Continuous flow magnetic separation chip and preparation method thereof

Abstract

The invention provides a continuous flow magnetic separation chip and a preparation method thereof.The chip 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 arranged on the chip bottom plate in an attached mode, a flow channel is formed between the flow channel cover plate and the chip bottom plate, and the flow channel cover plate is further provided with a flow channel inlet and a flow channel outlet; the chip bottom plate and the runner cover plate are fixedly clamped between the upper pressing plate and the lower pressing plate, soft magnetic strip materials are packaged in the chip bottom plate and the runner cover plate, and the soft magnetic strip materials are located on one side of the runner. The soft magnetic strip material patterned array is used, and the soft magnetic strip materials are packaged in the chip bottom plate and the flow channel cover plate, so that double-layer arrangement is formed in the vertical direction, magnetic force distribution with the inner side direction of the flow channel as the main direction and the longitudinal direction as the auxiliary direction is achieved, the cell wall attaching effect is effectively weakened, high-flux cell magnetic control screening can be achieved, and the cell magnetic control screening efficiency is improved. And the magnetic field screening efficiency is improved. In addition, during cell sorting, gradient grading regulation and control can be achieved in the aspect of magnetic field force.

Description

Technical Field

[0001] The invention relates to the technical field of magnetophoresis, and in particular to a continuous flow magnetic separation chip and a preparation method thereof. Background Art

[0002] Cell sorting is a key technology for cell subtype analysis. It is a method of screening specific cell populations from mixed phases through differences in the expression of cell surface membrane proteins. This technology can support a variety of biological studies and even medical clinical diagnosis. At present, the sorting method based on flow sorting adjusts the trajectory of droplets through electric field manipulation to achieve continuous flow cell sorting. This method usually faces problems such as high cell mortality and high recognition threshold, and also puts forward corresponding development needs in the field of cell sorting. The magnetic sorting method combines magnetic beads connected to antibodies with cells, and can adhere magnetic beads to the surface of specific cells through antigen-antibody specific recognition. This method can reduce cell mortality. However, the current magnetic sorting method faces the problems of low control accuracy, and the cells adsorbed by the magnetic field in the flow channel are severely adhered to the wall, which will also cause flow channel blockage and low recovery rate. Summary of the invention

[0003] In view of this, the present invention provides a continuous flow magnetic separation chip and a preparation method thereof to solve at least one of the above-mentioned problems.

[0004] In order to achieve the above object, the present invention adopts the following scheme:

[0005] According to a first aspect of the present invention, there is provided a continuous flow magnetic sorting chip, the chip comprising: an upper pressing plate, a lower pressing plate, a chip base plate and a flow channel cover plate, the flow channel cover plate is fitted on the chip base plate to form a flow channel therebetween, the flow channel cover plate is also provided with a flow channel inlet and a flow channel outlet, the upper pressing plate and the lower pressing plate fix and clamp the chip base plate and the flow channel cover plate in the middle, the chip base plate and the flow channel cover plate are both encapsulated with soft magnetic tape material, and the soft magnetic tape material is located on one side of the flow channel.

[0006] As an embodiment of the present invention, the chip base plate comprises a PDMS substrate, a patterned soft magnetic tape formed on the surface of the PDMS substrate, and an epoxy resin layer wrapping the soft magnetic tape.

[0007] As an embodiment of the present invention, the above-mentioned flow channel cover plate is entirely made of PDMS, and a patterned soft magnetic tape material is encapsulated inside, including a cover plate body and a flow channel wall.

[0008] As an embodiment of the present invention, the soft magnetic tape material encapsulated in the flow channel cover plate and the soft magnetic tape material encapsulated in the chip base plate overlap, and the area overlap rate is greater than 95%.

[0009] As an embodiment of the present invention, 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.

[0010] As an embodiment of the present invention, the soft magnetic tape is rectangular, with a length of 1.2 to 1.5 mm and a width of 400 to 600 μm.

[0011] As an embodiment of the present invention, the average width of the flow channel is 400-430 μm, and the height is 90-110 μm.

