Magnetic sorting chip for cell sorting and preparation method thereof

Abstract

The embodiment of the invention discloses a magnetic separation chip for cell separation and a preparation method thereof.The magnetic separation chip structurally comprises a sample layer, a functional layer, a clamp set and a magnetic field source, the sample layer is composed of a flow channel layer and a sealing film, and the flow channel layer is provided with a sheath fluid inlet, a sample inlet and a sample outlet; the sealing film is attached to the open surface of the runner layer to seal the runner; the functional layer is located below the sample layer and comprises a patterned substrate and an amorphous alloy soft magnetic strip material, the patterned substrate is provided with a counter bore groove, the amorphous alloy soft magnetic strip material is embedded into the counter bore groove, and the upper surface of the amorphous alloy soft magnetic strip material is flush with the upper surface of the substrate, right faces the sealing film and is arranged in the extending direction of the flow channel; the clamp group comprises an upper clamping plate and a lower clamping plate, the upper clamping plate is arranged above the sample layer, the lower clamping plate is arranged below the functional layer, the upper clamping plate is provided with runner holes corresponding to the inlets and the outlets, and the two are connected through bolts to clamp and fix the sample layer and the functional layer; the magnetic field source is arranged at the end where the sample outlet is located and on one side opposite to the functional layer, so that a gradient magnetic field which is gradually enhanced from the inlet to the outlet is formed in the flow channel.

Description

Technical Field

[0001] This application relates to the field of cell sorting technology, and in particular to a magnetic sorting chip for cell sorting and its preparation method. Background Technology

[0002] Magnetic cell sorting technology uses antibody-modified magnetic beads to specifically label target cells, and then uses an external magnetic field to efficiently capture the target cells. Due to its advantages such as simple operation and minimal cell damage, it is widely used in stem cell isolation, immune cell enrichment, and circulating tumor cell detection. However, existing technologies have the following limitations: the magnetic beads require a dedicated sorting column, which is costly, and the magnetic field source is bulky, resulting in cumbersome equipment.

[0003] Therefore, it is necessary to provide a cell sorting solution that enables device miniaturization and lower cost. Summary of the Invention

[0004] This specification provides a magnetic sorting chip for cell sorting and a method for its fabrication, in order to solve at least one of the technical problems mentioned above.

[0005] To solve the above-mentioned technical problems, the embodiments in this specification are implemented as follows:

[0006] According to a first aspect of the present invention, a magnetic sorting chip for cell sorting is provided, comprising:

[0007] The sample layer includes a flow channel layer and a sealing membrane. The flow channel layer is provided with a sheath fluid inlet, a sample inlet, and a sample outlet, and has flow channels machined inside. The sealing membrane is attached to the open surface of the flow channel layer to seal the flow channels inside the flow channel layer.

[0008] A functional layer is located below the sample layer. The functional layer includes a patterned substrate. A plurality of countersunk grooves are sequentially formed along the length of the patterned substrate. An amorphous alloy flexible magnetic tape is embedded in each countersunk groove, and the upper surface of the amorphous alloy flexible magnetic tape is flush with the upper surface of the patterned substrate, with the amorphous alloy flexible magnetic tape facing the sealing film. The amorphous alloy flexible magnetic tape is arranged sequentially along the length of the patterned substrate, forming an array distribution parallel to the flow channel extension direction of the sample layer.

[0009] The clamp assembly includes an upper clamping plate and a lower clamping plate. The upper clamping plate is disposed above the sample layer, and the lower clamping plate is disposed below the functional layer. The upper clamping plate has corresponding flow channel holes at the positions of the sheath fluid inlet, the sample inlet, and the sample outlet. The upper clamping plate and the lower clamping plate are connected by bolts to clamp and fix the sample layer and the functional layer.

[0010] A magnetic field source is located at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the outlet is formed in the flow channel.

[0011] In some optional embodiments, the patterned substrate is provided with a cross-shaped positioning mark and a triangular flow direction mark; wherein, the cross-shaped positioning mark is used for alignment when the sample layer and the functional layer are assembled, and the triangular flow direction mark points in the same direction as the fluid flow direction in the flow channel.

[0012] In some alternative embodiments, the cross-shaped positioning marks are disposed at the four corners of the patterned substrate, and the triangular flow direction marks are located at the flow channel inlet end of the patterned substrate.

[0013] In some alternative embodiments, the sealing film is made of polycarbonate and has a thickness in the micrometer range, and is bonded to the open surface of the flow channel layer by adhesive bonding or hot pressing; the flow channel layer is made of polycarbonate and is prepared by injection molding.

