Microfluidic chip and microfluidic system, method of using the same

Abstract

The microfluidic chip and microfluidic system disclosed herein, and their usage method, include a substrate layer, the substrate layer including a substrate substrate and a plurality of first micropillars on the substrate substrate, the plurality of first micropillars being arranged in multiple rows and columns, with each row of first micropillars being staggered by a predetermined distance; a cover layer, placed opposite the substrate layer, the cover layer including a first liquid inlet hole penetrating the thickness direction of the cover layer and at least two liquid outlet holes, the space between the cover layer and the substrate layer including a mixing chamber, a separation chamber and at least two liquid collection chambers, wherein the first liquid inlet hole is connected to the mixing chamber, the at least two liquid outlet holes are connected to the at least two liquid collection chambers, the separation chamber is connected to the mixing chamber and the at least two liquid collection chambers, the mixing chamber is configured to contain a sample liquid including exosomes and magnetic beads, the plurality of first micropillars are placed in the separation chamber, and different liquid collection chambers are configured to collect exosome-magnetic bead complexes of different sizes.

Description

Technical Field

[0001] This disclosure relates to the field of microfluidics, and in particular to a microfluidic chip and a microfluidic system, and a method of using the same. Background Technology

[0002] Exosomes are vesicle-like structures secreted by cells into the body, typically ranging in size from 30 nm to 150 nm. They are generally considered to be involved in vital processes such as apoptosis and intercellular communication. Because exosomes often carry information such as nucleic acids and proteins derived from maternal cells and are widely distributed in body fluids, they are increasingly attracting attention as biomarkers for liquid biopsies. Summary of the Invention

[0003] This disclosure provides a microfluidic chip and microfluidic system, and a method for using them, to simultaneously achieve multiple separations and purifications of exosomes, including specific and non-specific separations.

[0004] This disclosure provides a microfluidic chip, comprising:

[0005] The substrate layer includes a substrate substrate and a plurality of first micropillars on the substrate substrate, the plurality of first micropillars being arranged in multiple rows and columns, with each row of first micropillars being staggered by a predetermined distance.

[0006] A cover plate layer, positioned opposite the substrate layer, includes a first inlet hole extending through the thickness direction of the cover plate layer and at least two outlet holes. The space between the cover plate layer and the substrate layer includes a mixing chamber, a separation chamber, and at least two collection chambers. The first inlet hole communicates with the mixing chamber, the at least two outlet holes communicate with the at least two collection chambers, and the separation chamber communicates with the mixing chamber and the at least two collection chambers. The mixing chamber is configured to contain a sample solution including exosomes and magnetic beads. The plurality of first microcolumns are placed in the separation chamber, and different collection chambers are configured to collect exosome-magnetic bead complexes of different sizes.

[0007] In some embodiments, in the microfluidic chip provided in the present disclosure, the substrate layer further includes a plurality of second micropillars disposed in the mixing chamber, the plurality of second micropillars being arranged in multiple rows and columns, and each row of second micropillars being aligned along the column direction.

[0008] In some embodiments, in the microfluidic chip provided in the present disclosure, the arrangement density of each of the first micropillars is greater than the arrangement density of each of the second micropillars.

[0009] In some embodiments, in the microfluidic chip provided in the present disclosure, the diameter of the first micropillar is smaller than the diameter of the second micropillar, and the height of the first micropillar is greater than the height of the second micropillar.

[0010] In some embodiments, in the microfluidic chip provided in the present disclosure, the ratio of the preset distance to the diameter of the first micropillar is greater than or equal to 0.1 and less than or equal to 0.6.

[0011] In some embodiments, in the microfluidic chip provided in the present disclosure, a first groove, a second groove, and a third groove are sequentially connected on one side of the cover plate facing the substrate layer, wherein the first groove and the substrate form the mixing chamber, the second groove and the substrate form the separation chamber, and the third groove and the substrate form the liquid collection chamber.

[0012] In some embodiments, in the microfluidic chip provided in the present disclosure, a first flow channel groove and a second flow channel groove are further provided on the side of the cover plate facing the substrate layer, wherein the first flow channel groove connects the first groove and the second groove, and the second flow channel groove connects the second groove and the third groove.

[0013] In some embodiments, in the microfluidic chip provided in the present disclosure, the depths of the first groove, the second groove, the third groove, the first channel groove, and the second channel groove are equal.

[0014] In some embodiments, in the microfluidic chip provided in the present disclosure, the cover plate layer further includes a second liquid inlet hole, a third liquid inlet hole, and a fourth liquid inlet hole penetrating the thickness direction of the cover plate layer. The cover plate layer is also provided with a first liquid inlet groove, a second liquid inlet groove, a third liquid inlet groove, a fourth liquid inlet groove, and an outlet groove on the side facing the substrate layer. The first liquid inlet groove connects the first liquid inlet hole to the mixing chamber, the second liquid inlet groove connects the second liquid inlet hole to the mixing chamber, the third liquid inlet groove connects the third liquid inlet hole to the separation chamber, the fourth liquid inlet groove connects the fourth liquid inlet hole to the collection chamber, and the outlet groove connects the outlet hole to the collection chamber.

