baffles 2 are arranged in parallel and spaced apart, and adjacent two J-shaped baffles 2 are staggered, the distance between adjacent two J-shaped baffles 2 is 0.9mm, the length of each J-shaped baffle 2 is 9mm, and the width is 0.2mm; the fixed end of each J-shaped baffle 2 is connected with one side inner wall of the capture flow channel 1, and the arc-shaped free end of each J-shaped baffle 2 leaves a gap of 1mm with the other side inner wall of the capture flow channel 1; the arc-shaped free end is used to block the sample flow to form a local micro-vortex; the height of the J-shaped baffle 2 and the capture flow channel 1 is 50μm.

[0071] The magnetic separation chip is made of PDMS and bonded to a glass substrate, as shown in Figures 4-9 , wherein, Figure 7 , Figure 8 and Figure 9 are respectively Figure 6The local enlarged view of the region I, region II and region III of the magnetic separation chip comprises a separation flow channel 6 and a magnetic field device 10 arranged on one side of the separation flow channel 6, the height of the separation flow channel 6 is 50 μm, the output end of the separation flow channel 6 is provided with three U-shaped cavities 7, the three U-shaped cavities 7 are arranged side by side, and each U-shaped cavity 7 is provided with a sample outlet, the width of the output end of the separation flow channel 6 is 10 mm, the distance between the straight edge segment of the separation flow channel 6 far away from the one end of the U-shaped cavity 7 and the input end of the U-shaped cavity 7 is 10 mm, and the distance between the two straight edge segments closest to each other between the two adjacent U-shaped cavities 7 is 0.2 mm; the input end of the separation flow channel 6 is provided with the sample inlet, the sample inlet is opposite to the U-shaped cavity 7 located at the edge of the separation flow channel 6, the sample inlet is connected with a sample channel 8, and the width of the sample channel 8 is 0.1 mm; the input end of the separation flow channel 6 is further provided with the buffer inlet 9, the magnetic field device 10 is arranged on the side of the separation flow channel 6 far away from the sample inlet and closely arranged on the separation flow channel 6, the magnetic field device 10 is a neodymium-iron-boron permanent magnet, the model number is N35, the size of a single neodymium-iron-boron permanent magnet is 10*20*3 mm (cuboid), and the magnetic field strength of the magnetic field device 10 is 200 mT.

[0072] The embodiment also provides a preparation method of the microfluidic chip, which comprises the following steps:

[0073] The mold is taken out and the surface of the mold is blown clean by using nitrogen to reduce the influence of pollutants, and then the mold is gently placed in a glass dish, 30 μL of perfluorosilane is uniformly dropped at the gap between the mold and the edge of the glass dish, and then the perfluorosilane is naturally volatilized in a fume hood for 10 min. The PDMS pre-polymer and the curing agent are uniformly mixed in a mass ratio of 10:1, fully stirred, poured into the glass dish containing the mold, and air bubbles are removed by using a vacuum pump. After the internal air bubbles are fully removed, the glass dish is taken out and placed in a 60℃ oven for heating for 2 h. After curing, the glass dish is taken out and placed at room temperature, the PDMS is peeled off, and the PDMS and the glass substrate are covered with invisible adhesive tape to prevent being contaminated;

[0074] Bonding: in a super-clean room, the PDMS and the glass substrate are taken out, blown clean by using nitrogen, and then the PDMS flow channel surface and the front surface of the glass substrate are placed upwards in an oxygen plasma degumming machine, surface modification is performed under the condition of 60 W radio frequency power and 30 s reaction time. Subsequently, the PDMS flow channel surface and the front surface of the glass substrate are slowly contacted and bonded together;

[0075] Assembling: appropriate PTFE pipes with the same length are taken, one end of each PTFE pipe is connected with the needle of a disposable syringe by using a 3145 type epoxy adhesive to prevent liquid leakage, the other end of each PTFE pipe is connected with a sample injection needle, and the sample injection needle is vertically inserted into the hole of the PDMS chip which is previously punched;

[0076] Sealing: For the assembled microfluidic chip, first use pure water to clean at a flow rate of 200 μL / min for 10 min, then use pure water to clean at a flow rate of 25 μL / min for 10 min, and then use a 2% BSA / PBS solution to seal at a flow rate of 1000 μL / h for 1000 μL, and then use pure water to clean at a flow rate of 100 μL / min for 10 min to flush away the excess BSA.