[0014] In some optional embodiments, O-rings are provided between the sheath fluid inlet, sample inlet, and sample outlet of the flow channel layer and their respective flow channel holes on the upper clamping plate.

[0015] In some alternative embodiments, the depth of the countersunk groove is adapted to the thickness of the amorphous alloy flexible magnetic tape.

[0016] In some alternative implementations, the magnetic field source is a rectangular permanent magnet with its long side parallel to the flow channel direction.

[0017] In some optional embodiments, the sheath fluid inlet, sample inlet, and sample outlet are distributed sequentially along the flow channel layer; wherein the sheath fluid inlet and the sample inlet are both located in the upstream region of the flow channel layer, and the sample outlet is located at the downstream end of the flow channel layer.

[0018] According to a second aspect of the present invention, a method for fabricating a magnetic sorting chip for cell sorting is provided, comprising:

[0019] A countersunk hole groove, a cross-shaped positioning mark, and a triangular flow direction mark are processed on a patterned substrate. An amorphous alloy soft magnetic tape is then embedded in the countersunk hole groove, so that the upper surface of the amorphous alloy soft magnetic tape is flush with the upper surface of the patterned substrate, forming a microfluidic functional layer.

[0020] A flow channel layer with a sheath fluid inlet and a sample outlet is prepared by injection molding. A sealing film is then attached to the open surface of the flow channel layer by adhesive bonding or hot pressing to seal the flow channel, thus forming a microfluidic sample layer.

[0021] Flow channel holes are machined on the upper and lower clamping plates at the positions corresponding to the sheath fluid inlet, sample inlet and sample outlet, and O-rings are pre-installed at the interface between the flow channel holes on the upper clamping plate and the flow channel layer.

[0022] The microfluidic sample layer and the microfluidic functional layer are sequentially sandwiched between the upper clamping plate and the lower clamping plate, and the upper clamping plate and the lower clamping plate are connected by bolts to achieve interlayer clamping and fixation;

[0023] The magnetic field source is placed at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the outlet is formed in the flow channel.

[0024] One embodiment of this specification achieves at least the following beneficial effects: The magnetic sorting chip provided by this invention is designed collaboratively through a sample layer, a functional layer, a fixture assembly, and a magnetic field source. Specifically, the sheath fluid inlet of the flow channel layer in the sample layer introduces sheath fluid to form a stable boundary layer, ensuring the flow state of the sample within the flow channel. The sealing film, adhering to the open surface of the flow channel layer, achieves sealing while, due to its appropriate thickness design, reducing the weakening of the magnetic field, ensuring that the magnetic field can effectively act on the cells bound to magnetic beads within the flow channel. In the functional layer, the patterned substrate with recessed grooves embeds amorphous alloy soft magnetic tape, and its upper surface is flush with the array distribution parallel to the flow channel extension direction. Combined with the localized strengthening effect of the high relative permeability of the amorphous alloy on the magnetic field, it can work in conjunction with the magnetic field source to construct a gradient magnetic field that gradually increases from the inlet to the outlet within the flow channel. This allows cells with different numbers of bound magnetic beads to be captured successively at different positions within the flow channel, preventing cell accumulation at the magnetic field inlet and improving sorting efficiency. The upper and lower clamping plates of the fixture assembly clamp the sample layer and the functional layer together with bolts, ensuring a tight fit between the layers and preventing fluid leakage. Simultaneously, the flow channel holes in the upper clamping plate allow for effective connection of external pipelines to each inlet / outlet. A magnetic field source is positioned at the sample outlet end, directly opposite the functional layer, further enhancing the formation of the gradient magnetic field. Combined with the array distribution of amorphous alloys, this reduces the requirements for magnet size. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments or prior art of this specification, the drawings used in the description of the embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the structure of the magnetic sorting chip for cell sorting provided by the present invention;

[0027] Figure 2 This is a schematic diagram of the sample layer flow channel in the magnetic sorting chip for cell sorting provided by the present invention;

[0028] Figure 3 A planar diagram of a patterned base;

[0029] Figure 4 This is an assembly diagram of the magnetic sorting chip for cell sorting provided by the present invention;

[0030] Figure 5 This is a schematic diagram showing the location of the magnetic field source in the magnetic sorting chip for cell sorting provided by the present invention.

[0031] Figure 6 This is a flowchart illustrating the method for fabricating a magnetic sorting chip for cell sorting provided by the present invention.

