A target chip and a method for separating based on the target chip

Abstract

The application discloses a target chip and a separation method based on the target chip, and belongs to the technical field of micro-fluidic biochips and cell separation and purification. The target chip comprises a glass substrate layer, a PDMS structure layer attached to the glass substrate layer, a first-stage inertial separation channel and a second-stage magnetic separation channel arranged in the PDMS structure layer. The first-stage inertial separation channel comprises two inlets, a spiral focusing channel and two outlets. The second-stage magnetic separation channel comprises one inlet, a long straight channel, a plurality of cell capture zones arranged along the outer sidewall of the long straight channel, a spiral channel connected to the downstream of the long straight channel and two outlets. The second-stage magnetic separation channel is provided with a magnetic support embedded with a permanent magnet array on the periphery, so as to form a closed magnetic circuit surrounding the long straight channel and the cell capture zones through the magnetic support. The target chip is not limited to a specific disease model or a specific cell type, and has wide universality and clinical transformation potential.

Description

Technical Field

[0001] This application belongs to the field of microfluidic biochip and cell separation and purification technology, and specifically relates to a target chip and a separation method based on the target chip. Background Technology

[0002] Microfluidic chips, as a technology platform for precisely manipulating fluids within a micrometer-scale space, have been widely used in cell separation, purification, and functional analysis. Compared with traditional density gradient centrifugation and fluorescence activated cell sorting (FACS), microfluidic technology has significant advantages such as lower sample consumption, continuous operation, integrability, and less mechanical damage to cells, making it particularly suitable for the separation and enrichment of rare cells or specific functional cells in trace clinical samples.

[0003] Among various microfluidic cell separation methods, inertial microfluidics utilizes the inertial lift and Dean's force generated by fluid in microchannels to focus particles of different sizes to different equilibrium positions, thereby achieving size-dependent high-throughput separation. This method requires no external field, has a simple structure, and high throughput, but its separation resolution is limited, making it difficult to accurately distinguish cell subpopulations of similar sizes (such as neutrophils and lymphocytes, whose diameters differ by only 2–3 μm). On the other hand, immunomagnetic separation utilizes antibody-conjugated magnetic nanoparticles to specifically label cells, achieving selective enrichment or removal under the influence of an external magnetic field. This method has high specificity, but usually requires cumbersome label-elution steps, which may affect cell viability and function.

[0004] In recent years, researchers have combined inertial microfluidics with magnetic separation technology to develop inertial-magnetic microfluidic chips, aiming to utilize both the high throughput of inertial separation and the high selectivity of magnetic separation. For example, Chen Ni et al. designed an inertial-magnetic microfluidic sorter to separate tumor cells from malignant pleural effusions, achieving a purity of >70% at high flow rates. Zongjie Wang et al. developed MATIC technology to recover tumor-infiltrating lymphocytes from tumor tissue through quantitative immunomagnetic separation. Justin Myles et al. constructed a parallel immunomagnetic microfluidic device to directly separate basophils from whole blood with a purity of approximately 93%.

[0005] However, the aforementioned existing technologies still have the following common problems: 1) Force field coupling leads to a narrow operating window: Most reported inertial... Magnetic chips integrate inertial focusing and magnetic separation within the same channel, where the flow field and magnetic field interfere with each other. To balance these two mechanisms, it is often necessary to strictly limit the flow rate, magnet position, and channel geometry parameters. This results in an extremely narrow operating window, sensitivity to fluctuations in sample properties and experimental conditions, and difficulty in ensuring the stability and repeatability of the separation.

[0006] 2) Inadequate suitability for small amounts of whole blood and complex clinical samples: Many designs require large sample volumes (>1 mL), which are not suitable for pediatric, critical care, or rare samples (such as ≤50 μL of whole blood).

[0007] 3) The isolated cells are difficult to use directly for functional analysis: Most existing chips use a positive labeling strategy (directly labeling target cells). After isolation, magnetic beads need to be removed by enzyme digestion or competitive elution. This process may activate cells, change their phenotype or reduce their activity, thereby interfering with downstream functional detection such as chemotaxis, migration, deformation and proliferation.

[0008] 4) Lack of versatility in chip design: Most existing studies are optimized for a specific cell type (such as circulating tumor cells, basophils) and a specific disease model. Once the target cell or sample source is changed, the channel geometry and magnetic field layout need to be redesigned. There is a lack of a platform technology that can be flexibly applied to different cells and different diseases.

[0009] In summary, the problem to be solved is how to provide a chip and a separation method based on the chip that can continuously, gently, and with high purity separate multiple target cells from trace amounts of whole blood or other complex biological samples, and how to use the separated cells directly for downstream functional analysis without delabeling. Summary of the Invention

[0010] To address the aforementioned issues, this application provides a target chip and a target chip-based separation method, which enables continuous, gentle, and high-purity separation of multiple target cells from trace amounts of whole blood or other complex biological samples, and the separated cells can be directly used for downstream functional analysis without delabeling.

