An exosome separation device and method based on microfluidic technology

Abstract

The present invention provides an exosome separation device and method based on microfluidic technology. By constructing a water droplet-shaped microcolumn array with an asymmetric columnar structure in a microfluidic chip and adjusting relevant parameters of the microcolumn array, the separation of particles with larger particle sizes from exosomes is achieved by using deterministic lateral displacement technology. In addition, an alternating voltage source is electrically connected to the exosome sorting region, so as to apply a quantitative electrical stimulation to the microcolumn array to cause the microcolumn array to generate a highly non-uniform electric field, realizing the further separation of particles with a large difference in composition and structure from exosomes, thereby improving the separation purity of exosomes. This exosome separation device superimposes and synergistically combines the effects of deterministic lateral displacement technology and dielectrophoresis technology on the sorting mechanisms of different types of particles, effectively compensating for the defects existing in the individual actions of each technology and significantly improving the separation efficiency of exosomes.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome separation, and in particular, to an exosome separation device and method based on microfluidic technology. Background Art

[0002] The formation process of exosomes is that the cytoplasmic membrane invaginates to form endosomes, and the endosomal membrane further buds inward to form intraluminal vesicles, thus transforming into multivesicular bodies with dynamic subcellular structures. When the multivesicular bodies fuse with lysosomes, the vesicles inside the multivesicular bodies will be degraded, and when the multivesicular bodies fuse with the cell membrane, the vesicles inside the multivesicular bodies will invaginate again, forming granular small vesicles by inward budding and releasing them into the extracellular environment. Due to the non-uniformity during the invagination process of cells and the protein sorting mechanism existing during the maturation process of intracellular multivesicular bodies, there are significant differences in the size and contained contents of exosomes, that is, the contents carried by exosomes secreted by different tissue cells are different, and the exosome contents secreted by the same tissue in different states (such as: healthy or diseased, resting or exercising) also have significant differences. Due to the heterogeneous characteristics of exosomes, they are of great significance in the diagnosis, typing, staging, prognosis, and treatment of diseases.

[0003] The size of exosomes is generally 30nm - 150nm. There are a large number of different types of particles in the body fluids where they are located (such as extracellular vesicles with densities and structures very close to exosomes, such as ectosomes and apoptotic bodies) and various nanoscale particles that overlap with the exosome size range (such as lipoproteins and retroviruses, etc.). The conventional methods currently used for the separation of exosomes mainly include density gradient centrifugation, ultrafiltration centrifugation, and immunomagnetic bead methods. However, the above methods still have different degrees of defects in terms of equipment, reagent cost, operation complexity, damage degree to exosomes, and separation purity.

[0004] As an emerging technology for precisely manipulating trace amounts of liquids and micro-nano scale particles (such as cells, microspheres, biological macromolecules, etc.) therein, microfluidic technology has been widely applied in recent years in both basic life science research and clinical medical diagnosis and treatment directions. Using microfluidic technology to achieve the separation and capture of exosomes has gradually attracted the attention of researchers in this field. After years of technological development and accumulation in the microfluidic technology field, a series of mature technical solutions for manipulating microscale particles have been developed, such as dielectrophoresis technology and deterministic lateral displacement technology. Among them, it is difficult to separate extracellular nanoparticles similar in size to exosomes using deterministic lateral displacement technology, and compared with the separation of micron particles, the separation effect of dielectrophoresis technology is greatly weakened at the nanoscale. Therefore, how to combine the two technologies to make up for their respective advantages and disadvantages to improve the separation efficiency of exosomes has become an urgent problem to be solved. Summary of the Invention

[0005] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide an exosome separation device and method based on microfluidic technology, which are used to solve the problems of low separation efficiency and poor separation purity of exosomes in the prior art.

[0006] To achieve the above purpose and other related purposes, the present invention provides an exosome separation device based on microfluidic technology. The exosome separation device at least includes:

[0007] A microfluidic chip, the microfluidic chip includes an exosome sorting area, the exosome sorting area includes a micro-column channel, one end of the micro-column channel is connected to a sample inlet, the other end of the micro-column channel is connected to a first waste liquid outlet, a second waste liquid outlet and an exosome outlet, a micro-column array is arranged in the micro-column channel, the micro-column array contains a plurality of micro-columns with a water droplet-shaped cross-sectional morphology and the tips of the water droplet-shaped micro-columns point in the same direction;

[0008] A voltage source, the voltage source is electrically connected to the microfluidic chip, so as to form a high-gradient non-uniform electric field at the micro-column channel;

[0009] A PDMS encapsulation layer, the PDMS encapsulation layer is tightly bonded to the microfluidic chip.