[0032] Wherein, 1 represents the upper clamping plate, 2 represents the lower clamping plate, 3 represents the sheath fluid inlet, 4 represents the sample inlet, 5 represents the sample outlet, 6 represents the flow channel layer, 7 represents the sealing membrane, 8 represents the amorphous alloy soft magnetic tape material, and 9 represents the patterned substrate. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of one or more embodiments of this specification clearer, the technical solutions of one or more embodiments of this specification will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this specification, and not all of them. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of one or more embodiments of this specification.

[0034] It should be understood that although the terms first, second, third, etc., may be used in this application to describe various information, this information should not be limited to these terms. These terms are only used to distinguish information of the same type from one another.

[0035] like Figure 1 As shown, Figure 1 This is a schematic diagram of the structure of the magnetic sorting chip for cell sorting provided by the present invention. Figure 1The magnetic sorting chip comprises a sample layer, a functional layer, a fixture assembly, and a magnetic field source. The sample layer includes a flow channel layer 6 and a sealing membrane 7. The flow channel layer 6 has a sheath fluid inlet 3, a sample inlet 4, and a sample outlet 5, and internally has flow channels. The sealing membrane 7 is attached to the open surface of the flow channel layer 6 to seal the flow channels within the flow channel layer 6. The functional layer is located below the sample layer and includes a patterned substrate 9. Several countersunk holes are sequentially formed along the length of the patterned substrate 9. Each countersunk hole contains an amorphous alloy soft magnetic tape 8, the upper surface of which is flush with the upper surface of the patterned substrate 9, and the amorphous alloy soft magnetic tape 8 faces the sealing membrane 7. The amorphous alloy soft magnetic tape 8 is arranged sequentially along the length of the patterned substrate 9, forming an array distribution parallel to the flow channel extension direction of the sample layer. The clamp assembly includes an upper clamping plate 1 and a lower clamping plate 2. The upper clamping plate 1 is positioned above the sample layer, and the lower clamping plate 2 is positioned below the functional layer. The upper clamping plate 1 has corresponding flow channel holes at the positions of the sheath fluid inlet 3, sample inlet 4, and sample outlet 5. The upper clamping plate 1 and the lower clamping plate 2 are connected by bolts to clamp and fix the sample layer and the functional layer. A magnetic field source is positioned at the end where the sample outlet is located and on the side directly opposite the functional layer, creating a gradient magnetic field that gradually increases from the inlet to the outlet within the flow channel. Figure 5 As shown, Figure 5 This is a schematic diagram illustrating one possible placement of the magnetic field source in the magnetic sorting chip for cell sorting provided by the present invention.

[0036] Figure 2 This is a schematic diagram of the sample layer flow channel in the magnetic sorting chip for cell sorting provided by the present invention. Figure 2 The left side is a circular chamber. At the chamber outlet, i.e. the constriction section of the flow channel, a laminar flow structure with "sheath fluid on the outside and sample flow on the inside" is constructed to confine the sample flow to the center of the flow channel, preventing cells from deviating from the magnetic field area due to diffusion. The long strip-shaped main channel in the middle is the sorting core area. After the cells wrapped in laminar flow enter the main channel, cells with different magnetic bead loads are captured one after another at different positions in the flow channel due to the difference in magnetic field force. That is, cells with more magnetic beads are pulled towards the sealing membrane upstream of the main channel (the area with weaker magnetic field), while cells with fewer magnetic beads move downstream with the fluid to the area with stronger magnetic field until they are adsorbed by force. Unlabeled cells are discharged from sample outlet 5.