[0015] In some embodiments, in the microfluidic chip provided in the present disclosure, the cover plate layer further includes a first vent hole, a second vent hole, and a third vent hole penetrating the thickness direction of the cover plate layer. The cover plate layer is also provided with a first vent groove, a second vent groove, and a third vent groove on the side facing the substrate layer. The first vent groove connects the first vent hole to the mixing chamber, the second vent groove connects the second vent hole to the separation chamber, and the third vent groove connects the third vent hole to the liquid collection chamber.

[0016] Based on the same inventive concept, this disclosure provides a microfluidic system, including a microfluidic chip and a magnetizing component, wherein the microfluidic chip is the microfluidic chip provided in this disclosure, and the magnetizing component is configured to drive a magnetic bead to move on the side of the substrate layer opposite to the cover plate layer.

[0017] Based on the same inventive concept, this disclosure provides a method for using the above-mentioned microfluidic system, including:

[0018] A sample solution including exosomes and magnetic beads is added to a mixing chamber, such that the magnetic beads bind to at least a portion of the exosomes to form an exosome-magnetic bead complex.

[0019] Driven by the magnetic supply component, the exosome-magnetic bead complex in the mixing chamber is moved into the separation chamber, so as to separate exosome-magnetic bead complexes of different sizes using multiple first microcolumns in the separation chamber. The exosome-magnetic bead complexes of different sizes flow to different collection chambers and are collected.

[0020] In some embodiments, the above-described method of use provided in this disclosure further includes, before adding the sample solution comprising exosomes and magnetic beads to the mixing chamber:

[0021] Add the aqueous phase to the mixing chamber until the remaining space in the mixing chamber is used to hold the sample liquid, fill the separation chamber with the oil phase, and fill the collection chamber with the aqueous phase.

[0022] In some instances, in the above-described usage methods provided in the embodiments of this disclosure, the mixing chamber is kept open to the outside during the process of adding the aqueous phase and sample liquid to the mixing chamber; the separation chamber is kept open to the outside during the process of adding the oil phase to the separation chamber; and the collection chamber is kept open to the outside during the process of adding the aqueous phase to the collection chamber.

[0023] The beneficial effects of this disclosure are as follows:

[0024] The microfluidic chip and microfluidic system, and their usage method provided in this disclosure include a substrate layer, which includes a substrate substrate and a plurality of first micropillars on the substrate substrate. The plurality of first micropillars are arranged in multiple rows and columns, with each row of first micropillars staggered by a predetermined distance. A cover layer is placed opposite the substrate layer and includes a first liquid inlet hole and at least two liquid outlet holes penetrating the thickness direction of the cover layer. The space between the cover layer and the substrate layer includes a mixing chamber, a separation chamber, and at least two liquid collection chambers. The first liquid inlet hole is connected to the mixing chamber, the at least two liquid outlet holes are connected to the at least two liquid collection chambers, and the separation chamber is connected to the mixing chamber and the at least two liquid collection chambers. The mixing chamber is configured to contain a sample liquid including exosomes and magnetic beads. The plurality of first micropillars are placed in the separation chamber, and different liquid collection chambers are configured to collect exosome-magnetic bead complexes of different sizes. The magnetic beads are coated with antibodies, which can specifically bind to certain types of exosome surface proteins to form exosome-magnetic bead complexes, thereby achieving specific capture of exosomes by the magnetic beads. Multiple first microcolumns can effectively separate exosome-magnetic bead complexes of different sizes, achieving non-specific separation of exosomes. Therefore, the microfluidic chip provided in this disclosure can simultaneously achieve multiple separation and purification effects of specific and non-specific separation of exosomes. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the structure of a microfluidic chip provided in an embodiment of the present disclosure;

[0026] Figure 2 This is another schematic diagram of the structure of the microfluidic chip provided in the embodiments of this disclosure;

[0027] Figure 3 This is another schematic diagram of the structure of the microfluidic chip provided in the embodiments of this disclosure. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, to make the objectives, technical solutions, and advantages of the embodiments of this disclosure clearer, the technical solutions of the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, in the drawings, the thickness of layers, films, panels, regions, etc., is enlarged for clarity. Exemplary embodiments are described in this disclosure with reference to cross-sectional views as schematic diagrams of idealized embodiments. Thus, deviations from the shape of the figures will be expected as a result of, for example, manufacturing techniques and / or tolerances. Therefore, the embodiments described in this disclosure should not be construed as limited to the specific shape of the regions shown in this disclosure, but rather include deviations in shape caused, for example, by manufacturing processes. For example, a region illustrated or described as flat may typically have rough and / or non-linear characteristics; a sharp corner illustrated may be rounded, etc. Therefore, the regions shown in the figures are schematic in nature, and their dimensions and shapes are not intended to illustrate the precise shape of the regions or reflect true proportions; they are only intended to illustrate the content of this disclosure. Furthermore, the same or similar reference numerals are used throughout to denote the same or similar elements or elements having the same or similar functions. To keep the following description of the embodiments of this disclosure clear and concise, detailed descriptions of known functions and known components are omitted.