[0077] Pre-treatment: Before the microfluidic chip is sampled, first use pure water to clean at a flow rate of 200 μL / min for 3 min, and then use 10x PBS to clean at a flow rate of 200 μL / min for 3 min.

[0078] Example 2

[0079] The present embodiment provides a microfluidic chip system device, which comprises a capture chip and a magnetic separation chip for separating a captured sample;

[0080] The capture chip is made of PDMS and is bonded to a glass substrate, as shown in Figures 1-3 The capture chip comprises a capture flow channel 1, the input end of the capture flow channel 1 is provided adjacent to a sample inlet and a microsphere inlet, the sample inlet of the capture flow channel 1 is connected with a sample inlet channel 4, the microsphere inlet is connected with a magnetic bead inlet channel 3, and the sample inlet channel 4 and the magnetic bead inlet channel 3 are arranged vertically; the output end of the capture flow channel 1 is provided with a sample outlet 5;

[0081] The capture flow channel 1 is provided with five J-shaped baffles 2, the five J-shaped baffles 2 are arranged in parallel and at intervals, and adjacent two J-shaped baffles 2 are arranged alternately, the distance between adjacent two J-shaped baffles 2 is 4 mm, the length of each J-shaped baffle 2 is 9 mm, and the width is 0.2 mm; the fixed end of each J-shaped baffle 2 is connected with the inner wall of one side of the capture flow channel 1, and the arc-shaped free end of each J-shaped baffle 2 leaves a gap of 2 mm with the inner wall of the other side of the capture flow channel 1; the arc-shaped free end is used to block the sample flow to form a local micro-vortex; the height of the J-shaped baffle 2 and the height of the capture flow channel 1 are both 40 μm;

[0082] The magnetic separation chip is made of PDMS and is bonded to a glass substrate, as shown in Figures 4-9 , wherein Figure 7 , Figure 8 and Figure 9 are respectively Figure 6The local enlarged view of the region I, the region II and the region III of the magnetic separation chip comprises a separation flow channel 6 and a magnetic field device 10 arranged on one side of the separation flow channel 6, the height of the separation flow channel 6 is 40 μm, the output end of the separation flow channel 6 is provided with two U-shaped cavities 7, the two U-shaped cavities 7 are arranged side by side, and each U-shaped cavity 7 is provided with a sample outlet; the input end of the separation flow channel 6 is provided with the sample inlet, the sample inlet is opposite to the U-shaped cavity 7 located on the edge of the separation flow channel 6, the sample inlet is connected with a sample inlet channel 8, the width of the sample inlet channel 8 is 0.2 mm; the input end of the separation flow channel 6 is also provided with the buffer inlet 9, the magnetic field device 10 is arranged on the side of the separation flow channel 6 away from the sample inlet and closely arranged on the separation flow channel 6, the magnetic field device 10 is two neodymium-iron-boron permanent magnets, the model is N35, the size of a single neodymium-iron-boron permanent magnet is 10*20*3 mm (cuboid), and the magnetic field strength of the magnetic field device 10 is 100 mT.

[0083] The microfluidic chip system device is prepared by using the preparation method of the microfluidic chip in Example 1.

[0084] Example 3

[0085] The microfluidic chip system device comprises a capture chip and a magnetic separation chip for separating a captured sample.

[0086] The material of the capture chip is PDMS and is bonded on a glass substrate, as shown in Figures 1-3 The capture chip comprises a capture flow channel 1, the input end of the capture flow channel 1 is adjacent to a sample inlet and a microsphere inlet, the sample inlet of the capture flow channel 1 is connected with a sample inlet channel 4, the microsphere inlet is connected with a magnetic bead inlet channel 3, and the sample inlet channel 4 and the magnetic bead inlet channel 3 are arranged vertically; the output end of the capture flow channel 1 is provided with a sample outlet 5;

