Microfluidic device, use method and exosome separation and analysis method

Abstract

The invention belongs to the technical field of micro-nano-scale biological sample separation and analysis, and discloses a microfluidic device, a use method and an exosome separation and analysis method. The separation unit comprises a micro-channel, and the micro-channel is provided with an inlet channel and at least two outlet channels branched from the inlet channel; and the magnetic component is used for applying a magnetic field at a branch opening between the inlet channel and the outlet channel, and the magnetic field is used for enabling the magnetic particles injected into the micro-channel to form an accumulation body with a porous structure at the branch opening, and the accumulation body is used for filtering interception. According to the invention, exosomes or micro-nano particles in a sample can be filtered, intercepted and enriched, spatial distribution visualization of the exosomes is realized through three-dimensional reconstruction and a projection image of a three-dimensional reconstruction model, and a qualitative analysis method based on the image is provided; meanwhile, by quantitatively measuring the interception volume, the device is expected to be used as a concentration detection device.

Description

Technical Field

[0001] The present invention belongs to the technical field of micro-nanoscale biological sample separation and analysis, and specifically relates to a microfluidic device, a use method, and an exosome separation and analysis method. Background Art

[0002] Exosomes are a type of extracellular vesicles with a phospholipid bilayer, with a diameter of about 30-150nm and a density of 1.15-1.19g / mL. Studies have found that exosomes are stably present in various body fluids, such as blood, urine, saliva, tears, semen, amniotic fluid and even breast milk, and are involved in immune balance and regulation, tumorigenesis, metabolite processing and other processes. As a liquid biopsy marker, exosomes have biological information and data information as intermediate results, and the study of their separation and detection technology is crucial. Traditional exosome separation methods such as ultracentrifugation, ultrafiltration, polymer precipitation, affinity capture, etc. are cumbersome, time-consuming, sample damage, high cost and require a large amount of biological fluids, which limits their transformation into clinical applications.

[0003] Microfluidics is a technology that uses channels ranging from tens to hundreds of micrometers to manipulate or process tiny volumes (10 -9 ~10 -18 Microfluidics is the science and technology of fluids (μL). Microfluidics can integrate multiple functions, couple multiple processing steps in one chip, reduce processing time and sample loss, and improve analysis efficiency. The emerging microfluidics technology can enrich exosomes from various biological matrices and classify and separate exosomes and other micro- and nano-scale particles by precisely controlling and manipulating fluids in microchannels.

[0004] In the existing microfluidic technology, there are four major separation methods: the first is to chemically modify the surface of magnetic beads or other micron particles, capture exosomes with the help of binding force, and then use the microarray or microchamber structure in the microfluidic device to intercept and capture the magnetic beads or other micron particles, thereby achieving enrichment of exosomes; the second is to directly chemically modify the surface of the microfluidic channel to capture exosomes in situ; the third is to set up a nanopore-rich membrane in the microfluidic device to achieve the interception and enrichment of exosomes; the fourth is to construct a complex microfluidic channel to achieve the separation of exosomes with the help of surface acoustic waves, fluid inertial force, electric field force, etc.

[0005] However, these separation methods mainly rely on PDMS devices based on soft lithography technology, which require many processing procedures, complex chip structures, and specialized equipment. In addition, the specific separation process requires extremely professional and delicate operations, with many steps and inconvenient operations, and the microchamber channels are prone to frequent blockages due to their complex structures. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a microfluidic device, a method of use, and a method for separating and analyzing exosomes.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, a microfluidic device is provided, comprising:

[0009] at least one separation unit; the separation unit comprises a microfluidic channel having an inlet channel and at least two outlet channels branching from the inlet channel; and

[0010] The magnetic component applies a magnetic field at the branching port between the inlet channel and the outlet channel, and the magnetic field is used to make the magnetic particles injected into the microchannel form a pile with a porous structure for filtering and interception at the branching port.

[0011] Through the above technical scheme, the microchannel is used as a place for dynamic accumulation of magnetic particles. Under the joint driving action of the magnetic field applied by the magnetic component and the microchannel flow field, the magnetic particles injected into the microchannel can gather at the branch mouth of the T-shaped channel to form an accumulation body with filtering and interception function. The accumulation body can filter samples containing separation targets such as exosomes, thereby achieving the separation of the separation targets. The accumulation body formed by accumulation has good flexibility and plasticity, and the device structure is relatively simple, so that it also has the characteristics of simple structure, miniaturization, low cost, small sample amount and reusability.

[0012] In a possible implementation, the magnetic field applied by the magnetic component causes the accumulation body to extend into the outlet channel and form a blocking section 1 with a preset length.

[0013] Through the above-mentioned technical solution, under the action of the magnetic field, the accumulation body can extend into the outlet channel and form a blocking section with a preset length. Through this blocking section, the length of the accumulation body in the direction of the outlet channel can be extended to improve the filtering capacity, and the overall structure can be made more stable under the action of the magnetic field.

[0014] In a possible implementation, the magnetic field applied by the magnetic component causes the accumulation body to extend into the inlet channel and form a second blocking section with a preset length.

[0015] Through the above-mentioned technical solution, the second blocking section can have a preset length in the direction of the inlet channel, and the preset length can make the magnetic particle chain formed by it longer in this direction, further improving the filtering capacity of the accumulation body, and to a certain extent can form a T-shaped structure with the first blocking section, which can make the accumulation body and the microchannel relatively stable.

[0016] In a possible implementation, the direction in which the magnetic poles of the magnetic member point is perpendicular to the plane where the microchannel is located.

[0017] Through the above technical solution, the applicant has verified through multiple experiments that the magnetic pole orientation of the magnetic component has a great influence on the accumulation of magnetic particles. Different magnetic pole orientations will result in different magnetic particle accumulation methods and changes in the arrangement structure. By making the magnetic pole orientation direction of the magnetic component perpendicular to the plane where the microfluidic channel is located, the magnetic particles in the channel will form a complex, tightly arranged chain cluster structure along the direction of the magnetic field, which can completely block the outlet channel and accumulate more tightly and stably, and may have the best retention effect for nanoparticles and exosomes.

[0018] In a possible implementation, the magnetic particles are Fe3O4 magnetic nanoparticles.

[0019] Through the above technical solution, Fe3O4 magnetic nanoparticles are superparamagnetic nanoparticles, which are black crystals with magnetism, insoluble in water, soluble in acid solution, and have unique chemical and magnetic stability, low biological toxicity, low price and other advantages. In addition, the molecular arrangement inside them is regular, and the molecular current orientation is consistent, resulting in external magnetism. When a magnet is brought close to a ferromagnetic material, the molecular current inside the ferromagnetic material also tends to be regularly oriented under the magnetic effect of the magnet, and has magnetism. This process is called magnetization. By using Fe3O4 magnetic nanoparticles as the stacking material for forming the stack, the stack can be made more stable, and Fe3O4 magnetic nanoparticles of different sizes can be selected to stack into stacks with different filtering accuracies.

