A cell separation device, separation method and application based on a halbach permanent magnet array and a soft magnetic column array

Abstract

The application belongs to the technical field of biomedical detection, liquid biopsy and immunomagnetic separation, and particularly relates to a cell separation device based on a Halbach permanent magnet array and a soft magnetic column array, a separation method and application. The application realizes efficient removal of all-spectrum leukocytes through the synergistic effect (interaction effect P<0.01) of multi-target immunomagnetic bead combination and an inwardly enhanced Halbach permanent magnet array plus a radial soft magnetic column array: the leukocyte removal rate reaches 99.96% ± 0.23%, and the residual leukocytes are only 2.0×10 3 The CTC recovery rate is as high as 91.57% ± 1.15%, the purity reaches 24.4% ± 2.8%, the cell activity is greater than or equal to 90%, the device flux is high, the magnetic field utilization rate is high, and the non-contact magnetization is convenient for sealing and sterilization, and the device has good clinical practicability and industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical detection, liquid biopsy and immunomagnetic separation technology, specifically relating to a cell separation device, separation method and application based on Halbach permanent magnet array and soft magnetic column array. Background Technology

[0002] Circulating tumor cells (CTCs) are rare cells that detach from solid tumor tissue and enter the peripheral blood circulation. They are one of the core biomarkers in liquid biopsy and have important clinical value in early tumor screening, efficacy evaluation, recurrence monitoring, and research on drug resistance mechanisms. However, CTCs are extremely rare in peripheral blood (typically less than 10 per 100 cells). 6 ~10 9 Only a few to dozens of CTCs are present in each blood cell, while the background blood cells (especially red blood cells, lymphocytes, neutrophils, monocytes, and other white blood cells) are numerous, causing the CTC detection signal to be severely masked. Therefore, efficiently removing background blood cells, preserving CTCs to the maximum extent, and obtaining high-purity rare cells are prerequisites for achieving accurate CTC detection and downstream analysis.

[0003] Currently, immunomagnetic bead negative enrichment is one of the mainstream technical routes in clinical and research applications: CD45 immunomagnetic beads are used to specifically label leukocytes, and the labeled cells are adsorbed and retained under an external magnetic field, while unlabeled CTCs flow out with the fluid and are collected. Existing implementation schemes mainly include: conventional magnetic racks or separation columns (with divergent magnetic fields and low gradients, resulting in a leukocyte removal rate of only 90%–99%, leaving up to 10% of leukocytes remaining). 4 ~10 5 The methods employed include: packed magnetic separation devices (such as MACS, which are filled with ferromagnetic microspheres or steel needles and can generate a high-gradient magnetic field, but CTCs must pass through narrow and tortuous channels, leading to physical jamming, surface adsorption, and shear damage. CTC recovery rates are typically below 60%, and they are prone to clogging); and microfluidic chips (with narrow channels and extremely low throughput, requiring several hours to process 1 mL of sample, making them unsuitable for clinical 4 mL–10 mL whole blood samples). Furthermore, these methods generally overlook the significant differences in CD45 expression intensity among leukocyte subsets: high expression in lymphocytes, moderate expression in neutrophils and monocytes, significantly low expression in eosinophils, and extremely weak expression in basophils. Therefore, even with a high-gradient magnetic field, a single CD45 magnetic bead cannot achieve efficient clearance of eosinophils / basophils, resulting in a consistently limited total leukocyte removal rate.

[0004] In summary, existing technologies suffer from the following drawbacks: ① Incomplete removal of background immune cells, especially severe residue of eosinophils / basophils with weak CD45 expression; ② Physical barriers in packing or microfluidic devices cause significant retention, damage, and low recovery rates of CTCs; ③ Microfluidic chips have low throughput and are prone to clogging, making them unsuitable for clinical sample processing; ④ There is a lack of multi-target labeling strategies targeting the entire spectrum of leukocytes, and an inherent contradiction exists between magnetic field design and flow channel design, making it difficult to simultaneously achieve both high removal and high recovery rates. Therefore, developing a negative enrichment device and method that can achieve efficient removal of the entire spectrum of leukocytes, high CTC recovery rates, high throughput, and no physical retention is of significant clinical urgency and technical challenge. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a cell separation device, separation method and application based on Halbach permanent magnet array and soft magnetic column array.

