Continuous flow magnetic separation chip

Abstract

The application provides a continuous flow magnetic separation chip, which comprises an upper pressing plate, a lower pressing plate, a chip bottom plate and a flow channel cover plate, the flow channel cover plate is attached to the top of the chip bottom plate, a flow channel is formed between the two, the flow channel cover plate is further provided with a flow channel inlet and a flow channel outlet, the upper pressing plate and the lower pressing plate are used to fix and clamp the chip bottom plate and the flow channel cover plate in the middle, and the chip bottom plate and the flow channel cover plate are both encapsulated with soft magnetic strip material, and the soft magnetic strip material is located on one side of the flow channel. The application uses a patterned array of soft magnetic strip material, and the chip bottom plate and the flow channel cover plate are both encapsulated with soft magnetic strip material, so that a double-layer arrangement is formed in the vertical direction, the magnetic force distribution inside the flow channel is mainly lateral and auxiliary longitudinal, the cell adhesion effect is effectively weakened, high-throughput cell magnetic control screening can be realized, and the efficiency of magnetic field screening is improved. In addition, the application can realize gradient grading regulation in the size of the magnetic field force during cell sorting.

Description

Technical Field

[0001] This invention relates to the field of magnetophoresis technology, and in particular to a continuous flow magnetic separation chip and its fabrication method. Background Technology

[0002] Cell sorting is a key technology for cell subtype analysis. It separates specific cell populations from a mixed phase based on differences in the expression levels of cell surface membrane proteins. This technology supports various biological research and even clinical medical diagnosis. Currently, flow cytometry-based sorting methods use electric fields to manipulate droplet trajectories, achieving continuous flow cell sorting. However, this method typically faces challenges such as high cell mortality and high recognition thresholds, creating a need for further development in the field. Magnetic sorting, through the binding of antibody-linked magnetic beads to cells, allows for the adhesion of these beads to specific cell surfaces via antigen-antibody specificity. This method can reduce cell mortality. However, current magnetic sorting methods suffer from low manipulation precision, and the severe adhesion of cells adsorbed by the magnetic field in the flow channel can lead to channel blockage and low recovery rates. Summary of the Invention

[0003] In view of this, the present invention provides a continuous flow magnetic sorting chip and a method for fabricating the same, in order to solve at least one of the problems mentioned above.

[0004] To achieve the above objectives, the present invention adopts the following solution: According to a first aspect of the present invention, a continuous flow magnetic sorting chip is provided, the chip comprising: an upper pressure plate, a lower pressure plate, a chip substrate, and a flow channel cover plate, the flow channel cover plate being disposed in close contact with the chip substrate, forming a flow channel between the two, the flow channel cover plate also having a flow channel inlet and a flow channel outlet, the upper pressure plate and the lower pressure plate fixingly clamping the chip substrate and the flow channel cover plate in the middle, both the chip substrate and the flow channel cover plate being encapsulated with a flexible magnetic material, the flexible magnetic material being located on one side of the flow channel.

[0005] As an embodiment of the present invention, the chip substrate includes a PDMS substrate, a patterned flexible magnetic tape formed on the surface of the PDMS substrate, and an epoxy resin layer encapsulating the flexible magnetic tape.

[0006] As an embodiment of the present invention, the above-mentioned flow channel cover is made of PDMS and encapsulates patterned soft magnetic material inside, including the cover body and the flow channel wall.

[0007] In one embodiment of the present invention, the flexible magnetic tape encapsulated in the flow channel cover plate and the flexible magnetic tape encapsulated in the chip substrate overlap, and the area overlap rate is greater than 95%.

[0008] As an embodiment of the present invention, the material of the above-mentioned flexible magnetic tape is selected from any one of MATS-2010S, ZC600, 1J46, and VAC17, and the thickness is between 30 and 45 μm.

[0009] As an embodiment of the present invention, the above-mentioned flexible magnetic tape is rectangular, with a length between 1.2 and 1.5 mm and a width between 400 and 600 μm.

[0010] As an embodiment of the present invention, the average width of the flow channel is 400~430μm and the height is 90~110μm.