[0029] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this disclosure pertains. The terms “first,” “second,” and similar terms used in this disclosure and the claims do not indicate any order, quantity, or importance, but are merely used to distinguish different components. Terms such as “comprising” or “including” mean that the element or object preceding the word covers the element or object listed following the word and its equivalents, without excluding other elements or objects. Terms such as “connected” or “linked” are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. Terms such as “inner,” “outer,” “upper,” and “lower” are used only to indicate relative positional relationships, and these relative positional relationships may change accordingly when the absolute position of the described object changes.

[0030] In the following description, when an element or layer is referred to as "on" or "connected to" another element or layer, the element or layer may be directly on or directly connected to the other element or layer, or there may be intermediate elements or intermediate layers. When an element or layer is referred to as "located on one side of" another element or layer, the element or layer may be directly on or directly connected to the other element or layer, or there may be intermediate elements or intermediate layers. However, when an element or layer is referred to as "directly on" or "directly connected to" another element or layer, no intermediate elements or intermediate layers are present. The term "and / or" includes any and all combinations of one or more of the related listed items.

[0031] Exosome samples obtained directly are typically mixtures from various cell sources, necessitating sorting or purification to extract exosome vesicles of specific sizes or carrying specific antigens. Mainstream exosome separation methods include the gold standard ultracentrifugation, co-precipitation methods commonly used in reagent kits, and size exclusion chromatography. Microfluidic chip-based exosome separation technology, however, has seen rapid development in recent years due to its advantages such as low sample requirements, ease of operation, and suitability for automation and integration. Among these, the lateral displacement-based exosome separation method utilizes fluid dynamics, employing photolithography to construct an array of columns within microchannels to control the movement trajectories of exosomes of different sizes. The columns are staggered at a certain distance; smaller exosomes maintain their overall direction of movement with the laminar flow, while larger exosomes move along the predetermined path of the columns, undergoing lateral displacement during flow to achieve separation. This method is simple to operate and causes minimal damage to exosomes, but it is prone to clogging the flow channels and cannot achieve specific separation and purification of exosomes.

[0032] Current literature (Lab Chip, 2011, 11, 1747-1753) reports a technique for nucleic acid extraction using microfluidic chips based on surface tension-assisted immiscible filtration (IFAST). In this technique, cells to be extracted are mixed with lysis buffer and magnetic beads, then added to a reservoir located at the left end of the microfluidic chip. The magnetic beads bind to the nucleic acids and move under the influence of an external permanent magnet, sequentially passing through three reservoirs: an aqueous phase, an oil phase, and another aqueous phase. Hydrophobic impurities such as proteins are retained in the oil phase in the middle of the microfluidic chip, while hydrophilic nucleic acid-magnetic bead complexes pass through the oil phase and remain in the reservoir at the right end of the microfluidic chip for subsequent experimental operations. The oil and aqueous phase interfaces between the reservoirs maintain contact and coexistence without miscibility or stratification due to surface tension. This technique can also be used for screening vesicle-like microparticles such as cells. Furthermore, exosome sorting does not require the same level of bioactivity as cell sorting; therefore, IFAST technology is more suitable for exosomes. However, most of the IFAST chips reported in the literature are not encapsulated, making them prone to cross-contamination between the chambers and the environment; the mixing degree of the solution on the chip is poor, affecting the purification efficiency; at the same time, the overall function of the chip is limited, which further restricts its application in the field of liquid biopsy.

[0033] As can be seen from the above, the exosome separation and purification chips in related technologies have limited functionality and cannot simultaneously achieve multiple separation and purification effects, including specific and non-specific separation. Therefore, to solve the aforementioned technical problems in related technologies, this disclosure provides a microfluidic chip, such as... Figures 1 to 3 As shown, it includes:

[0034] The substrate layer 001 includes a substrate 101 and a plurality of first micropillars 102 on the substrate 101. The plurality of first micropillars 102 are arranged in multiple rows and columns, and the first micropillars 102 in each row are staggered by a predetermined distance. Optionally, the substrate 101 can be made of glass, silicon or other hard materials. The plurality of first micropillars 102 can be formed by deposition, etching or other methods.

[0035] A cover layer 002 is disposed opposite to the substrate layer 001. Optionally, the cover layer 002 and the substrate layer 001 are bonded and encapsulated by means of ultraviolet curing adhesive or surface oxygen plasma treatment. The cover layer 002 includes a first liquid inlet a and at least two liquid outlets (e.g., a first liquid outlet e and a second liquid outlet e') extending through its thickness direction. The space between the cover layer 002 and the substrate layer 001 includes a mixing chamber A, a separation chamber B, and at least two liquid collection chambers (e.g., a first liquid collection chamber C and a second liquid collection chamber C'). The first liquid inlet a is connected to the mixing chamber A, and the at least two liquid outlets (e.g., a first liquid outlet e and a second liquid outlet e') are connected to the at least two liquid collection chambers (e.g., a first liquid collection chamber C and a second liquid collection chamber C'). The separation chamber B is connected to the mixing chamber A and the at least two liquid collection chambers (e.g., a first liquid collection chamber C and a second liquid collection chamber C'). The collection chamber C' and the mixing chamber A are configured to contain a sample solution including exosomes and magnetic beads. Multiple first microcolumns 102 are placed in the separation chamber B. Different collection chambers (e.g., the first collection chamber C and the second collection chamber C') are configured to collect exosome-magnetic bead complexes of different sizes. The first inlet port a is used to inject the mixed solution of exosomes and magnetic beads, and the outlet port (e.g., the first outlet port e and the second outlet port e') is used to draw out the solution of exosome-magnetic bead complexes of different sizes. Optionally, the cover layer 002 can be made of a polymer plastic such as polydimethylsilane (PDMS). The first inlet port a and the outlet port (e.g., the first outlet port e and the second outlet port e') can be formed by drilling. The diameter of the first inlet port a and the diameter of the outlet port (e.g., the first outlet port e and the second outlet port e') can both be 1 mm.