[0012] According to a second aspect of the present invention, a method for preparing a continuous flow magnetic separation chip is provided, the method comprising preparing a chip base plate, preparing a flow channel cover plate and assembling the chip, wherein:

[0013] The preparation of the chip base plate includes:

[0014] Step S10: preparing a patterned soft magnetic tape with PDMS as the substrate;

[0015] Step S11: flattening the polypropylene film, spraying a release agent on the surface of the polypropylene film, and then dripping epoxy resin on the surface of the polypropylene film;

[0016] Step S12: pressing the patterned soft magnetic tape with the PDMS as the base onto the surface of the polypropylene film so that the surface of the soft magnetic tape is pressed toward the epoxy resin;

[0017] Step S13: After the epoxy resin is cured, the polypropylene film is peeled off to obtain a chip base plate encapsulating the soft magnetic tape material;

[0018] The preparation of the flow channel cover plate includes:

[0019] Step S20: preparing a patterned soft magnetic tape with PDMS as the substrate;

[0020] Step S21: Spin-coating a photoresist on a silicon wafer, and then obtaining a composite structure consisting of a silicon wafer and patterned photoresist by exposure and development;

[0021] Step S22: spin coating PDMS on the composite structure and performing a round of curing;

[0022] Step S23: pouring a PDMS solution onto the solid PDMS after one round of curing, pressing the patterned soft magnetic tape with the PDMS as the substrate into the PDMS solution, so that the plane where the soft magnetic tape is located is pressed on the solid PDMS surface obtained by one round of curing, and positioning is completed, and then a second round of curing is performed;

[0023] Step S24: peeling off the PDMS flow channel after the second round of curing from the composite structure to obtain a flow channel cover plate encapsulating the soft magnetic tape material;

[0024] The chip assembly comprises:

[0025] Step S31: Assembling the flow channel cover plate and the chip base plate by pressing, so that the PDMS flow channel of the flow channel cover plate and the epoxy resin plane of the chip base plate are combined;

[0026] Step S32: clamping the flow channel cover plate and the chip base plate in the middle by using an upper pressing plate and a lower pressing plate, and applying pressure to the upper pressing plate and the lower pressing plate by screwing.

[0027] As an embodiment of the present invention, the above method allows the soft magnetic tape material encapsulated in the flow channel cover plate and the soft magnetic tape material encapsulated in the chip base plate to overlap when the PDMS flow channel of the flow channel cover plate and the epoxy resin plane of the chip base plate are combined, and the area overlap rate is greater than 95%.

[0028] As an embodiment of the present invention, the step S10 and the step S20 of preparing a patterned soft magnetic tape with PDMS as the substrate include:

[0029] Step S40: spin coating liquid PDMS on the solid surface of PDMS, and pasting the soft magnetic tape onto the solid PDMS through the liquid PDMS;

[0030] Step S41: heating until the liquid PDMS is completely solidified, fixing the soft magnetic tape on the solid surface of PDMS;

[0031] Step S42: Spin-coating photosensitive paint on the surface of the soft magnetic tape and drying it;

[0032] Step S43: performing patterned exposure of the flow channel morphology on the surface of the photosensitive paint, and spraying with a sodium carbonate solution with a mass concentration of 0.8-1% after the exposure until the pattern boundary is clearly visible;

[0033] Step S44: spraying the etching solution until the boundary of the metal pattern is clearly visible, and then rinsing the sample with pure water to terminate the etching;

[0034] Step S45: Soak 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 soft magnetic tape falls off, and then rinse the sample with pure water to obtain a patterned soft magnetic tape with PDMS as the substrate.

[0035] It can be seen from the above technical scheme that the continuous flow magnetic sorting chip and its preparation method provided by the present invention use a patterned array of soft magnetic tape materials, and the chip bottom plate and the flow channel cover plate are both encapsulated with soft magnetic tape materials, so a double-layer arrangement is formed in the vertical direction, and a magnetic force distribution with the main lateral direction and the auxiliary longitudinal direction in the flow channel is realized, which effectively weakens the cell adhesion effect, can realize high-throughput cell magnetic control screening, and improves the efficiency of magnetic field screening. In addition, the cell sorting after utilizing the continuous flow magnetic sorting chip of the present invention can realize gradient graded regulation in terms of the magnitude of the magnetic field force, and realize the regulation of the magnitude of the local magnetic field force according to the difference in the distance between each soft magnetic pattern array and the magnet. The flow rate control is completed by coordinating the sequence of the flow field increasing channel by channel, and finally a design with gradually enhanced separation effect is realized, thereby completing the sorting function. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0037] Figure 1 It is a structural schematic diagram of a continuous flow magnetic separation chip provided in an embodiment of the present application;

[0038] Figure 2 It is a schematic diagram of the structure of the chip base plate provided in an embodiment of the present application;

[0039] Figure 3 is a schematic diagram of the structure of the flow channel cover plate provided in an embodiment of the present application;