[0011] According to a second aspect of the present invention, a method for fabricating a continuous flow magnetic sorting chip is provided, the method comprising fabricating a chip substrate, fabricating a flow channel cover plate, and assembling the chip, wherein: The fabrication of the chip substrate includes: Step S10: Fabricate a patterned flexible magnetic tape based on PDMS; Step S11: Flatten the polypropylene film, spray a release agent on the surface of the polypropylene film, and then drop epoxy resin onto the surface of the polypropylene film. Step S12: Press the patterned flexible magnetic tape based on PDMS onto the surface of the polypropylene film, so that the surface of the flexible magnetic tape is pressed against the epoxy resin; Step S13: After the epoxy resin has cured, peel off the polypropylene film to obtain the chip substrate with the encapsulated flexible magnetic tape. The preparation of the flow channel cover plate includes: Step S20: Fabricate a patterned flexible magnetic tape based on PDMS; Step S21: Spin-coat photoresist onto a silicon wafer, and then obtain a composite structure consisting of silicon wafer and patterned photoresist by exposure and development; Step S22: Spin-coat PDMS onto the composite structure and perform one round of curing; Step S23: Pour PDMS solution onto the solid PDMS after one round of curing, press the patterned flexible magnetic tape with PDMS as the substrate into the PDMS solution, so that the plane of the flexible magnetic tape is pressed onto the solid PDMS surface obtained after one round of curing and complete the positioning, and then perform a second round of curing. Step S24: Peel off the PDMS flow channel after the second curing from the composite structure to obtain the flow channel cover plate with the encapsulated soft magnetic tape material; The chip assembly includes: Step S31: Assemble the flow channel cover and the chip substrate by pressing, so that the PDMS flow channel of the flow channel cover and the epoxy resin plane of the chip substrate are bonded together; Step S32: Use the upper and lower pressure plates to clamp the flow channel cover and the chip base plate in the middle, and use the screw threads to tighten the upper and lower pressure plates to apply pressure.

[0012] As an embodiment of the present invention, when the PDMS channel of the flow channel cover and the epoxy resin plane of the chip substrate are bonded together, the soft magnetic tape material encapsulated in the flow channel cover and the soft magnetic tape material encapsulated in the chip substrate overlap, and the area overlap rate is greater than 95%.

[0013] As an embodiment of the present invention, the fabrication of the patterned flexible magnetic tape based on PDMS in steps S10 and S20 includes: Step S40: Spin-coat liquid PDMS onto the solid PDMS surface, and then attach the flexible magnetic tape to the solid PDMS via the liquid PDMS; Step S41: Heat until the liquid PDMS is completely solidified, and fix the soft magnetic tape on the PDMS solid surface; Step S42: Spin-coat photosensitive paint onto the surface of the flexible magnetic tape and then dry it; Step S43: Pattern the flow channel morphology on the surface of the photosensitive paint by exposure. After exposure, spray with a sodium carbonate solution with a mass concentration of 0.8~1% until the pattern boundary is clearly visible. Step S44: Spray the etching solution until the metal pattern boundary is clearly visible. After completion, rinse the sample with pure water to terminate the etching process. Step S45: Immerse the sample obtained in step S44 in a sodium hydroxide solution with a mass concentration of 4-6% until the photosensitive paint on the surface of the flexible magnetic tape falls off, and then rinse the sample with pure water to obtain a patterned flexible magnetic tape based on PDMS.