[0006] To achieve the above objectives, the present invention may employ the following technical solutions: This invention provides a cell separation device based on a Halbach permanent magnet array and a soft magnetic column array, comprising: an inwardly enhanced Halbach permanent magnet array (2) ring for providing a radially converging main magnetic field; a central separation cavity (3) disposed inside the Halbach permanent magnet array ring, the cavity having a sample inlet (5) and a central outlet (6); a soft magnetic column array (4) disposed inside the central separation cavity (3), consisting of multiple soft magnetic columns (1), the soft magnetic columns being arranged radially and uniformly in a regular polygonal pattern; the soft magnetic columns being disposed in non-contact with the inner wall of the Halbach permanent magnet array (2) ring, maintaining a gap between them; a cell flow channel being formed between adjacent soft magnetic columns, the minimum width of the channel being ≥0.8mm; the interior of the central separation cavity being composed of a unique magnetic trapping structure formed by the soft magnetic column array, the gap between adjacent soft magnetic columns being an unobstructed cell flow channel.

[0007] Preferably, in the above-mentioned cell separation device, the soft magnetic column (1) is made of a 3D printable soft magnetic metal material, which is at least one of mold steel, permalloy, iron-cobalt-vanadium alloy or iron-based amorphous soft magnetic alloy.

[0008] Preferably, in the above-mentioned cell separation device, the soft magnetic column (1) satisfies one or more of the following characteristics: the diameter of a single soft magnetic column is 0.5mm to 1.0mm; the number of soft magnetic columns is 8 to 16; the cross-sectional shape of the soft magnetic column is circular, hexagonal or conical; and the gap between the soft magnetic column and the inner wall of the Halbach permanent magnet array ring is 0.5mm to 5mm.

[0009] Preferably, in the above-mentioned cell separation device, the surface of the soft magnetic column (1) is provided with an anti-nonspecific adsorption treatment layer, the treatment layer including an electrochemical passivation layer and a hydrophilic antifouling coating covering the passivation layer; the hydrophilic antifouling coating is at least one of zwitterionic polymer coating, polyethylene glycol coating, polyvinylpyrrolidone coating, albumin coating, heparin coating or hyaluronic acid coating.

[0010] Another aspect of the present invention provides a cell separation method using the above-mentioned cell separation device, comprising the following steps: (1) multi-target immunomagnetic bead labeling: multi-target labeling of leukocytes in the sample to be tested, adding an immunomagnetic bead mixture to the sample, the mixture comprising at least anti-CD45 magnetic beads, and one or more selected from anti-CD66b magnetic beads, anti-CD15 magnetic beads, anti-CD14 magnetic beads, anti-CD49d magnetic beads, anti-CD125 magnetic beads, anti-CD123 magnetic beads, and anti-CD203c magnetic beads; (2) injecting the labeled sample from the peripheral inlet of the device, so that the sample flows radially through the soft magnetic column array, the magnetically labeled leukocytes are adsorbed on the surface of the soft magnetic columns, and the unlabeled target cells flow out from the central outlet with the fluid and are collected; (3) washing the chamber and collecting the washing solution.

[0011] Preferably, in the above cell separation method, the immunomagnetic bead mixture simultaneously comprises: anti-CD45 magnetic beads, anti-CD66b or anti-CD15 magnetic beads, anti-CD14 magnetic beads, anti-CD49d or anti-CD125 magnetic beads, and anti-CD123 or anti-CD203c magnetic beads.

[0012] Preferably, in the above cell separation method, the flow rate of sample injection in step (2) is 6 mL / min to 8 mL / min.

[0013] Preferably, the above cell separation method further includes an elution step: after removing the Halbach permanent magnet array ring, the elution solution is injected in reverse at a flow rate of 0.5 mL / min to 1.0 mL / min to collect the cells adsorbed on the surface of the soft magnetic column.

[0014] Preferably, the above-mentioned cell separation method further includes a two-stage separation mode: the effluent collected in the first stage is separated a second time by passing it through the same device.