[0040] Figure 4 is a flow chart of the steps for preparing a chip base plate provided in an embodiment of the present application;

[0041] Figure 5 is a flow chart of the preparation steps of the flow channel cover plate provided in an embodiment of the present application;

[0042] Figure 6 is a flowchart of the steps of chip assembly provided in an embodiment of the present application;

[0043] Figure 7 It is a flowchart of the steps of making a patterned soft magnetic tape material with PDMS as a substrate provided in an embodiment of the present application;

[0044] Figure 8(a)-Figure 8(e) It is a schematic diagram of the structural changes when making a patterned soft magnetic tape material with PDMS as a substrate provided in an embodiment of the present application;

[0045] Figure 9(a)-Figure 9(b)It is a schematic diagram of the structural changes in the process of preparing the chip base plate provided in the embodiment of the present application;

[0046] Figure 10(a)-Figure 10(f) is a schematic diagram of structural changes during the preparation process of the flow channel cover plate provided in an embodiment of the present application;

[0047] Fig.11 It is a schematic diagram of the assembly of the chip base plate and the flow channel cover plate provided in the embodiment of the present application;

[0048] Fig.12 It is a schematic diagram of the experimental results of Example 1 provided in the examples of the present application;

[0049] Fig.13 It is a schematic diagram of the experimental results of Comparative Example 1 provided in the embodiments of the present application. DETAILED DESCRIPTION

[0050] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. Here, the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0051] In order to achieve high-throughput and high-precision magnetic field regulation in a continuous flow so that cells can be specifically identified and sorted, the present invention proposes a continuous flow magnetic sorting chip and a preparation method thereof. The continuous flow magnetic sorting chip uses the characteristics of passive magnetic field regulation of array soft magnetic tape materials to control the magnetic field arrangement in the flow channel, and provides a lateral dragging magnetic force to the magnetic particles in the flow channel. The magnitude of the magnetic force can be adjusted, and the flow rate of the flow channel is also gradient-differentiated by design. The design of the double-layer soft magnetic tape material prevents the adhesion of the upper and lower surfaces of the flow channel due to excessive magnetic particles on the cell surface, thereby improving the recovery rate, and can be applied to high-throughput continuous flow magnetic sorting.

[0052] like Figure 1 The structure diagram of a continuous flow magnetic separation chip provided by an embodiment of the present application is shown, and the chip includes: an upper pressure plate (not shown), a lower pressure plate (not shown), a chip base plate 1 and a flow channel cover plate 2. The flow channel cover plate 2 is arranged on the chip base plate 1. Since a patterned flow channel 4 is formed on the surface of the flow channel cover plate 2, when the flow channel cover plate 2 and the chip base plate 1 are attached, a closed flow channel will be formed between the two. The flow channel cover plate 2 is also provided with a flow channel inlet 5 and a flow channel outlet 6. There can be multiple flow channel inlets 5 and flow channel outlets 6, such as Figure 1 There are two flow channel inlets and four flow channel outlets 6 .

[0053] The chip base plate 1 and the flow channel cover plate 2 are both encapsulated with a soft magnetic tape material 3. Figure 1It can be seen that the soft magnetic tape material 3 constitutes a graphic array, and each soft magnetic tape material 3 is located on one side of the flow channel 4. The figure only illustrates the structure of a flow channel and the layout of the soft magnetic tape material. In fact, the layout of the soft magnetic tape material 3 array and the layout of the flow channel can be adjusted accordingly according to different sorting needs, and the present application does not limit this.

[0054] When the chip base plate 1 and the flow channel cover plate 2 are fitted together, the upper pressure plate and the lower pressure plate can fix the chip base plate 1 and the flow channel cover plate 2 in the middle. In order to increase the pressure on the chip base plate 1 and the flow channel cover plate 2, threads can be set around the pressure plate and the lower pressure plate, and then the bolts are tightened and pressurized to increase the pressure on the chip base plate 1 and the flow channel cover plate 2, so that the flow channel can be closed and the liquid will not leak.

[0055] Preferably, Figure 2 As shown, the chip base plate 1 includes a PDMS substrate 11, an epoxy resin layer 12 and a soft magnetic tape material 3, wherein the soft magnetic tape material 3 is a pattern formed on the surface of the PDMS substrate 11, and the epoxy resin layer 12 covers the upper surface of the PDMS substrate 11 and completely wraps the soft magnetic tape material 3.