[0010] Optionally, the voltage source is an alternating voltage source.

[0011] Optionally, the frequency of the alternating voltage source is 10 kHz to 500 kHz.

[0012] Optionally, the threshold of the micro-column array is 150 nm and the row displacement fraction of the micro-column array is 0.1 to 0.2.

[0013] Optionally, the height of the water droplet-shaped micro-column is 2 μm to 5 μm, and the magnitude of the lateral displacement between adjacent two rows of the water droplet-shaped micro-columns satisfies ε = δ / λ, where ε is the row displacement fraction of the water droplet-shaped micro-column array, λ is the center-to-center spacing between adjacent two water droplet-shaped micro-columns, and δ is the lateral displacement between two rows of the water droplet-shaped micro-columns.

[0014] Optionally, the gap width of the water droplet-shaped micro-column satisfies D c = 1.4Gε 0.48 where D c is the threshold of the water droplet-shaped micro-column array, and G is the gap width of the water droplet-shaped micro-columns in the same column.

[0015] The present invention also provides an exosome separation method based on the above-mentioned exosome separation device based on microfluidic technology. The exosome separation method includes the following steps:

[0016] Etch on a silicon wafer to form a microfluidic chip containing an exosome sorting region, bond the microfluidic chip with a PDMS encapsulation layer, and form a power access hole in the PDMS encapsulation layer to expose the microfluidic chip;

[0017] Insert one end of an electrode into the power access hole to electrically connect with the microfluidic chip, and connect the other end to a voltage source with a wire;

[0018] Fluorescently label the buffer solution containing exosomes and use an injection pump to inject the buffer solution containing exosomes into the injection port at a certain speed;

[0019] Set a fluorescence microscope above the exosome sorting region to observe the separation effect of exosomes.

[0020] Optionally, the exosome sorting region includes a microcolumn channel, a microcolumn array is arranged in the microcolumn channel, and the microcolumn array contains a plurality of microcolumns with a water droplet-shaped cross-sectional morphology.

[0021] Optionally, the directions pointed by the tips of the water droplet-shaped microcolumns are the same and perpendicular to the flowing direction of the buffer solution containing exosomes in the microfluidic chip.

[0022] Optionally, control the gap size between individual microcolumns in the exosome sorting region and the frequency of the voltage source to achieve the superposition of deterministic lateral displacement and dielectrophoresis, and promote the separation of exosomes from other particles.

[0023] As described above, the exosome separation device and method based on microfluidic technology of the present invention have the following beneficial effects: By constructing a water droplet-shaped microcolumn array with an asymmetric columnar structure in the microfluidic chip and adjusting the relevant parameters of the microcolumn array, the separation of particles with larger particle size from exosomes is achieved by using the deterministic lateral displacement technology. In addition, an alternating voltage source is electrically connected in the exosome sorting region, so as to apply a quantitative electrical stimulus to the microcolumn array to generate a highly non-uniform electric field, and further separate the particles with a large difference in composition structure from exosomes, thereby improving the separation purity of exosomes. The exosome separation device superimposes and synergistically combines the effects of the deterministic lateral displacement technology and the dielectrophoresis technology on the sorting mechanisms of different types of particles, effectively making up for the defects existing in the individual actions of each technology and significantly improving the separation efficiency of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It shows a schematic structural diagram of the exosome separation device based on microfluidic technology of the present invention.

[0025] Figure 2It shows a schematic structural diagram of the sorting area of the exosome separation device based on microfluidic technology of the present invention.

[0026] Figure 3 It shows a scanning electron microscope image of the microcolumn channels in the exosome sorting area of the present invention.

[0027] Description of component numbers.

[0028] 101, microfluidic chip; 102, exosome sorting area; 1021, water-drop-shaped microcolumns; 103, sample inlet; 104, first waste liquid outlet; 105, exosome outlet; 106, second waste liquid outlet; 107, voltage source; 108, PDMS encapsulation layer; 109, injection pump; 110, collection device; 111, fluorescence microscope. Detailed implementation manners

[0029] The following uses specific specific examples to illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0030] For the convenience of description, spatial relationship terms such as "beneath", "below", "lower", "under", "above", "upper", etc. may be used herein to describe the relationship between one element or feature shown in the drawings and other elements or features. It will be understood that these spatial relationship terms are intended to encompass other directions of the device in use or operation in addition to the directions depicted in the drawings. In addition, when a layer is referred to as being "between" two layers, it can be the only layer between the two layers, or there can be one or more intervening layers.