[0020] In a possible implementation, the separation unit includes two transparent tubes that are perpendicular to each other and connected, and the inner cavities of the two transparent tubes together constitute the microchannel;

[0021] The separation unit and the magnetic component are arranged on a substrate.

[0022] Through the above technical solution, a transparent separation unit that is transparent and easy to observe can be produced by using a transparent tube, and the formed separation unit has a T-shaped microfluidic channel. The microfluidic channel with such a structure can be more conducive to the accumulation of magnetic particles and better play the role of the microfluidic channel. The separation channel is arranged on a substrate to form a chip. The chip has a simple structure and a low production cost, and its use method is also simpler.

[0023] In a possible implementation, one end of the outlet channel close to the inlet channel is a bent structure.

[0024] Through the above technical solution, the bending structure can increase the volume and path length of the accumulation formed by the aggregation of magnetic particles, complicate the channel path, increase the water flow range, increase the interception amount, and improve the interception efficiency, thereby improving the filtration capacity and better separating exosomes.

[0025] In a possible implementation, the separation unit is provided in plurality and the plurality of separation units are connected in series or in parallel;

[0026] When a plurality of the separation units are connected in series, the outlet channel of the upper separation unit is respectively connected to the inlet channel of one lower separation unit, and the filter aperture of the stack of the upper separation unit is larger than the filter aperture of the stack of the lower separation unit;

[0027] When a plurality of the separation units are connected in parallel, the inlet channel of each separation unit is simultaneously connected to a sample inlet pipeline, and the filter apertures of the stacks of each separation unit are the same.

[0028] Through the above-mentioned technical scheme, multiple separation units can be connected in series or in parallel according to actual needs, so as to facilitate multi-stage separation or same-stage batch separation, which can be beneficial to improve separation accuracy and realize batch separation processing. The series connection method can realize continuous multi-stage separation of samples (particles of different sizes in the sample are classified and separated step by step), improve the overall processing capacity and flexibility of the system, make the various separation steps more closely coordinated to improve the separation efficiency, and the parallel connection method can carry out multiple identical microfluidic experiments at the same time, improve the efficiency of sample processing and the ability of parallel operation, and is suitable for applications in high-throughput analysis and repetitive experiments.

[0029] In a second aspect, a method for using the microfluidic device according to any of the above technical solutions is also provided, comprising the following steps:

[0030] A multi-channel suction device is used, and the multi-channel suction device is connected to each outlet channel;

[0031] The solution containing magnetic particles is sucked from the inlet channel into the microchannel through the multi-channel suction device at least twice, and the flow rate of the solution sucked in each time is gradually reduced, and the magnetic particles form an accumulation at the branch port under the action of the magnetic field applied by the magnetic field component;

[0032] By suctioning the multi-channel suction device, the sample enters from the inlet channel of the microfluidic channel and passes through the stacking body for separation and enrichment.

[0033] Through the above technical scheme, the multi-channel suction method can be adapted to multiple outlet channels, and the magnetic particles can be more evenly docked at the outlet channel near the intersection with a relatively balanced flow rate and flow rate, and the waste of samples can be reduced by stacking at different and gradually decreasing flow rates, and the aggregation and sedimentation of magnetic particles in the sample tube can be prevented, shortening the experimental time, which is also to reduce the impact on the magnetic particles that have been accumulated at the T-shaped intersection, and at the same time, relying on the attraction of the magnet and the inertia of the fluid, the microchannel is gradually blocked to construct an accumulation body with a porous structure. Correspondingly, when injecting fluid, the multi-channel suction method can also enable the sample to be filtered more evenly through each branch of the accumulation body, and the separation effect is better. In addition, by the suction sampling method, the passive transport of the fluid can be realized, which can effectively avoid the problems of channel blockage, uneven sampling, poor operation stability and repeatability caused by the gravity sedimentation of other micro-nano substances such as magnetic particles and exosomes, thereby ensuring the reliability of the experiment, improving the efficiency of the experiment and reducing the consumption of samples and reagents.

[0034] In a third aspect, a method for separating and analyzing exosomes is also provided, based on a microfluidic device according to any of the above technical solutions, comprising the following steps:

[0035] Positioning fluorescent dyes at locations in the sample where exosomes can be observed;

[0036] The sample containing the exosomes is sent into the separation unit of the microfluidic device through a pumping device, so that the sample passes through the accumulation body formed in the microchannel for separation;

[0037] Using a microscope to obtain a fluorescent image of the exosomes trapped in the microfluidic device, and importing the image into an image processing software for image processing to obtain a processed image;

[0038] Perform qualitative and quantitative analysis on the processed images.

[0039] Through the above technical solution, real-time visualization of fluorescent dyed particle capture can be achieved through real-time observation under a microscope, and three-dimensional image reconstruction can be performed using software such as ImageJ and Imaris viewer to achieve stereoscopic display and quantitative analysis of the separation effect, thereby obtaining relevant data information about exosomes.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The microfluidic device of the present invention uses a microchannel with a T-shaped intersection as a place for dynamic accumulation of magnetic particles. Under the joint driving action of an external magnetic field and a microchannel flow field, magnetic nanoparticles aggregate to form an accumulation body with interconnected nano-sized pores, thereby being able to filter, intercept and enrich exosomes or micro-nanoparticles in the sample. It also has the characteristics of miniaturization, simple operation, low cost, good repeatability and good stability, and provides a reliable separation and analysis platform for broader biomedical applications of exosomes.

[0042] The method for using the microfluidic device of the present invention adopts a suction injection mode for sampling magnetic particles and exosome samples, which can realize passive fluid transport, and can effectively avoid channel blockage, uneven injection, and poor operational stability and repeatability caused by gravity sedimentation of other micro-nano substances such as magnetic particles and exosomes, thereby ensuring the reliability of the experiment, improving the efficiency of the experiment and reducing the consumption of samples and reagents. In addition, by successively aspirating at different and gradually decreasing flow rates to accumulate magnetic particles, it can reduce sample waste, prevent the aggregation and sedimentation of magnetic particles in the sample tube, and reduce the impact on the accumulated magnetic particles at the T-intersection, which is more conducive to the accumulation to form an accumulation body.

[0043] The exosome separation and analysis method of the present invention realizes real-time visualization of the capture of fluorescent dyed particles through real-time observation under a microscope, and uses image processing software to perform three-dimensional image reconstruction to realize stereoscopic display and quantitative analysis of the separation effect, thereby obtaining relevant data information about exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a three-dimensional schematic diagram of a fluid control unit of a microfluidic device;

[0045] Figure 2a This is a physical picture of the microfluidic device;

[0046] Figure 2b This is a schematic diagram of the connection of microfluidic devices in series. In this figure, inlet means import and outlet means export.

[0047] Figure 2c Schematic diagram of the comparison of the microfluidic device when it changes from a T-shaped straight channel structure to different bending structures;

[0048] Figure 3 The microscope images of Fe3O4 magnetic particle accumulation at different flow rates are as follows: the Fe3O4 flow rates are 2000 μL / h in process a, 4000 μL / h in process b, and 6000 μL / h in process c. The scale bar is 200 μm.

