Cell sorting micro-fluidic chip and application

Abstract

The invention discloses a cell sorting micro-fluidic chip and application, the micro-fluidic chip is obliquely arranged on an inclined plane, the micro-fluidic chip comprises a substrate, the substrate is provided with a slender groove with a triangular prism profile, and a micro-fluidic channel is embedded in the slender groove; the upper end and the lower end of the inclined surface respectively face the center and the edge of the turntable; one side of the microfluidic channel back to the substrate is parallel to the upper surface of the substrate, one end of the microfluidic channel is communicated with the fluid buffer cavity, the other end is communicated with the sorting channel, and the other end of the sorting channel is communicated with the plurality of sorting sub-channels; a soft magnet is further arranged on the microfluidic channel, and a permanent magnet is arranged on one side, back on to the microfluidic channel, of the soft magnet. According to the invention, two-parameter sorting of preferential separation of magnetic microspheres according to density and accurate positioning according to size can be realized, high-flux and high-precision two-parameter sorting is realized, the resolution is greatly improved, the energy consumption is low, and the device is suitable for portable equipment.

Description

Technical Field

[0001] This invention belongs to the field of flow cytometry cell sorting technology, specifically relating to a cell sorting microfluidic chip and its application. Background Technology

[0002] Magnetic beads are tiny particles used to label and separate specific cells. They are typically made of superparamagnetic materials, and specific antibodies can bind to their surface, enabling the specific recognition and sorting of cells carrying corresponding antigens. Current techniques involve mixing magnetic beads with cells, allowing the antibodies on the beads to bind to the antigens on the cell surface. The mixture is then placed in a magnetic field; cells with magnetic beads are attracted and retained in the magnetic field area, while unlabeled cells are removed. Washing removes unbound cells and impurities, and finally, the target cells labeled with the magnetic beads are collected. Magnetic beads play a crucial role in cell sorting based on flow cytometry, significantly improving the efficiency and accuracy of cell sorting through their specific and efficient labeling and separation capabilities.

[0003] To achieve the sorting of multiple target cells using magnetic beads, existing technology CN114100704A sets the sorting channels at different distances from a permanent magnet. Based on the direct proportionality between the volume of the magnetic bead and the magnetic force, larger magnetic beads are sorted into sorting channels closer to the permanent magnet, while smaller magnetic beads are sorted into sorting channels farther away from the permanent magnet. This enables the sorting of different target cells according to their size after mixing with magnetic beads of different sizes loaded with different antibodies.

[0004] The existing technology mentioned above relies solely on size sorting, so the number of multi-target cells that can be sorted simultaneously is limited by the size range of the magnetic beads, resulting in low sorting resolution in terms of size. Therefore, there is an urgent need to develop a solution to increase the number of multi-target cells that can be sorted. Summary of the Invention

[0005] This invention provides a cell sorting microfluidic chip that uses different centrifugal forces to allow magnetic beads of different densities and sizes to flow into sorting channels in different orders. By applying different magnetic forces to magnetic beads of different sizes based on a magnetic field, the magnetic beads are sorted into different sorting sub-channels, and the number of multi-target cells sorted is significantly increased.

[0006] To achieve the above objectives, the present invention provides a cell sorting microfluidic chip. The microfluidic chip is inclinedly disposed on an inclined surface. The microfluidic chip includes a substrate, and the substrate has an elongated groove with a triangular prism shape. A microfluidic channel is embedded in the elongated groove, and the outer periphery of the microfluidic channel cooperates with the elongated groove. The inclined surface is placed on a turntable, and the upper end and the lower end of the inclined surface face the center of the turntable and the edge of the turntable, respectively. The microfluidic channel is parallel to the upper surface of the substrate on the side facing away from the substrate. The starting end of the microfluidic channel is connected to the upper part of the inclined plane and the fluid buffer cavity. The ending end is connected to the lower part of the inclined plane and the sorting channel. The other end of the sorting channel is connected to multiple sorting sub-channels. The angle between the first facet of the inner wall of the microfluidic channel and the inclined plane is less than 45°. The second facet is parallel to the upper surface of the substrate. The width of the third facet is less than the width of the second facet. The microfluidic channel is also equipped with a soft magnet, which is arranged parallel to the second edge. Permanent magnets are arranged on the side of the soft magnet facing away from the microfluidic channel, and the side of the soft magnet close to the microfluidic channel is serrated.

