Multi-stage magnetic separation system and magnetic separation method

Abstract

A multi-stage magnetic separation system, comprising a multi-stage separation channel unit (1), a magnet array unit (2), and a magnetic filtration unit (3), wherein the multi-stage separation channel unit (1) comprises a plurality of separation channel layers, the plurality of separation channel layers are sequentially stacked from top to bottom, and each separation channel layer is provided with a main separation channel and two outlet channels, so as to implement separation of particles of different sizes; the magnet array unit (2) comprises at least two magnet arrays, wherein one magnet arrays is a magnetic filtration magnet array and is used for providing the magnetic filtration unit with a magnetic field required by magnetic filtration, and the other magnet array is a separation magnet array and is used for providing the multi-stage separation channel unit with a magnetic field required by negative magnetophoretic separation; the magnetic filtration unit (3) is connected to the multi-stage separation channel unit (1).

Description

Multi-stage magnetic separation system and magnetic separation method Technical Field

[0001] The present invention relates to a magnetic separation system, in particular to a multi-stage magnetic separation system and a magnetic separation method, belonging to the technical field of magnetic separation. Background Art

[0002] Magnetic sorting is divided into magnetophoresis sorting and negative magnetophoresis sorting. Magnetophoresis requires the use of magnetic beads to label the sample, and then uses the attraction of the magnetic field to the magnetic beads to sort the sample. This method requires additional magnetic bead labeling and elution steps for the sample. The principle of negative magnetophoresis is that under the action of an external magnetic field, the non-magnetic particles suspended in the magnetic solution are subjected to a repulsive force positively correlated with their volume, thereby achieving the sorting of particles of different sizes. This method has the advantages of not requiring labeling and not relying on complex external systems. Biological samples can be size-sorted using magnetic solutions. Due to the size properties of biological particle samples (usually at the micron or nanometer level), the above two magnetic sorting methods are mainly used in microfluidic chips.

[0003] In recent years, magnetic solutions represented by magnetic fluids (usually suspensions of magnetic nanoparticles) have been increasingly used in microfluidic chips. Magnetic fluids can be used to construct a local magnetic environment inside microfluidic chips to achieve various functions of magnetic fields in microchannels [1-2]. In addition, some magnetic fluids are now biocompatible, making them play an important role in the manipulation of biological samples. Negative magnetophoresis technology based on magnetic fluids has been applied to label-free magnetic sorting of various samples [3]. To date, microfluidic sorting based on negative magnetophoresis can be roughly divided into three stages: 1) sorting of micron-sized polystyrene particles and bacteria [4]; 2) cell sorting [5]; and 3) sorting of nanobiological samples [6].

[0004] The current negative magnetophoresis separation scheme has the following disadvantages:

[0005] (1) The structure of the sorting channel is relatively fixed. The sorting channel is designed for a fixed sample model, which lacks versatility. In addition, the flux of negative magnetophoresis sorting based on microfluidics cannot be improved, which limits its application range.

[0006] (2) A single negative magnetophoresis separation structure and parameters cannot sort complex samples of various sizes, such as whole blood samples.

[0007] (3) Currently, negative magnetophoresis sorting is mostly targeted at micron-sized particles. Although a small number of them can sort nanoparticles, they require complex structural processing steps and have extremely low processing throughput, which limits their application.

[0008] (4) Negative magnetophoresis sorting based on microfluidics requires complex processing steps and external equipment support, such as micro pumps, etc., which is not suitable for some special scenarios, such as use in harsh environments such as the wild.

[0009] (5) Although biocompatible magnetic fluids are currently used in negative magnetophoresis separation, which can reduce the damage of magnetic nanoparticles in magnetic fluids to biological samples to a certain extent, their impact cannot be completely eliminated.

[0010] The above references are as follows:

[0011] [1]Dunne P,Adachi T,Dev AA,et al.Liquid flow and control without solid walls.Nature,2020,581:58-62.

[0012] [2] Liu Y, Vieira RMS, Mao L. Simultaneous and Multimodal Antigen-Binding Profiling and Isolation of Rare Cells via Quantitative Ferrohydrodynamic Cell Separation. ACS Nano, 2023, 17: 94-110.

[0013] [3] Zhao W, Cheng R, Miller JR, et al. Label-free microfluidic manipulation of particles and cells in magnetic liquids. Advanced Functional Materials, 2016, 26: 3916-3932.

[0014] [4]Liang L, Zhang C, Xuan X. Enhanced separation of magnetic and diamagnetic particles in a dilute ferrofluid. Applied Physics Letters, 2013,102:234101.

[0015] [5] Zhao W, Zhu T, Cheng R, et al. Label-free and continuous-flow ferrohydrodynamic separation of HeLa cells and blood cells in biocompatible ferrofluids. Advanced Functional Materials, 2016, 26: 3990-3998.

[0016] [6] Zeng L, Chen X, Du J, et al. Label-free separation of nanoscale particles by an ultrahigh gradient magnetic field in a microfluidic device. Nanoscale, 2021, 13: 4029-4037.