[0014] As can be seen from the above technical solution, the continuous flow magnetic sorting chip and its preparation method provided by the present invention use a patterned array of soft magnetic materials, and both the chip substrate and the flow channel cover are encapsulated with soft magnetic materials. Therefore, a double-layer arrangement is formed in the vertical direction, achieving a magnetic force distribution that is predominantly lateral and supplemented by longitudinal force within the flow channel. This effectively reduces cell adhesion, enabling high-throughput magnetically controlled cell sorting and improving the efficiency of magnetic field sorting. Furthermore, cell sorting using the continuous flow magnetic sorting chip of the present invention allows for gradient-level control of the magnetic field force. Based on the difference in distance between each soft magnetic pattern array and the magnet, the magnitude of the local magnetic field force can be controlled. Combined with a sequential increase in flow field across each channel to achieve flow rate control, a design that gradually enhances the separation effect is ultimately realized, thereby completing the sorting function. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a schematic diagram of the structure of a continuous flow magnetic sorting chip provided in an embodiment of this application; Figure 2 This is a schematic diagram of the chip substrate provided in an embodiment of this application; Figure 3 This is a schematic diagram of the flow channel cover provided in an embodiment of this application; Figure 4 This is a flowchart of the fabrication steps of the chip substrate provided in the embodiments of this application; Figure 5 This is a flowchart of the preparation steps of the flow channel cover plate provided in the embodiments of this application; Figure 6 This is a flowchart of the chip assembly steps provided in the embodiments of this application; Figure 7 This is a flowchart illustrating the steps involved in manufacturing a patterned flexible magnetic tape based on PDMS, as provided in an embodiment of this application. Figures 8(a)-8(e) are schematic diagrams of structural changes when fabricating patterned flexible magnetic tape based on PDMS according to the embodiments of this application; Figures 9(a) and 9(b) are schematic diagrams of structural changes during the fabrication process of the chip substrate provided in the embodiments of this application; Figures 10(a)-10(f) are schematic diagrams of structural changes during the fabrication process of the flow channel cover provided in the embodiments of this application; Figure 11 This is an assembly diagram of the chip substrate and flow channel cover provided in an embodiment of this application; Figure 12 This is a schematic diagram of the experimental results of Embodiment 1 provided in this application; Figure 13 This is a schematic diagram of the experimental results of Comparative Example 1 provided in the embodiments of this application. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Here, the illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0017] To achieve high-throughput, high-precision magnetic field modulation in continuous flow, enabling specific cell recognition and sorting, this invention proposes a continuous flow magnetic sorting chip and its fabrication method. This chip utilizes the passive magnetic field modulation characteristics of an array of flexible magnetic materials to control the magnetic field arrangement within the flow channel and provides a lateral dragging magnetic force to the magnetic particles in the channel. The magnitude of the magnetic force is adjustable, and the flow velocity in the channel is also designed to achieve gradient differentiation. The design of the double-layer flexible magnetic material prevents adhesion to the upper and lower surfaces of the flow channel caused by excessive magnetic particles on the cell surface, thereby improving the recovery rate and making it suitable for high-throughput continuous flow magnetic sorting applications.

[0018] like Figure 1 The diagram shows a structural schematic of a continuous flow magnetic sorting chip according to an embodiment of this application. The chip includes: an upper pressure plate (not shown), a lower pressure plate (not shown), a chip substrate 1, and a flow channel cover plate 2. The flow channel cover plate 2 is attached to the chip substrate 1. Because patterned flow channels 4 are formed on the surface of the flow channel cover plate 2, a closed flow channel is formed between the two when the flow channel cover plate 2 and the chip substrate 1 are attached. The flow channel cover plate 2 also has a flow channel inlet 5 and a flow channel outlet 6. There can be multiple flow channel inlets 5 and outlets 6, for example... Figure 1 There are two inlets in the middle channel and four outlets in channel 6.

[0019] Both the chip substrate 1 and the flow channel cover 2 are encapsulated with flexible magnetic tape 3, which is made of Figure 1 As can be seen, the soft magnetic tape 3 forms a patterned array, with each soft magnetic tape 3 located on one side of the flow channel 4. The figure only illustrates one flow channel structure and soft magnetic tape layout. In fact, the layout of the soft magnetic tape array 3 and the flow channel layout can be adjusted according to different sorting needs. This application does not limit this.

[0020] After the chip substrate 1 and the flow channel cover 2 are attached, the upper pressure plate and the lower pressure plate can fix the chip substrate 1 and the flow channel cover 2 in the middle. In order to increase the pressure on the chip substrate 1 and the flow channel cover 2, threads can be set around the pressure plate and the lower pressure plate. Then, the pressure is increased by tightening the bolts, thereby increasing the pressure on the chip substrate 1 and the flow channel cover 2, so that the flow channel is sealed and the liquid will not leak.