[0037] The working principle of this invention is explained below. The functional layer in this invention functions to construct a locally enhanced magnetic field using the characteristics of the amorphous alloy soft magnetic tape 8, providing magnetic drive for cell sorting. Specifically, the amorphous alloy soft magnetic tape 8 in the functional layer, due to its high relative permeability, can efficiently concentrate and enhance the magnetic field generated by an external magnetic field source, forming a localized magnetic field concentrated in the sample layer flow channel region. This enhancement is not uniform but exhibits a gradient characteristic, and its intensity distribution is directly related to the position of the magnetic field source. That is, since the magnetic field source is located at the sample outlet end and on the side directly opposite the functional layer, the closer to the outlet end, i.e., the closer to the magnetic field source, the stronger the initial magnetic field generated by the magnetic field source itself. The enhancement effect of the amorphous alloy at the corresponding position on the magnetic field is also more significant, thus forming a gradient magnetic field that gradually strengthens from the flow channel inlet to the outlet. Conversely, the closer to the inlet of the sample layer, the weaker the initial magnetic field due to the greater distance from the magnetic field source, and the weaker the local enhancement effect of the amorphous alloy, resulting in a decrease in the localized magnetic field intensity. This locally enhanced magnetic field generates a downward magnetic force on cells bound to magnetic beads within the sample layer flow channel, towards the functional layer, thus pulling the cells toward the sealing membrane 7 of the microfluidic chip. As the cells flow within the channel, they are simultaneously subjected to this downward magnetic force and the frictional force from the fluid. The magnetic force attempts to attract the cells to the surface of the sealing membrane 7, while the frictional force attempts to propel the cells further along the flow channel. When the magnetic force exceeds the frictional force, the cells are fixed at specific positions on the sealing membrane 7. Simultaneously, due to the gradient distribution of the magnetic field (weak at the inlet and strong at the outlet), cells with a higher number of bound magnetic beads can obtain sufficient magnetic force in the weaker magnetic field at the inlet and are captured earlier. Cells with fewer bound magnetic beads need to continue moving within the channel to a region with a stronger magnetic field until the magnetic force is sufficient to overcome the frictional force and are captured. Through this dynamic balance between magnetic force and friction, cells with different magnetic bead loads are fixed at different positions on the sealing membrane 7, ultimately achieving precise cell sorting. In the above process, the amorphous alloy, through its high magnetic permeability, concentrates the magnetic field lines of the magnetic field source in its own region, thereby increasing the local magnetic field strength. This reduces the dependence on the volume of the magnetic field source, thus achieving equipment weight reduction. Furthermore, by cooperating with the position of the magnetic field source, it constructs a gradient magnetic field, which can solve the problems of cell accumulation and low sorting efficiency in traditional technologies.

[0038] Specifically, in the above process, after the sheath fluid enters through the sheath fluid inlet, it forms a fluid layer along the inner wall of the channel, enveloping the sample flow. This boundary layer effectively constrains the diffusion and turbulence of the sample flow, ensuring that the cells in the sample maintain an orderly linear motion within the channel, providing a stable fluid environment for subsequent magnetic field sorting. The heterogeneity of target cells, especially tumor cells, is reflected in significant individual differences within the cell population. Different cells express different levels of specific proteins on their surfaces. Magnetic beads used to label cells typically bind to specific proteins on the cell surface via antibodies. This specific interaction between antibodies and proteins results in a significant difference in the number of magnetic beads that can bind to different cell surfaces; cells with high surface protein expression levels bind more magnetic beads, while those with low expression levels bind fewer. When the target cells enter the chip with the sample flow, the gradient magnetic field, which gradually intensifies from the inlet to the outlet, begins to function. The cells with the most bound magnetic beads experience the strongest magnetic force and can overcome fluid friction in the relatively weak magnetic field at the channel inlet, thus being the first to be pulled towards the surface of the sealing membrane 7 for capture. Cells with fewer magnetic beads are less likely to be captured in the weak magnetic field at the inlet and will continue to move with the sample flow toward the outlet. As the magnetic field strength in the channel gradually increases, these cells will be subjected to sufficient magnetic force in turn and eventually be captured at the corresponding location. This successive capture mechanism based on the difference in the number of magnetic beads and the gradient magnetic field can effectively prevent cells from accumulating in a single location.

[0039] The magnetic sorting chip provided by this invention features a collaborative design of a sample layer, a functional layer, a fixture assembly, and a magnetic field source. In the sample layer, the sheath fluid inlet of the flow channel layer 6 introduces sheath fluid to form a stable boundary layer, ensuring the sample's flow within the channel. The sealing film 7, adhering to the open surface of the flow channel layer 6, achieves sealing while its appropriately designed thickness reduces the weakening of the magnetic field, ensuring the magnetic field effectively acts on the cells bound to magnetic beads within the channel. In the functional layer, the patterned substrate 9 has recessed slots into amorphous alloy soft magnetic tape 8, with its upper surface flush and arrayed parallel to the flow channel extension direction. Combined with the high relative permeability of the amorphous alloy, which locally enhances the magnetic field, this works in conjunction with the magnetic field source to construct a gradient magnetic field that gradually increases from the inlet to the outlet within the channel. This allows cells with varying numbers of bound magnetic beads to be captured sequentially at different locations within the channel, preventing cell accumulation at the magnetic field inlet and improving sorting efficiency. The upper and lower clamping plates of the fixture assembly clamp the sample layer and the functional layer together with bolts, ensuring a tight fit between the layers and preventing fluid leakage. Simultaneously, the flow channel holes in the upper clamping plate 1 allow for effective connection of external pipelines to each inlet / outlet. A magnetic field source is positioned at the sample outlet end, directly opposite the functional layer, further enhancing the formation of the gradient magnetic field. Combined with the array distribution of amorphous alloys, this reduces the requirements for magnet size.