[0017] Summary of the Invention

[0018] The purpose of the present invention is to solve the problems of the current negative magnetophoresis sorting, such as the single structure, relatively fixed sorting size, low sorting flux and low sorting resolution. The present invention provides a multi-stage magnetic sorting system. The system can continuously perform negative magnetophoresis sorting on particles of different sizes in a sample with high throughput. The particle size range covers from macroparticles to nanoparticles. The system can flexibly and freely combine sorting channels of different sizes to adapt to samples of various sizes. The negative magnetophoresis sorting resolution can reach the nanometer level. The system can effectively remove magnetic nanoparticles introduced during the negative magnetophoresis sorting process, completely eliminating the problem of biocompatibility in negative magnetophoresis sorting.

[0019] Another object of the present invention is to provide a magnetic separation method.

[0020] The purpose of the present invention can be achieved by taking the following technical solutions:

[0021] A multi-stage magnetic separation system includes a multi-stage separation channel unit, a magnet array unit and a magnetic filtration unit. The multi-stage separation channel unit includes several separation channel layers, which are stacked in sequence from top to bottom. Each separation channel layer is provided with a main separation channel and two outlet channels to achieve separation of particles of different sizes. The magnet array unit includes at least two groups of magnet arrays, one of which is a magnetic filtration magnet array, which is used to provide the magnetic field required for magnetic filtration to the magnetic filtration unit, and the remaining magnet arrays (i.e., one or more groups of magnet arrays) are separation magnet arrays, which are used to provide the magnetic field required for negative magnetophoresis separation to the multi-stage separation channel unit. The magnetic filtration unit is connected to the multi-stage separation channel unit.

[0022] Furthermore, in the multi-stage sorting channel unit, an intermediate layer is provided between every two adjacent sorting channel layers, which are separated and connected by the intermediate layer. The intermediate layer is provided with a sorting sample inlet and a sample outlet, and the sorting sample inlet and the sample outlet are respectively connected to the sorting sample outlets to which the two outlet channels of the previous sorting channel layer lead.

[0023] Furthermore, in the multi-level sorting channel unit, the two outlet channels of each sorting channel layer are respectively an outer outlet channel and an inner outlet channel. In the main sorting channel, large particles in the sample are repelled by the magnetic field at a farther distance and enter the outer outlet channel, while small particles are repelled by the magnetic field at a closer distance and enter the inner outlet channel.

[0024] Furthermore, a substrate is provided between each sorting channel layer of the multi-stage sorting channel unit and the sorting magnet array.

[0025] Furthermore, a metal layer is added to the substrate.

[0026] Furthermore, the first magnet array and the second magnet array are both composed of a plurality of magnets arranged in a preset order.

[0027] Furthermore, the magnetic filtration unit includes a sample recovery pool, a connecting tube, a magnetic filtration channel and a filtered sample outlet, the multi-stage sorting channel unit is connected to the sample recovery pool, and the sample recovery pool, connecting tube, magnetic filtration channel and filtered sample outlet are connected in sequence.

[0028] Furthermore, the magnetic filtration channel is a section of soft tube filled with micron-sized ferromagnetic powder, which is placed between two sets of magnet arrays. Under the clamping of the two sets of magnet arrays, the magnetic field magnetizes the micron-sized ferromagnetic powder in the soft tube and fixes it in the magnet area, forming a section of magnetic filtration channel with a porous structure.

[0029] Furthermore, it also includes a fixed bracket, and the multi-stage sorting channel unit, the magnet array unit and the magnetic filtration unit are fixed and supported by the fixed bracket.

[0030] Another object of the present invention can be achieved by adopting the following technical solutions:

[0031] A magnetic separation method is implemented based on the above-mentioned multi-stage magnetic separation system, the method comprising:

[0032] Mixing the sample to be sorted and the magnetic fluid or magnetic salt solution in a preset ratio;

[0033] The sample flows through each sorting channel layer in sequence through the injection pool, and the particles of different sizes are sorted in each sorting channel layer;

[0034] In the magnetic filtration unit, the sorted samples are recovered through the sample recovery pool. The samples in the sample recovery pool enter the magnetic filtration channel through the connecting tube. The strong magnetic field in the magnetic filtration channel adsorbs the magnetic nanoparticles in the magnetic fluid or magnetic salt solution, allowing the non-magnetic sample to flow out of the filtration sample outlet through the magnetic filtration channel, thereby achieving the filtration of magnetic nanoparticles in the sample.

[0035] The present invention has the following beneficial effects compared to the prior art:

[0036] 1. The present invention can achieve multi-stage negative magnetophoresis sorting through simple channel superposition, and the sorting stage can be selected according to the particle size and actual needs, thereby improving the processing throughput while also ensuring the size resolution of negative magnetophoresis sorting. Multi-stage sorting can avoid the mutual influence between particles of different sizes and can be adapted to the sorting of samples with particles of various sizes. At the same time, the present invention integrates a magnetic filtration unit, which can effectively remove magnetic nanoparticles in the sample while maintaining good biocompatibility. Therefore, it can be widely used for the efficient sorting of complex biological samples (such as whole blood) and micro-nanoparticles of different sizes.