[0036] In the microfluidic chip provided in this embodiment, the magnetic beads are coated with antibodies that can specifically bind to specific types of exosome surface proteins to form exosome-magnetic bead complexes, thereby achieving specific capture of exosomes by the magnetic beads. Multiple first micropillars 102 can effectively separate exosome-magnetic bead complexes of different sizes, achieving non-specific separation of exosomes. Therefore, the microfluidic chip provided in this disclosure can simultaneously achieve multiple separation and purification effects, including specific and non-specific separation of exosomes. Furthermore, the microfluidic chip provided in this disclosure has a closed chip structure composed of a substrate layer 001 and a cover layer 002, avoiding direct contact between the separation and purification system, including each chamber, and the environment, reducing cross-contamination between the chambers and the environment, and possessing the potential for high-throughput automated and integrated operation.

[0037] In some embodiments, in the microfluidic chip provided in the present disclosure, such as Figures 1 to 3As shown, the substrate layer 001 also includes a plurality of second micropillars 103 disposed within the mixing chamber A. The plurality of second micropillars 103 are arranged in multiple rows and columns, with each row of second micropillars 103 aligned along the column direction Y. In related technologies, IFAST chips suffer from poor sample liquid mixing, affecting purification efficiency. This disclosure addresses this issue by providing a plurality of second micropillars 103 within the mixing chamber A. This allows magnetic beads, guided by an external magnetic supply component (e.g., a permanent magnet), to pass through the micropillar array formed by the multiple second micropillars 103. The magnetic beads collide with the second micropillars 103, effectively agitating the sample liquid including the magnetic beads to an appropriate degree. This improves the mixing degree of the sample liquid, prevents insufficient mixing, and enhances separation and purification efficiency. Optionally, the plurality of second micropillars 103 can be formed by molding or deposition etching.

[0038] In some embodiments, in the microfluidic chip provided in the present disclosure, such as Figures 1 to 3 As shown, the arrangement density of each first microcolumn 102 is greater than the arrangement density of each second microcolumn 103. Since the first microcolumns 102 are mainly used to separate exosome-magnetic bead complexes of different sizes, the array spacing needs to match the size of the exosome-magnetic bead complexes; therefore, the arrangement density of the first microcolumns 102 is relatively high. The second microcolumns 103 are used to achieve uniform mixing; setting the arrangement density of the second microcolumns 103 to be smaller can enhance the macroscopic mixing efficiency. See also... Figures 1 to 3 As can be seen, the separation chamber B containing the first microcolumn 102 and the mixing chamber A containing the second microcolumn 103 are roughly the same size. Based on this, in this disclosure, the diameter of the first microcolumn 102 can be set smaller than the diameter of the second microcolumn 103, so that the arrangement density of the first microcolumn 102 is greater than the arrangement density of the second microcolumns 103. Optionally, the height of the first microcolumn 102 is greater than the height of the second microcolumn 103. The higher height of the first microcolumn 102 is equivalent to a deeper separation channel formed between the first microcolumns 102, which can better guide the exosome-magnetic bead complexes of different sizes to move along different separation channels, so that the first microcolumn 102 can better achieve effective separation of exosome-magnetic bead complexes of different sizes; while the sample liquid loses less kinetic energy after colliding with the lower-height second microcolumn 103, which is conducive to the sample liquid colliding back and forth between the second microcolumns 103, so as to better improve the mixing effect of the second microcolumn 103 on the sample liquid. In some embodiments, the second micropillar 103 has a diameter of 1 mm, a height of 0.2 mm, and a spacing of 2 mm; the first micropillar 102 has a diameter of 100 μm, a height of 0.45 mm, and a spacing of 0.05 μm to 0.1 μm.