[0087] The capture flow channel 1 is provided with 20 J-shaped baffles 2, the 20 J-shaped baffles 2 are arranged in parallel and at intervals, and adjacent two J-shaped baffles 2 are arranged alternately, the distance between adjacent two J-shaped baffles 2 is 2 mm, the length of each J-shaped baffle 2 is 9 mm, and the width is 0.2 mm; the fixed end of each J-shaped baffle 2 is connected with the inner wall of one side of the capture flow channel 1, and the arc-shaped free end of each J-shaped baffle 2 leaves a gap of 3 mm with the inner wall of the other side of the capture flow channel 1; the arc-shaped free end is used for blocking the sample flow to form a local micro-vortex; the height of the J-shaped baffle 2 and the capture flow channel 1 is 80 μm;

[0088] The material of the magnetic separation chip is PDMS and is bonded on a glass substrate, as shown in Figures 4-9As shown, where, Figure 7 , Figure 8 and Figure 9 They are respectively Figure 6 Enlarged views of regions I, II, and III show that the magnetic separation chip includes a separation channel 6 and a magnetic field device 10 disposed on one side of the separation channel 6. The separation channel 6 has a height of 80 μm. The output end of the separation channel 6 has three U-shaped cavities 7 arranged side-by-side, each with a sample outlet. The input end of the separation channel 6 has a sample inlet, which faces the U-shaped cavity 7 located at the edge of the separation channel 6. The inlet is connected to an inlet channel 8, the width of which is 0.3 mm. The input end of the separation channel 6 is also provided with a buffer inlet 9. The magnetic field device 10 is located on the side of the separation channel 6 away from the inlet and is closely attached to the separation channel 6. The magnetic field device 10 consists of 6 neodymium iron boron permanent magnets, model N35, each with dimensions of 10×20×3 mm (cubic parallelepiped). The magnetic field strength of the magnetic field device 10 is 300 mT.

[0089] This embodiment uses the microfluidic chip fabrication method described in Example 1 to prepare a microfluidic chip system device.

[0090] Comparative Example 1

[0091] This comparative example provides a device for capturing bulk phase, comprising a shaking incubator and a 1.5 mL EP tube.

[0092] Comparative Example 2

[0093] This comparative example provides a discrete chip, which differs from Example 1 only in that the magnetic field device is omitted.

[0094] Test Example 1

[0095] This test example provides a method for conducting a capture experiment using the capture chip described in Example 1. The method includes:

[0096] (1) Before extracting extracellular vesicles, remove serum extracellular vesicles: Add DMEM medium containing 1% penicillin and streptomycin to inactivated fetal bovine serum to prepare DMEM containing 30% FBS, centrifuge at 150,000g for 11h, and filter through a 0.22μm membrane before use; MCF-10A cell-specific medium is centrifuged at 150,000g for 11h to remove extracellular vesicles from the serum, and then filtered through a 0.22μm membrane before use;

[0097] (2) When the cells have been cultured to at least 3 passages, replace the culture medium with serum-free extracellular vesicles and culture for 48 hours. Collect the cell culture supernatant.

[0098] (3) Collect the supernatant under 800g centrifugal force at 4°C for 5 min to remove cells in the culture supernatant;

[0099] (4) Collect the supernatant under 2000g centrifugal force at 4°C for 20 min to remove cell debris in the culture supernatant;

[0100] (5) Filter the supernatant collected in step (4) through a 0.22 μm filter membrane to remove large vesicles;

[0101] (6) Introduce the cell culture supernatant collected in step (5) into the capture flow channel through the sample inlet, and introduce 100 μL of the biotinylated EGFR antibody modified microsphere sample into the capture flow channel through the microsphere inlet, and perform capture experiments at flow rates of 10, 30, 50, 70 and 90 μL / min, respectively. The capture results are shown in Figure 10 From the figure, it can be seen that as the flow rate increases, more extracellular vesicles are captured on the microspheres, and the residual extracellular vesicles in the supernatant decrease. The flow rate of 50 μL / min is the optimal capture flow rate;

[0102] (7) Wash the sample collected at the sample outlet with 1x PBS three times and store at 4°C. Extract as needed.

[0103] The capture efficiency is investigated by detecting the total protein amount of the un-captured vesicles in the sample supernatant using a BCA protein quantification kit (Solabio).

[0104] Comparative Test Example 1

[0105] This comparative test example uses the device described in Comparative Example 1 to perform a capture experiment, uses the same extracellular vesicles and microsphere sample as Test Example 1, and places it in a 1.5 mL EP tube for capture and investigation of different reaction times. The samples are collected at reaction times of 0 min, 30 min, 60 min and 90 min to investigate the capture efficiency.