[0049] Figure 4Microscope images of Fe3O4 magnetic particles of different sizes piled up in the same time process. The sizes of Fe3O4 are 50nm in process a, 100nm in process b, and 200nm in process c. d is a schematic diagram of L1 and L2 lengths and a columnar distribution diagram of magnetic particle piles of different sizes.

[0050] Figure 5 It is the accumulation process of magnetic particles under different external magnetic fields of cubic permanent magnet under the same time conditions, where process a is X-axis, process b is Y-axis, process c is Z-axis, and axis is translated as axis;

[0051] Figure 6 Figure 1 is a repeatability test of a magnetic nanoparticle stack, wherein Figure a is an optical image of a magnetic nanoparticle stack (n=10), Figure b is a schematic diagram of the length L1, L2 of the stack and the corresponding distribution area, Figure b1 is a bar graph of the length L1, L2 of the stack under 10 repeatability tests, and Figure b2 is a bar graph of the distribution area of ​​the stack under 10 repeatability tests, and area in the figure is translated as area;

[0052] Figure 7 The water impact test verifies the stability of the magnetic nanoparticle stack, where Figure a shows 50nm Fe3O4 magnetic particles, Figure b shows 200nm Fe3O4 magnetic particles, and the scale bar is 400μm;

[0053] Figure 8 Figure 2 is the effect of an external magnetic field on the separation performance of magnetic particle accumulation bodies, where Figure a is a confocal maximum intensity projection image (MIP) of different external magnetic fields, Figure b is a three-dimensional image, Figure c is a three-view image, and Figure d is a comparative schematic diagram of retention volume and separation efficiency under different external magnetic fields, with a scale of 200 μm; Merge in the figure is translated as superposition field, Fluorescence is translated as fluorescence field, Bright field is translated as bright field, Top view is translated as top view, frontview is translated as front view, Sidewiew is translated as side view, Orientation is translated as direction, Volume is translated as volume, and Separationefficiency is translated as separation efficiency;

[0054] Fig. 9 The effect of magnetic particle size and process flow on the separation performance of magnetic nanoparticle stacks, where Figure a is a confocal maximum brightness projection, Figure b is a three-dimensional image, Figure c and Figure d are comparisons of retention volume and separation efficiency, respectively, the scale bar is 200μm, and Merge in the figure is translated as superposition field, Fluorescence is translated as fluorescence field, Bright field is translated as bright field, Volume is translated as volume, and Separationefficiency is translated as separation efficiency;

[0055] Fig.10 Figure 1 is the effect of sample suction flow rate on the separation performance of magnetic particle accumulation, where Figure a is a confocal MIP image, Figure b is a schematic diagram of the change of characteristic value Lw with flow rate, and Figure c is a three-dimensional spatial distribution image (Q ex =400 μL / h), Figure d is a YZ fluorescence projection image; Figure e and Figure f are schematic diagrams of quantitative analysis of retention volume and separation efficiency, with a scale bar of 200 μm;

[0056] Fig.11 This is a study of the separation stability of the microfluidic device, where Figure a is a confocal MIP image, Figure b is a three-dimensional image, and Figures c and d are quantitative analyses of the retention volume and separation efficiency;

[0057] Fig.12 The microfluidic device is used to separate particles of different sizes. Figure a is a confocal MIP image, Figure b is a three-dimensional image, and Figures c and d are schematic diagrams of quantitative analysis of retention volume and separation efficiency. Chlorella in the figure is translated as Chlorella, and Sample is translated as Sample.

[0058] Fig.13 This is the separation and analysis of milk exosomes with different concentrations, where Figures a and b are TEM images and particle size distribution diagrams of milk exosomes, Figures c and d are three-dimensional spatial distribution images and co-localization images of fluorescently stained exosomes, and Figures e and f are schematic diagrams of quantitative analysis of retention volume and separation efficiency. In the figure, Size Distribution by Number is translated as size distributed by number, Percentage is translated as percentage, Exosomes is translated as exosomes, and Particles is translated as particles;

[0059] Fig.14 The microfluidic device is used for separation and analysis of milk exosomes and nanoparticles mixture, wherein Figure a is a three-dimensional spatial distribution image, Figures b, c and d are three-view projections, and Figures e and f are schematic diagrams of quantitative analysis of retention volume and separation efficiency, with a scale bar of 200 μm;

[0060] Fig.15 The separation of exosomes from clinical serum and urine samples, where Figure a is a three-dimensional fluorescence co-localization image of clinical serum, Figure b is a three-view projection, and Figure c is a volume diagram of trapped exosomes; Figure d is a three-dimensional fluorescence co-localization image of clinical serum, Figure e is a three-view projection, and Figure f is a volume diagram of trapped exosomes; the scale bar is 200μm, and Serumsample in the figure is translated as serum sample;

[0061] Fig.16These are MDA.MB.231 cells and HSC-3 cells. Figure a is a laser confocal microscope image, Figure b is a particle size distribution diagram, and Figure c is a 3D image. Frequency in the figure is translated as frequency, and Diameter is translated as diameter.

[0062] In the figure: 1-PP needle; 2-entrance channel; 3-slot; 4-substrate; 5-exit channel; 6-bending structure. DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0064] Please refer to Figure 1 As shown, an embodiment of the present application provides a microfluidic device, comprising: at least one separation unit; the separation unit comprises a microfluidic channel, the microfluidic channel having an inlet channel 2 and at least two outlet channels 5 branching from the inlet channel 2; and a magnetic component, applying a magnetic field at a branching port between the inlet channel 2 and the outlet channel 5, the magnetic field being used to cause the magnetic particles injected into the microfluidic channel to form an accumulation having a porous structure at the branching port for filtration and retention.

[0065] Among them, the microfluidic channel of the separation unit serves as the accumulation place of magnetic particles. Magnetic particles and samples containing separation targets such as exosomes enter the microfluidic channel and can flow out from the outlet channel 5. The outlet channel 5 of the microfluidic channel can be set to two or more, which branch out from the inlet channel 2, and form a flow channel structure with an angle between the outlet channel 5 and the inlet channel 2. Such a structure can form a flow field effect and facilitate the accumulation of magnetic particles at the branch port. At the same time, under the action of the magnetic field, the accumulation forms an accumulation body that can be filtered and intercepted. The magnetic component is used to form a magnetic field, which can cause the magnetic particles to be adsorbed and accumulated when passing through the branch port.

[0066] It can be understood that the angle between the outlet channel 5 and the inlet channel 2 can be a right angle, so that the microchannel forms a T-shaped structure, or it can be an acute angle or an obtuse angle, which complicates the channel path, increases the water flow range, and reduces the impact intensity of the water flow on the magnetic particle accumulation group, so that a relatively stable and effective accumulation body can be accumulated for separation.