[0039] The principle behind separating exosome-magnetic bead complexes of different sizes using multiple first micropillars 102 is deterministic lateral displacement (DLD), a particle size-based separation technique. The interaction between laminar fluid and an ordered array of micropillars forces particles to move along predetermined trajectories according to their different sizes. Assuming the overall fluid flow is vertically downward, the magnitude of the particle displacement perpendicular to the streamlines (i.e., lateral displacement) is determined by the arrangement of the array pattern: particles below a certain critical size (e.g., 100 nm for the exosome-magnetic bead complex in this disclosure) can follow the streamlines through the array gaps without obstruction and do not undergo lateral displacement; while particles exceeding this critical size are affected by the array pillars, undergoing lateral displacement at a predetermined angle based on the relative offset distance of each row of micropillars. Thus, as the fluid continues to flow, particles of different sizes gradually converge onto different paths, ultimately achieving separation. The critical size depends on the micropillar gaps and the relative offset of each row. In this disclosure, in order to enhance the separation effect of exosome-magnetic bead complexes of different sizes, the first microcolumn 102 in each row can be offset by 0.1 to 0.6 diameters compared to the first microcolumn 102 in the previous row along the flow direction. In other words, the ratio of the preset distance between the first microcolumns 102 in each row to the diameter of the first microcolumn 102 can be greater than or equal to 0.1 and less than or equal to 0.6, for example, it can be 1 / 10, 1 / 5, 1 / 4, 1 / 3, 1 / 2, 3 / 5, etc.

[0040] In some embodiments, in the microfluidic chip provided in the present disclosure, such as Figures 1 to 3 As shown, the cover layer 002 facing the substrate layer 001 has a first groove 201, a second groove 202, and a third groove (e.g., a first third groove 203 and a second third groove 203') that are connected in sequence. The first groove 201 and the substrate 101 form a mixing chamber A, the second groove 202 and the substrate 101 form a separation chamber B, and the third grooves (e.g., a first third groove 203 and a second third groove 203') and the substrate 101 form a liquid collection chamber (e.g., a first liquid collection chamber C and a second liquid collection chamber C'). This eliminates the need for grooves in the substrate layer 001, ensuring the surface flatness of the substrate layer 001 and facilitating sample liquid movement. Of course, in some embodiments, grooves can also be formed by providing grooves on the substrate layer 001, or by providing grooves on both the substrate layer 001 and the cover layer 002; this is not limited here.

[0041] In some embodiments, the first groove 201, the second groove 202, and the third groove (e.g., the first third groove 203 and the second third groove 203') of the cover layer 002 can be processed by molding or cutting. Optionally, the planar dimensions of the first groove 201 and the second groove 202 can both be 5mm × 10mm and the depth can be 0.5mm; the planar dimensions of the third groove (e.g., the first third groove 203 and the second third groove 203') can be 2.5mm × 5mm and the depth can be 0.5mm.

[0042] In some embodiments, in the microfluidic chip provided in this disclosure, to effectively guide the flow of sample liquid between different chambers, such as... Figures 1 to 3 As shown, a first flow channel groove 204 and a second flow channel groove (e.g., a first second flow channel groove 205 and a second second flow channel groove 205') can also be provided on the side of the cover plate layer 002 facing the substrate layer 001. The first flow channel groove 204 connects the first groove 201 and the second groove 202, and the second flow channel groove (e.g., the first second flow channel groove 205 and the second second flow channel groove 205') connects the second groove 202 and the third groove (e.g., the first third groove 203 and the second third groove 203'). Optionally, the first flow channel groove 204 and the second flow channel groove (e.g., the first second flow channel groove 205 and the second second flow channel groove 205') can be processed by molding or cutting, and the width of the first flow channel groove 204 and the width of the second flow channel groove (e.g., the first second flow channel groove 205 and the second second flow channel groove 205') can both be 0.5 mm.

[0043] In some embodiments of the microfluidic chip provided in this disclosure, to facilitate fabrication and ensure that the sample liquid moves at a near-uniform speed along the flow path, thereby improving the separation and purification effect, the depths of the first groove 201, the second groove 202, the third groove (e.g., the first third groove 203, the second third groove 203'), the first flow channel 204, and the second flow channel 205 (e.g., the first second flow channel 205, the second second flow channel 205') can be set to be equal. In this disclosure, "equal" can be understood as identical or within a 10% error range caused by factors such as fabrication and measurement.

[0044] In some embodiments, in the microfluidic chip provided in this disclosure, for better separation and purification of exosomes, such as... Figures 1 to 3As shown, the cover plate layer 002 may further include a second liquid inlet b, a third liquid inlet c, and a fourth liquid inlet (e.g., a first fourth liquid inlet d and a second fourth liquid inlet d'), wherein the second liquid inlet b is connected to the mixing chamber A, the third liquid inlet c is connected to the separation chamber B, the fourth liquid inlet (e.g., a first fourth liquid inlet d and a second fourth liquid inlet d') is connected to the collection chamber (e.g., a first collection chamber C and a second collection chamber C'), and the liquid outlet (e.g., a first liquid outlet e and a second liquid outlet e') is connected to the collection chamber (e.g., a first collection chamber C and a second collection chamber C'). The second inlet hole b can be used to inject the aqueous phase to fully dissolve the exosome-magnetic bead complex; the third inlet hole c can be used to inject the oil phase to trap hydrophobic proteins and other impurities in the solution; the fourth inlet hole (e.g., the first fourth inlet hole d and the second fourth inlet hole d') can be used to inject the aqueous phase to fully dissolve the exosome-magnetic bead complex, facilitating the complete removal of the exosome-magnetic bead complex from the collection chambers (e.g., the first collection chamber C and the second collection chamber C'). Optionally, the diameters of the second inlet hole b, the third inlet hole c, and the fourth inlet hole (e.g., the first fourth inlet hole d and the second fourth inlet hole d') can all be 1 mm.