[0106] The investigation method of capture efficiency is the same as that of Test Example 1, and the results are shown in Figure 11 From the figure, it can be seen that in the bulk phase (1.5 mL EP tube), 60 min is needed to reach 100% capture efficiency, i.e. after 30 min of reaction, there is no free extracellular vesicle sample in the sample supernatant. Under the same sample amount, the required time is 30 times that of the capture chip.

[0107] Test Example 2

[0108] This test example provides a method for performing a separation experiment using the magnetic separation chip described in Example 1, which comprises:

[0109] (1) Take the three kinds of captured extracellular vesicles microspheres obtained by the method of test example 1, including 2.8 μm magnetic beads, 5.78 μm magnetic beads and 4.95 μm microspheres, wherein the 2.8 μm magnetic beads and the 5.78 μm magnetic beads are both magnetic, and the 4.95 μm microspheres are non-magnetic: put into room temperature, vortex for 30 s, then ultrasonic for 20 min, vortex for 30 s, take 20 μL 2.8 μm magnetic beads in 200 μL 10x PBS, take 40 μL 5.78 μm magnetic beads in 200 μL 10x PBS, take 10 μL 4.95 μm microspheres in 200 μL 10x PBS, mix well; then, the magnetic bead sample is washed with 10x PBS on the magnetic stand for three times, and finally resuspended with 1000 μL 10x PBS; the microsphere sample is washed by centrifugation at 13800g for 3 min, repeated three times, and finally resuspended with 1000 μL 10x PBS. Take 10 μL of each resuspended sample and dilute to 1 mL with 10x PBS for standby; dilute the magnetic bead / microsphere solution to prepare a mixed sample in a volume ratio of 1:1:1;

[0110] (2) After vortex mixing the sample for 10 s, the mixed sample is introduced into the separation channel through the sample inlet, and PBS buffer is injected into the separation channel through the buffer inlet, and the flow rate ratio of the mixed sample to the buffer is 25 μL / min:75 μL / min, and the sample is collected from the sample outlet, wherein the three sample outlets are named as lateral displacement 2 mm sample outlet, lateral displacement 6 mm sample outlet and lateral displacement 9 mm sample outlet according to the vertical distance from the sample inlet.

[0111] The morphology of the 5.78 μm magnetic beads before and after capture is characterized, as shown in Figure 12 From the figure, it can be seen that there is no other substance on the surface of the magnetic beads before capture, and there is obviously a film-like substance attached to the surface of the magnetic beads after capture, and the size of the film-like substance is about 100 nm, which proves that the magnetic beads have successfully captured the extracellular vesicles. The samples collected from each sample outlet are counted and analyzed by flow cytometry, and the separation effect is evaluated by the proportion of each magnetic bead / microsphere in different sample outlets, and the results are shown in Figure 13 From the figure, it can be seen that under the condition of magnetic field, the three kinds of microparticles are effectively distinguished, and the proportion of 4.95 μm microspheres in the lateral displacement of 2 mm sample outlet is the highest; the proportion of 2.8 μm magnetic beads in the lateral displacement of 6 mm sample outlet is the highest; the proportion of 5.78 μm magnetic beads in the lateral displacement of 9 mm sample outlet is the highest.

[0112] Comparative test example 2

[0113] This comparative test example uses the device described in comparative example 2 to perform separation experiments, and uses the same mixed sample and buffer as test example 2, under the condition of no magnetic field, to investigate the separation effect.

[0114] The test method of the separation result is the same as that of Test Example 2, and the result is shown in the figure, from which it can be seen that the three kinds of magnetic beads / microspheres are difficult to be distinguished in the absence of a magnetic field; and the extracellular vesicles with different protein expressions can be effectively separated in the presence of a magnetic field. Figure 14

[0115] Application Example 1

[0116] This application example provides a method for capturing and separating a subpopulation of extracellular vesicles using the microfluidic chip system device described in Embodiment 1, comprising:

[0117] (1) 2 μL of biotinylated EGFR (biotin-EGFR) antibody stock solution was diluted in 1000 μL of 1x PBS, mixed well, and then 100 μL of 2.8 μm streptavidin-modified magnetic beads was added and incubated at room temperature for 30 min to obtain 2.8 μm magnetic beads with EGFR antibody; 20 μL of biotinylated CD133 (biotin-CD133) antibody stock solution was diluted in 1000 μL of 1x PBS, mixed well, and then 100 μL of 4.95 μm streptavidin-modified microspheres was added and incubated at room temperature for 30 min to obtain 4.95 μm microspheres with CD133 antibody; 50 μL of the above biotin-EGFR antibody solution and 50 μL of the above biotin-CD133 antibody solution were mixed well and then added to 100 μL of 5.78 μm streptavidin-modified magnetic beads and incubated at room temperature for 30 min to obtain 5.78 μm magnetic beads with both EGFR and CD133 antibodies; wherein the 2.8 μm magnetic beads and the 5.78 μm magnetic beads are both magnetic, and the 4.95 μm microspheres are non-magnetic, and then 1x PBS was used for three times of washing to wash away the excess unbound antibodies;

[0118] (2) The culture supernatant containing MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231 cell lines was obtained by centrifuging at 500-1000 g for 3-8 min first and then at 1500-2200 g for 15-25 min; 100 μL of the cell culture supernatant was introduced into the sample inlet of the capture chip, and 100 μL of the biotinylated antibody-modified three kinds of magnetic beads / microspheres sample was introduced into the microsphere inlet, and the capture experiment was carried out at a flow rate of 50 μL / min to obtain the captured sample;

[0119] ​(3) The capture sample is introduced into the separation flow channel through the sample inlet, and PBS buffer is injected into the separation flow channel through the buffer inlet, and the flow rate ratio of the capture sample to the buffer is 25 μL / min:75 μL / min, and the capture sample is subjected to different lateral displacements under the action of the magnetic field to separate, and the samples in different sample outlets are collected after separation, so that the capture and separation of the extracellular vesicle subpopulation are completed.

[0120] Detection: CD63 / CD9-FITC antibody was added to each sample, and fluorescence detection was performed using flow cytometry and fluorescence inverted microscope after incubation in the dark for 40 minutes to detect the expression of CD133 and EGFR proteins in extracellular vesicles from four different malignant cell lines, and the results are shown in FIG. 2. Figure 15 As can be seen from the figure, the expression of the two proteins in extracellular vesicles from normal breast cell lines is low, while the expression of the two proteins in extracellular vesicles from high-malignant cell lines is significantly increased, and the expression of EGFR protein increases with the increase of cell malignancy.

[0121] Application Example 2

[0122] The application example provides a method for capturing and separating extracellular vesicle subpopulations using the microfluidic chip system device described in Example 2, comprising:

[0123] (1) 2 μL of biotinylated EGFR (biotin-EGFR) antibody stock solution was diluted in 1000 μL of 1x PBS, mixed well, and then 100 μL of 2.8 μm streptavidin-modified magnetic beads was added and incubated at room temperature for 30 min to obtain 2.8 μm magnetic beads with EGFR antibody. 20 μL of biotinylated CD133 (biotin-CD133) antibody stock solution was diluted in 1000 μL of 1x PBS, mixed well, and then 100 μL of 4.95 μm streptavidin-modified microspheres was added and incubated at room temperature for 30 min to obtain 4.95 μm microspheres with CD133 antibody. The 2.8 μm magnetic beads have magnetic properties, and the 4.95 μm microspheres have no magnetic properties, and then 1x PBS is used for three times of washing to wash away the excess unbound antibody;

[0124] (2) The culture supernatant containing MCF-10A and MCF-7 cell lines was obtained by centrifuging at 500-1000 g for 3-8 min and then centrifuging at 1500-2200 g for 15-25 min. 100 μL of the culture supernatant was introduced through the sample inlet, and 100 μL of the biotinylated antibody-modified 2 kinds of magnetic beads / microspheres sample was introduced through the microsphere inlet, and the capture experiment was carried out at a flow rate of 20 μL / min to obtain a capture sample.

[0125] (3) The capture sample is introduced into the separation flow channel through the sample inlet, and PBS buffer is injected into the separation flow channel through the buffer inlet, and the flow rate ratio of the capture sample to the buffer is 25 μL / min:25 μL / min, and the capture sample is subjected to different lateral displacement under the action of the magnetic field to separate, and the samples in different sample outlets are collected after separation, so that the capture and separation of the extracellular vesicle subpopulation are completed.

