DLD chip and method based on high-flow-speed composite flow channel multi-target sorting

Abstract

The invention discloses a DLD chip and method based on high-flow-speed composite flow channel multi-target sorting. The chip comprises a sample inlet, a sheath fluid inlet, a rectification module and a two-stage deterministic lateral displacement DLD sorting module, the rectification module adopts a circular microcolumn array to optimize fluid distribution, and the first-stage module realizes primary sorting through a microfluid channel and is connected with the second-stage module; and the second-stage module is communicated with three sample outlets after being accurately sorted through a microfluid channel II. The chip is made of a PDMS material through a soft photoetching process, and the two stages of sorting modules are designed to be of a rectangular structure connected end to end and are respectively composed of elliptical micro-columns with opposite deflection angles. Under the high-flow-rate working condition, stable separation of cells can be kept, blockage is not prone to occurring, the good sorting effect on various cells of different sizes is achieved, the flow channel layout is compact, the chip manufacturing process is simplified, and the chip is suitable for the sorting scene with the high-flux requirement.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidic deterministic lateral displacement (DLD) technology, and particularly relates to a DLD chip and method based on high-flow-rate composite flow channel multi-target sorting. Background Art

[0002] Microfluidic systems operate on single cells at the micron scale with high sensitivity and precision. Through unique microchannel design and external field effects, precise sorting can be achieved based on physical properties such as cell size, density, and charge. In addition, microfluidic systems can effectively reduce reagent usage, improve experimental efficiency, and have the potential for miniaturization and low cost. They will play an increasingly important role in future biomedical research. As a type of microfluidic technology, DLD technology achieves efficient separation by precisely controlling the flow of fluids in microchannels and utilizing the differences in forces exerted on particles of different sizes, densities, or surface properties in the flow field. Compared to screening methods with lower precision and slower speeds, or magnetic sorting and dielectrophoresis that require labeling, DLD technology only requires injecting samples into the chip to achieve high-throughput, high-precision sorting of biological cells. Its resolution can even reach the nanometer level, and particles can be accurately classified according to their geometric size.

[0003] Since DLD technology was first proposed, a lot of research results have been achieved so far. This technology has been applied to certain medical scenarios such as plasma separation and circulating tumor cell separation. Like other microfluidic passive separation methods, DLD requires laminar flow with a low Re (Reynolds number). This is a prerequisite for the formation of definite laminar flow lines and predictable movement of particles through the micropillar array. Traditional DLD technology usually uses circular micropillars. Although this structure is easy to design and manufacture, it results in relatively low fluid flow rates, which limits separation performance. On the other hand, as Re increases, the streamlines will become more dispersed, the particles will be more significantly affected by the wall force of the micropillar, and will be more prone to clogging. Summary of the Invention

[0004] To solve the above problems, the present invention discloses a DLD chip based on high-flow composite flow channel multi-target sorting. By using elliptical microcolumns with a certain aspect ratio (a / b), the streamline period is reduced to accurately control the critical diameter of sorting, so that cells of similar sizes can be stably separated. In addition, the use of elliptical microcolumns also reduces the wall force, making the movement trajectory of cells in the flow channel smoother and reducing the blockage of intermediate-sized cells. Furthermore, by changing the inclination angle (Angles-of-inclination, Aoi) of the microcolumns, the particle trajectory and fluid distribution in the microchannel within the chip can be flexibly controlled, so that the DLD chip can maintain a good cell separation effect at a higher flow rate, effectively improve the throughput, simplify the sorting steps, and have a low manufacturing cost.

[0005] To achieve the above object, the technical solution of the present invention is as follows: A DLD chip for multi-target sorting based on a high-flow-rate composite flow channel includes a sample inlet, a sheath liquid inlet, a rectifying module at the inlet, and a two-stage deterministic lateral displacement (DLD) sorting module.