[0040] Based on the foregoing technical solutions, this invention also provides some more specific technical solutions, which are described below.

[0041] In an optional embodiment, the patterned substrate 9 may be provided with a cross-shaped positioning mark and a triangular flow direction mark; wherein, the cross-shaped positioning mark is used for alignment when the sample layer and the functional layer are assembled, and the triangular flow direction mark points in the same direction as the fluid flow direction in the flow channel.

[0042] In this embodiment, the cross-shaped positioning mark on the patterned substrate 9 is used to achieve precise alignment during the assembly of the sample layer and the functional layer, ensuring that the flow channels of the sample layer and the amorphous alloy soft magnetic tape array of the functional layer are accurately matched in spatial position. The triangular flow direction mark points in the same direction as the fluid flow direction in the flow channel, providing intuitive guidance for the orientation matching of each component during chip assembly, pipeline connection, and magnetic field source arrangement, avoiding misalignment of the magnetic field gradient and the fluid movement direction due to incorrect orientation.

[0043] In an optional embodiment, the cross-shaped positioning marks are disposed at the four corners of the patterned substrate 9, and the triangular flow direction marks are located at the flow channel inlet end of the patterned substrate 9.

[0044] In this embodiment, cross-shaped positioning marks are placed at the four corners of the patterned substrate 9. These marks ensure precise alignment of the sample layer and functional layer during assembly, guaranteeing accurate spatial correspondence between the flow channels of the sample layer and the amorphous alloy soft magnetic tape array of the functional layer. This prevents misalignment between layers from affecting the magnetic field's effect on the labeled cells. Triangular flow direction marks are located at the inlet of the flow channels on the substrate, pointing in the same direction as the fluid flow within the channels. This provides clear directional guidance during chip assembly, pipeline connection, and magnetic field source placement, ensuring that each component is installed according to the preset fluid flow direction and magnetic field gradient direction. This guarantees the synergy between the gradient magnetic field and the cell movement path, thereby achieving effective cell sorting.

[0045] like Figure 3 As shown, Figure 3 The patterned substrate is a planar diagram. The patterned substrate can be made of silica glass. Taking advantage of the good chemical stability and easy control of processing precision of silica glass, countersunk grooves are constructed on the surface of the substrate through laser processing. Figure 3In the patterned silica glass substrate planar diagram, the white-filled area corresponds to the laser-processed area. Four cross-shaped markers are placed within this area as positioning marks during the assembly of the substrate with other functional layers of the chip (such as the sample layer and upper clamping plate). The spatial alignment using these cross-shaped markers ensures precise fit between the amorphous alloy flexible magnetic tape placement area and the sample layer flow channels, magnetic field source, and other structures, preventing misalignment between layers that could cause the magnetic field application area to shift. Simultaneously, the triangular markers within the area serve as flow direction indicators, clearly indicating the flow direction of sheath fluid, sample fluid, and cells within the microfluidic chip. The rectangular area is the placement area for the amorphous alloy flexible magnetic tape. The depth of the countersunk groove is precisely matched to the thickness of the amorphous alloy flexible magnetic tape. When the amorphous alloy flexible magnetic tape is bonded to the patterned substrate, its upper surface remains flush with the substrate's upper surface. This flush design ensures tight adhesion between functional layers during subsequent chip lamination and packaging, preventing gaps caused by height differences from interfering with fluid flow or magnetic field conduction. In addition, amorphous alloy soft magnetic tape can also be prepared by laser cutting. By utilizing the high precision of laser processing, the size and shape of the amorphous alloy soft magnetic tape can be matched with the rectangular area of ​​the substrate recess, ensuring that the amorphous alloy soft magnetic tape is stably embedded in the substrate.

[0046] In an optional embodiment, the sealing film 7 may be made of polycarbonate, and the thickness of the sealing film 7 is on the micrometer level and is bonded to the open surface of the flow channel layer 6 by adhesive or hot pressing; the flow channel layer 6 may also be made of polycarbonate and prepared by injection molding.