[0067] In some scenarios, such as during manufacturing, the product formed may only include a separation unit with a microfluidic channel and a magnetic component. When in use, the product may also include configured magnetic particles, without limitation.

[0068] Through the above technical scheme, the microchannel is used as a place for dynamic accumulation of magnetic particles. Under the joint driving action of the magnetic field applied by the magnetic component and the microchannel flow field, the magnetic particles injected into the microchannel can gather at the branch mouth of the T-shaped channel to form an accumulation body with filtering and interception function. The accumulation body can filter samples containing separation targets such as exosomes, thereby achieving the separation of the separation targets. The accumulation body formed by accumulation has good flexibility and plasticity, and the device structure is relatively simple, so that it also has the characteristics of simple structure, miniaturization, low cost, small sample amount and reusability.

[0069] In order to obtain a stable stack with a stacking arrangement structure that is more suitable for filtration, the applicant has conducted multiple experimental verifications and found that the magnetic pole orientation of the magnetic component has an impact on the stacking mode of magnetic particles at the branch mouth of the microchannel, and that by changing the magnetic pole orientation to regulate the spatial distribution of the magnetic field, the stacking mode of the magnetic particles can be controlled.

[0070] By applying a magnetic field through the magnetic component to extend the accumulation body into the outlet channel 5 and form a blocking section with a preset length, an accumulation body that can meet the separation requirements and has a preset length can be obtained. This length can improve the filtering capacity and help make the overall structure more stable under the action of the magnetic field.

[0071] On this basis, the magnetic field applied by the magnetic member can also be used to extend the deposit into the inlet channel 2 and form a second plugging section with a preset length. The second plugging section can have a preset length in the direction of the inlet channel 2, and the preset length can make the magnetic particle chain formed by it longer in this direction, further improving the filtering capacity of the deposit, and to a certain extent, it can form a T-shaped structure with the first plugging section, which can make the deposit and the microchannel relatively stable.

[0072] In some specific implementation processes, the magnetic components can be configured according to actual needs. For example, a cubic neodymium iron boron permanent magnet (4mm×4mm×4mm, magnetic field strength of 3520gs) can be used as a magnetic component, or an electromagnet that can generate a magnetic field with equivalent strength can be used as a magnetic field component, or an electromagnetic coil can be used. There is no limitation and the configuration can be selected according to actual conditions or actual needs.

[0073] Preferably, the magnetic pole pointing direction of the magnetic component is arranged in a layout perpendicular to the plane where the microfluidic channel is located. In three-dimensional space, a cube permanent magnet is used as a magnetic component, and its six faces are classified according to X, Y, and Z axes, specifically divided into X-axis direction surface (X-axis), Y-axis direction surface (Y-axis) and Z-axis direction surface (Z-axis). Wherein, the X-axis direction surface of the magnet refers to the direction pointed to by the N pole and S pole of the magnet, that is, the direction of the magnetic pole points to the entrance channel direction of the microfluidic channel. It was found that the magnetic particles were tightly packed in a smooth arc under the X-axis. Under the Y-axis, the magnet has strong magnetism at two vertices. Most of the magnetic particles are still accumulated at the intersection of the T-channel, and the magnetic particles near the entrance end are sparsely arranged in a chain along the direction of the magnetic field. And the magnetic particles in the channel under the Z-axis form a complex, tightly arranged chain cluster structure along the direction of the permanent magnet magnetic field, which can completely block the channel. The results show that when the Z-axis of the magnet, that is, the direction in which the magnetic poles of the magnetic component point is perpendicular to the plane where the microfluidic channel is located, the magnetic particles are stacked more tightly and stably than in other axial directions, and the retention effect on nanoparticles and exosomes may be the best.

[0074] Specifically, the magnetic component can be easily disassembled, for example, a magnet is used as the magnetic component and is installed in a slot 3 on the front side of the separation unit by glue.

[0075] In a preferred embodiment of the magnetic particles, the magnetic particles are Fe3O4 magnetic nanoparticles.

[0076] Fe3O4 magnetic nanoparticles are a kind of superparamagnetic nanoparticles, which are black crystals with magnetism. They are insoluble in water but soluble in acid solution. They have unique chemical and magnetic stability, low biological toxicity, low price and other advantages. In addition, the molecular arrangement inside them is regular, and the molecular current orientation is consistent, which leads to external magnetism. When a magnet is brought close to a ferromagnetic material, the molecular current inside the ferromagnetic material also tends to be regularly oriented under the magnetic effect of the magnet, and becomes magnetic. This process is called magnetization. By using Fe3O4 magnetic nanoparticles as the stacking material to form a stack, the stack can be made more stable, and Fe3O4 magnetic nanoparticles of different sizes can be selected to stack into stacks with different filtration accuracies.

[0077] Of course, other magnetic particles that can achieve the same stacking and separation effects may also be used.

[0078] In one embodiment, in combination Figure 2a As shown, the separation unit may include two mutually perpendicular and connected transparent tubes, and the inner cavities of the two transparent tubes together constitute the microchannel; the separation unit and the magnetic component are arranged on a substrate 4.

[0079] In this way, a separation unit can be made by using a transparent tube, which is convenient for observation and analysis, and the formed separation unit has a T-shaped microchannel. The microchannel with such a structure can be more conducive to the accumulation of magnetic particles and better play the role of the microchannel. The separation channel is arranged on the substrate 4 to form a chip. The chip has a simple structure and low manufacturing cost, and its use method is also simpler. PP needles 1 can be respectively arranged at the ends of the inlet channel 2 and the outlet channel 5 formed by the transparent tube to facilitate connection with the pumping device. The substrate 4 can be a glass slide.

[0080] In order to further improve the separation capability of the accumulation formed in the microchannel, the end of the outlet channel 5 close to the inlet channel 2 can be set as a bending structure 6, and the bending structure 6 increases the volume and path length of the accumulation formed by the aggregation of magnetic particles.

[0081] Combination Figure 2c As shown, there are roughly two types of bending structures 6 and are not limited to these two types. One is that one end of the outlet channel 5 near the branch port or intersection is set to be inclined, even if the angle relationship between the end and the inlet channel 2 is set from a vertical angle to an acute angle relationship, so that the volume of the accumulation body per unit length in the horizontal direction is larger, the extension path is longer, and the water flow formation is also increased; the other is that one end of the outlet channel 5 near the branch port or intersection is set to an S-shaped bending structure 6, which can complicate the channel path, increase the water flow range, increase the interception amount, and improve the interception efficiency.

[0082] In some application scenarios, combined with Figure 2b As shown, in order to achieve batch separation of samples, or in order to achieve continuous multi-stage separation of samples, multiple separation units can be provided and the multiple separation units can be connected in series or in parallel, and the corresponding separation requirements can be achieved through the series or parallel connection.