[0031] It should be understood that using terms such as "first" and "second" to limit components is only for the convenience of distinguishing the above-mentioned components. Without otherwise stating, the above terms have no special meanings, so they cannot be understood as limiting the protection scope of the present invention.

[0032] In this application, unless otherwise clearly specified and limited, terms such as "installed", "connected", "connected to", "fixed" and other terms should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. It can be the internal connection of two components or the interaction relationship between two components, unless otherwise clearly limited. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0033] Please refer to Figures 1 to 3 . It should be noted that the illustrations provided in this embodiment only schematically illustrate the basic concept of the present invention. Therefore, only the components related to the present invention are shown in the illustrations, rather than being drawn according to the number, shape, and size of the components in actual implementation. The type, quantity, and ratio of each component in actual implementation can be arbitrarily changed, and the component layout type may also be more complex.

[0034] In one embodiment of the present invention, an exosome separation device based on microfluidic technology is provided. The exosome separation device includes: a microfluidic chip 101, a voltage source 107, and a PDMS encapsulation layer 108.

[0035] As shown in Figure 1 , the microfluidic chip 101 includes an exosome sorting region 102, and a microcolumn channel is formed in the exosome sorting region 102. One end of the microcolumn channel is connected to a sample injection port 103 for injecting a buffer solution containing exosomes. In this embodiment, in addition to exosomes, the buffer solution at least contains other types of particles such as ectosomes, lipoproteins, and retroviruses. Among them, the particle size of exosomes is 30nm - 150nm, and the particle size of ectosomes is 200nm - 1000nm. The other end of the microcolumn channel is connected to a first waste liquid outlet 104, a second waste liquid outlet 106, and an exosome outlet 105, and collection devices 110 are respectively arranged at the corresponding outlets. As the buffer solution flows, the exosomes in the exosome sorting region 102 gradually move away from the bottom side and approach the top side, and finally flow to the exosome outlet 105, realizing the separation, purification, and collection of exosomes.

[0036] As an example, the microcolumn array includes a plurality of microcolumns 1021 with a cross-sectional shape of a water droplet, and the tips of the water droplet-shaped microcolumns 1021 point in the same direction.

[0037] As shown in Figure 2 , in this embodiment, the microcolumn array includes a plurality of microcolumns 1021 with a cross-sectional shape of a water droplet, and the tips of the microcolumns point in the same direction, so that the particles in the buffer solution can continuously collide with the water droplet-shaped microcolumns 1021 when passing through the microcolumn array, thereby changing the movement trajectory.

[0038] As an example, the height of the water droplet-shaped microcolumn 1021 is 2μm - 5μm, and the magnitude of the lateral displacement between adjacent two rows of water droplet-shaped microcolumns 1021 satisfies ε = δ / λ, where ε is the row displacement fraction of the water droplet-shaped microcolumn array, λ is the center-to-center distance between adjacent two water droplet-shaped microcolumns 1021, and δ is the lateral displacement between two rows of the water droplet-shaped microcolumns 1021.

[0039] As shown in Figure 3As shown, the height of a single droplet-shaped micro-post 1021 in the micro-post array is 2 μm to 5 μm. For example, it can be 2 μm, 3 μm, 4 μm, or 5 μm. In this embodiment, as Figure 2 shown, ε is the row displacement fraction of the droplet-shaped micro-post array, and the magnitude of the row displacement fraction is 0.1 to 0.2. λ is the center-to-center spacing between adjacent droplet-shaped micro-posts 1021, and δ is the lateral displacement between two rows of the droplet-shaped micro-posts 1021. The magnitude of the lateral displacement between adjacent two rows of droplet-shaped micro-posts 1021 should satisfy the setting of ε = δ / λ so that particles in the buffer can continuously collide with the droplet-shaped micro-posts 1021 when passing through the micro-post array.

[0040] As an example, the gap width of the droplet-shaped micro-post 1021 satisfies D c = 1.4Gε 0.48 , where D c is the threshold of the droplet-shaped micro-post array, G is the gap width of the droplet-shaped micro-posts 1021 in the same column, and the threshold D c of the micro-post array is 150 nm.