[0037] 2. The present invention utilizes the principle of negative magnetophoresis to sort micro-nanoparticles of different sizes. Negative magnetophoresis refers to the fact that non-magnetic particles in a magnetic solution (such as a magnetic fluid or a magnetic salt solution) can be repelled by the repulsive force of the magnetic field (negative magnetophoresis force) and move away from areas with high magnetic field gradients, i.e., they are repelled by the magnet. The negative magnetophoresis force is proportional to the volume of the particles, as well as to the magnetic field gradient and magnetic field strength. Therefore, the principle of negative magnetophoresis can be used to perform label-free sorting of particles according to size. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] 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 description of the prior art. 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 the structures shown in these drawings without paying any creative work.

[0039] FIG1 is a structural diagram of a multi-stage magnetic separation system according to Example 1 of the present invention.

[0040] FIG2 is a three-dimensional structural diagram of a three-stage sorting channel unit according to Example 1 of the present invention from one angle.

[0041] FIG3 is a three-dimensional structural diagram of the three-stage sorting channel unit according to Example 1 of the present invention from another angle.

[0042] FIG4 is a front structural diagram of a three-stage sorting channel unit according to Example 1 of the present invention.

[0043] FIG5 is a top view of the structure of the three-stage sorting channel unit according to Example 1 of the present invention.

[0044] FIG6 is a side structural diagram of a three-stage sorting channel unit according to Example 1 of the present invention.

[0045] FIG7 is a structural diagram of the channel upper and lower parts in the three-stage sorting channel unit according to Example 1 of the present invention.

[0046] FIG8 is a structural diagram of a first-stage sorting channel layer in a three-stage sorting channel unit according to Example 1 of the present invention.

[0047] FIG9 is a structural diagram of a first-level intermediate layer in a three-level sorting channel unit according to Example 1 of the present invention.

[0048] FIG10 is a structural diagram of the secondary sorting channel layer in the three-stage sorting channel unit of Example 1 of the present invention.

[0049] FIG11 is a structural diagram of the secondary intermediate layer in the three-stage sorting channel unit of Example 1 of the present invention.

[0050] FIG12 is a structural diagram of a three-level sorting channel layer in a three-level sorting channel unit according to Example 1 of the present invention.

[0051] FIG13 is a structural diagram of the bottom of the channel in the three-stage sorting channel unit of Example 1 of the present invention.

[0052] FIG14 is a structural diagram of the side substrate of Example 1 of the present invention.

[0053] FIG15 is a structural diagram of the nano-scale sorting side substrate of Example 1 of the present invention.

[0054] FIG16 is a dimensional diagram of the side substrate of the nano-scale sorting in Example 1 of the present invention.

[0055] FIG17 is a schematic diagram of the surface pattern of the nanoscale sorting base metal layer according to Example 1 of the present invention.

[0056] FIG18 is a flow chart of the production process of the nano-scale sorting side substrate according to Example 1 of the present invention.

[0057] FIG19 is a three-dimensional structural diagram of the magnet array unit and the fixing bracket according to Example 1 of the present invention.

[0058] FIG20 is a front structural diagram of the magnet array unit and the fixing bracket according to Example 1 of the present invention.

[0059] FIG21 is a top view of the structure of the magnet array unit and the fixing bracket according to Example 1 of the present invention.

[0060] FIG22 is a side structural diagram of the magnet array unit and the fixing bracket according to Example 1 of the present invention.

[0061] FIG23 is a structural diagram of the fixing bracket of Example 1 of the present invention.

[0062] FIG24 is a three-dimensional structural diagram of the first form of the magnet array unit of Example 1 of the present invention.

[0063] FIG25 is a front view of the first form of the magnet array unit of Example 1 of the present invention.

[0064] FIG26 is a side structural diagram of the first form of the magnet array unit of Example 1 of the present invention.

[0065] FIG27 is a top view of the first form of the magnet array unit of Example 1 of the present invention.

[0066] FIG28 is a three-dimensional structural diagram of the second form of the magnet array unit of Example 1 of the present invention.

[0067] FIG29 is a front view of the second form of the magnet array unit of Example 1 of the present invention.

[0068] FIG30 is a side structural diagram of the second form of the magnet array unit of Example 1 of the present invention.

[0069] FIG31 is a top view of the second form of the magnet array unit of Example 1 of the present invention.

[0070] FIG32 is a three-dimensional structural diagram of the third form of the magnet array unit of Example 1 of the present invention.

[0071] FIG33 is a front view of the structure of the third form of the magnet array unit of Example 1 of the present invention.

[0072] FIG34 is a side view of the third form of the magnet array unit of Example 1 of the present invention.

[0073] FIG35 is a top view of the third form of the magnet array unit of Example 1 of the present invention.

[0074] FIG36 is a three-dimensional structural diagram of the magnet array unit and the magnetic filter unit according to Example 1 of the present invention.

[0075] FIG37 is a front view of the structure of the magnet array unit and the magnetic filter unit according to the first embodiment of the present invention.

[0076] FIG38 is a side structural diagram of the magnet array unit and the magnetic filter unit of Example 1 of the present invention.

[0077] FIG39 is a top view of the structure of the magnet array unit and the magnetic filter unit according to Example 1 of the present invention.