[0006] The inlet rectification module is composed of a circular micro-column array and is only present in the sample inlet channel and the sheath liquid inlet channel; The two-stage deterministic lateral displacement (DLD) sorting module includes a main channel, a first-stage deterministic lateral displacement sorting module, and a second-stage deterministic lateral displacement sorting module; The first-stage deterministic lateral displacement sorting module includes a first-stage sorting microcolumn array, a sample outlet channel I, and a sample outlet I; The second-stage deterministic lateral displacement sorting module includes a second-stage sorting micro-pillar array, a sample outlet channel II, a sample outlet II, a sample outlet channel III, and a sample outlet III; The first-stage sorting micro-pillar array and the second-stage sorting micro-pillar array are arranged in the main channel, and a partition is provided in the main channel to separate the first-stage sorting micro-pillar array from the second-stage sorting micro-pillar array; The sample inlet and the sheath fluid inlet merge into the inlet of the first-stage sorting microcolumn array. The upper end of the first-stage deterministic lateral displacement sorting module is connected to the sample outlet channel I, and the lower end is connected to the second-stage deterministic lateral displacement sorting module. The second-level deterministic lateral displacement sorting module is connected to the sample outlet channel II and the sample outlet channel III.

[0007] Furthermore, all microchannels of the chip are made of PDMS material and are manufactured through a soft photolithography casting process.

[0008] Furthermore, the rectifying modules at the microchannel inlet are composed of circular microcolumn arrays with a diameter of 60 μm and a spacing of 80 μm. The angle between the connecting line of each row of microcolumn array and the central axis corresponding to the deterministic lateral displacement microchannel connected to the sample inlet and the sheath fluid inlet is 15°, and the critical size is 30 μm.

[0009] Furthermore, the microchannel sheath fluid inlet uses a gradually expanding microfluidic channel, and the sample inlet uses a contracting microfluidic channel.

[0010] Furthermore, the sample outlet I and the sample outlet II in the deterministic lateral displacement sorting module use slow closing.

[0011] Furthermore, the first-level deterministic lateral displacement sorting module micropillar array of the chip is composed of several elliptical micropillars, with a long axis length of 60 μm, a short axis length of 30 μm, a spacing of 68.8 μm, an angle between the long axis of the micropillar and the central axis of the microfluidic channel of -45°, an angle between the line connecting each row of the micropillar array and the central axis of the deterministic lateral displacement microfluidic channel of 11°, and a critical size of 27 μm; except for the angle between the long axis of the micropillar and the central axis of the microfluidic channel of 45°, the other parameters of the micropillar array of the second-level deterministic lateral displacement sorting module are consistent with those of the micropillar array of the first-level deterministic lateral displacement sorting module.

[0012] Furthermore, in the deterministic lateral displacement sorting module, the angle between the sample inlet and the sheath liquid inlet is 46°, the angle between the sample outlet I and the sample outlet II is 47°, and the angle between the sample outlet II and the sample outlet III is 47°.

[0013] Furthermore, the second-stage deterministic lateral displacement sorting module is directly connected to the first-stage lateral displacement sorting module, and the flow channel size is narrower than that of the first-stage lateral displacement sorting module, and the flow channel length is longer than that of the first-stage lateral displacement sorting module.

[0014] Furthermore, in order to facilitate the structural design of the deterministic lateral displacement sorting module, the micropillar array migrates downward by one row when each cycle is satisfied.

[0015] Furthermore, the end of the first-level deterministic lateral displacement sorting module is integrated with the second-level deterministic lateral displacement sorting module and the sample outlet I.

[0016] The present invention also provides a method for sorting cells in a high flow rate environment using the above-mentioned two-stage deterministic lateral displacement sorting module, comprising the following steps: S1. Connect the syringe pump to the sheath fluid inlet on the DLD chip through a hose. Turn on the syringe pump. When the buffer passes through the rectifying microcolumn array, impurities and bubbles are filtered out. Wait for the buffer to completely expel the bubbles in the chip to maintain a laminar flow state. S2. Mix the cells evenly and draw them into the syringe, which is then connected to the sample inlet via a flexible tube. S3. Push the sample into the chip. After the cells pass through the narrowed sample inlet channel, the flow rate increases and focuses at the bottom of the first-stage deterministic lateral displacement sorting module inlet, and then enters the first-stage deterministic lateral displacement sorting module. At this time, the deflection angle of the elliptical microcolumn is negative 45°. The reaction force of the microcolumn on the fluid makes it easier for the fluid to flow over the microcolumn. Smaller cells are more likely to make zigzag movements, reducing blockage, while larger cells always collide with the microcolumn, quickly distance themselves from the smaller cells, and therefore quickly separate from the mixed cells and enter the sample outlet. Although the smaller cells have also begun to separate, the period of their streamlines is much greater than that of the larger cells, so that they cannot make sufficient lateral displacement before approaching the wall. They are still in the microchannel, waiting to enter the second-stage deterministic lateral displacement sorting module for further separation. S4. After the cells enter the second-level deterministic lateral displacement sorting module, the deflection angle of the elliptical microcolumns changes to 45°, which is opposite to the first-level sorting microcolumn array. As a result, the actual movement period of the remaining two sizes of smaller cells in the flow channel is greatly reduced compared to the first-level sorting flow channel, making them more susceptible to lateral displacement and being quickly sorted into their respective corresponding sample outlet channels.