[0047] The following explains why the thickness of the sealing film 7 in the embodiment is so thin, it must be at the micrometer level. Considering that in the technical solution of this invention, the magnetic field in the chip sample layer does not come directly from an external magnetic field source, but is formed by the amorphous alloy soft magnetic tape 8 in the functional layer strengthening the local magnetic field, the strength of this magnetic field exhibits a clear spatial characteristic, that is, it is negatively correlated with the distance from a certain position in the sample layer to the amorphous alloy in the functional layer. The closer to the amorphous alloy soft magnetic tape 8, the stronger the magnetic field; the farther away, the weaker the magnetic field. Because the magnetic field generated by the amorphous alloy has significant locality and its range of action is relatively concentrated, the negative correlation between the magnetic force at each location in the sample layer and the distance from that location to the amorphous alloy soft magnetic tape 8 is very strong. Specifically, the magnetic field strength weakens rapidly and significantly with even a small change in distance. This characteristic places strict requirements on the thickness of the sealing film 7 in the sample layer. If the sealing film 7 is too thick, it will significantly increase the distance between the cells and their attached magnetic beads in the sample layer and the amorphous alloy soft magnetic tape 8, causing the magnetic field strength to decrease sharply and failing to generate sufficient magnetic force on the magnetic beads for effective cell capture and sorting. Therefore, the thickness of the sealing film 7 must be controlled at the micrometer level to minimize the weakening of the magnetic field strength by reducing the distance, ensuring that the magnetic field can exert an effective force on the magnetic beads, thereby guaranteeing the cell sorting function.

[0048] Meanwhile, in this embodiment, the sealing membrane 7 is made of polycarbonate and is bonded to the open surface of the flow channel layer 6 by adhesive bonding or hot pressing. This choice of material and connection method ensures the sealing performance of the flow channel layer, preventing fluid leakage, and also reduces the weakening of the magnetic field due to the thinness of the film, ensuring that the magnetic field can effectively act on the cells with magnetic beads bound inside the flow channel. Moreover, the flow channel layer 6 is also made of polycarbonate and is prepared by injection molding. This material is suitable for injection molding to form the required flow channel structure and can achieve good adhesion with the sealing membrane 7 of the same material through bonding.

[0049] In an optional embodiment, an O-ring is provided between the sheath fluid inlet 3, sample inlet 4, and sample outlet 5 of the flow channel layer 6 and the corresponding flow channel holes on the upper clamping plate.

[0050] In this embodiment, O-rings are provided between the sheath fluid inlet 3, sample inlet 4, and sample outlet 5 of the flow channel layer 6 and the corresponding flow channel holes on the upper clamping plate. The O-rings can achieve a seal between the upper clamping plate 1 and the flow channel layer 6, preventing leakage of fluids such as sheath fluid and sample suspension when they flow through the inlet or outlet, ensuring that the fluid flows stably in the flow channel according to the preset path, so that the flow state of the sample and the sorting environment during the cell sorting process are not affected by external leakage.

[0051] In an optional embodiment, the depth of the countersunk groove can be adapted to the thickness of the amorphous alloy soft magnetic tape 8.

[0052] In this embodiment, the depth of the countersunk groove is adapted to the thickness of the amorphous alloy soft magnetic tape 8, so that after the amorphous alloy soft magnetic tape 8 is embedded in the countersunk groove, its upper surface that is bonded to the substrate can be flush with the upper surface of the patterned substrate 9. This structural design can ensure the flatness of the functional layer surface, thereby avoiding uneven stress on the sealing film 7 of the sample layer or deformation of the flow channel due to the protrusion or depression of the amorphous alloy soft magnetic tape 8 when the functional layer is bonded to the sample layer. This ensures the stable flow of fluid in the flow channel and the uniform strengthening effect of the amorphous alloy soft magnetic tape 8 on the local magnetic field, ensuring that the magnetic field can effectively act on the cells with magnetic beads bonded in the flow channel.

[0053] In an optional embodiment, the magnetic field source is a rectangular permanent magnet, with its long side parallel to the flow channel direction.

[0054] In this embodiment, the magnetic field source is a rectangular permanent magnet with its long side parallel to the flow channel direction. This arrangement helps the magnetic field to form a stable distribution along the flow channel extension direction. Combined with the amorphous alloy soft magnetic tape material 8 arranged along the flow channel direction in the functional layer, it can better construct a gradient magnetic field that gradually increases from the inlet to the outlet in the flow channel, so that cells with different numbers of magnetic beads are captured one after another at different positions in the flow channel.

[0055] In an optional embodiment, the sheath fluid inlet 3, the sample inlet 4, and the sample outlet 5 are sequentially distributed along the flow channel layer 6; wherein the sheath fluid inlet 3 and the sample inlet 4 are both located in the upstream region of the flow channel layer 6, and the sample outlet 5 is located at the downstream end of the flow channel layer.