[0045] In some embodiments, in the microfluidic chip provided in the present disclosure, such as Figures 1 to 3As shown, a first liquid inlet groove 206, a second liquid inlet groove 207, a third liquid inlet groove 208, a fourth liquid inlet groove (e.g., a first fourth liquid inlet groove 209, a second fourth liquid inlet groove 209'), and an outlet groove (e.g., a first outlet groove 210, a second outlet groove 210') can be provided on the side of the cover plate layer 002 facing the substrate layer 001. The first liquid inlet groove 206 connects the first liquid inlet hole a to the mixing chamber A, the second liquid inlet groove 207 connects the second liquid inlet hole b to the mixing chamber A, and the third liquid inlet groove 208... The third inlet hole c is connected to the separation chamber B. The fourth inlet groove (e.g., the first fourth inlet groove 209, the second fourth inlet groove 209') is connected to the fourth inlet hole (e.g., the first fourth inlet hole d, the second fourth inlet hole d') and the collection chamber (e.g., the first collection chamber C, the second collection chamber C'). The outlet groove (e.g., the first outlet groove 210, the second outlet groove 210') is connected to (e.g., the first outlet hole e, the second outlet hole e') and the collection chamber (e.g., the first collection chamber C, the second collection chamber C'). The above-mentioned inlet grooves can buffer the solution injected through the inlet hole, avoiding splashing and overflow of the solution that may occur when the solution is directly injected into each chamber through the inlet hole, thus wasting the sample solution. The outlet grooves can buffer the solution drawn out through the outlet hole, avoiding spraying of the solution that may occur when the solution is directly drawn out of the collection chamber. Optionally, for ease of manufacturing, the depth of the inlet groove and the depth of the outlet groove can be set to be equal to the depth of the first groove 201, the depth of the second groove 202, and the depth of the third groove (e.g., the first third groove 203 and the second third groove 203'), for example, all being 0.5mm. Optionally, the width of the inlet groove and the width of the outlet groove are both 0.5mm.

[0046] In some embodiments, in the microfluidic chip provided in the present disclosure, such as Figures 1 to 3As shown, the cover layer 002 may further include a first vent f, a second vent g, and a third vent (e.g., a first third vent h and a second third vent h') penetrating the thickness direction of the cover layer 002. The first vent f is connected to the mixing chamber A, the second vent g is connected to the separation chamber B, and the third vent (e.g., a first third vent h and a second third vent h') is connected to the liquid collection chamber (e.g., a first liquid collection chamber C and a second liquid collection chamber C'). During the process of adding solution to the chamber through the inlet hole, there may be excessive gas pressure inside the chamber, preventing the solution from smoothly entering the chamber. The vent holes facilitate the discharge of gas during solution addition, balancing the gas pressure inside the chamber with the environment, allowing the solution to smoothly enter the chamber. Correspondingly, during the process of drawing the solution out of the chamber through the outlet, there may be a problem of excessive air pressure in the chamber, which may prevent the solution from being drawn out of the chamber smoothly. The setting of the vent hole helps to release gas during the process of drawing out the solution, balance the air pressure inside the chamber with the environment, and enable the solution to be drawn out of the chamber smoothly.

[0047] See also Figures 1 to 3 As can be seen, the cover plate layer 002 facing the substrate layer 001 is also provided with a first venting groove 211, a second venting groove 212, and a third venting groove (e.g., the first third venting groove 213 and the second third venting groove 213'). The first venting groove 211 connects the first venting hole f to the mixing chamber A; the second venting groove 212 connects the second venting hole g to the separation chamber B; and the third venting grooves (e.g., the first third venting groove 213 and the second third venting groove 213') connect the third venting hole (e.g., the first third venting hole h and the second third venting hole h') to the liquid collecting chamber (e.g., the first liquid collecting chamber C and the second liquid collecting chamber C'). Optionally, for ease of manufacturing, the depth of each venting groove can be set to be equal to the depth of the first groove 201, the depth of the second groove 202, and the depth of the third groove (e.g., the first third groove 203 and the second third groove 203').

[0048] Based on the same inventive concept, this disclosure provides a microfluidic system, including a microfluidic chip and a magnetizing component. The microfluidic chip is the same as described above, and the magnetizing component is configured to drive a magnetic bead to move on the side of the substrate layer opposite to the cover plate layer. Since the principle by which this microfluidic system solves the problem is similar to that of the microfluidic chip described above, the implementation of this microfluidic system can refer to the implementation of the microfluidic chip described above, and repeated details will not be elaborated further.

[0049] Based on the same inventive concept, this disclosure provides a method for using the above-described microfluidic system, which may include the following steps:

[0050] A sample solution including exosomes and magnetic beads is added to a mixing chamber, such that the magnetic beads bind to at least a portion of the exosomes to form an exosome-magnetic bead complex; in some embodiments, a magnetic supply component may be moved back and forth in the mixing chamber to drive the magnetic beads to fully mix the sample solution.