[0083] When multiple separation units are connected in series, the outlet channel of the upper separation unit is connected to the inlet channel 2 of a lower separation unit, and the filter aperture of the stack of the upper separation unit is larger than the filter aperture of the stack of the lower separation unit. Such a series connection method can realize continuous multi-stage separation of samples (different-size particles in the sample are classified and separated step by step), improve the overall processing capacity and flexibility of the system, and make the various separation steps more closely coordinated to improve the separation efficiency.

[0084] When multiple separation units are connected in parallel, the inlet channel 2 of each separation unit is connected to a sample injection pipeline at the same time, and the filter pore size of the stack of each separation unit is the same. The parallel connection mode can simultaneously carry out multiple identical microfluidic experiments, improve the efficiency of sample processing and the ability of parallel operation, and is suitable for the application of high-throughput analysis and repetitive experiments.

[0085] Specific, combined Figure 2bAs shown, magnetic particles of the same size are first accumulated at the magnets b and c through the inlet, and then magnetic particles of another size are accumulated at the magnet a. After the accumulation is completed, a series connection is formed, and then the sample to be separated is pumped in. When parallel connection is required, magnet 1 is not set and accumulation is not performed.

[0086] In the specific implementation process, the magnetic particles can be dissolved in a solution to form a magnetic particle solution, which can be easily injected into the microchannel, such as a Fe3O4 magnetic particle solution. In order to meet the separation goals of different sizes, the magnetic particles can be selected in different sizes according to the needs to obtain different separation effects, such as a Fe3O4 magnetic particle solution with a particle size of 50nm, 100nm or 200nm. The applicant has found through multiple experiments that the larger the particle size of the magnetic particles, the greater the suction force on the magnetic particles and the denser the accumulation. Therefore, samples with different separation accuracies can be separated by magnetic particles of different sizes.

[0087] The embodiments of the present application also provide a method for using any of the above-mentioned microfluidic devices, which involves separation of nanoscale particles, comprising the following steps:

[0088] Step S1: using a multi-channel suction device, the multi-channel suction device is connected to each outlet channel.

[0089] In this step, the multi-channel suction device can be implemented in the liquid suction working mode, so that the solution containing magnetic particles can be passively transported, which is more conducive to accumulation, and the multi-channel suction device can make the sample passing through each outlet channel pass at a more balanced flow rate and flow rate, so that the accumulation body is more symmetrical and the molding quality is improved. The multi-channel suction device can be a dual-channel micro-injection pump.

[0090] Step S2: The solution containing magnetic particles is sucked from the inlet channel into the microchannel through a multi-channel suction device at least twice, and the flow rate of the solution is gradually reduced each time, and the magnetic particles form an accumulation at the branch port under the action of the magnetic field applied by the magnetic field component.

[0091] In this step, the amount of magnetic particles accumulated in the channel increases with the increase of injection speed and passage time. In order to reduce the waste of samples, and to prevent the aggregation and sedimentation of magnetic particles in the sample tube and shorten the experimental time, it can be injected at least twice with a decreasing flow rate, the purpose is to reduce the impact on the accumulated magnetic particles at the T-intersection, and at the same time rely on the attraction of the magnet and the inertia of the fluid to gradually block the microchannel to build an accumulation body with a porous structure. Finally, these two combined flow rate modes are used as the final flow rate. For example, 6000μL / h is first selected as the initial speed of the magnetic particles, and the passage time is 90s. Then, the injection pump continues to pass 2000μL / h for 90s. Such a two-stage combined flow rate mode is used as the final flow rate.

[0092] Step S3: Through suction by a multi-channel suction device, the sample enters from the inlet channel of the microfluidic channel and passes through the stacking body for separation.

[0093] In this step, the multi-channel suction device realizes the passive transport of biological liquid samples, improves the experimental efficiency and reduces the consumption of samples and reagents. In addition, the multi-channel suction device allows samples passing through each outlet channel to pass at a more balanced flow rate and flow rate, thereby improving the filtering effect.

[0094] After the separation is completed, the microfluidic device can be used to directly perform in situ analysis without desorption.

[0095] If it is necessary to desorb or recover micro-nano particles such as exosomes, the external magnetic field can be removed and the syringe pump can be operated in the liquid discharge mode to recover the magnetic particles and exosome samples at the same time. Subsequently, the magnet can be used to adsorb the magnetic particles in the recovery liquid to obtain the recovered exosome samples.

[0096] In some application scenarios, the device can also be used in the separation and detection of other micron-sized biological particles such as GY-D12 protein core Chlorella, tumor cells, etc., and also has significant effects.

[0097] The present application also provides an exosome separation and analysis method based on a microfluidic device according to any of the above technical solutions, comprising the following steps:

[0098] Step S1: Position the fluorescent dye at the site where the exosomes in the sample can be observed.

[0099] In this step, the exosomes in the sample are located by fluorescent dyes, which can facilitate subsequent observation and further related image processing and analysis. The location method can be to use dual fluorescent dyes for fluorescence detection, and the two dyes are located in the membrane and on the membrane of the exosomes respectively.

[0100] Step S2: The sample containing exosomes is sent into the separation unit of the microfluidic device through a pumping device, so that the sample passes through the accumulation body formed in the microchannel for separation.

[0101] In this step, the sample containing exosomes can be separated through the pre-stacked stack under the action of suction, such as a dual-channel microinjection pump, so that the exosomes can be intercepted and confined in the gaps between the magnetic particles.

[0102] Step S3: Use a microscope to obtain a fluorescent image of the exosomes trapped in the microfluidic device, and import the image into an image processing software for image processing to obtain a processed image.

[0103] In this step, the fluorescently stained exosomes and other model particles are observed in real time by combining laser confocal microscopy, and three-dimensional image reconstruction is performed using software such as ImageJ and Imaris viewer to obtain the processed image. Specifically, after the z-stack series of fluorescent images are obtained using a confocal microscope, they can be imported into ImageJ for processing, and the area (ImageJ->Stacks->Measure Stack) and volume (ImageJ->Stacks-statistics) of the fluorescent area can be measured by selection or division. In addition, the image can be further operated and analyzed by installing plug-ins or custom development. To achieve three-dimensional visualization of fluorescent images, ImageJ's 3D visualization plug-ins: such as 3D Viewer (Plugins->3D Viewer) and Volume Viewer (Plugins->Volume Viewer). Preferably, the Imaris viewer software uses advanced image rendering technology, which can efficiently process large-scale data sets and achieve fast rendering speed and smooth interactive experience. It provides a wealth of advanced visualization functions, including volume rendering, surface rendering, projection rendering, transparency control and lighting effects. The rendering effect can be flexibly adjusted as needed to display the details and structures in the image data. In addition, Imaris also supports the three-dimensional display of fluorescence co-localization.

[0104] Step S4: Perform qualitative and quantitative analysis on the processed image.

[0105] In this step, the processed image can be quantitatively analyzed to obtain data information including at least the number and concentration of exosomes.