[0056] In this embodiment, the sheath fluid inlet 3, sample inlet 4, and sample outlet 5 are distributed sequentially along the flow channel layer. The sheath fluid inlet 3 and sample inlet 4 are both located in the upstream region of the flow channel layer 6, and the sample outlet 5 is located at the downstream end of the flow channel layer 6. This distribution allows the sheath fluid and sample suspension to converge in the upstream region. The sheath fluid can form a stable boundary layer in the flow channel to encapsulate the sample flow. Subsequently, the mixed fluid flows along the flow channel to the sample outlet located at the downstream end. During the flow, cells with different numbers of magnetic beads are successively captured under the action of the gradient magnetic field. The uncaptured fluid is eventually discharged from the sample outlet, thereby achieving orderly sorting of cells in conjunction with the gradient magnetic field in the flow channel.

[0057] like Figure 4 As shown, Figure 4 For actual production processes, the following is an assembly diagram (magnetic field source not shown) of the magnetic sorting chip for cell sorting provided by this invention. Figure 4The assembly diagram is described below. The upper clamping plate uses a brown frame as its carrier, with bolt holes at the four corners. Bolts connect it to the lower clamping plate, applying uniform clamping force to the sample layer and functional layer in the middle. This ensures a tight fit between the sealing membrane and the flow channel layer and functional layer, while also compressing the interlayer air, improving the penetration efficiency of the magnetic field from the functional layer to the sample layer. Three sets of fluid interfaces are distributed on the frame surface: the symmetrical purple and pink interfaces at the top correspond to the sheath fluid inlet and sample inlet of the sample layer. Both inlets are located in the upstream region of the flow channel, where the sheath fluid and sample suspension enter and converge into a laminar flow state of "sheath flow enveloping sample flow" within the flow channel layer. The yellow interface at the bottom corresponds to the sample outlet, located at the downstream end of the flow channel, used to discharge the sorted fluid. The interfaces feature a threaded design for easy external piping, and the flow channel holes on the inner side of the interfaces are precisely aligned with the inlet / outlet of the sample layer. Combined with the O-ring sealing design between the flow channel holes and the inlet / outlet, fluid leakage is prevented, ensuring stable fluid flow within the flow channel.

[0058] The purple area in the middle is the exposed outline of the sample layer flow channel layer. The sealing film covers the open surface of the flow channel. Its spatial dimensions are adapted to the patterned substrate of the functional layer to ensure that the flow channel extension direction is consistent with the array arrangement direction of the amorphous alloy soft magnetic tape material when stacked. That is, the amorphous alloy gathers the magnetic flux of the magnetic field source along the flow channel direction, thereby constructing a gradient magnetic field with "weak inlet and strong outlet". Combined with sheath flow constraint, it realizes the sorting logic of capturing cells with more magnetic beads first and capturing cells with fewer magnetic beads in turn.

[0059] This invention also provides a method for fabricating the magnetic sorting chip for cell sorting mentioned above, such as... Figure 6 As shown, Figure 6 This is a flowchart illustrating the fabrication method of the magnetic sorting chip for cell sorting provided by the present invention. (Refer to...) Figure 6 The method may include the following steps:

[0060] Step S1: Process countersunk grooves, cross positioning marks, and triangular flow direction marks on a patterned substrate, and embed the amorphous alloy soft magnetic tape into the countersunk grooves so that the upper surface of the amorphous alloy soft magnetic tape is flush with the upper surface of the patterned substrate to form a microfluidic functional layer.

[0061] Step S2: Prepare a flow channel layer with a sheath fluid inlet and a sample outlet by injection molding process, and seal the flow channel by adhesive bonding or hot pressing to attach a sealing film to the open surface of the flow channel layer to form a microfluidic sample layer;

[0062] Step S3: Machine flow channel holes on the upper and lower clamping plates at the positions corresponding to the sheath fluid inlet, sample inlet, and sample outlet, and pre-install O-rings at the interface between the flow channel holes on the upper clamping plate and the flow channel layer;

[0063] Step S4: The microfluidic sample layer and the microfluidic functional layer are sequentially sandwiched between the upper clamping plate and the lower clamping plate, and the upper clamping plate and the lower clamping plate are connected by bolts to achieve interlayer clamping and fixation;

[0064] Step S5: Place the magnetic field source at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the outlet is formed in the flow channel.