[0078] Figure 40 is a three-dimensional structural diagram of the magnetic filter unit of Example 1 of the present invention.

[0079] Figure 41 is a front view structural diagram of the magnetic filter unit of Example 1 of the present invention.

[0080] Figure 42 is a side view of the magnetic filter unit of Example 1 of the present invention.

[0081] Figure 43 is a top view of the magnetic filter unit of Example 1 of the present invention.

[0082] Figure 44 is a structural diagram of the multi-stage magnetic separation system of Example 2 of the present invention.

[0083] Figure 45 is a three-dimensional structural diagram of one angle of the three-stage sorting channel unit of Example 2 of the present invention.

[0084] FIG46 is a three-dimensional structural diagram of the three-stage sorting channel unit of Example 2 of the present invention from another angle.

[0085] Figure 47 is a structural diagram of the channel upper and lower parts in the three-level sorting channel unit of Example 2 of the present invention.

[0086] FIG48 is a structural diagram of the first-level sorting channel layer in the three-level sorting channel unit of Example 2 of the present invention.

[0087] Figure 49 is a structural diagram of the first-level intermediate layer in the three-level sorting channel unit of Example 2 of the present invention.

[0088] Figure 50 is a structural diagram of the secondary sorting channel layer in the three-stage sorting channel unit of Example 2 of the present invention.

[0089] Figure 51 is a structural diagram of the secondary intermediate layer in the three-stage sorting channel unit of Example 2 of the present invention.

[0090] Figure 52 is a structural diagram of the three-level sorting channel layer in the three-level sorting channel unit of Example 2 of the present invention.

[0091] Figure 53 is a structural diagram of the bottom of the channel in the three-level sorting channel unit of Example 2 of the present invention.

[0092] Figure 54 is a structural diagram of the fixing bracket of embodiment 2 of the present invention.

[0093] Figure 55 is a three-dimensional structural diagram of the magnet array unit of Example 2 of the present invention.

[0094] Figure 56 is a front view structural diagram of the magnet array unit of Example 2 of the present invention.

[0095] Figure 57 is a side view of the magnet array unit of Example 2 of the present invention.

[0096] Figure 58 is a top view of the magnet array unit of Example 2 of the present invention.

[0097] Among them, 1-multi-stage sorting channel unit, 2-magnet array unit, 3-magnetic filtration unit, 4-fixed bracket, 5-sample injection pool, 6-first-level sample outlet, 7-second-level sample outlet, 8-third-level sample outlet, 9-fourth-level sample outlet, 10-channel upper bottom, 11-first-level sorting channel layer, 12-first-level middle layer, 13-second-level sorting channel layer, 14-second-level middle layer, 15-third-level sorting channel layer, 16-channel lower bottom, 17-micron-level sorting side substrate, 18-nanoscale sorting side substrate, 19-first-level sorting sample inlet, 20-second-level sorting sample inlet, 21-third-level sorting sample inlet, 22-first-level sorting sample outlet A, 23-first-level sorting sample outlet B, 24-second-level sorting Sample selection outlet A, 25-secondary sorting sample outlet B, 26-tertiary sorting sample outlet A, 27-tertiary sorting sample outlet B, 28-main sorting channel, 29-inner outlet channel, 30-outer outlet channel, 31-nanoscale sorting base metal layer, 32-nanoscale sorting base PDMS layer, 33-nanoscale sorting base metal layer surface pattern, 34-sorting magnet array, 35-magnetic filtering magnet array, 36-sample recovery pool, 37-connecting tube, 38-magnetic filtration channel, 39-filtered sample outlet; W1-first-level sorting channel width, W2-secondary sorting channel width, W3-tertiary sorting channel width, W4-nanoscale sorting base PDMS layer thickness, W5-nanoscale sorting base metal layer thickness, W6-nanoscale sorting base metal layer surface pattern cutting depth. DETAILED DESCRIPTION

[0098] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0099] Example 1:

[0100] The sorting levels of this embodiment range from 1 to n levels, and can be freely selected and combined according to needs. The first-level sorting refers to a sorting channel layer, including a sample inlet and two sample outlets, which can divide the particles into two levels according to size, or completely sort out the particles in the sample; the second-level sorting refers to two sorting channel layers, including a sample inlet and three sample outlets. After the first-level sorting channel sorts the larger particles, the remaining samples can enter the second-level sorting channel through the middle layer to further subdivide the particle size.

[0101] This embodiment uses a three-stage magnetic separation system to separate small extracellular vesicles from whole blood as an example. First, whole blood is mixed with a small amount of magnetic fluid. The red blood cells and white blood cells (both with a particle size >6 μm) in the whole blood can be removed through the primary separation channel, allowing the red blood cells and white blood cells to flow out of outlet B, while the serum / plasma containing platelets flows out of outlet A and enters the secondary separation channel. In the secondary separation channel, platelets (>1 μm) can be removed and flow out of outlet B, while platelet-free serum / plasma is retained from outlet A, ultimately achieving serum / plasma separation from whole blood. If it is necessary to further separate the extracellular vesicles (<1 μm) in the serum / plasma by size, the platelet-free serum / plasma can be further separated through the three-stage separation channel to separate the extracellular vesicles therein into microvesicles (>200 nm) and small extracellular vesicles (<200 nm) by size. For nanoscale particles (<1μm), it is necessary to enhance the magnetic field gradient and magnetic field strength by adding a patterned metal layer to the side substrate to achieve high-resolution negative magnetophoresis sorting; after the required sample is sorted, the sample is passed through a magnetic filtration unit to remove the magnetic nanoparticles (usually 10nm) in the magnetic fluid. The final sample will not contain any exogenous substances and therefore will not have any impact on back-end detection or treatment.