[0007] The width of the microfluidic channel is the sum of the widths of the multiple sorting sub-channels.

[0008] The width of the microfluidic channel is 10 micrometers to 1000 micrometers.

[0009] The sharp corners of the soft magnet face the microfluidic channel and tilt toward the fluid buffer chamber.

[0010] The soft magnet consists of a soft magnet embedding pit disposed in a substrate and a metal hybrid adhesive filled in the soft magnet embedding pit. The metal hybrid adhesive consists of PDMS premixed adhesive and nickel powder or nickel-iron alloy powder incorporated into the PDMS premixed adhesive. The sharp corner of the soft magnet embedding pit faces the microfluidic channel and is inclined to the fluid buffer chamber side.

[0011] The substrate is made of glass or transparent resin material, and the microfluidic chip is made of polydimethylsiloxane, glass, or transparent resin material.

[0012] The turntable rotates at a set speed, with the rotation direction pointing from the side with the smaller height between the first and third facets of the triangular prism inner wall of the microfluidic channel to the side with the larger height.

[0013] An application of a cell sorting microfluidic chip, wherein the cell sorting microfluidic chip is used for cell separation and enrichment.

[0014] Using a cell sorting microfluidic chip for cell separation and enrichment includes the following steps: A mixture of nonmagnetic microbeads, nonmagnetic microbead cells, and magnetic fluid containing various nonmagnetic microbeads is injected from the inlet of the fluid buffer chamber. The inlet of the sealed fluid buffer chamber and the outlet of the sorting sub-channel are closed, and the drive turntable is rotated at a set speed. The rotation direction is from the side with the smaller height of the triangular prism inner wall of the microfluidic channel 3 to the side with the larger height. By applying a stepwise increasing centrifugal velocity, a stepwise increasing centrifugal force is generated, causing non-magnetic beads to migrate towards the sorting channel, and non-magnetic microbeads to be intercepted at different positions in the transverse cross section of the microfluidic channel.

[0015] The critical value of centrifugal force satisfies the following formula: Where Δρ is the density difference between the nonmagnetic microspheres and the liquid, V is the volume of the nonmagnetic microspheres, ω is the centrifugal angular velocity, r is the centrifugal radius, g is the gravitational constant, θ is the horizontal angle of the chip, and F 磁 This refers to the force exerted by the magnetohydrodynamic fluid in the direction of centrifugal force.

[0016] Beneficial effects: This invention attracts magnetic beads to the initial position of the sorting channel using a magnetic field, and then increases the centrifugal force in sequence to make magnetic beads with higher density or larger size overcome the magnetic field and migrate to the buffer channel at the entrance of the sorting channel. Then, through the lateral magnetic field, the magnetic beads are migrated to sorting channels at different distances from the lateral magnetic field according to their size. The dual-parameter sorting of priority separation by density and precise positioning by size achieves high throughput and high precision dual-parameter sorting, with significantly improved resolution, low energy consumption, and suitability for portable equipment. Attached Figure Description

[0017] Figure 1 This is a top view of a microfluidic chip; Figure 2 This is a schematic diagram of the cross-section of the triangular prism-shaped inner wall of a microfluidic channel; Figure 3 This is a schematic diagram of the microfluidic chip from the left. Figure 4 for Figure 3 A magnified view of a portion of the image. Detailed Implementation

[0018] The technical solution of the present invention will be described below through specific embodiments.

[0019] like Figure 1-4 As shown, a cell sorting microfluidic chip is provided. The microfluidic chip 1 is inclinedly disposed on an inclined surface 8. The microfluidic chip 1 includes a substrate 2, on which microfluidic channels 3 are provided. The starting end of the microfluidic channel 3 of the microfluidic chip 1 faces the upper end of the inclined surface 8, and the ending end of the microfluidic channel 3 of the microfluidic chip 1 faces the lower end of the inclined surface 8. The inclined surface 8 is placed on a turntable 10, and the upper end and the lower end of the inclined surface 8 face the center of the turntable 10 and the edge of the turntable 10, respectively.