[0021] Preferred, such as Figure 2 As shown, the chip substrate 1 includes a PDMS substrate 11, an epoxy resin layer 12, and a flexible magnetic tape 3. The flexible magnetic tape 3 is a pattern formed on the surface of the PDMS substrate 11, while the epoxy resin layer 12 covers the upper surface of the PDMS substrate 11 and completely encapsulates the flexible magnetic tape 3.

[0022] In this embodiment, the size of the PDMS substrate 11 is 30mm × (30~40)mm × 1mm. In another embodiment of this application, the PDMS substrate 11 can also be replaced by a glass substrate or a transparent rigid plate material such as PMMA.

[0023] Preferably, the material of the flexible magnetic tape 3 can be selected from any one of MATS-2010S, ZC600, 1J46, and VAC17, and the thickness is between 30 and 45 μm. More preferably, the flexible magnetic tape 3 is rectangular, with a length between 1.2 and 1.5 mm and a width between 400 and 600 μm.

[0024] Preferred, such as Figure 3 As shown, the channel cover 2 is entirely composed of PDMS and internally encapsulates a patterned flexible magnetic tape material 3. It includes a cover body 13 and a channel wall 14. The channel wall 14 is a raised pattern formed on one side surface of the cover body 13. When the channel cover 2 and the chip substrate 1 are bonded together, a closed channel can be formed. More preferably, the average width of the channel is 400~430μm and the height is 90~110μm.

[0025] Preferably, when the flow channel cover plate 2 and the chip substrate 1 are attached, the soft magnetic tape material encapsulated in the flow channel cover plate 2 and the soft magnetic tape material encapsulated in the chip substrate 1 need to overlap as much as possible, and the area overlap rate needs to be greater than 95%.

[0026] The fabrication of the above-mentioned continuous flow magnetic separation chip is described below. The fabrication method includes three parts: the fabrication of the chip substrate, the fabrication of the flow channel cover plate, and the chip assembly. These are described separately below.

[0027] like Figure 4 As shown, the fabrication of the chip substrate may include the following steps: Step S10: Create a patterned flexible magnetic tape based on PDMS.

[0028] Step S11: Flatten the polypropylene film, spray a release agent on the surface of the polypropylene film, and then drop epoxy resin onto the surface of the polypropylene film.

[0029] Step S12: Press the patterned flexible magnetic tape based on PDMS onto the surface of the polypropylene film, so that the surface of the flexible magnetic tape is pressed against the epoxy resin.

[0030] Step S13: After the epoxy resin has cured, peel off the polypropylene film to obtain the chip substrate with the encapsulated flexible magnetic tape.

[0031] like Figure 5 As shown, the fabrication of the flow channel cover plate may include the following steps: Step S20: Create a patterned flexible magnetic tape based on PDMS.

[0032] Step S21: Spin-coat photoresist onto a silicon wafer, and then obtain a composite structure consisting of silicon wafer and patterned photoresist by exposure and development.

[0033] Step S22: Spin-coat PDMS onto the composite structure and perform one round of curing.

[0034] Step S23: Pour PDMS solution onto the solid PDMS after one round of curing, press the patterned flexible magnetic tape with PDMS as the substrate into the PDMS solution, so that the plane of the flexible magnetic tape is pressed onto the solid PDMS surface obtained after one round of curing and complete the positioning, and then perform a second round of curing.

[0035] Step S24: Peel off the PDMS channel after the second curing from the composite structure to obtain the channel cover plate of the encapsulated soft magnetic tape.

[0036] like Figure 6 As shown, chip assembly may include the following steps: Step S30: Assemble the flow channel cover and the chip substrate by pressing, so that the PDMS flow channel of the flow channel cover and the epoxy resin plane of the chip substrate are bonded together.

[0037] Preferably, when the PDMS channel of the channel cover plate is bonded to the epoxy resin plane of the chip substrate, the soft magnetic tape material encapsulated in the channel cover plate and the soft magnetic tape material encapsulated in the chip substrate need to overlap, and the area overlap rate is greater than 95%.

[0038] Step S31: Use the upper and lower pressure plates to clamp the flow channel cover and the chip base plate in the middle, and use the screw threads to tighten the upper and lower pressure plates to apply pressure.