[0015] In another aspect, the present invention provides a system for negative enrichment of circulating tumor cells, comprising: the cell separation device described above; and a multi-target immunomagnetic bead mixture, said mixture comprising at least anti-CD45 magnetic beads and one or more selected from anti-CD66b, anti-CD15, anti-CD14, anti-CD49d, anti-CD125, anti-CD123, and anti-CD203c magnetic beads.

[0016] The beneficial effects of this invention include: Through the synergistic effect (interaction effect P<0.01) of a multi-target immunomagnetic bead combination (containing at least one or more of anti-CD45, anti-CD66b / CD15, anti-CD14, anti-CD49d / CD125, and anti-CD123 / CD203c) and an inward-enhancing Halbach permanent magnet array plus a radial soft magnetic column array (inter-column channel ≥0.8mm, no filler balls or filter membrane, non-contact gap 1mm-3mm, surface passivated and hydrophilic coated, water contact angle ≤30°, protein adsorption reduced ≥95%), this invention achieves highly efficient clearance of the entire spectrum of leukocytes: the leukocyte removal rate reaches 99.96%±0.23%, and the residual leukocytes are only 2.0×10⁻⁶. 3 Within one sample (better than 10 of existing technologies) 4 ~10 5 (Number of samples not specified); Simultaneously, due to the absence of physical retention and wide-channel design, the CTC recovery rate is as high as 91.57% ± 1.15% (far exceeding the <60% of packed-bed devices), with a purity of 24.4% ± 2.8% and cell viability ≥90%; the device has high throughput (10mL sample processing time <30 minutes) and high magnetic field utilization (Halbach array magnetic force is 76 times that of conventional NS pairs, with a peak ∇B of 2.5 × 10⁻⁶ after loading soft magnetic columns). 3 T / m-4.0×10 3 (T / m), and non-contact magnetization facilitates sealing and sterilization, possessing good clinical applicability and industrialization prospects. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of the device of the present invention, showing the assembly relationship of the Halbach permanent magnet array ring, the central separation cavity, and the soft magnetic column array; Figure 2 This is a schematic cross-sectional view of the overall structure of the device of the present invention; Figure 3 This is a schematic diagram of the cross-sectional distribution of the soft magnetic column array within the separation cavity, showing a radially uniform arrangement; Figure 4 This is a schematic diagram of the horizontal cross-section and flow channel of the soft magnetic column, showing the fluid paths of peripheral injection, radial flow, and central outlet; Figure 5 This is a schematic diagram illustrating the principle of high gradient adsorption and non-destructive passage of CTC. Figure 6 A schematic diagram of multi-target magnetic bead combination labeling of leukocytes; Figure 7 The pseudo-color image of the magnetic field gradient ∇B across the cross-section of the separated cavity when a soft magnetic column is added (the soft magnetic column does not affect the magnetic field distribution, the magnetic field at a fixed point is symmetrical, and the values ​​shown represent the magnetic force at half the magnetic field position). Figure 8A bar chart comparing the removal rate of residual white blood cells with and without soft magnetic column separation efficiency; Figure 9 A bar chart comparing the CTC recovery rates with and without soft magnetic column separation efficiency; Figure 10 A bar chart comparing the purity with and without soft magnetic column separation efficiency; Figure 11 Comparative data on leukocyte removal rates with and without a soft magnetic column for single magnetic beads and five-target combination; Figure 12 The interaction diagram of the 2×2 factorial experiment shows the synergistic effect of the antibody combination and the soft magnetic field on the removal rate. Figure 13 This is a schematic diagram of the entire process of the device of the present invention.

[0018] In the figure, 1: soft magnetic column; 2: Halbach permanent magnet array; 3: central separation chamber; 4: soft magnetic column array; 5: sample inlet; 6: central outlet. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0022] Example 1: Enrichment of CTCs in Peripheral Blood of Non-Small Cell Lung Cancer Patients (1) Sample collection and preprocessing 4 mL of peripheral venous blood was collected from healthy adults, anticoagulated with EDTA, and infused with 300 human non-small cell lung cancer A549 cells. Red blood cells were lysed (ACKBuffer, 1:10, room temperature for 10 minutes), centrifuged at 300×g, 4℃ for 5 minutes, and the supernatant was discarded. This process was repeated once to obtain a nucleated cell suspension (approximately 0.8 mL).