[0102] The whole blood separation process is described in detail below with reference to the illustrations. A three-stage magnetic separation system is proposed to separate blood cells (including white blood cells, red blood cells, and platelets) and microvesicles from the whole blood sample, ultimately obtaining a small extracellular vesicle sample in serum / plasma.

[0103] As shown in Figure 1, the multi-stage magnetic separation system of this embodiment includes a multi-stage separation channel unit 1, a magnet array unit 2, a magnetic filtration unit 3 and a fixed bracket 4. The multi-stage separation channel unit 1, the magnet array unit 2 and the magnetic filtration unit 3 are fixed and supported by the fixed bracket 4. Since the multi-stage magnetic separation system of this embodiment is a three-stage magnetic separation system, the multi-stage separation channel unit 1 is a three-stage separation channel unit. For other multi-stage magnetic separation systems, it is only necessary to increase or decrease the number of layers of the multi-stage separation channel unit 1, and then select a magnet of appropriate length according to the number of layers of the multi-stage separation channel unit 1. Finally, according to the final size of each unit, a fixed bracket 4 is made by 3D printing or mold injection molding, and the various units are combined together.

[0104] As shown in Figures 1 to 13, the three-level sorting channel unit includes three sorting channel layers, which are stacked in sequence from top to bottom, namely, a primary sorting channel layer 11, a secondary sorting channel layer 13 and a tertiary sorting channel layer 15; above the primary sorting channel layer 11 is a channel upper bottom 10, which includes a primary sorting sample inlet 19, which is connected to the injection pool 5; each sorting channel layer is provided with a main sorting channel 28 and two outlet channels to achieve the sorting of particles of different sizes, the two outlet channels are an inner outlet channel 29 and an outer outlet channel 30, the inner outlet channel 29 and the outer outlet channel 30 of the primary sorting channel layer 11 lead to the primary sorting sample outlet A22 and the primary sorting sample outlet B23 respectively, the secondary sorting channel layer 1 3 leads to the secondary sorting sample outlet A24 and the secondary sorting sample outlet B25, respectively. The inner outlet channel 29 and the outer outlet channel 30 of the tertiary sorting channel layer 15 lead to the tertiary sorting sample outlet A26 and the tertiary sorting sample outlet B27, respectively. In the main sorting channel 28, large particles in the sample will be repelled by the negative magnetophoretic force and enter the outer outlet channel 30, while small particles will be repelled a shorter distance and enter the inner outlet channel 29. An intermediate layer is provided between every two adjacent sorting channel layers, which are separated and connected by the intermediate layer. The intermediate layer between the primary sorting channel layer 11 and the secondary sorting channel layer 13 is the primary intermediate layer 12, and the intermediate layer between the secondary sorting channel layer 13 and the secondary sorting channel layer 14 is the primary intermediate layer 14.

[0105] In this embodiment, a whole blood sample is first mixed with a magnetic fluid, which is a suspension of magnetic nanoparticles, in a certain proportion. The magnetic nanoparticles are added to the whole blood at a volume concentration of 0.00001% to 10%, calculated based on the volume ratio of the nanoparticles. The mixed sample is then injected into the injection pool 5, where the whole blood is driven downward by gravity. The flow rate of the whole blood sample in the sorting channel can also be adjusted by adding an external syringe pump or by adding an oily liquid (immiscible with blood) above the whole blood. The volume of the injection pool 5 can be adjusted according to the sample volume. The sample volume processed by the multi-stage sorting channel unit 1 ranges from 1pL to 100L.

[0106] Furthermore, the whole blood sample in the sample inlet 5 enters the primary sorting channel layer 11 through the primary sorting sample inlet 19 in the channel bottom 10, and undergoes primary magnetic sorting in the main sorting channel 28. The white blood cells and red blood cells (the largest particles) therein will enter the outer outlet channel 30, while the serum / plasma with platelets and extracellular vesicles will enter the inner outlet channel 29; below the primary sorting channel layer 11 is the primary intermediate layer 12, which includes a secondary sorting sample inlet 20 connected to the primary sorting sample outlet A22, and a primary sample outlet 6 connected to the primary sorting sample outlet B23. The white blood cells and red blood cells in the whole blood sample will flow out from the primary sample outlet 6, while the serum / plasma with platelets and extracellular vesicles will enter the secondary sorting channel layer 13 through the secondary sorting sample inlet 20; in the secondary sorting channel layer 13, the same negative magnetophoresis sorting principle is used to achieve the negative magnetophoresis sorting by reducing the width W2 of the main sorting channel 28. The platelets in the sample are separated, passing through the outer outlet channel 30 of the layer into the secondary sorting sample outlet B25, and ultimately flowing out of the secondary sample outlet 7 in the secondary intermediate layer 14 below the secondary sorting channel layer 13. The serum / plasma containing extracellular vesicles enters the secondary sorting sample outlet A24 through the inner outlet channel 29 of the layer, and enters the tertiary sorting channel layer 15 through the tertiary sorting sample inlet 21 in the secondary intermediate layer 14. In the tertiary sorting channel layer 15, the same negative magnetophoresis sorting principle is used, and the width W3 of the main sorting channel 28 is reduced to achieve separation of microvesicles and small extracellular vesicles in the sample. Ultimately, the larger microvesicles enter the tertiary sorting sample outlet B27 through the outer outlet channel 30 of the layer and flow out of the tertiary sample outlet 8 on the lower channel bottom 16, while the small extracellular vesicles enter the tertiary sorting sample outlet A26 through the inner outlet channel 29 of the layer, and ultimately flow out of the quaternary sample outlet 9 on the lower channel bottom 16.