[0039] Preferred, such as Figure 7 As shown, the fabrication of the patterned flexible magnetic tape based on PDMS in steps S10 and S20 may further include: Step S40: Spin-coat liquid PDMS onto the solid PDMS surface and attach the flexible magnetic tape to the solid PDMS through the liquid PDMS.

[0040] Step S41: Heat until the liquid PDMS is completely solidified, and fix the soft magnetic tape on the PDMS solid surface.

[0041] Step S42: Spin-coat the surface of the flexible magnetic tape with photosensitive paint and then dry it.

[0042] Step S43: Pattern the flow channel morphology on the surface of the photosensitive paint by exposure. After exposure, spray with a sodium carbonate solution with a mass concentration of 0.8~1% until the pattern boundary is clearly visible.

[0043] Step S44: Spray the etching solution until the metal pattern boundary is clearly visible. After completion, rinse the sample with pure water to terminate the etching process.

[0044] Step S45: Immerse the sample obtained in step S44 in a sodium hydroxide solution with a mass concentration of 4-6% until the photosensitive paint on the surface of the flexible magnetic tape falls off, and then rinse the sample with pure water to obtain a patterned flexible magnetic tape based on PDMS.

[0045] The fabrication process of the above-mentioned continuous flow magnetic sorting chip will be further explained below through a specific embodiment: (1) Fabrication of patterned soft magnetic tape based on PDMS (1-1) Take a 2mm thick solid PDMS, spin coat it with liquid PDMS at a speed of 450~500 rpm, and then attach a 3cm×5cm flexible magnetic tape to the solid PDMS using the liquid PDMS. Heat at 80℃ for 1.5~2h until the PDMS is completely cured, and fix the flexible magnetic tape to the surface of the solid PDMS. The sample obtained after this step is shown in Figure 8(a).

[0046] (1-2) Photosensitive paint was spin-coated onto the surface of the soft magnetic tape obtained in (1-1) at a speed of 3500~4000 rpm for 50~55s, and then dried at 75℃ for 30~45min until the photosensitive paint was dry. The sample obtained after this step is shown in Figure 8(b).

[0047] (1-3) The photosensitive paint surface of the sample obtained in (1-2) was subjected to ultraviolet exposure for flow channel morphology determination using a MicroWriter ML®3 laser direct writing machine. The laser wavelength was 360~370nm, and the laser energy was 120~160 mJ / cm2. Alternatively, patterned exposure could be achieved using an ultraviolet exposure machine with a mask according to the above parameters. After exposure, a sodium carbonate solution with a mass concentration of 0.8~1% was sprayed until the pattern boundaries were clearly visible. The sample obtained after this step is shown in Figure 8(c).

[0048] (1-4) Spray the sample obtained in (1-3) into the etching solution until the boundary of the metal pattern is clearly visible. The mass ratio of the etching solution is pure water: ferric chloride hexahydrate: hydrochloric acid = (35~42):(16~24):1. After the etching is completed, rinse the sample with pure water to stop the etching. The sample obtained after this step is shown in Figure 8(d).

[0049] (1-5) Immerse the sample obtained in (1-4) in a sodium hydroxide solution with a mass concentration of 4-6%, maintaining the temperature at 45-55℃ for 10-15 minutes, until the photosensitive paint on the metal surface falls off. Then rinse the sample with pure water. The sample obtained after this step is shown in Figure 8(e). The sample obtained at this time is a patterned soft magnetic tape based on PDMS.

[0050] (2) Fabrication of the chip substrate

[0051] (2-1) A patterned soft magnetic tape based on PDMS is obtained through the steps (1-1)-(1-5) above.

[0052] (2-2) Flatten the polypropylene film, spray a release agent on its surface, and then drop 2-3 ml of epoxy resin onto the surface.

[0053] (2-3) Press the PDMS substrate and the flexible magnetic tape obtained in (2-1) onto the surface of (2-2), so that the PDMS surface where the flexible magnetic tape is located is pressed against the epoxy resin, and the pressure is controlled at 8~11 kPa. A schematic diagram of this step (2-2) and (2-3) can be seen in Figure 9(a).