[0023] (2) Multi-target immunomagnetic bead labeling Add the following immunomagnetic bead mixture (bead size 50nm–200nm) to the cell suspension: anti-CD45 magnetic beads (10μL), anti-CD66b magnetic beads (10μL), anti-CD14 magnetic beads (10μL), anti-CD49d magnetic beads (10μL), and anti-CD123 magnetic beads (10μL); incubate at 2℃–8℃ in the dark for 25 minutes, gently stirring every 5 minutes. Add 1mL of PBS containing 0.5% BSA, centrifuge at 300×g, 4℃ for 5 minutes, and discard the supernatant. Resuspend in 2mL of separation buffer (0.5% BSA, 2mM EDTA, PBS) for later use.

[0024] (3) Structure of the separation device See Figures 1 to 5 The separation device used in this embodiment includes: Inward-enhancing Halbach permanent magnet array ring: inner diameter Φ20mm, outer diameter Φ40mm, composed of 12 N52 neodymium magnets arranged in Halbach pattern, inner wall magnetic flux density 0.7T, providing a radially inward converging main magnetic field.

[0025] Central separation chamber: Located inside the Halbach ring, it is made of biocompatible material and has a peripheral sample inlet and a central outlet.

[0026] Soft magnetic pillar array: Internally assembled with 12 MS1 mold steel soft magnetic pillars (0.6mm in diameter, 20mm in length). MS1 material is 18Ni300 / 1.2709 mold steel with a relative permeability of 200-500 and a saturation magnetization of approximately 1.6T. The soft magnetic pillars are formed by laser powder bed fusion (LPBF) 3D printing, achieving a density ≥99%. The 12 soft magnetic pillars are evenly arranged radially in a regular dodecagonal pattern (see...). Figure 3 The minimum channel width between adjacent soft magnetic pillars is 0.9 mm. The gap between the soft magnetic pillars and the inner wall of the Halbach permanent magnet array ring is 2 mm (non-contact magnetization).

[0027] Surface anti-nonspecific adsorption treatment: The surface of the soft magnetic pillars is first electrochemically passivated (anodic oxidation in 0.5M H2SO4 to form a dense oxide layer), and then coated with a hydrophilic antifouling coating (MPC polymer, concentration 1 mg / mL, soaked at room temperature for 2 hours, thickness 20 nm to 50 nm). After treatment, the surface water contact angle is ≤30°, and the protein adsorption is reduced by ≥95%.

[0028] Fluid control components: including injection pump, flow rate controller, sample pretreatment and collection interface.

[0029] (4) Separation operation process 1) A suspension of cell samples labeled with magnetic nanoparticles was injected into the outer inlet of the separation device at a flow rate of 7.6 mL / min, and the sample flowed radially through the soft magnetic column array (see...). Figure 4 Flow path design).

[0030] 2) Magnetically labeled leukocytes are adsorbed onto the surface of the soft magnetic column, while unlabeled CTCs flow out from the central outlet and are collected.

[0031] 3) After the sample loading is complete, wash the chamber with 5 mL of separation buffer at the same flow rate (7.6 mL / min), and collect the washing solution together.

[0032] 4) After removing the Halbach permanent magnet array rings, inject the elution buffer in reverse at a flow rate of 0.76 mL / min to collect the adsorbed cells.

[0033] Specifically, refer to Figure 5 In the device state on the left side of this figure, the soft magnetic column array is magnetized. Under the action of external air pressure at the gas port of the soft magnetic column, the peripheral blood sample is processed and incubated with magnetic nanoparticles such as CD45. It enters the soft magnetic adsorption column from the liquid port of the soft magnetic column. Under the action of strong magnetic potential under the external magnetic field, the immune cells coupled with the magnetic nanoparticles are adsorbed on the surface of the magnetized soft magnetic column array. After standing or flowing out directly from the liquid port of the soft magnetic column, the immune cells labeled with magnetic nanoparticles are separated from the CTCs without magnetic nanoparticles. In the device state on the right side of this figure, the soft magnetic column array is demagnetized. Under the action of external air pressure at the gas port (5) of the soft magnetic column, the eluent is drawn in through the liquid port of the soft magnetic column (central outlet 6). Under the external air pressure, the eluent washes the surface of the soft magnetic column array from top to bottom, realizing the cleaning of the immune cells labeled with magnetic nanoparticles.