[0020] The substrate 2 has an elongated groove extending from the starting end to the ending end, the groove having a triangular prism shape. The microfluidic channel 3 has a triangular prism-shaped inner wall and outer periphery defining the elongated cavity. After the microfluidic channel 3 is embedded in the groove on the substrate 2, the side of the microfluidic channel 3 facing away from the substrate 2 is parallel to the upper surface of the substrate 2. The starting end of the microfluidic channel 3 is connected to the fluid buffer chamber 4, and the ending end of the microfluidic channel 3 is connected to the sorting channel 5. The other end of the sorting channel 5 is connected to multiple sorting sub-channels 6, each of which has a channel outlet. The width of the microfluidic channel 3 is the sum of the widths of the multiple sorting sub-channels 6.

[0021] The first facet 31 of the inner wall of the microfluidic channel 3 forms an acute angle with the inclined plane 8, the angle being less than 45°. The second facet 32 ​​is parallel to the upper surface of the substrate 2. The width of the third facet 33 is less than the width of the second facet 32. The height between the second facet 32 ​​and the first facet 31 gradually decreases from the junction with the third facet 33 to the intersection with the first facet 31. By utilizing the gradually decreasing height of the microfluidic channel 3, non-magnetic microspheres of corresponding sizes are intercepted at different positions in the transverse cross-section of the microfluidic channel 3, making the intercepted non-magnetic microspheres... The distance between the microsphere and the interface of the first facet 31 and the second facet 32 ​​decreases with the size of the non-magnetic microsphere. The intercepted non-magnetic microspheres of different sizes flow from the transverse cross-sections of different heights in the microfluidic channel into different sorting sub-channels of the microfluidic channel. The function of the microfluidic channel is to sort the non-magnetic microspheres according to their size. The channel width is 10-1000 micrometers. The three sorting sub-channels 6 sort the non-magnetic microspheres of the same density into three categories according to their size. The number of sorting sub-channels is the same as the number of sizes of the non-magnetic microspheres to be sorted.

[0022] A soft magnet 9 is also provided on the microfluidic channel 3. The soft magnet 9 is arranged parallel to the second facet 32 ​​of the microfluidic channel 3. Permanent magnets 11 are respectively provided on the side of the soft magnet 9 facing away from the microfluidic channel 3. The side of the soft magnet 9 near the microfluidic channel 3 is serrated. The sharp corners of the soft magnet on the upper surface of the microfluidic channel 3 face the microfluidic channel 3 and are inclined towards the fluid buffer cavity 4. This will further increase the density of magnetic induction lines. In this embodiment, the distance between the sharp corners and the sidewall of the microfluidic channel 3 can be adjusted as needed to optimize the distribution of magnetic field strength.

[0023] The soft magnet can concentrate the magnetic field formed by the external permanent magnet, thus drawing the denser magnetic field lines of the permanent magnet closer to the microfluidic channel 3. This strengthens the magnetic induction of the magnetic liquid within the microfluidic channel 3, resulting in a greater magnetic force on the magnetic liquid and causing the non-magnetic microspheres to move in the opposite direction due to the reaction force of the magnetic liquid. The soft magnet consists of a soft magnet embedding pit 12 disposed on the chip substrate and a metal hybrid adhesive filled within the soft magnet embedding pit 12. The metal hybrid adhesive is composed of PDMS premixed adhesive and nickel powder or nickel-iron alloy powder incorporated into the PDMS premixed adhesive. The density of the nano-nonmagnetic microspheres is lower than that of the magnetic liquid, so they are buoyed in the fluid buffer chamber 4 and remain in the buffer chamber when not subjected to centrifugal force. When the turntable starts to rotate, they are subjected to centrifugal force and immediately enter the upper part of the microfluidic channel 3. After leaving the microfluidic channel 3, the nano-nonmagnetic microspheres are no longer subjected to magnetic force and are only affected by centrifugal force, moving towards the sorting sub-channel 6.