[0106] For example, exosomes extracted from milk were selected as a model to evaluate the separation performance of the microfluidic device based on magnetic nanoparticle stacks on exosomes. Dual fluorescent dyes were used for fluorescence detection, and the two dyes were located in the membrane and on the membrane of the exosomes, respectively. 6 particles / mL to 3.14×10 11 Exosomes with 6 different concentrations in the range of particles / mL were introduced into the chip. Fig.13 The three-dimensional fluorescence images obtained by image processing can directly show the retention of exosomes, with a concentration as low as 10 6 Pieces / mL.

[0107] The following experimental examples are provided for the relevant performance parameters and usage of the microfluidic device:

[0108] Fabrication of a microfluidic device integrating magnetic nanoparticle stacks. Figure 1 As shown in the figure, a T-shaped microfluidic device is designed. The size of the device is 25mm×75mm, and it is mainly composed of a T-shaped square glass capillary and a glass slide. With the glass slide as the base, the T-shaped structure is composed of two square glass capillaries (0.20mmID×0.10mm Wall) with lengths of 1cm and 1.5cm. The microfluidic device includes an inlet and two outlets. The magnetic particle and exosome sample injection method adopts a dual-channel aspiration mode, which can realize the passive transport of fluids, improve the efficiency of the experiment and reduce the waste of samples and reagents. A PMMA card slot is fixed with glue at the intersection of the T-shaped square tube. The card slot is used to place a cubic neodymium iron boron permanent magnet (4mm×4mm×4mm, magnetic field strength of 3520gs). At the same time, the magnet can be removed at any time when cleaning the device. Figure 2a As shown. Under the combined driving force of magnetic field and fluid power, the magnetic field induces the magnetic nanoparticles to gradually assemble into stacks in the T-shaped microchannel.

[0109] Example 1: Controllable construction of a microfluidic device for magnetic particle accumulation

[0110] 1. Flow rate and accumulation time of magnetic particles: To study the accumulation of magnetic particles at different flow rates and accumulation times in the channel, magnetic particle solutions with different flow rates (2000μL / h, 4000μL / h and 6000μL / h) were pumped into the channel. The results showed that the amount of magnetic particle accumulation in the T-type channel increased with the increase of the suction flow rate and suction time. Figure 3 As shown. In order to reduce sample waste, prevent the aggregation and sedimentation of magnetic particles in the sample tube and shorten the experimental time, 6000μL / h is selected as the initial velocity of the magnetic particles. For example, the magnetic particles are first pumped at a flow rate of 6000μL / h for 90s, and then at a flow rate of 2000μL / h for 90s, named E90s-90s. This will reduce the impact of the fluid on the accumulated magnetic particles to a certain extent, which will help to achieve a tighter stacking of the magnetic particles in the T-shaped position. This fixed process will be used to construct magnetic nanoparticle deposits in the future.

[0111] 2. Magnetic particle size: To study the accumulation of magnetic particles of different sizes in the channel, select solutions of magnetic particles of different sizes (50nm, 100nm and 200nm) and pump them into the channel. Figure 4 As shown, within a certain size range, the larger the magnetic particle size, the more magnetic particles accumulate per unit time.

[0112] 3. External magnetic field and magnetic particle accumulation method

[0113] In three-dimensional space, the six faces of a cube permanent magnet are classified according to the X, Y, and Z axes, specifically divided into the X-axis direction face (X-axis), the Y-axis direction face (Y-axis), and the Z-axis direction face (Z-axis). Figure 5 As shown. The X-axis direction of the magnet refers to the direction pointed by the N pole and S pole of the magnet, that is, the direction of the magnetic pole points to the entrance channel of the microfluidic channel. The results show that the magnetic particles are tightly packed in a smooth arc shape under the X-axis, as shown in Figure 5 As shown in part a of the figure. Under the Y-axis, the magnetism at the two vertices of the magnet is stronger. Most of the magnetic particles are still accumulated at the intersection of the T-shaped channel, and the magnetic particles near the entrance are arranged in a sparse chain along the direction of the magnetic field, as shown in Figure 1. Figure 5 As shown in part b. The magnetic particles in the channel under the Z-axis form a complex chain cluster structure along the magnetic field direction of the permanent magnet, which can completely block the channel, as shown in Figure 5 As shown in part c, the results show that the magnetic particles are more compact and stable under the Z-axis of the magnet, which may have the best retention effect on nanoparticles and exosomes.

[0114] Example 2: Microfluidic device and stability test

[0115] In order to study the reproducibility of magnetic particle stacking structure, the same microfluidic device was used under the same experimental conditions to pump 200nm Fe3O4 magnetic particle solution to build stacking bodies, and the experiment was repeated 10 times. The stacking effect image taken under a stereo microscope, and the length and area of ​​the magnetic particle stacking bodies were measured by ImageJ and statistical software OriginPro 2024, as shown in the figure. Figure 6 The lengths L1, L2 and distribution areas of magnetic particle accumulation in the microchannel of 10 repetitive experiments are basically consistent, indicating that the microfluidic device exhibits good detection repeatability.

[0116] Example 3: Stability test of magnetic nanoparticle stack

[0117] The stacking stability of Fe3O4 magnetic particles of different sizes (50nm, 100nm and 200nm) under the Z-axis of a permanent magnet was tested. PBS solutions with different flow rates were introduced into the channel in sequence: (1) 400μL / h for 6min; (2) 600μL / h for 4min; (3) 800μL / h for 3min; (4) 1000μL / h for 2min; (5) 2000μL / h for 2min; (6) 4000μL / h for 2min. Figure 7 a and Figure 7As shown in b, the larger 200nm Fe3O4 magnetic particles have stronger stacking stability. When the suction flow rate is lower than 6000μL / h, the shape of the stack remains almost unchanged. The results show that the stack assembled by magnetic particles with larger diameters is more solid and has better stability.

[0118] Example 4: Microfluidic device for separation and analysis of particle samples

[0119] To determine the relationship between the magnetic field arrangement and the interception effect of the magnetic particle stack, the interception of the magnetic particle stack on 30nm PS fluorescent nanospheres was observed under a laser confocal microscope. The T-type microfluidic device extracted the same flow rate of 0.05% concentration of 200nm Fe3O4 solution (E90s-90s). After the magnetic particle stack was stable, the 30nm green fluorescent PS particle solution was pumped at a flow rate of 400μL / h for 12min (concentration of 6.73×10 12 The retention distribution of PS particles can be seen by confocal fluorescence tomography, such as Figure 8 a. Importing z-stack images into Imaris viewer software can realize three-dimensional visualization of fluorescence within the channel ( Figure 8 b) and three-view projection ( Figure 8 c). Statistical analysis results show that the volume of PS particles trapped along the X-axis, Y-axis, and Z-axis are 488 992.74 μm 3 、140 873.22μm 3 and 738 822.68 μm 3 , the separation efficiencies were 9.88%, 2.85% and 14.93% respectively. Figure 8 As shown in d, the Z-axis retained the most 30 nm PS particles and had the best retention effect, so the Z-axis of the magnet was used in subsequent experiments.