[0034] In addition, a schematic diagram of multi-target magnetic bead combination labeling of leukocytes is shown below. Figure 6 As shown.

[0035] (5) Result detection CTCs were identified by immunofluorescence staining (CK8 / 18 / 19-FITC, CD45-PE, DAPI) (CK+ / CD45- / DAPI+). Trypan blue staining was used to assess activity.

[0036] The test results are as follows: Residual white blood cell count: approximately 1000–1200 cells / sample; CTC (circulating tumor cells) recovery rate: approximately 90.8%–92.3%; CTC purity: approximately 22.7%–27.7%; Cell viability: ≥90%.

[0037] Example 2: Simulated CTC enrichment in peripheral blood of breast cancer patients (1) Sample collection and preprocessing Four mL of peripheral venous blood was collected from a breast cancer patient, anticoagulated with EDTA, and infused with 300 human breast cancer MCF7 cells. Red blood cells were lysed as in Example 1 to obtain a nucleated cell suspension.

[0038] (2) Multi-target immunomagnetic bead labeling (four targets) Add the following to the cell suspension: anti-CD45 magnetic beads (10 μL), anti-CD15 magnetic beads (10 μL, replacing CD66b), anti-CD14 magnetic beads (10 μL), and anti-CD125 magnetic beads (10 μL, targeting eosinophils) (basophil-specific magnetic beads were not used (because their proportion is <1%, which can be compensated by enhancing magnetic field capture)); labeling conditions are the same as in Example 1.

[0039] (3) Separation device A Φ30mm Halbach annular array (16 N52 magnets) is fitted with 16 MS1 soft magnetic pillars (0.5mm in diameter, PEG-coated surface), arranged radially in a regular hexagonal pattern, with 0.8mm channels between the pillars. The gap between the soft magnetic pillars and the inner wall of the Halbach is 2.5mm. The rest is the same as in Example 1.

[0040] (4) Separation operation (two-way mode) First stage: Inject the sample at a flow rate of 6.0 mL / min and collect the eluent; Second stage: Pass the collected solution from the first stage through the same device again (flow rate 6.0 mL / min) and collect the eluent; Washing and elution are the same as in Example 1.

[0041] (5) Results Total residual white blood cell count: approximately 1.2 × 10⁻⁶ 3 ~2.2×10 3 Quantity / sample; CTC recovery rate: approximately 90.7%–92.8%; CTC purity: approximately 20.4%–22.9%; Cell viability: ≥92%.

[0042] The two-pass mode improves the capture rate of weakly labeled cells by about 15% compared to the single-pass mode, but the processing time increases to about 25 minutes.

[0043] Comparative Example 1 (1) Process A microfluidic chip (channel width 50 μm, soft magnetic micropillar diameter 30 μm, permanent magnets arranged on both sides) was fabricated according to the scheme disclosed in prior art CN102360010A. The same breast cancer cell line (MCF-7) was processed by incorporating healthy blood samples (300 MCF-7 cells in 4 mL of whole blood, approximately 5 × 10⁶ white blood cells). 7 indivual).

[0044] (2) Results The processing time was >2 hours, and the chip became clogged in the first hour of operation, resulting in a CTC recovery rate of only 30% and a purity of approximately 6%. In comparison with Example 2, the processing time was <25 minutes, there was no clogging, the CTC recovery rate was 90.7%, and the purity was 20.4%.

[0045] Comparative Example 2 (1) Apparatus and process The same apparatus and operating procedures as in Example 1 were used, but only a single CD45 magnetic bead was used for sample pretreatment (without adding anti-CD66b, anti-CD14, anti-CD49d, and anti-CD123 magnetic beads). The same sample treatment was used, and other procedures were the same as in Example 1.

[0046] (2) Results The results showed that approximately 1.2 × 10⁻⁶ residual white blood cells were present. 4 The CTC recovery rate was approximately 68%, and the CTC purity was approximately 6%.