[0056] In this embodiment, the size of the PDMS substrate 11 is 30 mm×(30-40) mm×1 mm, and in another embodiment of the present application, the PDMS substrate 11 can also be replaced by a glass base, or a transparent hard plate material such as PMMA.

[0057] Preferably, the material of the soft magnetic tape material 3 can be selected from any one of MATS-2010S, ZC600, 1J46, and VAC17, and the thickness is between 30 and 45 μm. Further preferably, the soft magnetic tape material 3 is rectangular, with a length of between 1.2 and 1.5 mm and a width of between 400 and 600 μm.

[0058] Preferably, Figure 3 As shown, the flow channel cover plate 2 is made of PDMS as a whole, and a patterned soft magnetic tape material 3 is encapsulated inside, including a cover plate body 13 and a flow channel wall 14. The flow channel wall 14 is a raised pattern formed on one side surface of the cover plate body 13. When the flow channel cover plate 2 and the chip base plate 1 are attached, a closed flow channel can be formed. Further preferably, the average width of the flow channel here is 400-430μm, and the height is 90-110μm.

[0059] Preferably, when the flow channel cover plate 2 and the chip base plate 1 are attached, the soft magnetic tape material packaged in the flow channel cover plate 2 and the soft magnetic tape material packaged in the chip base plate 1 need to overlap as much as possible, and the area overlap rate needs to be greater than 95%.

[0060] The preparation of the above-mentioned continuous flow magnetic separation chip is described below. The preparation method includes three parts: preparation of the chip base plate, preparation of the flow channel cover plate and chip assembly, which are described below respectively.

[0061] like Figure 4 As shown, the preparation of the chip base plate may include the following steps:

[0062] Step S10: preparing a patterned soft magnetic tape with PDMS as the substrate.

[0063] Step S11: flatten the polypropylene film, spray a release agent on the surface of the polypropylene film, and then drop epoxy resin on the surface of the polypropylene film.

[0064] Step S12: Press the patterned soft magnetic tape with the PDMS as the base onto the surface of the polypropylene film so that the surface of the soft magnetic tape is pressed toward the epoxy resin.

[0065] Step S13: After the epoxy resin is cured, the polypropylene film is peeled off to obtain a chip base plate encapsulating the soft magnetic tape.

[0066] like Figure 5 As shown, the preparation of the flow channel cover plate may include the following steps:

[0067] Step S20: manufacturing a patterned soft magnetic tape with PDMS as the substrate.

[0068] Step S21: Spin-coating photoresist on the silicon wafer, and then obtaining a composite structure consisting of silicon wafer-patterned photoresist through exposure and development.

[0069] Step S22: spin-coating PDMS on the composite structure and performing a round of curing.

[0070] Step S23: Pour PDMS solution onto the solid PDMS after one round of curing, press the patterned soft magnetic tape based on PDMS into the PDMS solution, press the plane where the soft magnetic tape is located on the solid PDMS surface obtained by one round of curing, and complete the positioning, and then perform a second round of curing.

[0071] Step S24: peeling off the PDMS flow channel after the second round of curing from the composite structure to obtain a flow channel cover plate encapsulating the soft magnetic tape material.

[0072] like Figure 6 As shown, chip assembly may include the following steps:

[0073] Step S30: Assemble the flow channel cover plate and the chip base plate by pressing, so that the PDMS flow channel of the flow channel cover plate and the epoxy resin plane of the chip base plate are combined.

[0074] Preferably, when the PDMS channel of the channel cover plate is combined with the epoxy resin plane of the chip base plate, the soft magnetic tape material packaged in the channel cover plate and the soft magnetic tape material packaged in the chip base plate need to overlap, and the area overlap rate is greater than 95%.

[0075] Step S31: clamping the flow channel cover plate and the chip base plate in the middle by using an upper pressing plate and a lower pressing plate, and applying pressure to the upper pressing plate and the lower pressing plate by screwing.

[0076] Preferably, Figure 7 As shown, the preparation of the patterned soft magnetic tape with PDMS as the substrate in the above step S10 and the step S20 may further include:

[0077] Step S40: Spin-coating liquid PDMS on the solid surface of PDMS, and pasting the soft magnetic tape onto the solid PDMS through the liquid PDMS.

[0078] Step S41: heating until the liquid PDMS is completely solidified, and fixing the soft magnetic tape on the solid surface of PDMS.

[0079] Step S42: Spin-coat photosensitive paint on the surface of the soft magnetic tape and dry it.

[0080] Step S43: performing patterned exposure of the flow channel morphology on the surface of the photosensitive paint, and spraying with a sodium carbonate solution with a mass concentration of 0.8-1% after the exposure until the pattern boundary is clearly visible.