[0041] Specifically, the gap width of the droplet-shaped micro-post 1021 is set to a certain size, and the gap width of the droplet-shaped micro-post 1021 should satisfy D c = 1.4Gε 0.48 , where D c is the threshold of the droplet-shaped micro-post array. In this embodiment, G is the gap width of the droplet-shaped micro-posts 1021 in the same column. Combining with the row displacement fraction of the droplet-shaped micro-post array designed when forming the microfluidic chip 101, the gap width of the formed droplet-shaped micro-posts 1021 is determined. Only when the particle size in the buffer exceeds the threshold, the threshold of the micro-post array is 150 nm, that is, particles exceeding 150 nm can continuously collide with the droplet-shaped micro-posts 1021 and continuously generate lateral displacement. When the particles are small, that is, not exceeding 150 nm, they mainly travel along a zigzag trajectory in the gaps of the micro-post array along the flow direction of the buffer, thereby realizing the separation of exosomes from larger-sized extracellular particles.

[0042] As an example, the voltage source 107 is an alternating voltage source and the frequency of the alternating voltage source is 10 kHz to 500 kHz.

[0043] Specifically, as Figure 1As shown, the voltage source 107 is electrically connected to the microfluidic chip 101. The voltage source 107 used is an alternating voltage source, and the frequency of the alternating voltage source is set to be 10 kHz to 500 kHz. For example, 10 kHz, 200 kHz, or 500 kHz, so as to form a high-gradient non-uniform electric field at the microcolumn channel. Since there are particles in the buffer solution with sizes similar to those of exosomes, such as lipoproteins and retroviruses, whose particle sizes are 20 nm to 150 nm, it is impossible to separate them from exosomes using deterministic lateral displacement technology. However, the component structures of lipoproteins and retroviruses are quite different from those of exosomes, so there are significant differences in their electrical properties. By forming a high-gradient non-uniform electric field and adjusting the frequency of the voltage source 107, further separation of exosomes from lipoproteins and retroviruses can be achieved.

[0044] In another embodiment of the present invention, there is also provided an exosome separation method based on the exosome separation device based on microfluidic technology described in the above embodiment. The exosome separation method includes the following steps:

[0045] S1: Etch on a silicon wafer to form a microfluidic chip 101 containing an exosome sorting region 102, bond the microfluidic chip 101 with a PDMS encapsulation layer 108, and form a power access hole in the PDMS encapsulation layer 108 to expose the microfluidic chip 101;

[0046] S2: Insert one end of the electrode into the power access hole to be electrically connected to the microfluidic chip 101, and connect the other end to the voltage source 107 with a wire;

[0047] S3: Fluorescently label the buffer solution containing exosomes and use an injection pump 109 to inject the buffer solution containing exosomes into the injection port 103 at a certain speed;

[0048] S4: Set a fluorescence microscope 111 above the exosome sorting region 102 to observe the separation effect of exosomes.

[0049] Specifically in implementation, as a preferred implementation manner of the present invention, the chip substrate of the microfluidic used in the exosome separation method is a silicon wafer, and its manufacturing process is as follows: First, rinse the silicon wafer with acetone and deionized water and blow it dry with nitrogen to remove surface impurities; then pour an appropriate amount of photoresist in the center of the silicon wafer, and finally set the rotation speed and time according to the required height of the chip to make the photoresist evenly cover the surface of the silicon wafer, and through processes such as exposure and development, an exosome sorting region 102 including a microcolumn channel is formed on the microfluidic chip 101, and a microcolumn array formed by a plurality of microcolumns 1021 with a cross-sectional shape of a water droplet is arranged in the microcolumn channel.

[0050] Due to the presence of other particles in the buffer that are similar in size or significantly different in size from exosomes, such as lipoproteins and retroviruses with a particle size of 20 nm to 150 nm and ectosomes with a particle size of 200 nm to 1000 nm, the separation efficiency using only deterministic lateral displacement technology is low. Therefore, an alternating voltage source is electrically connected to the microfluidic chip 101, so as to utilize the superimposed synergistic effect of deterministic lateral displacement technology and dielectrophoresis technology to achieve the separation and purification of exosomes.

[0051] The separation principle of the exosome sorting region 102 based on deterministic lateral displacement technology is as follows: By designing a microcolumn array structure containing a plurality of microcolumns 1021 with a cross-sectional shape of a water droplet, particles with a larger particle size in the buffer (such as ectosomes with a particle size of 200 nm to 1000 nm) are screened, so that other particles with a particle size larger than the threshold of 150 nm of the microcolumn array will collide when passing through the microcolumns, resulting in continuous lateral displacement and finally flowing to the first waste liquid outlet 104.