[0107] In this embodiment, the side substrates of the three-level sorting channel layer 15 and the previous two-level sorting channel layers (the primary sorting channel layer 11 and the secondary sorting channel layer 13) are different. Since the side substrate is the partition between the magnet array unit 2 and the multi-level sorting channel unit 1, its thickness directly affects the magnetic field strength of the magnet array in the sorting channel. The magnetic field strength in the sorting channel can be enhanced by reducing the thickness of the side substrate. However, for nano-scale particles, simply relying on enhancing the magnetic field strength cannot provide a sufficiently large negative magnetophoretic force. It is necessary to add a patterned metal substrate layer to further enhance the magnetic field gradient and magnetic field strength to achieve nano-scale high resolution. Negative magnetophoresis sorting; The first two sorting channel layers of this embodiment (the first sorting channel layer 11 and the second sorting channel layer 13) are used to separate white blood cells, red blood cells and platelets. Their sizes are all in the micron level, so a conventional micron-level sorting side substrate 17 can be used, and its thickness range is 1μm-1cm; in the third sorting channel layer 15, nano-scale microvesicles and small extracellular vesicles need to be sorted. At this time, a nano-level sorting side substrate 18 is required to enhance its negative magnetophoresis force, as shown in Figure 14; the nano-level sorting side substrate 18 includes a nano-level sorting substrate metal layer 31 and a nano-level sorting substrate PDMS (polydimethylsiloxane) ) layer 32, as shown in Figures 15 to 17, the material of the nano-scale sorting base metal layer 31 is ferromagnetic materials such as iron, nickel, iron oxide, and permalloy. A groove array is cut on its surface by a femtosecond laser or a picosecond laser to form a square, polygonal or circular microarray structure on its surface. This embodiment takes a square as an example, that is, the surface pattern 33 of the nano-scale sorting base metal layer. On the one hand, the addition of the metal layer can increase the magnetic field strength in the sorting channel. On the other hand, by patterning the metal layer, the surface of the metal layer has a microarray, which can increase the magnetic field gradient in the sorting channel layer, thereby greatly increasing the negative magnetophoretic force on the particles. The thickness W4 of the nano-level sorting base PDMS layer 32 (i.e., the distance from the nano-level sorting base metal layer 31 to the sorting channel) ranges from 10 nm to 1 mm, the thickness W5 of the nano-level sorting base metal layer 31 ranges from 1 μm to 1 cm, and the surface cutting depth W6 of the nano-level sorting base metal layer 31 ranges from 10 nm to 1 mm (not cut through). The side length of the resulting square or polygon or the diameter of the circle ranges from 1 μm to 100 μm. The surface pattern 33 of the nano-level sorting base metal layer needs to cover the range of the main sorting channel 28 in the three-level sorting channel layer 15, so that particles in the main sorting channel 28 can be subjected to enhanced negative magnetophoretic force.The fabrication process for the nanoscale sorting side substrate 18 is shown in FIG18 . First, a metal substrate is laser-cut to form a metal substrate with a surface microarray pattern. A liquid PDMS layer is then spin-coated on the patterned metal surface. The thickness of the layer can be adjusted by the spin-coating speed. Finally, the PDMS layer is dried to form a PDMS+metal bilayer substrate, completing the fabrication of the nanoscale sorting side substrate 18.

[0108] Furthermore, the materials of the sorting channel layer, the upper and lower bottoms of the channel, the middle layer and the micron-scale sorting side substrate are plastic, glass, resin or polymer (such as polydimethylsiloxane, PDMS), etc., and the various layers and the side substrates are sealed by bonding, pressure bonding, plasma bonding, etc. The structure of the channel layer and the middle layer can be produced by laser cutting, injection molding, photolithography and other methods. In this example, the width of the main sorting channel 28 of each level decreases step by step from top to bottom, that is, W1>W2>W3. The width of the main sorting channel 28 is set according to the particle size. The larger the size of the sorted particles, the wider the width, which is in the range of 10nm-10cm. The width of the two sorting outlet channels can be adjusted according to the particle size, and the sum of the widths of the two channels is equal to the main sorting channel. The thickness of the sorting channel layer (i.e., the height of the sorting channel) can be adjusted according to the sample amount, which is in the range of 10nm-1m. The sorting channel layers can be connected by an intermediate layer or directly connected without an intermediate layer.