[0051] Driven by the magnetic supply component, the exosome-magnetic bead complex in the mixing chamber is moved into the separation chamber, so that exosome-magnetic bead complexes of different sizes can be separated by multiple first microcolumns in the separation chamber. The exosome-magnetic bead complexes of different sizes flow to different collection chambers and are collected.

[0052] In some embodiments, in the above-described method of use provided in this disclosure, before adding the sample solution including exosomes and magnetic beads to the mixing chamber, the following steps may be performed to better achieve specific separation of exosomes:

[0053] Add the aqueous phase to the mixing chamber until the remaining space in the mixing chamber is used to hold the sample liquid. Fill the separation chamber with the oil phase and fill the collection chamber with the aqueous phase.

[0054] In some instances, in the above-described usage methods provided in the embodiments of this disclosure, in order to balance the air pressure inside the chamber and the environment and facilitate the smooth injection of solution into each chamber, it is also necessary to keep the mixing chamber connected to the outside during the process of adding the aqueous phase and sample solution to the mixing chamber; keep the separation chamber connected to the outside during the process of adding the oil phase to the separation chamber; and keep the collection chamber connected to the outside during the process of adding the aqueous phase to the collection chamber.

[0055] To better understand the method for separating exosomes using the microfluidic system provided in the embodiments of this disclosure, the separation process is described in detail below.

[0056] Step 1, Liquid Filling: First, seal all through holes except for the second inlet hole b and the first vent hole f. Then, add aqueous phase through the second inlet hole b until it almost fills the mixing chamber A, but reserve a small volume for adding the sample solution to be separated and purified (including magnetic beads and exosomes). Optionally, the volume of the aqueous phase can be adjusted according to the sample volume, as long as the total volume of both fills the mixing chamber A, for example, the ratio of aqueous phase to sample solution volume is 4:1 or higher. Subsequently, seal all through holes except for the third inlet hole c and the second vent hole g, and add the aqueous phase through the second inlet hole b until it almost fills the mixing chamber A. Add oil phase through the three inlet holes (c) until the separation chamber B is filled; finally, seal all through holes except the fourth inlet hole (e.g., the first fourth inlet hole d, the second fourth inlet hole d') and the third vent hole (e.g., the first third vent hole h, the second third vent hole h'). Add aqueous phase through the fourth inlet holes (e.g., the first fourth inlet hole d, the second fourth inlet hole d') until the collection chamber (e.g., the first collection chamber C, the second collection chamber C') is filled. Optionally, the aqueous phase is 1×PBS solution, and the oil phase is mineral oil. It should be noted that there are several methods for sealing the through holes. A simple method is to directly attach a plastic film with good sealing properties to the through hole; a slightly more complex method is to use the adapters that are often used in chip inlet / outlet ports. The adapter is glued to the through hole with curing adhesive, and the other end is connected to the injection pump through a conduit. The injection pump controls the opening and closing.

[0057] The second step is sample addition and mixing: Seal all through holes except for the first inlet hole a and the first vent hole f. Add the mixture of the exosome sample to be separated and the magnetic beads through the first inlet hole a until the mixing chamber A is full. The surface of the magnetic beads is coated with antibodies that can specifically bind to specific types of exosome surface proteins, thereby capturing the exosomes and generating an exosome-magnetic bead complex. The antibody can be anti-CD63. After adding the sample solution, place the strip-shaped permanent magnet under the chip and move the permanent magnet back and forth in the mixing chamber A to drive the magnetic beads in the chamber to mix thoroughly.

[0058] The third step is the multiple separation and purification of exosomes: Driven by a permanent magnet, the exosome-magnetic bead complex from mixing chamber A is slowly moved into separation chamber B through the first flow channel 204, and continues to pass through the microcolumn array in separation chamber B at a low speed. At this time, the smaller volume of exosome-magnetic bead complex does not undergo lateral displacement and eventually flows into the second collection chamber C', while the larger volume of exosome-magnetic bead complex undergoes lateral displacement to the left and eventually flows into the first collection chamber C. After the exosome-magnetic bead complexes of different sizes have completely entered the first collection chamber C and the second collection chamber C', the third vent (e.g., the first third vent h and the second third vent h') and the outlet (e.g., the first outlet e and the second outlet e') are opened. The solution in the collection chamber (e.g., the first collection chamber C and the second collection chamber C') is aspirated from the outlet (e.g., the first outlet e and the second outlet e'). Subsequent magnetic bead elution and other operations are then performed to complete the purification and separation steps of the exosomes.

[0059] Although preferred embodiments have been described in this disclosure, it should be understood that those skilled in the art can make various modifications and variations to the embodiments of this disclosure without departing from the spirit and scope of the embodiments of this disclosure. Therefore, this disclosure is also intended to include such modifications and variations if they fall within the scope of the claims of this disclosure and their equivalents.