[0081] Step S44: spray the etching solution until the boundary of the metal pattern is clearly visible, and then rinse the sample with pure water to terminate the etching.

[0082] Step S45: Soak 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 soft magnetic tape falls off, and then rinse the sample with pure water to obtain a patterned soft magnetic tape with PDMS as the substrate.

[0083] The preparation process of the above-mentioned continuous flow magnetic separation chip is further described below through a specific embodiment:

[0084] (1) Preparation of patterned soft magnetic tape based on PDMS

[0085] (1-1) Take a 2 mm thick PDMS solid, spin-coat liquid PDMS on its surface at a speed of 450-500 rpm, and stick a 3 cm × 5 cm soft magnetic tape on the solid PDMS through the liquid PDMS. Heat at 80°C for 1.5-2 hours until the PDMS is completely cured, and the soft magnetic tape is fixed on the PDMS solid surface. The sample obtained after this step is shown in Figure 8(a).

[0086] (1-2) Spin-coat the surface of the soft magnetic tape of the sample obtained in (1-1) with a photosensitive paint at a speed of 3500-4000 rpm for 50-55 seconds, and then dry at 75°C for 30-45 minutes until the photosensitive paint is dry. The sample obtained after this step is shown in FIG8(b).

[0087] (1-3) Place the photosensitive paint surface of the sample obtained in (1-2) on a MicroWriter 3. Use a laser direct writer to perform ultraviolet exposure of the flow channel morphology. The laser wavelength is 360-370nm, and the laser energy is 120-160mJ / cm2. Patterned exposure can also be achieved by using an ultraviolet exposure machine in conjunction with a mask plate according to the above parameters. After exposure, spray with a sodium carbonate solution with a mass concentration of 0.8-1% until the pattern boundary is clearly visible. The sample obtained after this step is shown in Figure 8(c).

[0088] (1-4) Spray the sample obtained in (1-3) in an 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 completion, rinse the sample with pure water to terminate etching. The sample obtained after this step is shown in FIG8(d).

[0089] (1-5) Soak the sample obtained in (1-4) in a sodium hydroxide solution with a mass concentration of 4 to 6%, and maintain the temperature at 45 to 55°C for 10 to 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). At this time, the sample obtained is a patterned soft magnetic tape with PDMS as the substrate.

[0090] (2) Preparation of chip base plate

[0091] (2-1) A patterned soft magnetic tape with PDMS as the substrate is obtained through the above steps (1-1) to (1-5).

[0092] (2-2) Flatten the polypropylene film, spray a release agent on its surface, and then drop 2 to 3 ml of epoxy resin on the surface.

[0093] (2-3) Press the combination of the PDMS substrate and the soft magnetic tape obtained in (2-1) onto the surface of (2-2), so that the PDMS surface where the soft magnetic tape is located is pressed against the epoxy resin, and the pressure is controlled at 8 to 11 kPa. The schematic diagram of steps (2-2) and (2-3) can be seen in Figure 9 (a).

[0094] (2-4) Place (2-3) in air for 15 to 24 hours until the epoxy resin is cured, peel off the polypropylene film, and obtain a packaged chip base plate. The distance between the soft magnetic tape and the upper surface of the epoxy resin can be controlled to be 15 to 25 μm. The chip base plate obtained after this step is shown in FIG9(b).

[0095] (3) Preparation of flow channel cover plate

[0096] (3-1) A patterned soft magnetic tape with PDMS as the substrate is obtained through the above steps (1-1) to (1-5). It should be noted that the sample obtained in this step is a mirror image of the sample obtained in (2-1).

[0097] (3-2) Spin SU8 2075 photoresist on the polished surface of a 4-inch <1,1,1> silicon wafer at a speed of 2000-2100 rpm for 50-55 seconds. The sample obtained by this step is shown in FIG10( a ).

[0098] (3-3) Pre-baking: Bake at 60-65°C for 8-10 minutes and then bake at 93-97°C for 10-13 minutes and cool to room temperature.

[0099] (3-4) Exposure: In MicroWriter 3. Use a laser direct writer to perform ultraviolet exposure of the flow channel morphology. The laser wavelength is 360-370nm, and the laser energy is 280-320mJ / cm2. Patterned exposure can also be achieved by using an ultraviolet exposure machine in conjunction with a mask plate according to the above parameters.