[0065] Those skilled in the art will understand that the accompanying drawings are merely schematic diagrams of one embodiment, and the modules or processes shown in the drawings are not necessarily essential for implementing the present invention.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A magnetic sorting chip for cell sorting, characterized in that, include: The sample layer includes a flow channel layer and a sealing membrane. The flow channel layer is provided with a sheath fluid inlet, a sample inlet, and a sample outlet, and has flow channels machined inside. The sealing membrane is attached to the open surface of the flow channel layer to seal the flow channels inside the flow channel layer. A functional layer is located below the sample layer. The functional layer includes a patterned substrate. A plurality of countersunk grooves are sequentially formed along the length of the patterned substrate. An amorphous alloy flexible magnetic tape is embedded in each countersunk groove, and the upper surface of the amorphous alloy flexible magnetic tape is flush with the upper surface of the patterned substrate, with the amorphous alloy flexible magnetic tape facing the sealing film. The amorphous alloy flexible magnetic tape is arranged sequentially along the length of the patterned substrate, forming an array distribution parallel to the flow channel extension direction of the sample layer. The clamp assembly includes an upper clamping plate and a lower clamping plate. The upper clamping plate is disposed above the sample layer, and the lower clamping plate is disposed below the functional layer. The upper clamping plate has corresponding flow channel holes at the positions of the sheath fluid inlet, the sample inlet, and the sample outlet. The upper clamping plate and the lower clamping plate are connected by bolts to clamp and fix the sample layer and the functional layer. A magnetic field source is located at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the outlet is formed in the flow channel.

2. The magnetic sorting chip for cell sorting according to claim 1, characterized in that, The patterned substrate is provided with a cross-shaped positioning mark and a triangular flow direction mark; wherein, the cross-shaped positioning mark is used for alignment when the sample layer and the functional layer are assembled, and the triangular flow direction mark points in the same direction as the fluid flow direction in the flow channel.

3. The magnetic sorting chip for cell sorting according to claim 2, characterized in that, The cross-shaped positioning marks are set at the four corners of the patterned substrate, and the triangular flow direction marks are located at the inlet end of the flow channel of the patterned substrate.

4. The magnetic sorting chip for cell sorting according to claim 1, characterized in that, The sealing membrane is made of polycarbonate and has a thickness in the micrometer range. It is bonded to the open surface of the flow channel layer by adhesive or hot pressing. The flow channel layer is made of polycarbonate and is prepared by injection molding.

5. The magnetic sorting chip for cell sorting according to claim 1, characterized in that, O-rings are provided between the sheath fluid inlet, sample inlet, and sample outlet of the flow channel layer and the corresponding flow channel holes on the upper clamping plate.

6. The magnetic sorting chip for cell sorting according to claim 1, characterized in that, The depth of the countersunk groove is adapted to the thickness of the amorphous alloy soft magnetic tape.

7. The magnetic sorting chip for cell sorting according to claim 1, characterized in that, The magnetic field source is a rectangular permanent magnet, with its long side parallel to the flow channel direction.

8. The magnetic sorting chip for cell sorting according to claim 1, characterized in that, The sheath fluid inlet, sample inlet, and sample outlet are distributed sequentially along the flow channel layer; wherein the sheath fluid inlet and the sample inlet are both located in the upstream region of the flow channel layer, and the sample outlet is located at the downstream end of the flow channel layer.

9. A method for preparing a magnetic sorting chip for cell sorting, characterized in that, Includes the following steps: A countersunk hole groove, a cross-shaped positioning mark, and a triangular flow direction mark are processed on a patterned substrate. An amorphous alloy soft magnetic tape is then embedded in the countersunk hole groove, so that the upper surface of the amorphous alloy soft magnetic tape is flush with the upper surface of the patterned substrate, forming a microfluidic functional layer. A flow channel layer with a sheath fluid inlet and a sample outlet is prepared by injection molding. A sealing film is then attached to the open surface of the flow channel layer by adhesive bonding or hot pressing to seal the flow channel, thus forming a microfluidic sample layer. Flow channel holes are machined on the upper and lower clamping plates at the positions corresponding to the sheath fluid inlet, sample inlet and sample outlet, and O-rings are pre-installed at the interface between the flow channel holes on the upper clamping plate and the flow channel layer. The microfluidic sample layer and the microfluidic functional layer are sequentially sandwiched between the upper clamping plate and the lower clamping plate, and the upper clamping plate and the lower clamping plate are connected by bolts to achieve interlayer clamping and fixation; The magnetic field source is placed at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the outlet is formed in the flow channel.

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