[0024] Cell sorting is performed using the cell sorting microfluidic chip described above. The sorting steps include: S1. A mixture of nano-nonmagnetic microbeads containing various nonmagnetic microbeads and magnetic fluid is injected from the inlet of the fluid buffer chamber 4. The nonmagnetic microbeads are suspended and aggregated in the inlet area. The nonmagnetic microbeads are composed of several nonmagnetic microbeads of different sizes. Each nonmagnetic microbead of the same size has the same magnetic composition. S2. Seal the inlet of the fluid buffer chamber 4 and the outlet of the sorting sub-channel 6, and drive the turntable 10 to rotate at a set speed, with the rotation direction from the side with the smaller height of the triangular prism inner wall of the microfluidic channel 3 to the side with the larger height. Applying a stepped-increasing centrifugal velocity generates a stepped-increasing centrifugal force; When non-magnetic microspheres overcome the component of the magnetic force in the direction of the microfluidic channel and migrate towards the sorting channel in the microfluidic channel, the critical value of the centrifugal force satisfies the following formula: Where Δρ is the density difference between the nonmagnetic microspheres and the liquid, V is the volume of the nonmagnetic microspheres, ω is the centrifugal angular velocity, r is the centrifugal radius, g is the gravitational constant, θ is the horizontal angle of the chip, and F_magnetic is the force exerted by the magnetohydrodynamic fluid in the direction of centrifugal force.

[0025] Non-magnetic microspheres in a magnetic liquid are moved away from the soft magnet by negative magnetophoresis force. The force they experience is proportional to their volume. Therefore, larger particles experience greater negative magnetophoresis force and thus greater lateral displacement, while smaller particles experience less force and thus less lateral displacement.

[0026] When the mixed non-magnetic microspheres have different sizes and densities, the increasing centrifugal force causes the non-magnetic microspheres with higher densities to overcome the component force of the magnetofluid in the direction of the microfluidic channel 3 and migrate towards the sorting channel 5 in the microfluidic channel 3. Non-magnetic microspheres of the same density are shifted towards the side with the smaller height in the inner wall of the triangular prism under the action of inertia. The corresponding non-magnetic microspheres are intercepted at different positions in the transverse section of the microfluidic channel 3 by the gradient decreasing height of the microfluidic channel 3. The distance between the intercepted non-magnetic microspheres and the junction of the first and second facets decreases with the size of the non-magnetic microspheres. The intercepted non-magnetic microspheres of different sizes flow from the transverse section of the microfluidic channel at different heights into different sorting sub-channels at the outlet of the microfluidic channel. Therefore, at one centrifugal speed, non-magnetic microspheres of different densities and sizes can be sorted. By setting different centrifugal speeds multiple times, non-magnetic microspheres of different densities and sizes can be sorted.

[0027] When the mixed non-magnetic microspheres have the same density but different sizes, the non-magnetic microspheres of the same density will shift towards the side with the smaller height in the inner wall of the triangular prism under the action of inertia. The non-magnetic microspheres of the corresponding sizes will be intercepted at different positions in the transverse section of the microfluidic channel 3 by the gradient decreasing height of the microfluidic channel 3. The distance between the intercepted non-magnetic microspheres and the junction of the first and second facets decreases with the size of the non-magnetic microspheres. The intercepted non-magnetic microspheres of different sizes flow from the transverse section of the microfluidic channel at different heights into different sorting sub-channels at the outlet of the microfluidic channel. When the mixed non-magnetic microspheres have different densities but the same size, the increasing centrifugal force causes the non-magnetic microspheres with higher density to overcome the component force of the magnetofluid in the direction of the microfluidic channel 3 and then migrate to the sorting channel 5 in the microfluidic channel 3. Therefore, at one centrifugal speed, non-magnetic microspheres with the same density and size can be sorted. By setting different centrifugal speeds multiple times, non-magnetic microspheres with different densities but the same size can be sorted.

[0028] The height of the transverse cross-section of the microfluidic channel decreases from at least greater than the size of the largest non-magnetic microbead to less than the size of the smallest non-magnetic microbead. The interception positions of any two adjacent sizes of non-magnetic microbeads in the transverse cross-section of the microfluidic channel 3 are at least 0.5 mm apart within the transverse cross-section.

[0029] It should be understood that the above description of the preferred embodiments is quite detailed, but it should not be considered as a limitation on the scope of protection of this invention. Those skilled in the art, under the guidance of this invention, can make substitutions or modifications without departing from the scope of protection of the claims of this invention, and all such substitutions or modifications fall within the scope of protection of this invention. The scope of protection of this invention should be determined by the appended claims.