[0011] In a first aspect, embodiments of this application provide a target chip, comprising: Glass substrate; PDMS structural layer bonded to the glass substrate; The PDMS structure layer is provided with a first-stage inertial separation channel and a second-stage magnetic separation channel: The first-stage inertial separation channel includes: two inlets, a spiral focusing channel, and two outlets; the two inlets of the first-stage inertial separation channel include: an outer sample inlet and an inner sheath fluid inlet; the two outlets of the first-stage inertial separation channel include: a first outlet and a second outlet, the first outlet being used to collect the enriched target cell population, and the second outlet being used to discharge non-target particles of abnormal size. The second-stage magnetic separation channel includes: an inlet, a long straight channel, multiple cell capture zones arranged along the outer wall of the long straight channel, a spiral channel connected downstream of the long straight channel, and two outlets. The two outlets of the second-stage magnetic separation channel include: a third outlet and a fourth outlet. The third outlet is used to collect purified target cells, and the fourth outlet is used to discharge magnetically labeled non-target cells that have not been captured. The periphery of the second-stage magnetic separation channel is provided with a magnetic support embedded with a permanent magnet array, so as to form a closed magnetic circuit surrounding the long straight channel and the cell capture area through the magnetic support. The target chip is a cascaded cell-oriented sorting and concentration chip based on inertial and magnetic coupling effects.

[0012] Optionally, the first-stage inertial separation channel is a spiral focusing channel with 3 spiral turns, a channel width of 0.5 mm, a channel height of 0.1 mm, a first pitch of 1.5 mm, an inner radius of 8 mm, and the first outlet and the second outlet are separated at a 65° angle.

[0013] Optionally, the width of the long straight channel of the second-stage magnetic separation channel is 0.5 mm, the height of the long straight channel of the second-stage magnetic separation channel is 0.1 mm, the inlet width of the cell capture zone is 0.55 mm, the inlet depth of the cell capture zone is 0.415 mm, the interval between adjacent cell capture zones is 1.15 mm, and each capture zone forms a stable reflux vortex under the drive of the flow field.

[0014] Optionally, the permanent magnet array is composed of four neodymium magnets embedded in the four sides of a 3D-printed nylon magnetic frame, wherein the size of any one of the four neodymium magnets is 20 mm × 5 mm × 10 mm, and the size of the magnetic frame is 38.2 mm × 38.2 mm × 15 mm.

[0015] Optionally, the downstream spiral of the second-stage magnetic separation channel has two turns, the second pitch is 1.5 mm, and the end forks at 60° to form the third outlet and the fourth outlet.

[0016] Secondly, embodiments of this application provide a separation method based on a target chip, the method comprising: Obtain target biological samples containing target cells, including: whole blood, bone marrow aspiration fluid, pleural effusion, ascites, tissue digestion fluid, and cultured cell suspension; Obtain the dimensions of target cells and non-target cells in the target biological sample; Whether to use red blood cell lysis buffer depends on whether the red blood cells are enlarged due to disease, which would affect the sorting effect; By mixing and incubating a mixture of negatively selected antibodies targeting non-target cell surface markers with magnetic nanoparticles, non-target cells in the target biological sample are magnetically labeled, resulting in the corresponding magnetically labeled target biological sample. The magnetically labeled target biological sample and sheath fluid are simultaneously injected into the first-stage inertial separation channel, and the flow rate is controlled so that cell populations that meet the target size are enriched and flow out from the first outlet, while cell populations smaller than the target size are discharged from the second outlet; the cell suspension collected from the first outlet is injected into the second-stage magnetic separation channel, and the flow rate is controlled so that the inertial force and magnetic force are balanced. Under the closed magnetic field generated by the permanent magnet array, the magnetically labeled non-target cells are deviated from the inertial focusing trajectory by the transverse magnetic force, are captured in the long straight channel and remain in the reflux vortex of the cell capture area, or are deflected towards the fourth outlet and flow out in the internal spiral channel. Unlabeled target cells maintain their inertia and focus trajectory, flowing out from the third outlet to obtain a purified and concentrated target cell suspension.

[0017] Optionally, the target cells are selected from cell types that can be decontaminated by negative magnetic labeling, including neutrophils, T lymphocytes, B lymphocytes, monocytes, and circulating tumor cells.

[0018] Optionally, the sample volume of the target biological sample is 50 μL; the shear stress on the cells in the first-stage inertial separation channel and the second-stage magnetic separation channel is less than the cell shear stress tolerance threshold, and the activity of the separated target cells is greater than or equal to the preset value.

[0019] Optionally, the method further includes: The inertial and magnetic cascade separation chip is set as a disposable chip.

[0020] Optionally, the method further includes: Obtain the isolated target cells; The downstream functions of the isolated target cells were directly analyzed to obtain the analysis results. The downstream functions include chemotactic migration, deformation, proliferation, apoptosis, and drug sensitivity detection.