[0100] (3-5) Post-baking: bake at 60-65°C for 5-8 minutes and then bake at 93-97°C for 16-18 minutes and cool to room temperature.

[0101] (3-6) Development: Soak the room temperature sample obtained in (2-2-4) in 80-100 ml of MF 319 developer, shake slowly for 6-7 minutes, take out the sample, soak and shake the sample in 30-40 ml of isopropanol solution for 10-15 seconds, take out the sample, and finally soak and shake it in 100-150 ml of distilled water for 2 minutes. After taking out the sample, let it stand until the distilled water is air-dried. A composite structure consisting of a silicon wafer and a patterned photoresist is obtained. The exposure process changes the properties of the photoresist in some parts. After soaking in the developer, the photoresist in the unexposed part dissolves into the developer, and the exposed part does not dissolve into the developer. Therefore, the remaining silicon wafer-patterned photoresist composite structure retains the morphological characteristics of the designed flow channel. The sample obtained by steps (3-3)-(3-6) is shown in Figure 10(b).

[0102] (3-7) Hardening the film: Place the composite structure obtained in (2-6) into a baking oven and slowly heat it from room temperature to 125-150°C at a heating rate of 1-1.2°C / min. After maintaining the temperature at 125-150°C for 40-50 min, close the baking oven and allow the sample to cool to room temperature along with the oven to ensure that the cured photoresist has high strength and is firmly bonded to the silicon wafer.

[0103] (3-8) Surface treatment: The silicon wafer-photoresist composite structure obtained in (3-7) is smoked in TMCS vapor for 20 to 25 minutes. Surface treatment can weaken the adhesion between the silicon wafer and the photoresist and PDMS, making it easier to demold the subsequent process.

[0104] (3-9) PDMS channel mold: On the silicon wafer-patterned photoresist composite structure obtained in (3-8), PDMS is spin-coated on the upper surface at a speed of 500-550 rpm and cured at 80°C for 1.5-2 hours. This process is called one round of 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 from flowing out. 35-32 ml of PDMS solution is poured onto the upper surface of the solid PDMS cured in one round, and the patterned soft magnetic tape with PDMS as the substrate obtained in (3-1) is pressed into the PDMS solution so that the plane where the soft magnetic tape is located is pressed on the solid PDMS surface obtained in the first round of curing, and the positioning is completed. The schematic diagram of this step can be seen in Figure 10(d). The sample is placed in an 80°C drying condition for 1.5-2 hours, which is called the second round of 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 the soft magnetic tape material, that is, the flow channel cover plate shown in FIG. 10( f ) is obtained.

[0105] (4) Chip assembly

[0106] The chip floor obtained in (2-4) and the flow channel cover plate obtained in (3-9) are Fig.11 Specifically, the assembly is performed by pressing, so that the flow channel obtained by (3-9) is combined with the plane where the epoxy resin of (2-4) is located, and the soft magnetic tape materials encapsulated by the upper and lower structures are overlapped as much as possible, and the area overlap rate needs to exceed 95%. Pressing is achieved by upper and lower pressing plates made of PMMA material, and pressurization is achieved by tightening the threads.

[0107] The following is a description of the use of the continuous flow magnetic separation chip in the cell separation process. The use requirements are as follows: The use of the chip needs to meet the following conditions:

[0108] (1) Configure a magnetic field source, i.e., a permanent magnet, and the size and position of the permanent magnet must be strictly designed according to the present invention.

[0109] (1-1) The permanent magnet used in the present invention is 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), whose size is 10mm×10mm×30mm, and its magnetization direction is perpendicular to the surface of 10mm×30mm.

[0110] (1-2) Spatial position of the permanent magnet: The magnetization direction of the permanent magnet is horizontal. The permanent magnet is located on the positive side of the chip and the distance from the center of the chip is 4 to 4.5 cm.

[0111] (2) Solution sample preparation

[0112] (2-1) The solution sample is a 1×PBS solution doped with MCF-7 cells (human breast cancer cells), which contains sodium fluorescein for flow field tracking.

[0113] (2-2) Add 2.5-5 μl of a 10 mg / ml magnetic bead solution to 1-1.1 ml of the sample of (2-1), and incubate with shaking at 37°C for 50-60 min. The surface of the magnetic beads is bound with anti-EpCAM antibodies, which can specifically bind to MCF-7 cells expressing EpCAM.