[0120] 2. Effects of magnetic particle size and suction time on the separation performance of magnetic particle stacks

[0121] By selecting a stack of magnetic particles of different sizes, it is expected that exosomes of different size ranges can be trapped. Fig. 9 a. Fig. 9As shown in Figure b, under the same suction operation process E90s-90s, the spatial distribution of PS particles basically conforms to the theoretical mechanical analysis. The 200nm magnetic particles have a larger particle size, and the greater the magnetic field suction force they are subjected to, the more firmly they can be stacked in the channel. According to the analysis of ImageJ and statistical software, the separation efficiency of the accumulation of 50nm Fe3O4 magnetic particles for 30nm PS particles is only 4.35%, while the separation efficiency of the accumulation of 200nm Fe3O4 magnetic particles is as high as 15.29%. The experimental results show that 200nm Fe3O4 magnetic particles can accumulate more effectively and have the strongest ability to intercept 30nm PS particles. Therefore, 200nm Fe3O4 is used in subsequent experiments to obtain the best separation effect.

[0122] In order to explore the relationship between the magnetic particle suction operation process and the separation performance of the magnetic particle accumulation body, 200nm magnetic particles were selected as the basic particles of the magnetic particle accumulation body, and the process procedures were: (1) E90s-90s; (2) E60s-30s. Fig. 9 c. Fig. 9 d shows that the separation efficiency of the magnetic particle stack constructed by the E60s-30s process is only 9.48%. The research results show that the separation efficiency of the 200nm Fe3O4 magnetic particle stack constructed by the E90s-90s process is the highest, reaching 15.29%.

[0123] 3. Effect of sample aspiration flow rate on the separation performance of magnetic particle stacks

[0124] The sample suction flow rate affects the stability of the magnetic particle stack, which in turn affects the retention of fluorescent nanoparticles and exosomes and their spatial distribution in the stack. The 200nm Fe3O4 solution was introduced into the device for magnetic particle stacking with a process flow of E60s-30s. The 30nm PS particle sample (concentration of about 6.73×10 12 The total amount of aspiration was kept consistent, and the sample injection speed was changed to 400μL / h, 800μL / h, 1200μL / h, 1600μL / h and 2000μL / h, and the corresponding injection time was 12min, 6min, 4min, 3min and 2min25s respectively. Fig.10 As shown in a.

[0125] As the suction flow rate increases, the characteristic dimension Lw value decreases, such as Fig.10 As shown in b; Fig.10 As shown in d, the width of the front view brightness projection distribution becomes wider. Fig.10 As shown in Figure e, the retention volume of PS particles is not much different, but its distribution has changed. Fig.10As shown in Figure 5, the separation efficiencies are 9.48%, 5.48%, 6.63%, 6.82%, and 5.18% when the suction flow rates are 400 μL / h, 800 μL / h, 1200 μL / h, 1600 μL / h, and 2000 μL / h, respectively. For a fixed volume of PS particle sample, the separation rate is the highest at a flow rate of 400 μL / h.

[0126] 4. Study on separation stability of microfluidic device

[0127] In order to evaluate the repeatability and stability of the microfluidic device for constructing magnetic nanoparticle stacks during separation experiments, the same volume of monodisperse 30 nm PS particle solution was aspirated and the experiment was repeated three times. Fig.11 As shown, the separation rates of the three repeated experiments were 15.29%, 14.39% and 14.93%, respectively, indicating that the device has good separation stability.

[0128] 5. Microfluidic devices for separation and analysis of particles of different sizes

[0129] To evaluate the ability of the microfluidic device to separate particles of different sizes, 30 nm (green, 6.73 × 10 12 particles / mL,λ ex / λ em =488 / 520nm), 250nm (green, 1.16×10 10 particles / mL,λ ex / λ em =488 / 520nm), 1μm (red, 1.82×10 8 particles / mL,λ ex / λ em =528 / 580nm) fluorescent PS particles and GY-D12 protein core Chlorella vulgaris (4.6×10 6 particles / mL) and were pumped at a flow rate of 400 μL / h for 12 min. Fig.12 a and Fig.12 As shown in b, these nanoparticles of different size ranges correspond to the sizes of subpopulations of extracellular vesicles including exosomes (30-150nm), microvesicles (150-1000nm) and apoptotic bodies (1-5μm).

[0130] The volumes of 30 nm, 250 nm, 1000 nm PS and 4 μm Chlorella chloroplasts in the channels were measured by ImageJ and were 712084.95 μm 3 、755 872.52μm 3 , 2 240 699.36μm 3and 717 690.77 μm 3 ,like Fig.12 c and Fig.12 As shown in d. Since the natural fluorescent volume of Chlorella chloroplasts accounts for 16.25% of the total volume, the actual volume of Chlorella trapped is 8013200 μm 3 Therefore, the corresponding separation efficiencies of 30nm, 250nm, 1000nmPS and 4μm Chlorella are 14.39%, 15.27%, 45.27% and 55.12% respectively. The experimental results show that the separation efficiency of particles in the range of 30nm-5μm increases with the increase of particle size.

[0131] Example 5: Microfluidic device for separation and analysis of milk exosomes

[0132] 1. Exosomes extracted from milk were selected as a model to evaluate the separation performance of the microfluidic device based on magnetic nanoparticle stacks on exosomes. Dual fluorescent dyes were used for fluorescence detection, and the two dyes were located in the membrane and on the membrane of the exosomes, respectively. 6 particles / mL to 3.14×10 11 Six different concentrations of exosomes in the range of particles / mL were introduced into the microfluidic device at a flow rate of 200 μL / h, and the continuous aspiration time was 24 min. Fig.13 As shown, the three-dimensional spatial distribution image and co-localization map of fluorescent double-stained milk exosomes. Through fluorescence co-localization analysis, combined with Fig.13 c and Fig.13 As shown in d, the interception volumes of the six concentrations of milk exosomes were 1 582 424.12 μm 3 、75150.20μm 3 、22 487.40μm 3 、819.54μm 3 、190.66μm 3 and 12.30 μm 3 The co-localized fluorescence volume and the red fluorescent dye distribution have good consistency, indicating that the separation and enrichment of exosomes have been successfully achieved. The separation efficiency of the device for milk exosomes was 10.47%, 4.97%, 14.88%, 5.42%, 12.62% and 8.14%, respectively. The spatial distribution of the entrapment of milk exosomes can be directly seen through three-dimensional fluorescence images, with a concentration as low as 3.14×10 6 As can be seen from the above, there is a certain correspondence between the retention volume and concentration of milk exosomes. By quantitatively measuring the retention volume, it is expected to be used as a concentration detection device.

[0133] 2. Microfluidic device for separation and analysis of exosome-nanoparticle mixtures

[0134] To evaluate the ability of the microfluidic device to separate nanoparticle mixtures, a combination of green fluorescent particles (30nmPS, 250nmPS, 100nmSiO2) and milk exosomes dyed with red dye was used, and the mixture was pumped at a flow rate of 400μL / h for 12min. The concentrations of 30nm PS, 250nm PS, 100nm SiO2 and milk exosomes were 3.37×10 12 particles / mL、5.80×10 9 particles / mL、4.34×10 10 particles / mL and 2.62×10 11 particles / mL.