[0021] Compared with the prior art, this application has the following advantages: The target chip provided in this application embodiment can continuously, gently, and with high purity separate multiple target cells from trace amounts of whole blood or other complex biological samples, and the separated cells can be directly used for downstream functional analysis without delabeling.

[0022] Other features and advantages of this application will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the application. The objectives and other advantages of this application may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 A schematic diagram of the overall structure of the target chip according to an embodiment of this application is shown; Figure 2 A design diagram of the target chip according to an embodiment of this application is shown; Figure 3 A schematic diagram showing the specific design parameters of the inertial chip; Figure 4 A schematic diagram showing the specific design parameters of the magnetic chip; Figure 5 Here is a simulation diagram of the target chip, where, Figure 5 In the diagram, A represents the simulation graph of the inertial chip's flow rate. Figure 5 In the image, B represents a simulation of the magnetic flux density of a magnetic chip. Figure 5 C in the diagram represents the streamline diagram within the circular capture area; Figure 6 The figure shows the experimental results of separation using microspheres, where... Figure 6 Figure A shows the proportions of the three particle types (O1 and O3) at the outlet after applying an external magnetic field. The left column in A represents the result of O1 exiting, and the right column represents the result of O3 exiting after applying the magnetic field. Figure 6 B in the figure is a schematic diagram of the proportion of the three particles at the O1 and O3 exits without a magnetic field. The left column in the figure is the result of the O1 exit, and the right column is the result of the O3 exit without a magnetic field. Figure 7 This is a diagram showing the experimental results of separating target cells using blood samples. Figure 7 In the figure, A represents the proportion of neutrophils (target cells) and other cells at the O1 outlet of the target chip and the O3 outlet of the target chip after applying an external magnetic field. The left bar in the figure represents the result at the O1 outlet, and the right bar represents the result at the O3 outlet after applying a magnetic field. Figure 7 B in the image represents a comparison of cell viability obtained through centrifugation and enrichment using this chip. Figure 1The following are the target cell categories: 1. Leukocyte enrichment exit; 2. Erythrocyte impurity exit; 3. Neutrophil enrichment exit; 4. Non-target cell exit. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] This application provides a method for separating a target chip and a target chip-based separation method, which will be described below with reference to the accompanying drawings.

[0027] The target chip provided in this application is a cascaded cell-oriented sorting and concentration chip based on inertial and magnetic coupling effects. This application also proposes a force field decoupling inertial... This magnetic cascade separation chip separates cell enrichment (first-stage inertial separation) and target cell purification (second-stage magnetic separation) into two independent modules, each with optimized flow rate and magnetic field conditions. Employing a negative-selective magnetic labeling strategy (labeling non-target cells while keeping target cells unlabeled), combined with a unique vortex-assisted magnetic capture structure, this chip enables continuous, gentle, and high-purity separation of various target cells (such as neutrophils, T cells, circulating tumor cells, and fetal cells) from trace amounts (≤50 μL) of whole blood or other complex biological samples. The separated cells can be directly used for downstream functional analysis without delabeling. This chip is not limited to specific diseases or cell types, possessing broad versatility and clinical translational potential.

[0028] The embodiments of this application aim to provide an inertial-magnetic cascade separation chip with a universal structure, flexible operation, and decoupled force field, which can be applied to the purification and concentration of various target cells by simply "changing the magnetic labeling antibody" and "adjusting the flow rate parameters", thus breaking through the limitation of existing chips that are only applicable to a single cell type.

[0029] The target chip provided in this application embodiment is an inertial-magnetic cascade separation chip, which adopts a two-stage independent separation structure, decoupling inertial enrichment and magnetic purification in two series-connected channel modules, and optimizing their respective fluid and magnetic field conditions.

[0030] like Figure 1 A schematic diagram of the overall structure of the target chip according to an embodiment of this application is shown; For example Figure 1The overall structure of the target chip is described as follows: The target chip is an inertial-magnetic chip, comprising a glass substrate and a PDMS structure layer bonded thereon. The PDMS structure layer contains: First-stage inertial separation channel (W-Channel); second-stage magnetic separation channel (N-Channel).

[0031] like Figure 2 As shown, a design diagram of a target chip according to an embodiment of this application is illustrated.

[0032] like Figure 2 The target chip shown is a cascaded cell-oriented sorting and concentration chip based on the inertial-magnetic coupling effect, containing a base layer and a PDMS structural layer. The PDMS structural layer includes a first-stage inertial separation channel (W-Channel) and a second-stage magnetic separation channel (N-Channel). The first-stage channel, relying on the inertial focusing effect, removes impurity cells / particles with large size differences from complex biological samples such as whole blood, enriching the target cell population. The second-stage channel introduces an external permanent magnet array based on the inertial flow field, applying a lateral magnetic force to magnetically labeled non-target cells, causing them to deviate from their inertial trajectory and be captured in the circular cell capture area on the channel sidewall. The internal inertial channel further separates the uncaptured non-target cells from the target cells. Unlabeled target cells flow out from the target cell outlet along their original inertial trajectory, achieving high-purity, high-activity, label-free separation and concentration. The chip is designed with force field decoupling to achieve two-level independent control, which can continuously separate various target cells from trace samples of ≤50 μL under low shear stress, with a separation purity of ≥83% and activity of ≥93%. The separated cells can be directly used for downstream functional analysis, which is highly versatile and not limited to specific diseases or cell types.