[0114] (3) Liquid sample flow rate configuration

[0115] When a blood sample is introduced into the continuous flow magnetic separation chip mentioned in the present invention, a peristaltic pump, a thrust pump or a pressure pump can be used as a liquid sample pumping device. During the experiment, the flow rate of the liquid sample at the four outlets needs to be controlled to be 600-1000 μl / h for stable sample flow. The flow rate of the other buffer liquid delivery port is 2000-3200 μl / h.

[0116] (4) Observation of results

[0117] During the process of passing the sample through the continuous flow magnetic sorting chip mentioned in the present invention, the outlet and the inlet maintain a stable flow rate, and the stained MCF-7 cells can be observed to separate from the original flow field in the fluorescence channel and be discharged from other channel outlets. Therefore, the phenotypic differences of different cells can be detected based on this, which is used for high-throughput cell sorting.

[0118] The above sorting process is further described below through an embodiment and a pair of ratios:

[0119] Example 1: Separation of MCF-7 cells from a continuous sample flow

[0120] Sample preparation: Resuspend MCF-7 cells in 1xPBS to a concentration of 10,000 cells / ml. Add 5 μl of 10 mg / ml magnetic bead solution to 1 ml of MCF-7 cell suspension and incubate at 37°C with shaking for 60 min. The surface of the magnetic beads is bound to anti-EpCAM antibodies, which can specifically bind to cells expressing EpCAM protein on the surface. Finally, add 0.02 g of sodium fluorescein powder and shake well to complete the sample preparation.

[0121] Use a syringe pump and syringe to extract liquid at 600 μl / h at each of the four outlets; use a syringe pump and syringe to inject 1x PBS into the flow channel at a flow rate of 2000 μl / h at the buffer inlet. Place the sample inlet in the sample to automatically extract the sample.

[0122] The experimental results are as follows Fig.12 As shown, it can be observed that fluorescent and non-fluorescent colors represent samples and buffers. The area where the sample is located is the bright area in the figure, and the dark area is the buffer. However, some fluorescently stained cells in the sample (circled in the figure) penetrate the interface between the sample and the buffer under the action of the magnetic field, enter the buffer, and are washed to other outlets by the buffer.

[0123] Comparative Example 1

[0124] Sample preparation: Resuspend MCF-7 cells in 1xPBS to a concentration of 10,000 cells / ml. Add 5 μl of 10 mg / ml magnetic bead solution to 1 ml of MCF-7 cell suspension and incubate at 37°C with shaking for 60 min. The surface of the magnetic beads is bound to anti-EpCAM antibodies, which can specifically bind to cells expressing EpCAM protein on the surface. Finally, add 0.02 g of sodium fluorescein powder and shake well to complete the sample preparation.

[0125] Use a syringe pump and syringe to extract liquid at 300 μl / h at each of the four outlets; use a syringe pump and syringe to inject 1x PBS into the flow channel at a flow rate of 1000 μl / h at the buffer inlet. Place the sample inlet in the sample to automatically extract the sample.

[0126] The experiment also shows a sorting effect, but because the ratio of flow rate to magnetic field strength becomes smaller, the lateral adsorption effect of the magnetic field is too strong, and the problem of cells and magnetic beads sticking to the side wall of the flow channel will occur (such as Fig.13 ), proving that the parameters need to be optimized.

[0127] It can be seen from the above technical scheme that the continuous flow magnetic sorting chip and its preparation method provided by the present invention use a patterned array of soft magnetic tape materials, and the chip bottom plate and the flow channel cover plate are both encapsulated with soft magnetic tape materials, so a double-layer arrangement is formed in the vertical direction, and a magnetic force distribution with the main lateral direction and the auxiliary longitudinal direction in the flow channel is realized, which effectively weakens the cell adhesion effect, can realize high-throughput cell magnetic control screening, and improves the efficiency of magnetic field screening. In addition, the cell sorting after utilizing the continuous flow magnetic sorting chip of the present invention can realize gradient graded regulation in terms of the magnitude of the magnetic field force, and realize the regulation of the magnitude of the local magnetic field force according to the difference in the distance between each soft magnetic pattern array and the magnet. The flow rate control is completed by coordinating the sequence of the flow field increasing channel by channel, and finally a design with gradually enhanced separation effect is realized, thereby completing the sorting function.

[0128] The present invention uses specific embodiments to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present invention.

Claims

1. A continuous flow magnetic separation chip, characterized in that: 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 fitted on 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 a flow channel outlet. 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. Soft magnetic tape material is encapsulated in the chip base plate and the flow channel cover plate, and the soft magnetic tape material is located on one side of the flow channel.