[0126] Application Example 3

[0127] The application example provides a method for capturing and separating an extracellular vesicle subpopulation by using the microfluidic chip system device of the embodiment 1, comprising:

[0128] (1) 2 μL of biotinylated EGFR (biotin-EGFR) antibody stock solution is diluted in 1000 μL of 1x PBS, and after mixing, 100 μL is taken and mixed with 100 μL of 2.8 μm streptavidin modified magnetic beads, and incubated at room temperature for 30 min to obtain 2.8 μm magnetic beads with EGFR antibody; 20 μL of biotinylated CD133 (biotin-CD133) antibody stock solution is diluted in 1000 μL of 1x PBS, and after mixing, 100 μL is taken and mixed with 100 μL of 4.95 μm streptavidin modified microspheres, and incubated at room temperature for 30 min to obtain 4.95 μm microspheres with CD133 antibody; 50 μL of the above biotin-EGFR antibody solution and 50 μL of the above biotin-CD133 antibody solution are mixed, and then mixed with 100 μL of 5.78 μm streptavidin modified magnetic beads, and incubated at room temperature for 30 min to obtain 5.78 μm magnetic beads with EGFR and CD133 antibodies; wherein the 2.8 μm magnetic beads and the 5.78 μm magnetic beads are magnetic, and the 4.95 μm microspheres are non-magnetic, and then washed with 1x PBS for three times to wash away the excess unbound antibodies;

[0129] (2) The culture supernatant containing MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231 cell lines is obtained by centrifuging at 500-1000 g once for 3-8 min, and then centrifuging at 1500-2200 g twice for 15-25 min; 100 μL of the cell culture supernatant is introduced through the sample inlet, and 100 μL of the biotinylated antibody modified 3 kinds of magnetic beads / microspheres sample is introduced through the microsphere inlet, and the capture experiment is carried out at a flow rate of 90 μL / min to obtain a capture sample;

[0130] (3) The above-mentioned capture sample is introduced into the separation flow channel through the sample inlet, and PBS buffer is injected into the separation flow channel through the buffer inlet, and the flow rate ratio of the capture sample to the buffer is 25 μL / min: 100 μL / min, and the capture sample is subjected to different lateral displacement under the action of the magnetic field to separate, and the samples in different sample outlets are collected after separation, so that the capture and separation of the extracellular vesicle subpopulation are completed.

[0131] In the specific embodiment of the present application, the cell lines used: MCF-10A, MCF-7, MDA-MB-468 and MDA-MB-231 are purchased from the China National Experimental Cell Resource Sharing Platform. The magnetic beads / micro spheres used are streptavidin modified magnetic beads / micro spheres, wherein the 2.8 μm magnetic beads are purchased from Thermo Fisher Company (M-270), the 5.78 μm magnetic beads are purchased from Bangs Laboratories Company (UMC0101), and the 4.95 μm micro spheres are purchased from Bangs Laboratories Company (CP01N). The biotinylated CD133 used is purchased from American Tianyi Company, and the biotinylated EGFR antibody is purchased from Sino Biological Company. The CD63-FITC and CD9-FITC antibodies used are purchased from Biolegend Company and Abeam Company, respectively. The instruments used are: ultracentrifuge (OPTIMA XPN-100), microplate reader (SpectraMax i3), 120KV transmission electron microscope (HT-7700), flow cytometer (BD Accuri C6), plasma degumming machine (SY type 500W), split injection pump (WH-SSP-08), handheld digital tesla meter (TD8620).

[0132] The applicant declares that the above-mentioned is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A microfluidic chip system device, characterized by The microfluidic chip system device includes a capture chip and a magnetic separation chip for separating the captured sample; The capture chip includes a capture channel and a J-shaped baffle disposed within the capture channel. The input end of the capture channel is provided with a sample inlet and a microsphere inlet adjacent to each other, and the output end of the capture channel is provided with a sample outlet. The fixed end of the J-shaped baffle is connected to one side of the inner wall of the capture channel, and a gap is left between the arc-shaped free end of the J-shaped baffle and the other side of the inner wall of the capture channel. The arc-shaped free end is used to block the sample flow to form a local micro-vortex. The magnetic separation chip includes a separation channel and a magnetic field device disposed on one side of the separation channel. The separation channel is provided with a sample inlet, a buffer inlet and at least two sample outlets. The microspheres introduced through the microsphere inlet are at least two types of microspheres, wherein the at least two types of microspheres have different antibodies, and at least one type of microsphere is magnetic.