[0033] like Figure 3 The diagram shown is a schematic of the specific design parameters of the inertial chip.

[0034] The inertial chip corresponds to the first-stage inertial separation channel, which includes: a sample inlet, a sheath fluid inlet, a spiral focusing channel, a first outlet, and a second outlet. After the sample and sheath fluid converge at a certain angle, they enter the spiral channel. Under the combined action of inertial lift and Dean's force, particles of different sizes are focused to different lateral equilibrium positions. Larger cell populations (including target cells and large non-target cells) focus near the inner wall and flow out from the first outlet; smaller red blood cells, platelets, and cell debris migrate to the vicinity of the outer wall and are discharged from the second outlet, thus achieving the initial enrichment of target cell populations and the removal of large-volume impurities.

[0035] It should be noted that the first-stage inertial separation relies solely on cell size differences and does not depend on any surface markers. As long as the target cell size is larger than that of red blood cells (typically >8μm), it can effectively remove and enrich a large number of red blood cell impurities, and is applicable to the vast majority of cells.

[0036] like Figure 4 The diagram shows the specific design parameters of the magnetic chip.

[0037] The second-stage magnetic separation channel corresponding to the inertial chip includes: a cell inlet, a long straight channel, multiple cell capture zones spaced apart along the outer wall of the long straight channel, a downstream spiral channel, and a third and fourth outlet.

[0038] The channel is surrounded by a permanent magnet array (four neodymium magnets embedded in the four sides of a nylon magnetic frame), forming a closed magnetic circuit that surrounds the long straight channel and the cell capture area. The linear distance between the magnets and the sidewall of the channel is 500 μm.

[0039] It should be noted that the second-stage magnetic separation employs a negative selection strategy: for different non-target cell types in different samples, simply changing the corresponding antibody mixture (e.g., using CD66b, CD14, CD19 to label non-T cells in whole blood; adding CD45 and mesothelial cell markers to pleural effusion) allows this target chip to be applied to the separation of any target cells.

[0040] This target chip employs a negative magnetic labeling strategy, which is applicable only if non-target cells (impure cells) in the sample can be specifically magnetically labeled (e.g., through magnetic nanoparticles coupled with corresponding antibodies), while target cells remain unlabeled. Under this premise, this target chip is not limited to specific disease models or specific cell types, but provides a general technical platform for the continuous, mild, and label-free separation and concentration of target cells from complex biological samples. It can also be further coupled with downstream cell function analysis, making it suitable for basic medical research, clinical diagnosis, and regenerative medicine.

[0041] During operation, the cell suspension collected at the first outlet is injected into the second-stage channel through the cell inlet, where a stable inertial focusing flow field is re-established in the long, straight channel. The cells in the sample are divided into two categories: Magnetically labeled non-target cells: These cells are pre-coupled to magnetic nanoparticles via a mixture of negative selection antibodies. As they flow through the magnetic field region, they are subjected to a transverse magnetic force. This magnetic force competes with the combined force of inertial lift, Dean's force, and fluid resistance. By precisely controlling the flow rate (1.1 mL / min), the magnetic force is sufficient to drive the labeled cells away from their original inertial focusing trajectory and move towards the outer wall of the channel.

[0042] Unlabeled target cells: almost unaffected by magnetic force, they maintain their original inertial focusing trajectory and flow downstream along the main channel.

[0043] Multiple cell-capturing zones on the outer wall of the channel utilize geometric expansion structures to form stable reflux vortices. Magnetically labeled cells entering these zones are captured and retained within the vortices, unable to return to the main flow. Simultaneously, continuous magnetic force further pulls the cells towards the boundary of the capture zone, enhancing retention stability. Uncaptured residual magnetically labeled cells continue to flow downstream and are eventually discharged from the fourth outlet under the combined action of inertial and magnetic forces. Unlabeled target cells maintain their inertial focusing trajectory, flowing through the downstream spiral channel and exiting from the third outlet, resulting in a purified and concentrated target cell suspension.

[0044] In a specific application scenario, the structure of the target chip is as follows: Glass substrate; PDMS structural layer bonded to the glass substrate; The PDMS structure layer contains a first-stage inertial separation channel and a second-stage magnetic separation channel. The first-stage inertial separation channel includes: two inlets, a spiral focusing channel, and two outlets; the two inlets of the first-stage inertial separation channel include: an outer sample inlet and an inner sheath fluid inlet; the two outlets of the first-stage inertial separation channel include: a first outlet and a second outlet, the first outlet is used to collect the enriched target cell population, and the second outlet is used to discharge non-target particles with abnormal size. The second-stage magnetic separation channel includes: an inlet, a long straight channel, multiple cell capture zones arranged along the outer wall of the long straight channel, a spiral channel connected downstream of the long straight channel, and two outlets. The two outlets of the second-stage magnetic separation channel include: a third outlet and a fourth outlet. The third outlet is used to collect purified target cells, and the fourth outlet is used to discharge magnetically labeled non-target cells that have not been captured. The second-stage magnetic separation channel is surrounded by a magnetic scaffold with an embedded permanent magnet array to form a closed magnetic circuit surrounding the long straight channel and the cell capture area. The target chip is a cascaded cell-oriented sorting and concentration chip based on inertial and magnetic coupling effects.