[0017] The present invention has the following beneficial effects: The DLD chip uses a rectifier module to completely eliminate bubbles. Combined with a slow-closing outlet design, it ensures that cells remain active during the sorting process. The secondary sorting module utilizes a reverse 45° microcolumn deflection angle to create a complementary sorting flow field, resulting in a superimposed separation effect on cell trajectories. The increased length and narrowing of the second-stage flow channel significantly improves the separation resolution of small-sized cells, extending the sorting path for small-sized cells and avoiding localized blockages caused by brief retention.

[0018] Traditional DLD chips are prone to flow channel blockage due to the accumulation of intermediate particles at low flow rates. However, the present invention designs a micro-pillar array of specific shapes and deflection angles to change the streamline distribution pattern and period, reducing the risk of blockage. This enables the chip to stably sort cells of different sizes even at higher flow rates, significantly improving the chip's sample processing speed and making it suitable for high-throughput sorting needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the chip structure of the present invention.

[0020] Figure 2 This is a schematic diagram of the rectification module at the inlet of the present invention.

[0021] Figure 3 This is a structural schematic diagram of the first-level deterministic lateral displacement sorting module on the left side of the present invention.

[0022] Figure 4 This is a schematic diagram of the second-stage deterministic lateral displacement sorting module and partition structure on the right side of the present invention.

[0023] Figure 5 This is a partial enlargement of the micropillar array of the present invention.

[0024] Figure 6 Schematic diagram of the sample outlet channel and sample outlet of the present invention.

[0025] Figure 7 Schematic diagram of the partition structure of the present invention.

[0026] Figure 8 Schematic diagram of the cell sorting effect of the first-level sorting micro-pillar array.

[0027] Figure 9 Schematic diagram of the cell sorting effect of the second-level sorting micro-pillar array.

[0028] List of Figure Symbols: 1. Sample inlet, 11. Sample inlet channel, 2. Sheath fluid inlet, 21. Sheath fluid inlet channel, 3. Rectifying microcolumn array, 4. Deterministic lateral displacement sorting module, 41. First-stage sorting microcolumn array, 42. Second-stage sorting microcolumn array, 43. Partition, 51. Sample outlet channel I, 52. Sample outlet I, 53. Sample outlet channel II, 54. Sample outlet II, 55. Sample outlet channel III, 56. Sample outlet III. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0030] like Figure 1 As shown, the present invention provides a DLD chip based on high-flow rate composite flow channel multi-target sorting, which mainly adopts a multi-stage sorting module integrated on a single flow channel (main channel), including a first-stage deterministic lateral displacement sorting module and a second-stage deterministic lateral displacement sorting module.

[0031] like Figure 1 As shown, the rectification module at the entrance is mainly composed of a sample inlet 1, a sample inlet channel 11, a sheath liquid inlet 2, a sheath liquid inlet channel 21, and a rectification microcolumn array 3. The rectification microcolumn array 3 is located in the sample inlet channel 11 close to the sample inlet 1. The layout of the rectification microcolumn array at the sheath liquid inlet channel is consistent with that at the sample inlet. The sample inlet channel 11 and the sheath liquid inlet channel 21 are connected in front of the deterministic lateral displacement sorting module 4.

[0032] The rear end of the sample inlet channel 11 is narrowed for ease of processing and matches the sample inlet size required for the sorting effect of the chip, so that the cell flow is focused at the bottom of the chip, providing sufficient lateral displacement distance for larger cells.

[0033] like Figure 1 As shown, the deterministic lateral displacement sorting module 4 includes a first-level deterministic lateral displacement sorting module at the front end (left side) and a second-level deterministic lateral displacement sorting module at the back end (right side).