[0054] (2-4) Place the entire assembly (2-3) in the air and wait 15-24 hours until the epoxy resin cures. Then peel off the polypropylene film to obtain the encapsulated chip substrate. The distance between the flexible magnetic tape and the upper surface of the epoxy resin can be controlled at 15-25 μm. The chip substrate obtained after this step is shown in Figure 9(b).

[0055] (3) Preparation of flow channel cover plate

[0056] (3-1) A patterned soft magnetic tape based on PDMS is obtained through the steps (1-1)-(1-5) above. It should be noted that the sample obtained in this step is a mirror image of the sample obtained in (2-1).

[0057] (3-2) SU8 2075 photoresist was spin-coated on the polished surface of a 4-inch <1,1,1> silicon wafer at a speed of 2000~2100 rpm for 50~55s. The sample obtained by this step is shown in Figure 10(a).

[0058] (3-3) Pre-baking: Bake at 60~65℃ for 8~10 minutes, then bake at 93~97℃ for 10~13 minutes, and then cool to room temperature in the oven.

[0059] (3-4) Exposure: Ultraviolet exposure is performed on the flow channel morphology using a MicroWriter ML®3 laser direct writing machine. The laser wavelength is 360~370nm, and the laser energy is 280~320 mJ / cm2. Patterned exposure can also be achieved using an ultraviolet exposure machine with a mask according to the above parameters.

[0060] (3-5) Post-baking: Bake at 60~65℃ for 5~8 minutes, then bake at 93~97℃ for 16~18 minutes, and then cool to room temperature in the oven.

[0061] (3-6) Development: Immerse the room temperature sample obtained in (2-2-4) in 80-100 ml of MF 319 developer, shake slowly for 6-7 min, remove the sample, then immerse it in 30-40 ml of isopropanol solution and shake for 10-15 s, remove the sample, and finally immerse it in 100-150 ml of distilled water and shake for 2 min. After removing the sample, let it stand until the distilled water is air-dried. A composite structure consisting of silicon wafer and patterned photoresist is obtained. The exposure process changes the properties of the photoresist in some areas. After immersion in the developer, the photoresist in the unexposed areas dissolves into the developer, while the exposed areas do not dissolve into the developer. Therefore, the residual silicon wafer-patterned photoresist composite structure retains the morphological characteristics of the designed flow channels. The sample obtained through steps (3-3)-(3-6) is shown in Figure 10(b).

[0062] (3-7) Hardening: The composite structure obtained in (2-6) is placed in the baking oven and slowly heated from room temperature to 125-150℃ at a heating rate of 1-1.2℃ / min. After maintaining the temperature at 125-150℃ for 40-50min, the baking oven is turned off and the sample is cooled to room temperature with the oven to ensure that the cured photoresist has high strength and is firmly bonded to the silicon wafer.

[0063] (3-8) Surface treatment: The silicon wafer-photoresist composite structure obtained in (3-7) is fumigated in TMCS vapor for 20-25 minutes. Surface treatment can weaken the adhesion between the silicon wafer and photoresist and PDMS, making it easier for subsequent demolding.

[0064] (3-9) PDMS Channel Molding: PDMS is spin-coated onto the upper surface of the silicon wafer-patterned photoresist composite structure obtained in (3-8) at a rotation speed of 500-550 rpm and cured at 80℃ for 1.5-2 hours. This process is called the first curing. The sample obtained after this step is shown in Figure 10(c). Then, a baffle with a height of 3-4 mm is added to its side to prevent subsequent PDMS outflow. 35-32 ml of PDMS solution is poured onto the upper surface of the first-cured solid PDMS. The patterned flexible magnetic tape with PDMS as the substrate obtained in (3-1) is pressed into the PDMS solution, so that the plane of the flexible magnetic tape is pressed against the surface of the first-cured solid PDMS and the positioning is completed. A schematic diagram of this step can be seen in Figure 10(d). The entire sample is dried at 80℃ for 1.5-2 hours, which is called the second curing. The sample obtained after this step is shown in Figure 10(e). After secondary curing, the flow channel is peeled off from the upper surface of the photoresist-silicon wafer to obtain the PDMS flow channel encapsulated with soft magnetic tape, which is the flow channel cover plate shown in Figure 10(f).