Claims

1. A microfluidic chip, characterized in that, include: The substrate layer includes a substrate substrate and a plurality of first micropillars on the substrate substrate, the plurality of first micropillars being arranged in multiple rows and columns, with each row of first micropillars being staggered by a predetermined distance. A cover plate layer, positioned opposite the substrate layer, includes a first inlet hole extending through the thickness direction of the cover plate layer and at least two outlet holes. The space between the cover plate layer and the substrate layer includes a mixing chamber, a separation chamber, and at least two collection chambers. The first inlet hole communicates with the mixing chamber, the at least two outlet holes communicate with the at least two collection chambers, and the separation chamber communicates with the mixing chamber and the at least two collection chambers. The mixing chamber is configured to contain a sample solution including exosomes and magnetic beads. The plurality of first microcolumns are placed in the separation chamber, and different collection chambers are configured to collect exosome-magnetic bead complexes of different sizes.

2. The microfluidic chip as described in claim 1, characterized in that, The substrate layer also includes a plurality of second micropillars disposed in the mixing chamber, the plurality of second micropillars being arranged in multiple rows and columns, with each row of second micropillars aligned along the column direction.

3. The microfluidic chip as described in claim 2, characterized in that, The arrangement density of each of the first micropillars is greater than the arrangement density of each of the second micropillars.

4. The microfluidic chip as described in claim 2, characterized in that, The diameter of the first micropillar is smaller than the diameter of the second micropillar, and the height of the first micropillar is greater than the height of the second micropillar.

5. The microfluidic chip as described in claim 1, characterized in that, The ratio of the preset distance to the diameter of the first micropillar is greater than or equal to 0.1 and less than or equal to 0.

6.

6. The microfluidic chip as described in claim 1, characterized in that, The cover plate has a first groove, a second groove, and a third groove that are connected in sequence on one side facing the substrate layer. The first groove and the substrate form the mixing chamber, the second groove and the substrate form the separation chamber, and the third groove and the substrate form the liquid collection chamber.

7. The microfluidic chip as described in claim 6, characterized in that, The cover plate surface is further provided with a first flow channel groove and a second flow channel groove on the side facing the substrate layer, wherein the first flow channel groove connects the first groove and the second groove, and the second flow channel groove connects the second groove and the third groove.

8. The microfluidic chip as described in claim 7, characterized in that, The depths of the first groove, the second groove, the third groove, the first flow channel groove, and the second flow channel groove are equal.

9. The microfluidic chip according to any one of claims 1 to 8, characterized in that, The cover plate layer further includes a second liquid inlet hole, a third liquid inlet hole, and a fourth liquid inlet hole penetrating the thickness direction of the cover plate layer. The cover plate layer is also provided with a first liquid inlet groove, a second liquid inlet groove, a third liquid inlet groove, a fourth liquid inlet groove, and an outlet groove on the side facing the substrate layer. The first liquid inlet groove connects the first liquid inlet hole to the mixing chamber, the second liquid inlet groove connects the second liquid inlet hole to the mixing chamber, the third liquid inlet groove connects the third liquid inlet hole to the separation chamber, the fourth liquid inlet groove connects the fourth liquid inlet hole to the collection chamber, and the outlet groove connects the outlet hole to the collection chamber.

10. The microfluidic chip as described in claim 9, characterized in that, The cover plate layer further includes a first vent hole, a second vent hole, and a third vent hole penetrating the thickness direction of the cover plate layer. The cover plate layer also has a first vent groove, a second vent groove, and a third vent groove on the side facing the substrate layer. The first vent groove connects the first vent hole to the mixing chamber, the second vent groove connects the second vent hole to the separation chamber, and the third vent groove connects the third vent hole to the liquid collection chamber.

11. A microfluidic system, characterized in that, The invention includes a microfluidic chip and a magnetizing component, wherein the microfluidic chip is the microfluidic chip as described in any one of claims 1 to 10, and the magnetizing component is configured to drive a magnetic bead to move on the side of the substrate layer opposite to the cover plate layer.

12. A method of using the microfluidic system as described in claim 11, characterized in that, include: A sample solution including exosomes and magnetic beads is added to a mixing chamber, such that the magnetic beads bind to at least a portion of the exosomes to form an exosome-magnetic bead complex. Driven by the magnetic supply component, the exosome-magnetic bead complex in the mixing chamber is moved into the separation chamber, so as to separate exosome-magnetic bead complexes of different sizes using multiple first microcolumns in the separation chamber. The exosome-magnetic bead complexes of different sizes flow to different collection chambers and are collected.

13. The method of use as described in claim 12, characterized in that, Before adding the sample solution, including exosomes and magnetic beads, to the mixing chamber, the following steps are also included: Add the aqueous phase to the mixing chamber until the remaining space in the mixing chamber is used to hold the sample liquid, fill the separation chamber with the oil phase, and fill the collection chamber with the aqueous phase.

14. The method of use as described in claim 13, characterized in that, During the process of adding the aqueous phase and sample solution to the mixing chamber, the mixing chamber is kept open to the outside; during the process of adding the oil phase to the separation chamber, the separation chamber is kept open to the outside; during the process of adding the aqueous phase to the collection chamber, the collection chamber is kept open to the outside.

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