[0045] The chip's flow rate parameters are optimized to suit most 8–20 μm cells. For cells of extreme sizes, they can be fine-tuned within the scope of the claims without redesigning the chip structure.

[0046] In one example, the first-stage inertial separation channel is a spiral focusing channel with 3 spiral turns, a channel width of 0.5 mm, a channel height of 0.1 mm, a first pitch of 1.5 mm, an inner radius of 8 mm, and a first outlet and a second outlet separated at a 65° angle.

[0047] In one example, the width of the long straight channel of the second-stage magnetic separation channel is 0.5 mm, the height of the long straight channel of the second-stage magnetic separation channel is 0.1 mm, the inlet width of the cell capture zone is 0.55 mm, the inlet depth of the cell capture zone is 0.415 mm, the interval between adjacent cell capture zones is 1.15 mm, and each capture zone forms a stable reflux vortex under the drive of the flow field.

[0048] In one example, the permanent magnet array consists of four neodymium magnets embedded in the four sides of a 3D-printed nylon magnetic frame. The dimensions of any one of the four neodymium magnets are 20 mm × 5 mm × 10 mm, and the dimensions of the magnetic frame are 38.2 mm × 38.2 mm × 15 mm.

[0049] In one example, the downstream spiral of the second-stage magnetic separation channel has two turns, the second pitch is 1.5 mm, and the end forks at 60° to form a third and fourth outlet.

[0050] This application provides a separation method based on a target chip, which includes the following steps: Step S1: Obtain target biological samples containing target cells. Target biological samples include: whole blood, bone marrow aspiration fluid, pleural effusion, ascites, tissue digestion fluid, and cultured cell suspension.

[0051] Step S2: Obtain the size of the target cells and the size of the non-target cells in the target biological sample.

[0052] Step S3: Determine whether to use red blood cell lysis buffer based on whether the red blood cells are enlarged due to disease, which would affect the sorting effect.

[0053] Step S4: By mixing and incubating a mixture of negatively selected antibodies against non-target cell surface markers with magnetic nanoparticles, non-target cells in the target biological sample are magnetically labeled to obtain the corresponding magnetically labeled target biological sample.

[0054] Step S5: Simultaneously inject the magnetically labeled target biological sample and sheath fluid into the first-stage inertial separation channel, and control the flow rate so that cell populations that meet the target size are enriched and flow out from the first outlet, while cell populations smaller than the target size are discharged from the second outlet; inject the cell suspension collected from the first outlet into the second-stage magnetic separation channel, and control the flow rate so that the inertial force and magnetic force are balanced.

[0055] Step S6: Under the closed magnetic field generated by the permanent magnet array, the magnetically labeled non-target cells are deviated from their inertial focusing trajectory by the transverse magnetic force, are captured in the long straight channel and remain in the reflux vortex of the cell capture area, or are deflected towards the fourth outlet and flow out in the internal spiral channel; the unlabeled target cells maintain their inertial and focusing trajectory and flow out from the third outlet, obtaining a purified and concentrated target cell suspension.

[0056] In one example, the target cells are selected from cell types that can be decontaminated by negative magnetic labeling, including neutrophils, T lymphocytes, B lymphocytes, monocytes, and circulating tumor cells.

[0057] In one example, the sample volume of the target biological sample is 50 μL; the shear stress on the cells in both the first-stage inertial separation channel and the second-stage magnetic separation channel is less than the cell shear stress tolerance threshold, the shear stress on the cells in both the first-stage inertial separation channel and the second-stage magnetic separation channel is less than 100 dyn / cm², the activity of the separated target cells is greater than or equal to the preset value, and the activity of the separated target cells is greater than the activity of the cells after centrifugation.

[0058] In a specific application scenario, the above preset value is set based on the traditional method. The preset value can be set to 90%. There is no specific limitation on this preset value. It can be fine-tuned according to the needs of different application scenarios, which will not be elaborated here.

[0059] In one example, the separation method based on the target chip provided in this application embodiment further includes the following steps: The inertial and magnetic cascade separation chip is set as a disposable chip.

[0060] In one example, the separation method based on the target chip provided in this application embodiment further includes the following steps: Obtain the isolated target cells; The downstream functions of the isolated target cells were directly analyzed to obtain the results. The downstream functions include chemotaxis, migration, deformation, proliferation, apoptosis, and drug sensitivity detection.