[0065] (4) Chip assembly

[0066] The chip floor obtained in (2-4) and the flow channel cover obtained in (3-9) are arranged according to... Figure 11 Assembly is performed in a pressing manner. Specifically, it is assembled by pressing, so that the flow channel obtained by (3-9) is combined with the plane of epoxy resin in (2-4), and the soft magnetic tape materials of the upper and lower structures are overlapped as much as possible, with an area overlap rate of more than 95%. Pressing is achieved by upper and lower pressure plates made of PMMA material, and pressure is applied by tightening with threads.

[0067] The following describes the application of the aforementioned continuous flow magnetic sorting chip in the cell sorting process. Its application requires the following conditions: (1) Configure a magnetic field source, i.e. a permanent magnet, and the size and position of the permanent magnet need to be strictly designed according to the present invention.

[0068] (1-1) The permanent magnet used in this invention is an N52 permanent magnet (other specifications such as N42, N35, etc. can also be used, but their size and spatial position need to be designed separately), its size is 10mm×10mm×30mm, and its magnetization direction is perpendicular to the 10mm×30mm surface.

[0069] (1-2) Spatial position of permanent magnet: The magnetization direction of the permanent magnet is horizontal, and the permanent magnet is located on the right side of the chip, with a distance of 4~4.5cm from the center of the chip.

[0070] (2) Solution sample preparation

[0071] (2-1) The solution sample was a 1×PBS solution containing MCF-7 cells (human breast cancer cells) and sodium fluorescein, which was used for flow field tracing.

[0072] (2-2) Add 2.5-5 μl of magnetic bead solution with a mass concentration of 10 mg / ml to 1-1.1 ml of sample (2-1), and incubate with shaking at 37°C for 50-60 min. The magnetic beads are coated with anti-EpCAM antibody, which can specifically bind to MCF-7 cells expressing EpCAM.

[0073] (3) Liquid sample flow rate configuration

[0074] When introducing blood samples into the continuous flow magnetic separation chip mentioned in this invention, a peristaltic pump, thrust pump, or pressure pump can be used as the liquid sample pumping device. During the experiment, the flow rate of the liquid sample at all four outlets needs to be controlled at 600~1000 μl / h for stable sample introduction. The flow rate at the other buffer solution inlet is 2000~3200 μl / h.

[0075] (4) Observation of results

[0076] During sample passage in the continuous flow magnetic sorting chip mentioned in this invention, a stable flow rate is maintained at both the inlet and outlet. It can be observed in the fluorescence channel that stained MCF-7 cells detach from the original flow field and exit from other channel outlets. Therefore, this can be used as a basis for detecting phenotypic differences among different cells, enabling high-throughput cell sorting.

[0077] The sorting process described above will be further illustrated below through an example and a pair of proportions: Example 1: Isolation of MCF-7 cells from a continuous sample stream Sample preparation: Resuspend MCF-7 cells in 1xPBS to a concentration of 10,000 cells / ml. Add 5 μl of magnetic bead solution (10 mg / ml) to 1 ml of MCF-7 cell suspension and incubate at 37°C with shaking for 60 min. The magnetic beads have an anti-EpCAM antibody on their surface, which can specifically bind to cells expressing EpCAM protein. Finally, add 0.02 g of sodium fluorescein powder, shake well, and the sample preparation is complete.

[0078] Using a syringe pump and syringe, draw liquid out at a flow rate of 600 μl / h from each of the four outlets; using a syringe pump and syringe, inject 1x PBS into the flow channel at a flow rate of 2000 μl / h from the buffer inlet. Place the sample inlet in the sample container to allow it to automatically extract the sample.

[0079] Experimental results are as follows Figure 12As shown, the fluorescent and non-fluorescent colors represent the sample and buffer solution, respectively. The sample area is the bright area in the figure, and the dark area is the buffer solution. However, some fluorescently stained cells in the sample (circled in the figure) penetrate the sample-buffer interface under the influence of the magnetic field, enter the buffer solution, and are washed out by the buffer solution to other outlets.