[0052] The separation principle of the exosome sorting region 102 based on dielectrophoresis technology is as follows: The alternating voltage source is electrically connected to the microfluidic chip 101, so as to form a high-gradient non-uniform electric field at the microcolumn array, and further separate particles such as lipoproteins and retroviruses that cannot be separated by deterministic lateral displacement technology, and finally make lipoproteins and retroviruses flow to the second waste liquid outlet 106.

[0053] In summary, the present invention provides an exosome separation device and method based on microfluidic technology. By constructing an asymmetric columnar structure of a water droplet-shaped microcolumn array in the microfluidic chip and adjusting the relevant parameters of the microcolumn array, the separation of particles with a larger particle size from exosomes is achieved by using deterministic lateral displacement technology. In addition, an alternating voltage source is electrically connected to the exosome sorting region, so as to apply a quantitative electrical stimulation to the microcolumn array to generate a highly non-uniform electric field, realizing the further separation of particles with a large difference in composition structure from exosomes, thereby improving the separation purity of exosomes. This exosome separation device superimposes and synergizes the functions of deterministic lateral displacement technology and dielectrophoresis technology for sorting different types of particles, effectively making up for the defects existing in the individual actions of each technology and significantly improving the separation efficiency of exosomes. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

[0054] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. An exosome separation device based on microfluidic technology, characterized in that, The exosome separation device at least includes: A microfluidic chip, the microfluidic chip includes an exosome sorting region, the exosome sorting region includes a micro-column channel, one end of the micro-column channel is connected to a sample inlet, the other end of the micro-column channel is connected to a first waste liquid outlet, a second waste liquid outlet and an exosome outlet, a micro-column array is arranged in the micro-column channel, the threshold of the micro-column array is 150 nm and the row displacement fraction of the micro-column array is 0.1 to 0.2, the micro-column array contains a plurality of micro-columns with a cross-sectional shape of a water droplet, and the directions pointed by the tips of the water droplet-shaped micro-columns are the same, the height of the water droplet-shaped micro-columns is 2 μm to 5 μm, and the magnitude of the lateral displacement between adjacent two rows of the water droplet-shaped micro-columns satisfies ε = δ / λ, where ε is the row displacement fraction of the water droplet-shaped micro-column array, λ is the center-to-center spacing between adjacent two water droplet-shaped micro-columns, δ is the lateral displacement between two rows of the water droplet-shaped micro-columns, and the gap width of the water droplet-shaped micro-columns satisfies D c = 1.4Gε 0.48 , where D c is the threshold of the water droplet-shaped micro-column array, and G is the gap width of the water droplet-shaped micro-columns in the same column; A voltage source, which is electrically connected to the microfluidic chip, so as to form a high-gradient non-uniform electric field at the micro-column channel; A PDMS encapsulation layer, which is tightly bonded to the microfluidic chip.

2. The exosome separation device according to claim 1, wherein: The voltage source is an alternating voltage source.

3. The exosome separation device according to claim 2, wherein: The frequency of the alternating voltage source is 10 kHz to 500 kHz.

4. An exosome separation method for the exosome separation device based on microfluidic technology according to any one of the above claims 1-3, characterized in that: Etching is carried out on a silicon wafer to form a microfluidic chip containing an exosome sorting area, bonding the microfluidic chip with a PDMS encapsulation layer, and forming a power access hole in the PDMS encapsulation layer to expose the microfluidic chip; Insert one end of an electrode into the power access hole to be electrically connected to the microfluidic chip, and connect the other end to a voltage source with a wire; Fluorescently label the buffer solution containing exosomes and use an injection pump to inject the buffer solution containing exosomes into the injection port at a certain speed; A fluorescence microscope is arranged above the exosome sorting area to observe the separation effect of exosomes.

5. The exosome isolation method according to claim 4, characterized in that: The exosome sorting area includes a micro-column channel, in which a micro-column array is arranged, and the micro-column array contains a plurality of micro-columns with a water-droplet-shaped cross-sectional morphology.

6. The exosome isolation method according to claim 5, characterized in that: The directions pointed by the tips of the water-droplet-shaped micro-columns are the same and perpendicular to the flow direction of the buffer solution containing exosomes in the microfluidic chip.

7. The exosome isolation method according to claim 5, wherein: Control the gap size of a single micro-column in the exosome sorting area and the frequency of the voltage source to achieve the superposition of deterministic lateral displacement and dielectrophoresis, and promote the separation of exosomes from other particles.

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