[0061] Example 1: Chip fabrication: 1) Design the chip structure using SolidWorks and fabricate the SU-8 silicon mold using standard soft lithography.

[0062] 2) Mix PDMS prepolymer and curing agent at a ratio of 10:1, pour into the mold, and cure at 70°C for 1 hour.

[0063] 3) After demolding, use a 1.5 mm punch to punch holes to form the inlet and outlet.

[0064] 4) The PDMS structure is bonded to the glass substrate after oxygen plasma treatment, and then baked at 90°C for 12 hours to restore hydrophobicity.

[0065] 5) The magnetic frame is 3D printed (nylon material), with neodymium magnets (20×5×10 mm) embedded on all four sides to form a closed magnetic circuit.

[0066] Figure 5 Here is a simulation diagram of the target chip, where, Figure 5 In the diagram, A represents the simulation graph of the inertial chip's flow rate. Figure 5 In the image, B represents a simulation of the magnetic flux density of a magnetic chip. Figure 5 In the diagram, C represents the streamline diagram within the circular capture region; as shown... Figure 5 As shown, this illustrates that particles entering this region (the circular capture area in C) will be captured and find it difficult to escape.

[0067] against Figure 5 The following explanation is provided for Figure A in the figure: The left side of Figure A shows the simulation results of the velocity field distribution in the inertial spiral microfluidic channel. The background grayscale represents the magnitude of the fluid velocity. The channel velocity gradually increases from the wall to the center. Due to the viscous no-slip condition at the channel wall, the velocity approaches zero. The velocity is highest in the central region of the channel. The right side shows a partial magnified view of the simulation results of the inlet and outlet. The inlet velocity S1 is set to 0.4 m / s, and the inlet velocity B1 is set to 0.1 m / s. The upper partial magnified view is the velocity field of the inlet, and the lower partial magnified view is the velocity field of the outlet.

[0068] against Figure 5 The following explanation is provided for Figure B in the diagram: Figure B shows the simulation results of the magnetic flux density distribution generated by the magnet array. The background grayscale represents the magnitude of the magnetic flux density in Tesla (T). The arrows indicate the direction of the magnetic field, and the direction the arrows point to is the vector direction of the magnetic field at that point. The streamlines are magnetic field lines, used to visually show the overall direction of the magnetic field.

[0069] against Figure 5 The following explanation is provided for diagram C in the figure: Figure C shows the simulation results of the velocity field distribution in the main channel and the capture zone. The background grayscale represents the magnitude of the fluid velocity. The flow velocity in the channel gradually increases from the wall to the center. Due to the viscous no-slip condition at the channel wall, the flow velocity approaches zero, and the flow velocity is highest in the central region of the channel. The interior of the capture zone is a low-velocity region with a flow velocity significantly lower than that in the mainstream region. Streamlines represent the trajectory of the fluid, and their tangent direction corresponds to the direction of the fluid velocity.

[0070] Example 2: Microsphere Simulation Separation Experiment: 1) Prepare the mixed microsphere suspension: Add 10 μL of each of the following microspheres: 15 μm green fluorescent magnetic microspheres (simulating magnetically labeled leukocytes), 10 μm blue fluorescent non-magnetic microspheres (simulating neutrophils), and 5 μm red fluorescent magnetic microspheres (simulating magnetically labeled erythrocytes). Add 2.95 mL of PBS and 20 μL of Tween-20 (mix thoroughly and incubate overnight at room temperature to prevent microsphere adhesion).

[0071] 2) First-stage separation: Blood inlet flow rate 5.5 mL / min, sheath fluid inlet flow rate 6 mL / min, collect the first outlet product.

[0072] 3) Second-stage separation: Inject the first outlet product into the second-stage channel at a flow rate of 1.1 mL / min and turn on the magnetic field.

[0073] 4) Results: The recovery rate of 10 μm microbeads collected from the third outlet reached 87.97%; while the recovery rates of 5 μm and 15 μm microbeads were only 8.56% and 4.47%, respectively, proving efficient separation.

[0074] Figure 6 The figure shows the experimental results of separation using microspheres, where... Figure 6 In the diagram, A represents the proportions of the three types of particles at the O1 and O3 outlets after an external magnetic field is applied. Figure 6 B in the diagram represents the proportions of the three types of particles at the O1 and O3 outlets without a magnetic field.

[0075] like Figure 6 The diagram shows a comparison of the concentrations of 5µm, 15µm magnetic particles, and 10µm non-magnetic particles at the outlets of target chip O1 and target chip O3. The comparison reveals that the concentration of non-magnetic particles significantly increases after magnetization, indicating the adsorption of magnetic particles by the magnetic field. The percentages represent the changes in the concentration of the three types of particles at the outlet of target chip O3 compared to the outlet of target chip O1.

[0076] Example 3: Isolation of neutrophils from whole blood: 1) Take 50 μL of whole blood from a healthy person, add 5 μL of negative selection antibody mixture and 5 μL of magnetic nanoparticles, then add 150 μL of erythrocyte lysis buffer, and incubate at 4℃ for 10 min.