Claims

1. A cell sorting microfluidic chip, characterized in that, The microfluidic chip (1) is tilted on the inclined surface (8). The microfluidic chip (1) includes a substrate (2). The substrate (2) has a slender groove with a triangular prism shape. The microfluidic channel (3) is embedded in the slender groove. The outer periphery of the microfluidic channel (3) is engaged with the slender groove. The inclined surface (8) is placed on the turntable (10). The upper end and the lower end of the inclined surface (8) face the center of the turntable (10) and the edge of the turntable (10) respectively. The microfluidic channel (3) is parallel to the upper surface of the substrate (2) on the side facing away from the substrate (2). The starting end of the microfluidic channel (3) is connected to the upper end of the inclined plane (8) and the fluid buffer cavity (4). The ending end is connected to the lower end of the inclined plane (8) and the sorting channel (5). The other end of the sorting channel (5) is connected to multiple sorting sub-channels (6). The angle between the first facet (31) of the inner wall of the microfluidic channel (3) and the inclined plane (8) is less than 45°. The second facet (32) is parallel to the upper surface of the substrate (2). The width of the third facet (33) is less than the width of the second facet (32). The microfluidic channel (3) is also provided with a soft magnet (9), which is arranged parallel to the second facet (32). A permanent magnet (11) is provided on the side of the soft magnet (9) facing away from the microfluidic channel (3), and the side of the soft magnet (9) close to the microfluidic channel (3) is set in a sawtooth shape.

2. The cell sorting microfluidic chip according to claim 1, characterized in that, The width of the microfluidic channel (3) is the sum of the widths of the multiple sorting sub-channels (6).

3. The cell sorting microfluidic chip according to claim 1, characterized in that, The width of the microfluidic channel (3) is 10 micrometers to 1000 micrometers.

4. The cell sorting microfluidic chip according to claim 1, characterized in that, The sharp corner of the soft magnet (9) faces the microfluidic channel (3) and tilts towards the fluid buffer cavity (4).

5. A cell sorting microfluidic chip according to claim 1, characterized in that, The soft magnet (9) consists of a soft magnet embedding pit disposed in the substrate and a metal mixed adhesive filled in the soft magnet embedding pit. The metal mixed adhesive consists of PDMS premixed adhesive and nickel powder or nickel-iron alloy powder incorporated into the PDMS premixed adhesive. The sharp corner of the soft magnet embedding pit faces the microfluidic channel (3) and is inclined toward the fluid buffer cavity (4).

6. The cell sorting microfluidic chip according to claim 1, characterized in that, The substrate (2) is made of glass or transparent resin material, and the microfluidic chip (1) is made of polydimethylsiloxane, glass or transparent resin material.

7. A cell sorting microfluidic chip according to claim 1, characterized in that, The turntable (10) rotates at a set speed, and the rotation direction is from the side with a smaller height between the first facet (31) and the third facet (33) of the triangular prism inner wall of the microfluidic channel (3) to the side with a larger height.

8. An application of the cell sorting microfluidic chip as described in claim 1, characterized in that, The cell sorting microfluidic chip is used for cell separation and enrichment.

9. The application of the cell sorting microfluidic chip according to claim 8, characterized in that, Using a cell sorting microfluidic chip for cell separation and enrichment includes the following steps: A mixture of nonmagnetic microbeads, nonmagnetic microbead cells, and magnetic fluid containing various nonmagnetic microbeads is injected from the inlet of the fluid buffer chamber (4); The inlet of the closed fluid buffer chamber (4) and the outlet of the sorting sub-channel (6) are closed, and the drive turntable (10) is rotated at a set speed. The rotation direction is from the side with a smaller height of the triangular prism inner wall of the microfluidic channel 3 to the side with a larger height. Applying a step-increasing centrifugal velocity generates a step-increasing centrifugal force, causing non-magnetic beads to migrate toward the sorting channel (5), and non-magnetic microbeads to be intercepted at different positions in the transverse section of the microfluidic channel 3.

10. The application of the cell sorting microfluidic chip according to claim 8, characterized in that, The critical value of centrifugal force satisfies the following formula: Where Δρ is the density difference between the nonmagnetic microspheres and the liquid, V is the volume of the nonmagnetic microspheres, ω is the centrifugal angular velocity, r is the centrifugal radius, g is the gravitational constant, θ is the horizontal angle of the chip, and F 磁 This refers to the force exerted by the magnetohydrodynamic fluid in the direction of centrifugal force.

Images