[0135] Combination Fig.14 As shown in the figure, the microfluidic separation efficiency of nanoparticle mixtures was quantitatively obtained by ImageJ: M#1 (30nmPS: 10.29%; exosomes: 18.96%), M#2 (250nmPS: 14.58%; exosomes: 15.13%), and M#3 (100nmSiO2: 36.64%; exosomes: 15.37%). The experimental results show that the microfluidic device has the potential to separate nanoparticles of different sizes and mixtures. In addition, Fig.13 The brightness projection of the side view shown in d shows that the mixing of hard particles has little effect on the separation of softer exosomes, and the separated exosomes have good stability in spatial distribution. The pore size of the magnetic particle stack can be adjusted as needed, which is more flexible in different applications.

[0136] Example 6: Microfluidic device for separation and analysis of exosomes in serum and urine

[0137] Studies have shown that the number of exosomes in serum or urine is an intermediate result. By obtaining information about intermediate results, it can be helpful as a potential indicator for early diagnosis and prognosis evaluation of tumors. The microfluidic device with integrated magnetic particle stacks was used to separate and detect exosomes in clinical samples. Serum and urine specimens were first centrifuged at 3000g for 10 minutes at 4°C to remove cell debris. Then, they were incubated with fluorescent dye at 37°C and protected from light for 30 minutes. Fig.15 As shown, the fluorescence signal distribution of exosomes in serum and urine stained with fluorescence double staining. The difference in fluorescence signal distribution can be seen through three-view projection ( Fig.15b, e), for exosomes in serum samples, the side view brightness projection difference between tumor patients S1, S2 and healthy person S3 is the most obvious, and the interception volume differs by two times; while the projection distribution of tumor patients S1 and S2 is almost the same. For urine specimens, the side view brightness projection distribution of tumor patients U1 and U2 is wider, while the projection distribution of healthy person U3 is narrower. The joint detection of serum and urine specimens shows that the side view projection trend of normal people is consistent. However, there is a small difference in the side view brightness projection of urine samples compared with serum, which may be caused by dietary differences between individuals, and the chemical composition in urine is also relatively unstable. Therefore, the new method based on this graphic analysis may be more suitable for analyzing serum samples. The above results show that the microfluidic device can effectively intercept exosomes in serum and urine, and realizes an image-based qualitative analysis method through three-dimensional reconstruction and three-dimensional projection, which can be used as a new method for separation and analysis of exosomes in body fluids.

[0138] Example 7: Microfluidic device for separation and analysis of tumor cells

[0139] In order to evaluate the separation performance of the microfluidic device for micron-sized biological particles such as tumor cells, MDA.MB.231 human breast cancer cells (19 μm) and HSC-3 human oral squamous cell carcinoma cells (14 μm) labeled with green fluorescent dye were selected as model cells. Fig.16 As shown in a. MDA.MB.231 cells (concentration 1.4×10 5 / mL) with an aspiration volume of about 50 μL and a retention volume of 12377 534.16 μm 3 , the separation efficiency is 47.36%. Fig.16 b and Fig.16 As shown, for HSC-3 cells, even at low concentrations (10 3 The fluorescence signal can still be detected even with a small sample volume of 1000 cells / mL. The experimental results show that the device has the potential to separate CTCs (circulating tumor cells) from blood samples and can be further quantitatively analyzed.

[0140] In summary, a microfluidic device according to an embodiment of the present application also has a significant effect in obtaining information on intermediate results when applied to the separation and detection of other micron-sized biological particles such as GY-D12 protein-core Chlorella, tumor cells, etc.

[0141] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A microfluidic device, characterized in that: include: at least one separation unit; The separation unit comprises a microfluidic channel having an inlet channel and at least two outlet channels branching from the inlet channel; as well as The magnetic component applies a magnetic field at the branching port between the inlet channel and the outlet channel, and the magnetic field is used to make the magnetic particles injected into the microchannel form a pile with a porous structure for filtering and interception at the branching port.

2. A microfluidic device according to claim 1, characterized in that: The magnetic field applied by the magnetic component causes the accumulation body to extend into the outlet channel and form a blocking section 1 with a preset length.

3. A microfluidic device as claimed in claim 2, characterized in that: The magnetic field applied by the magnetic component causes the accumulation body to extend into the inlet channel and form a second blocking section with a preset length.

4. A microfluidic device as claimed in claim 1, characterized in that: The magnetic pole of the magnetic component points in a direction perpendicular to the plane where the microchannel is located.

5. A microfluidic device as claimed in claim 1, characterized in that: The magnetic particles are Fe3O4 magnetic nanoparticles.

6. A microfluidic device as claimed in claim 1, characterized in that: The separation unit comprises two mutually perpendicular and connected transparent tubes, and the inner cavities of the two transparent tubes together constitute the microchannel; The separation unit and the magnetic component are arranged on a substrate.

7. A microfluidic device as claimed in claim 1, characterized in that: One end of the outlet channel close to the inlet channel is a bent structure.

8. A microfluidic device according to any one of claims 1 to 7, characterized in that: The separation unit is provided in plurality and the plurality of separation units are connected in series or in parallel; When a plurality of the separation units are connected in series, the outlet channel of the upper separation unit is respectively connected to the inlet channel of one lower separation unit, and the filter aperture of the stack of the upper separation unit is larger than the filter aperture of the stack of the lower separation unit; When a plurality of the separation units are connected in parallel, the inlet channel of each separation unit is simultaneously connected to a sample inlet pipeline, and the filter apertures of the stacks of each separation unit are the same.

9. A method for using the microfluidic device according to any one of claims 1 to 8, characterized in that: The following steps are involved: A multi-channel suction device is used, and the multi-channel suction device is connected to each outlet channel; The solution containing magnetic particles is sucked from the inlet channel into the microchannel through the multi-channel suction device at least twice, and the flow rate of the solution sucked in each time is gradually reduced, and the magnetic particles form an accumulation at the branch port under the action of the magnetic field applied by the magnetic field component; By suctioning the multi-channel suction device, the sample enters from the inlet channel of the microfluidic channel and passes through the stacking body for separation.

10. A method for separation and analysis of exosomes, based on the microfluidic device according to any one of claims 1 to 8, characterized in that: The following steps are involved: Positioning fluorescent dyes at locations in the sample where exosomes can be observed; The sample containing the exosomes is sent into the separation unit of the microfluidic device through a pumping device, so that the sample passes through the accumulation body formed in the microchannel for separation; Using a microscope to obtain a fluorescent image of the exosomes trapped in the microfluidic device, and importing the fluorescent image into an image processing software for image processing to obtain a processed image; Perform qualitative and quantitative analysis on the processed images.

Images