[0077] 2) Dilute with PBS containing 5% FBS to 3 mL.

[0078] 3) Run the chip according to the flow rate conditions of Example 2.

[0079] 4) Collect the third exit product and analyze it using a cell counter and fluorescence microscope.

[0080] 5) Results: Neutrophil purity was 83.77%, viability was 93%, and other cells (monocytes, lymphocytes, etc.) accounted for only 16.23%. Compared with density gradient centrifugation (viability 81%), the viability of this target chip was significantly improved.

[0081] Figure 7 This is a diagram showing the experimental results of separating target cells using blood samples. Figure 7 In the diagram, A represents the proportion of neutrophils (target cells) and other cells at the O1 outlet of the target chip and the O3 outlet of the target chip after applying an external magnetic field. Figure 7 B in the figure is a comparison of cell activity obtained by centrifugation and enrichment using this chip.

[0082] Example 4 Downstream Functional Coupling Analysis: 1) The neutrophil suspension obtained in Example 3 was directly injected into a six-unit migration chip (SU). 6 In the chip).

[0083] 2) Cell migration was captured in real time under the fMLP chemotactic gradient, and the number of migrating cells and the linear migration distance were calculated using automated analysis software.

[0084] 3) Results: After separation, the cells exhibited normal chemotactic responses in the migration chip, indicating that the separation process of this target chip did not damage cell function.

[0085] The target chip provided in this application embodiment can continuously, gently, and with high purity separate multiple target cells from trace amounts of whole blood or other complex biological samples, and the separated cells can be directly used for downstream functional analysis without delabeling.

[0086] The target chip provided in this application embodiment also has other advantages, as detailed below: Advantage 1: Force field decoupling, independent control, and wide operating window.

[0087] Inertial enrichment and magnetic purification are separated into two independent channels, with their respective flow rates and magnetic field conditions optimized to avoid mutual interference between the flow field and the magnetic field. The first stage can use a higher flow rate to achieve high-throughput enrichment, while the second stage uses a lower flow rate to ensure magnetic capture efficiency. This significantly widens the operating window and enhances the system's robustness.

[0088] Advantage 2: High-purity separation of trace samples.

[0089] High-purity target cells can be obtained with only 50 μL of trace sample (such as whole blood, puncture fluid, or tissue digestion fluid). It is particularly suitable for the detection and analysis of pediatric, critically ill, elderly patients or rare clinical samples (such as cerebrospinal fluid, synovial fluid, or fine-needle aspiration samples).

[0090] Advantage 3: Low shearing, high activity, and does not activate cells.

[0091] The shear stress on cells within the first and second level channels is controlled below 100 dyn / cm² (within the physiological range). The viability of the separated cells remains ≥93%, and they have not undergone any positive labeling or elution treatment, thus avoiding magnetic bead-induced cell activation or phenotypic changes.

[0092] Advantage 4: Vortex-assisted magnetic trapping improves separation purity.

[0093] The unique cell capture zone design utilizes a reflux vortex to stably capture magnetically labeled non-target cells, preventing them from returning with the mains or mixing into the target cell suspension from the outlet, thus significantly improving separation purity.

[0094] Advantage 5: Negative selection strategy, target cells are unlabeled and can be directly used for downstream functional analysis.

[0095] The target cells were not labeled with any magnetic beads or antibodies during the entire separation process. After separation, no delabeling step was required, and they could be directly injected into downstream chips for functional detection such as chemotaxis, migration, deformation, proliferation, and drug sensitivity, thus avoiding the influence of human intervention on the natural state of the cells.

[0096] Advantage 6: High versatility, applicable to a variety of diseases and cell types.

[0097] This chip is not limited to specific diseases or cell types. By changing the magnetically labeled antibody mixture, it can be flexibly applied to: the separation of immune cells such as neutrophils, T cells, B cells, and monocytes from whole blood; the separation of circulating tumor cells from malignant effusions; and the separation of stem cells or infiltrating lymphocytes from tissue digestion fluids. A single chip design can be used in multiple application scenarios, demonstrating excellent platform characteristics.

[0098] Advantage 7: Compact structure, mature technology, and easy to mass-produce.

[0099] Manufactured using standard PDMS soft lithography, it boasts low cost and high repeatability. The chip is for single use only, avoiding cross-contamination and meeting clinical testing standards.

[0100] Advantage 8: Separation and concentration are completed simultaneously.

[0101] While purifying target cells, simultaneous concentration is achieved through volume reduction (e.g., from 3 mL enrichment suspension to a final volume of 2 mL), which facilitates subsequent detection and analysis of low-concentration samples.

[0102] Advantage 9: Compatible with automation and standardization.

[0103] With a fixed chip structure and standardized operating procedures, it can be automated by using an injection pump or a pneumatic pump, facilitating standardized operations and result comparison in clinical laboratories.