[0080] Comparative Example 1

[0081] Sample preparation: Resuspend MCF-7 cells in 1xPBS to a concentration of 10,000 cells / ml. Add 5 μl of magnetic bead solution (10 mg / ml) to 1 ml of MCF-7 cell suspension and incubate at 37°C with shaking for 60 min. The magnetic beads have an anti-EpCAM antibody on their surface, which can specifically bind to cells expressing EpCAM protein. Finally, add 0.02 g of sodium fluorescein powder, shake well, and the sample preparation is complete.

[0082] Using a syringe pump and syringe, draw liquid out at a flow rate of 300 μl / h from each of the four outlets; using a syringe pump and syringe, inject 1x PBS into the flow channel at a flow rate of 1000 μl / h into the buffer inlet. Place the sample inlet in the sample container to allow it to automatically extract the sample.

[0083] The experiment also showed sorting effects, but due to the smaller ratio of flow velocity to magnetic field strength, the lateral adsorption effect of the magnetic field was too strong, leading to problems such as cells and magnetic beads adhering to the sidewalls of the flow channel (e.g., Figure 13 This proves that the parameters need to be optimized.

[0084] As can be seen from the above technical solution, the continuous flow magnetic sorting chip and its preparation method provided by the present invention use a patterned array of soft magnetic materials, and both the chip substrate and the flow channel cover are encapsulated with soft magnetic materials. Therefore, a double-layer arrangement is formed in the vertical direction, achieving a magnetic force distribution that is predominantly lateral and supplemented by longitudinal force within the flow channel. This effectively reduces cell adhesion, enabling high-throughput magnetically controlled cell sorting and improving the efficiency of magnetic field sorting. Furthermore, cell sorting using the continuous flow magnetic sorting chip of the present invention allows for gradient-level control of the magnetic field force. Based on the difference in distance between each soft magnetic pattern array and the magnet, the magnitude of the local magnetic field force can be controlled. Combined with a sequential increase in flow field across each channel to achieve flow rate control, a design that gradually enhances the separation effect is ultimately realized, thereby completing the sorting function.

[0085] Specific embodiments have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of this invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this invention. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A continuous-flow magnetic separation chip, characterized by, The chip includes an upper pressure plate, a lower pressure plate, a chip base plate, and a flow channel cover plate. The flow channel cover plate is attached to the chip base plate to form a flow channel between the two. The flow channel cover plate is also provided with a flow channel inlet and two or more flow channel outlets. The upper pressure plate and the lower pressure plate fix and clamp the chip base plate and the flow channel cover plate in the middle. Both the chip base plate and the flow channel cover plate are encapsulated with soft magnetic material. The soft magnetic material is located on one side of the flow channel to provide lateral dragging magnetic force to the magnetic particles in the flow channel. The flow channel is a honeycomb array composed of multiple interconnected hexagonal units, and the soft magnetic tape is a patterned array arranged along the outline of the honeycomb array. The soft magnetic tape encapsulated in the flow channel cover plate overlaps with the soft magnetic tape encapsulated in the chip substrate, and the area overlap rate is greater than 95%.

2. The continuous-flow magnetic separation chip of claim 1, wherein, The chip substrate includes a PDMS substrate, a patterned flexible magnetic tape formed on the surface of the PDMS substrate, and an epoxy resin layer encapsulating the flexible magnetic tape.

3. The continuous-flow magnetic separation chip of claim 1, wherein, The flow channel cover is made entirely of PDMS and encapsulates patterned soft magnetic material inside, including the cover body and the flow channel wall.

4. The continuous-flow magnetic separation chip of claim 1, wherein, The material of the soft magnetic tape is selected from any one of MATS-2010S, ZC600, 1J46, and VAC17, and the thickness is between 30 and 45 μm.

5. The continuous-flow magnetic separation chip of claim 1, wherein, The soft magnetic tape is rectangular, with a length between 1.2 and 1.5 mm and a width between 400 and 600 μm.

6. The continuous-flow magnetic separation chip of claim 1, wherein, The average width of the flow channel is 400~430μm and the height is 90~110μm.

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