2. The microfluidic chip system device of claim 1, wherein, The sample inlet of the capture channel is connected to a sample entry channel, and the microsphere inlet is connected to a magnetic bead entry channel. The sample entry channel and the magnetic bead entry channel are arranged perpendicularly.

3. The microfluidic chip system device according to claim 1, characterized in that, The capture channel is provided with at least two J-shaped baffles.

4. The microfluidic chip system device according to claim 1, characterized in that, The capture channel is equipped with 5-20 J-shaped baffles.

5. The microfluidic chip system device according to claim 3, characterized in that, The at least two J-shaped stops are arranged in parallel at intervals.

6. The microfluidic chip system device according to claim 3, characterized in that, Two adjacent J-shaped baffles are staggered within the capture channel.

7. The microfluidic chip system device according to claim 3, characterized in that, The distance between two adjacent J-shaped stops is 0.2-4mm.

8. The microfluidic chip system device according to claim 1, characterized in that, The gap between the arc-shaped free end of the J-type stop and the inner wall of the capture channel is 0.8-3mm.

9. The microfluidic chip system device according to claim 1, characterized in that, The heights of the J-shaped baffle and the capture channel are independently 20-80 μm.

10. The microfluidic chip system device according to claim 1, characterized in that, The height of the J-shaped baffle and the capture channel are equal.

11. The microfluidic chip system device according to claim 1, characterized in that, The output end of the separation channel is provided with at least two U-shaped cavities, and each U-shaped cavity is provided with a sample outlet.

12. The microfluidic chip system device according to claim 11, characterized in that, The at least two U-shaped cavities are arranged side by side.

13. The microfluidic chip system device according to claim 11, characterized in that, The inlet is provided at the input end of the separation channel, and the inlet is directly opposite the U-shaped cavity located at the edge of the separation channel.

14. The microfluidic chip system device according to claim 11, characterized in that, The inlet of the separation channel is connected to an injection channel, the width of which is 0.08-0.3 mm.

15. The microfluidic chip system device according to claim 11, characterized in that, The input end of the separation channel is also provided with the buffer inlet.

16. The microfluidic chip system device according to claim 11, characterized in that, The height of the separation channel is 20-80 μm.

17. The microfluidic chip system device according to claim 1, characterized in that, The magnetic field device is located on the side of the separation channel away from the inlet and is positioned close to the separation channel.

18. The microfluidic chip system device according to claim 1, characterized in that, The magnetic field strength of the magnetic field device is 100-300mT.

19. The microfluidic chip system device according to claim 1, characterized in that, The magnetic field strength of the magnetic field device is 180-203 mT.

20. A method for capturing and separating extracellular vesicle subsets, characterized in that, The capture-separation method is performed using the microfluidic chip system device according to any one of claims 1-19, and the capture-separation method includes: (1) An extracellular vesicle sample and at least two types of microspheres are introduced into a capture channel through a sample inlet and a microsphere inlet, respectively, for capture, and then the captured sample is obtained through a sample outlet; wherein, the at least two types of microspheres have different antibodies, and at least one type of microsphere is magnetic; (2) Buffer solution is injected into the separation channel through the buffer inlet, and the captured sample is introduced into the separation channel through the injection port. The captured sample is separated under the action of a magnetic field.

21. The capture and separation method according to claim 20, characterized in that, The antibodies include EGFR and / or CD133.

22. The capture and separation method according to claim 20, characterized in that, The injection flow rate of the extracellular vesicle sample was 10-90 μL / min.

23. The capture and separation method according to claim 20, characterized in that, The capture time in the capture channel is 1-10 minutes.

24. The capture and separation method according to claim 20, characterized in that, The flow rate ratio of the captured sample to the buffer solution is 1:(1-4).

25. An application of the microfluidic chip system device according to any one of claims 1-19, characterized in that, The microfluidic chip system is used to capture and isolate extracellular vesicles from breast cancer cells.

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