[0104] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A target chip, characterized in that, include: Glass substrate; A PDMS structural layer bonded to the glass substrate; The PDMS structure layer is provided with a first-stage inertial separation channel and a second-stage magnetic separation channel: The first-stage inertial separation channel includes: two inlets, a spiral focusing channel, and two outlets; the two inlets of the first-stage inertial separation channel include: an outer sample inlet and an inner sheath fluid inlet; the two outlets of the first-stage inertial separation channel include: a first outlet and a second outlet, the first outlet being used to collect the enriched target cell population, and the second outlet being used to discharge non-target particles of abnormal size. The second-stage magnetic separation channel includes: an inlet, a long straight channel, multiple cell capture zones arranged along the outer wall of the long straight channel, a spiral channel connected downstream of the long straight channel, and two outlets. The two outlets of the second-stage magnetic separation channel include: a third outlet and a fourth outlet. The third outlet is used to collect purified target cells, and the fourth outlet is used to discharge magnetically labeled non-target cells that have not been captured. The periphery of the second-stage magnetic separation channel is provided with a magnetic support embedded with a permanent magnet array, so as to form a closed magnetic circuit surrounding the long straight channel and the cell capture area through the magnetic support. The target chip is a cascaded cell-oriented sorting and concentration chip based on inertial and magnetic coupling effects.

2. The target chip according to claim 1, characterized in that, The first-stage inertial separation channel is a spiral focusing channel with 3 spiral turns. The channel width of the first-stage inertial separation channel is 0.5 mm, the channel height of the first-stage inertial separation channel is 0.1 mm, the first pitch is 1.5 mm, the inner radius of the first-stage inertial separation channel is 8 mm, and the first outlet and the second outlet are separated at a 65° angle.

3. The target chip according to claim 1, characterized in that, The width of the long straight channel of the second-stage magnetic separation channel is 0.5 mm, the height of the long straight channel of the second-stage magnetic separation channel is 0.1 mm, the inlet width of the cell capture zone is 0.55 mm, the inlet depth of the cell capture zone is 0.415 mm, the interval between adjacent cell capture zones is 1.15 mm, and each capture zone forms a stable reflux vortex under the drive of the flow field.

4. The target chip according to claim 1, characterized in that, The permanent magnet array consists of four neodymium magnets embedded in the four sides of a 3D-printed nylon magnetic frame. The dimensions of any one of the four neodymium magnets are 20 mm × 5 mm × 10 mm, and the dimensions of the magnetic frame are 38.2 mm × 38.2 mm × 15 mm.

5. The target chip according to claim 1, characterized in that, The downstream of the second-stage magnetic separation channel has two spiral turns, a second pitch of 1.5 mm, and the end forks at 60° to form the third and fourth outlets.

6. A method for separating a target chip as described in any one of claims 1 to 5, the method comprising: Obtain target biological samples containing target cells, including: whole blood, bone marrow aspiration fluid, pleural effusion, ascites, tissue digestion fluid, and cultured cell suspension; Obtain the dimensions of target cells and non-target cells in the target biological sample; Whether to use red blood cell lysis buffer depends on whether the red blood cells are enlarged due to disease, which would affect the sorting effect; By mixing and incubating a mixture of negatively selected antibodies targeting non-target cell surface markers with magnetic nanoparticles, non-target cells in the target biological sample are magnetically labeled, resulting in the corresponding magnetically labeled target biological sample. The magnetically labeled target biological sample and sheath fluid are simultaneously injected into the first-stage inertial separation channel, and the flow rate is controlled so that cell populations that meet the target size are enriched and flow out from the first outlet, while cell populations smaller than the target cell size are discharged from the second outlet; the cell suspension collected from the first outlet is injected into the second-stage magnetic separation channel, and the flow rate is controlled so that the inertial force and magnetic force are balanced. Under the closed magnetic field generated by the permanent magnet array, the magnetically labeled non-target cells are deviated from the inertial focusing trajectory by the transverse magnetic force, are captured in the long straight channel and remain in the reflux vortex of the cell capture area, or are deflected towards the fourth outlet and flow out in the internal spiral channel. Unlabeled target cells maintain their inertia and focus trajectory, flowing out from the third outlet to obtain a purified and concentrated target cell suspension.

7. The separation method according to claim 6, characterized in that, The target cells are selected from cell types that can be removed by negative magnetic labeling, including neutrophils, T lymphocytes, B lymphocytes, monocytes, and circulating tumor cells.

8. The separation method according to claim 6, characterized in that, The sample volume of the target biological sample is 50 μL; the shear stress on the cells in the first-stage inertial separation channel and the second-stage magnetic separation channel is less than the cell shear stress tolerance threshold, and the activity of the separated target cells is greater than or equal to the preset value.

9. The separation method according to claim 6, characterized in that, The method further includes: The inertial and magnetic cascade separation chip is set as a disposable chip.

10. The separation method according to claim 8, characterized in that, The method further includes: Obtain the isolated target cells; The downstream functions of the isolated target cells were directly analyzed to obtain the analysis results. The downstream functions include chemotactic migration, deformation, proliferation, apoptosis, and drug sensitivity detection.

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