[0047] Comparative Example 3 The same sample as in Example 1 was used (4 mL of whole blood from a healthy person with approximately 300 GFP-labeled A549 cells and a white blood cell background of approximately 5 × 10⁻⁶ cells). 7 The method used a combination of five target magnetic beads and a single (soft magnetic pillar) array, but the separation device was a Halbach array only (soft magnetic pillars were removed, and the separation cavity was empty); otherwise, it was the same as in Example 1. The results are shown in Table 1.

[0048] Table 1 Test results of Example 1 and Comparative Example 3

[0049] II. Magnetic Field Optimization Simulation and Performance Verification (1) Comparison of magnetic force between Halbach array and general magnetic field (theoretical simulation) Static magnetic field simulation was performed using COMSOL Multiphysics 6.0. Three magnetic circuit designs were compared: Design A: Conventional NS two-pole permanent magnet pair (same magnet size, 20mm spacing, 1.4T surface remanence). Design B: Single-sided permanent magnet + back iron (same magnet size, back iron thickness 5mm). Design C: 12 Halbach ring arrays (inner diameter Φ20mm, outer diameter Φ40mm, each magnet size 10×10×20mm) 3 (N52 neodymium magnet).

[0050] The results (magnetic flux density B and gradient ∇B at 1 mm from the inner wall on the central cross-section) are shown in Table 2 below.

[0051] Table 2 Magnetic properties of different designs

[0052] The results show that the Halbach array generates approximately 76 times the magnetic force of a conventional NS pair within the separation cavity, and its magnetic field uniformity (coefficient of variation <10%) is far superior to other designs.

[0053] (2) Comparison of magnetic force distribution before and after loading of soft magnetic materials MS1 soft magnetic pillars (12 pillars, Φ0.6mm, radially arranged) were loaded inside the Halbach array, and the magnetic field gradient distribution before and after loading was compared. The magnetic field gradient at a distance of 1μm from the surface of the soft magnetic pillars was measured.

[0054] Without soft magnetic pillars: The maximum ∇B within the cavity is approximately 210 T / m (near the Halbach inner wall), while the central region has ∇B < 50 T / m, exhibiting numerous low-gradient regions. With soft magnetic pillars: Localized high-gradient regions are generated on the surface of each soft magnetic pillar, with a peak ∇B reaching 2.5 × 10⁻⁶. 3 ~4.0×10 3 T / m, the high gradient region covers more than 95% of the entire separation cavity. The channel region ∇B between the soft magnetic pillars remains at 200T / m~500T / m (see Figure 7 ).

[0055] A bar chart comparing the separation efficiency with and without soft magnetic columns is shown below. Figure 8 , Figure 9 and Figure 10 As shown, comparative data on leukocyte removal rate, CTC recovery rate, and purity are presented.

[0056] (3) Independent contribution analysis of antibody combination and soft magnetic field (2×2 factorial experiment) Design a 2×2 factorial experiment: Factor A (antibody strategy): Level A1 = single CD45 magnetic bead; Level A2 = five-target combination.

[0057] Factor B (Magnetic Field Device): Level B1 = Halbach array only (without soft magnetic pillars); Level B2 = Halbach array + soft magnetic pillar array (standard device of this invention).

[0058] The sample is the same as before.

[0059] The white blood cell removal rate (%) was measured, and the results are shown in Table 3 below (mean ± SD).

[0060] Table 3 Independent contribution analysis of antibody combination and soft magnetic field

[0061] It can be seen from Table 3 above: Main effect of antibody strategy: A2 improved the removal rate by an average of 1.89% compared with A1; Main effect of the magnetic field device: B2 improves the removal rate by an average of 1.26% compared to B1; Interaction: The combined effect of A2 and B2 (99.98%) was greater than the maximum contribution of each alone (99.03%), indicating that the antibody combination and the soft magnetic field have a synergistic effect (see...). Figure 11 and Figure 12 The complete flowchart of this invention can be found here. Figure 13 .