[0109] As shown in Figures 1 and 19 to 23, the magnet array unit 2 includes two groups of magnet arrays, which are the first magnet array and the second magnet array respectively. The first magnet array is the sorting magnet array 34, and the second magnet array is the magnetic filtering magnet array 35. A part of the sorting magnet array 34 (shown as the upper part in the figure) provides the multi-stage sorting channel unit 1 with the magnetic field required for negative magnetophoresis sorting, and the other part (shown as the lower part in the figure) and the magnetic filtering magnet array 35 together provide the magnetic field required for magnetic filtration to the magnetic filtration unit 3; the two groups of magnet arrays are fixed by a fixed bracket, and the distance W7 between them ranges from 100μm to 1m. The width of the magnet array ranges from 1mm to 10m. The height of the magnet array can be adjusted according to the height of the multi-stage sorting channel unit 1 and the magnetic filtration unit 3, and ensure that its magnetic field range can cover the above two units.

[0110] This embodiment lists three types of magnet array arrangements. The first type of array is denoted as A array, as shown in Figures 24 to 27. The second type of array is denoted as B array, as shown in Figures 28 to 31. The third type of array is denoted as C array, as shown in Figures 32 to 35. The dotted arrows in the figures represent the magnetic field directions of the magnets. If magnets of the same grade are used for arrangement, the magnetic field strength generated by array A is greater than that of array B, while the magnetic field strength of array C is the smallest. However, the structure of array C is relatively simple and the easiest to implement. A suitable magnet array can be selected according to the size of the sorted particles, and is not limited to the three magnet arrays given in the figures.

[0111] The sorted sample flowing out of the multi-stage sorting channel unit 1 contains magnetic nanoparticles. Although biocompatible magnetic nanoparticles are already present, in order to eliminate the impact of magnetic nanoparticles on biological samples and subsequent detection, treatment and other processes, this embodiment also integrates a magnetic filtration unit 3, the structure of which is shown in Figures 36 to 43. The magnetic filtration unit 3 includes a sample recovery pool 36, a connecting tube 37, a magnetic filtration channel 38 and a filtered sample outlet 39, wherein the magnetic filtration channel 38 is a hose of the same length as the width of the magnet array, with an inner diameter ranging from 100μm to 1m and a wall thickness ranging from 10μm to 1. 0cm, the hose is filled with micron-sized ferromagnetic powder, such as iron particles or iron oxide particles, with a particle size range of 1μm-100μm; the magnetic filtration channel 38 is placed between the sorting magnet array 34 and the magnetic filtration magnet array 35 through the fixed bracket 4, and is clamped by the two sets of magnet arrays; the magnetic field can magnetize the ferromagnetic powder in the hose and fix it in the magnet area, forming a porous magnetic filtration channel. Since it is filled with magnetized ferromagnetic powder, the magnetic filtration channel 38 has extremely high magnetic field strength and magnetic field gradient, which can adsorb magnetic nanoparticles in the sample.

[0112] Furthermore, the sample recovery pool 36 can collect samples from the four sample outlets as needed. In this embodiment, in order to collect small extracellular vesicle samples, the sample recovery pool 36 is used to recover the small extracellular vesicle samples flowing out of the fourth sample outlet 9, and then a negative pressure drive is provided at the filtration sample outlet 39 (for example, suction by a syringe), or a positive pressure drive is provided at the end of the recovery pool 36. The sample in the recovery pool 36 will enter the magnetic filtration channel 38 through the connecting tube 37. The strong magnetic field in the magnetic filtration channel 38 can adsorb the magnetic nanoparticles in the magnetic fluid, and the non-magnetic sample can flow out of the filtration sample outlet 39 through the porous structure, thereby realizing the filtration of the magnetic nanoparticles in the sample and obtaining a pure small extracellular vesicle sample.

[0113] Example 2:

[0114] As shown in Figures 44 to 58, compared with Example 1, this embodiment has two groups of magnet arrays arranged on both sides of the multi-stage sorting channel unit 1, and the number of main sorting channels is correspondingly increased in the sorting channel layer. Negative magnetophoresis sorting is performed together by the cooperation of the two groups of magnet arrays. The advantage of the two groups of magnet arrays is that the magnetic field acting on the sorting channel is stronger, thereby faster sorting speed, and the number of main sorting channels is increased, thereby improving the sorting flux of samples, and the overall sorting efficiency can be improved.

[0115] Those skilled in the art will appreciate that, in addition to arranging two sets of magnet arrays on both sides, magnet arrays may also be arranged on all four sides of the multi-stage sorting channel unit 1, so that large particles are affected by the negative magnetophoretic force on all sides and flow out from the middle of the sorting channel, while small particles flow out from all sides of the sorting channel.

[0116] Example 3:

[0117] In this embodiment, in addition to being self-driven into the sorting channel from top to bottom, the sample can also be injected from bottom to top through external pressure; in addition, the placement of the magnet array can be adjusted according to needs, for example, the magnet array is placed on the top and the sorting channel layer is located below, or the magnet array is placed below and the sorting channel layer is located on the top.