2. The continuous flow magnetic separation chip according to claim 1, characterized in that: The chip base plate comprises a PDMS substrate, a patterned soft magnetic tape material formed on the surface of the PDMS substrate, and an epoxy resin layer wrapping the soft magnetic tape material.

3. The continuous flow magnetic separation chip according to claim 1, characterized in that: The flow channel cover plate is made of PDMS as a whole, and a patterned soft magnetic tape material is encapsulated inside, including a cover plate body and a flow channel wall.

4. The continuous flow magnetic separation chip according to claim 1, characterized in that: The soft magnetic tape material packaged in the flow channel cover plate overlaps with the soft magnetic tape material packaged in the chip bottom plate, and the area overlap rate is greater than 95%.

5. The continuous flow magnetic separation chip according to claim 1, characterized in that: 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.

6. The continuous flow magnetic separation chip according to claim 1, characterized in that: The soft magnetic tape material is rectangular, with a length of 1.2 to 1.5 mm and a width of 400 to 600 μm.

7. The continuous flow magnetic separation chip according to claim 1, characterized in that: The average width of the flow channel is 400-430 μm, and the height is 90-110 μm.

8. A method for preparing a continuous flow magnetic separation chip, characterized in that: The preparation method includes the preparation of a chip base plate, the preparation of a flow channel cover plate and chip assembly, wherein: The preparation of the chip base plate includes: Step S10: preparing a patterned soft magnetic tape with PDMS as the substrate; Step S11: flattening the polypropylene film, spraying a release agent on the surface of the polypropylene film, and then dripping epoxy resin on the surface of the polypropylene film; Step S12: pressing the patterned soft magnetic tape with the PDMS as the base onto the surface of the polypropylene film so that the surface of the soft magnetic tape is pressed toward the epoxy resin; Step S13: After the epoxy resin is cured, the polypropylene film is peeled off to obtain a chip base plate encapsulating the soft magnetic tape material; The preparation of the flow channel cover plate includes: Step S20: preparing a patterned soft magnetic tape with PDMS as the substrate; Step S21: Spin-coating a photoresist on a silicon wafer, and then obtaining a composite structure consisting of a silicon wafer and patterned photoresist by exposure and development; Step S22: spin coating PDMS on the composite structure and performing a round of curing; Step S23: pouring a PDMS solution onto the solid PDMS after one round of curing, pressing the patterned soft magnetic tape with the PDMS as the substrate into the PDMS solution, so that the plane where the soft magnetic tape is located is pressed on the solid PDMS surface obtained by one round of curing, and positioning is completed, and then a second round of curing is performed; Step S24: peeling off the PDMS flow channel after the second round of curing from the composite structure to obtain a flow channel cover plate encapsulating the soft magnetic tape material; The chip assembly comprises: Step S31: Assembling the flow channel cover plate and the chip base plate by pressing, so that the PDMS flow channel of the flow channel cover plate and the epoxy resin plane of the chip base plate are combined; Step S32: clamping the flow channel cover plate and the chip base plate in the middle by using an upper pressing plate and a lower pressing plate, and applying pressure to the upper pressing plate and the lower pressing plate by screwing.

9. The method for preparing a continuous flow magnetic separation chip according to claim 8, characterized in that: When the PDMS flow channel of the flow channel cover plate and the epoxy resin plane of the chip base plate are combined, the soft magnetic tape material packaged in the flow channel cover plate and the soft magnetic tape material packaged in the chip base plate overlap, and the area overlap rate is greater than 95%.

10. The method for preparing a continuous flow magnetic separation chip according to claim 8, characterized in that: The step S10 and the step S20 of preparing the patterned soft magnetic tape with PDMS as the substrate include: Step S40: spin coating liquid PDMS on the solid surface of PDMS, and pasting the soft magnetic tape onto the solid PDMS through the liquid PDMS; Step S41: heating until the liquid PDMS is completely solidified, fixing the soft magnetic tape on the solid surface of PDMS; Step S42: Spin-coating photosensitive paint on the surface of the soft magnetic tape and drying it; Step S43: performing patterned exposure of the flow channel morphology on the surface of the photosensitive paint, and spraying with a sodium carbonate solution with a mass concentration of 0.8-1% after the exposure until the pattern boundary is clearly visible; Step S44: spraying the etching solution until the boundary of the metal pattern is clearly visible, and then rinsing the sample with pure water to terminate the etching; Step S45: Soak 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 soft magnetic tape falls off, and then rinse the sample with pure water to obtain a patterned soft magnetic tape with PDMS as the substrate.

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