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A cell separation device based on a Halbach permanent magnet array and a soft magnetic column array, characterized in that, include: An inward-enhancing Halbach permanent magnet array (2) ring is used to provide a radially inward-converging main magnetic field; A central separation chamber (3) is located inside the Halbach permanent magnet array ring, and the chamber has a sample inlet (5) and a central outlet (6). The soft magnetic column array (4) is located inside the central separation cavity (3) and is composed of multiple soft magnetic columns (1). The soft magnetic columns are arranged radially in a regular polygonal pattern. The soft magnetic column is set in a non-contact manner with the inner wall of the Halbach permanent magnet array (2) ring, and a gap is maintained between them; Cell flow channels are formed between adjacent soft magnetic pillars, and the minimum width of the channels is ≥0.8mm; The central separation cavity is equipped with a unique magnetic trapping structure consisting of the array of soft magnetic pillars, and the gaps between adjacent soft magnetic pillars serve as unobstructed cell flow channels.

2. The cell separation device according to claim 1, characterized in that, The soft magnetic column (1) is made of a 3D printable soft magnetic metal material, which is at least one of mold steel, permalloy, iron-cobalt-vanadium alloy or iron-based amorphous soft magnetic alloy.

3. The cell separation device according to claim 1 or 2, characterized in that, The soft magnetic pillar (1) satisfies one or more of the following characteristics: The diameter of a single soft magnetic column is 0.5mm to 1.0mm; The number of soft magnetic pillars is 8 to 16; The cross-sectional shape of the soft magnetic column is circular, hexagonal, or conical; The gap between the soft magnetic column and the inner wall of the Halbach permanent magnet array ring is 0.5mm to 5mm.

4. The cell separation device according to claim 1 or 2, characterized in that, The surface of the soft magnetic column (1) is provided with an anti-nonspecific adsorption treatment layer, the treatment layer including an electrochemical passivation layer and a hydrophilic antifouling coating covering the passivation layer; The hydrophilic antifouling coating is at least one of the following: zwitterionic polymer coating, polyethylene glycol coating, polyvinylpyrrolidone coating, albumin coating, heparin coating, or hyaluronic acid coating.

5. A cell separation method using the cell separation apparatus according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Multi-target immunomagnetic bead labeling: Leukocytes in the sample to be tested are labeled with multiple targets by adding an immunomagnetic bead mixture to the sample. The mixture contains at least anti-CD45 magnetic beads and one or more selected from anti-CD66b magnetic beads, anti-CD15 magnetic beads, anti-CD14 magnetic beads, anti-CD49d magnetic beads, anti-CD125 magnetic beads, anti-CD123 magnetic beads, and anti-CD203c magnetic beads. (2) The labeled sample is injected from the peripheral inlet of the device, so that the sample flows radially through the soft magnetic column array. The magnetically labeled white blood cells are adsorbed onto the surface of the soft magnetic columns, and the unlabeled target cells flow out from the central outlet with the fluid and are collected. (3) Wash the chamber and collect the washing liquid.

6. The cell separation method according to claim 5, characterized in that, The immunomagnetic bead mixture simultaneously comprises: anti-CD45 magnetic beads, anti-CD66b or anti-CD15 magnetic beads, anti-CD14 magnetic beads, anti-CD49d or anti-CD125 magnetic beads, and anti-CD123 or anti-CD203c magnetic beads.

7. The cell separation method according to claim 5 or 6, characterized in that, In step (2), the sample injection flow rate is 6 mL / min to 8 mL / min.

8. The cell separation method according to claim 5 or 6, characterized in that, The cell separation method also includes an elution step: after removing the Halbach permanent magnet array ring, the elution solution is injected in reverse at a flow rate of 0.5 mL / min to 1.0 mL / min to collect the cells adsorbed on the surface of the soft magnetic column.

9. The cell separation method according to claim 5 or 6, characterized in that, Cell separation methods also include a two-pass separation mode: the effluent collected in the first pass is passed through the same device for a second separation.

10. A system for negative enrichment of circulating tumor cells, characterized in that, include: The cell separation apparatus according to any one of claims 1 to 4; as well as A multi-target immunomagnetic bead mixture, the mixture comprising at least anti-CD45 magnetic beads and one or more selected from anti-CD66b, anti-CD15, anti-CD14, anti-CD49d, anti-CD125, anti-CD123, and anti-CD203c magnetic beads.

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