[0118] In summary, the multi-stage magnetic separation system of the present invention has the following advantages:

[0119] 1) A multi-level stacked magnetic separation channel structure is designed, which can select the separation level and freely combine the separation channel layers according to the needs. Therefore, it can handle complex samples with various particle sizes, such as whole blood, and has wide versatility.

[0120] 2) Through the combination of macrochannels and microchannels, the sample processing throughput can be effectively improved.

[0121] 3) A patterned metal layer substrate was designed to effectively increase the negative magnetophoretic force on the particles, achieving high-resolution sorting of nanoparticles. The processing flux can be freely adjusted by the channel height, and the flux is no longer limited.

[0122] 4) The integrated magnetic filtration unit can effectively remove the magnetic nanoparticles introduced during negative magnetophoresis separation, completely eliminating their impact on biological samples.

[0123] 5) The entire system adopts a passive design, relying on a magnet array to provide sorting power. No external power supply is required, and the sample can be self-driven into the sorting channel. Therefore, it has wide applicability and can be used in harsh environments such as the wild.

[0124] The above description is only a preferred embodiment of the present invention, but the implementation of the present invention is not limited to the above embodiment. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A multi-stage magnetic separation system, characterized in that, It includes a multi-stage sorting channel unit, a magnet array unit, and a magnetic filtration unit. The multi-stage sorting channel unit includes several sorting channel layers which are stacked in sequence from top to bottom. Each sorting channel layer is provided with a main sorting channel and two outlet channels to achieve the sorting of particles of different sizes. The magnet array unit includes at least two groups of magnet arrays. One group of magnet arrays is a magnetic filtration magnet array for providing the magnetic field required for magnetic filtration to the magnetic filtration unit, and the remaining magnet arrays are sorting magnet arrays for providing the magnetic field required for negative magnetophoresis sorting to the multi-stage sorting channel unit. The magnetic filtration unit is connected to the multi-stage sorting channel unit.

2. The multi-stage magnetic separation system according to claim 1, wherein In the multi-stage sorting channel unit, an intermediate layer is provided between every two adjacent sorting channel layers, which is partitioned and connected through the intermediate layer. The intermediate layer is provided with a sorting sample inlet and a sample outlet, and the sorting sample inlet and the sample outlet are respectively connected to the sorting sample outlets leading to the two outlet channels of the previous sorting channel layer.

3. The multi-stage magnetic separation system according to claim 1, characterized in that, In the multi-stage sorting channel unit, the two outlet channels of each sorting channel layer are respectively an outer outlet channel and an inner outlet channel. In the main sorting channel, the large particles in the sample are repelled by the magnetic field to a farther distance and enter the outer outlet channel, while the small particles are repelled by the magnetic field to a closer distance and enter the inner outlet channel.

4. The multi-stage magnetic separation system according to claim 1, characterized in that, A substrate is provided between each sorting channel layer of the multi-stage sorting channel unit and the sorting magnet array.

5. The multi-stage magnetic separation system according to claim 4, wherein, A metal layer is added to the substrate.

6. The multi-stage magnetic separation system according to claim 1, characterized in that, Each group of magnet arrays is composed of several magnets arranged in a preset order.

7. The multi-stage magnetic separation system according to claim 1, characterized in that, The magnetic filtration unit includes a sample recovery pool, a connecting pipe, a magnetic filtration channel, and a filtered sample outlet. The multi-stage sorting channel unit is connected to the sample recovery pool, and the sample recovery pool, the connecting pipe, the magnetic filtration channel, and the filtered sample outlet are connected in sequence.

8. The multi-stage magnetic separation system according to claim 7, wherein, The magnetic filtration channel is a flexible hose filled with micron-sized ferromagnetic powder. The magnetic filtration channel is placed between two groups of magnet arrays. Under the clamping of the two groups of magnet arrays, the magnetic field magnetizes the micron-sized ferromagnetic powder in the hose and fixes it in the magnet region, forming a magnetic filtration channel with a porous structure.

9. The multi-stage magnetic separation system according to any one of claims 1-8, characterized in that, It also includes a fixing bracket, and the multi-stage sorting channel unit, the magnet array unit, and the magnetic filtration unit are fixed and supported by the fixing bracket.

10. A magnetic separation method implemented based on the multi-stage magnetic separation system according to any one of claims 1-9, characterized in that, The method includes: Mixing the sample to be sorted with a magnetic fluid or a magnetic salt solution in a preset ratio; Flowing through each sorting channel layer in sequence through the sample injection pool and achieving the sorting of particles of different sizes in each sorting channel layer; In the magnetic filtration unit, recovering the sorted sample through the sample recovery pool. The sample in the sample recovery pool enters the magnetic filtration channel through the connecting pipe. The strong magnetic field in the magnetic filtration channel adsorbs the magnetic nanoparticles in the magnetic fluid or the magnetic salt solution, and enables the non-magnetic sample to flow out from the filtered sample outlet through the magnetic filtration channel, thereby realizing the filtration of the magnetic nanoparticles in the sample.

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