[0034] like Figure 1 、 Figure 6 As shown in the figure, the first-stage deterministic lateral displacement sorting module mainly includes a first-stage sorting micro-column array 41 and a partition 43. Cells are focused from the sample inlet channel 11 and enter the Figure 3 In the first-stage sorting microcolumn array shown, larger cells are sorted out first, enter the sample outlet channel I 51 , and reach the sample outlet I 52 ; the partition 43 separates the second-stage sorting microcolumn array from the sample outlet channel I 51 .

[0035] like Figure 1 、 Figure 6 As shown, the second-stage deterministic lateral displacement sorting module includes a second-stage sorting micropillar array 42, a partition 43, a sample outlet channel II 53, a sample outlet II 54, a sample outlet channel III 55, and a sample outlet III 56. The sorted larger diameter cells (20 μm) have already entered sample outlet channel I 51 along the partition 43 and reached sample outlet I 52. Cells with remaining diameters leave the first-stage sorting micropillar array 41 and enter the second-stage sorting micropillar array 42. After sorting, medium diameter cells (10 μm) enter sample outlet channel III 54 and reach sample outlet III 55. Small diameter cells (5 μm) enter sample outlet channel II 53 and reach sample outlet II 52.

[0036] The diameters of the sample inlet 1, the sheath liquid inlet 2, the sample outlet I 52, the sample outlet 53 and the sample outlet II 54 in the deterministic lateral displacement sorting chip are the same.

[0037] The micro-pillar deflection angle of the first-stage sorting micro-pillar array 41 is opposite to that of the second-stage sorting micro-pillar array 42, but the shape and distribution are the same. Figure 5 shown.

[0038] All microchannels of the chip are made of PDMS material and cast using a soft photolithography process.

[0039] The rectifying modules at the microchannel inlet are composed of circular rectifying microcolumn arrays 3, with a diameter of 60 μm and a spacing of 80 μm. The angle between the connecting line of each row of microcolumn arrays and the central axis corresponding to the deterministic lateral displacement microchannel (sorting module) connected to the sample inlet 1 and the sheath liquid inlet 2 is 15°, and the critical size is 30 μm.

[0040] The chip's first-level deterministic lateral displacement sorting module, a micropillar array, consists of several elliptical micropillars with a major axis of 60 μm, a minor axis of 30 μm, and a spacing of 68.8 μm. The angle between the major axis of the micropillar and the central axis of the microfluidic channel is -45°, and the angle between the line connecting each row of the micropillar array and the central axis of the deterministic lateral displacement microfluidic channel is 11°. The critical dimension is 27 μm. The second-level deterministic lateral displacement sorting module, a micropillar array, has a 45° angle between the major axis of the micropillar and the central axis of the microfluidic channel. Due to the asymmetric arrangement of the columnar array in the DLD chip, the fluid forms specific streamlines around the pillars. When large-sized cells encounter a micropillar, due to their larger size, they cannot move smoothly along the original streamline and are squeezed onto adjacent streamlines, moving in a lateral displacement direction perpendicular to the original direction. Small-sized cells, however, are smaller and can move along the original streamline, following it through the micropillar array.

[0041] In the deterministic lateral displacement sorting module, the angle between the sample inlet 1 and the sheath liquid inlet 2 is 46°, the angle between the sample outlet I 52 and the sample outlet II 54 is 47°, and the angle between the sample outlet II 54 and the sample outlet III 56 is 47°.

[0042] The deterministic lateral displacement sorting module 4 is designed to facilitate structural design, and the micro-pillar array migrates downward by one row when each cycle is met.

[0043] The specific separation process includes the following steps: S1. Connect the syringe on the syringe pump to the sheath liquid inlet 2 on the DLD chip through a hose. Turn on the syringe pump. When the buffer passes through the rectifying micro-pillar array 3, impurities and bubbles are filtered out. Wait for the buffer to completely expel the bubbles in the chip to maintain the buffer in a laminar flow state. S2, after uniformly mixing the cells, draw them into the syringe, and connect the syringe to the sample inlet 1 through a flexible tube; S3. Push the sample into the chip. After the cells pass through the narrowed sample inlet channel, the flow rate increases and focuses at the bottom of the first-stage deterministic lateral displacement sorting module inlet, and then enters the first-stage deterministic lateral displacement sorting module. At this time, the deflection angle of the elliptical microcolumn is negative 45°. The reaction force of the microcolumn on the fluid makes it easier for the fluid to flow over the microcolumn. Smaller cells are more likely to make zigzag movements, reducing blockage, while larger cells always collide with the microcolumn, quickly distance themselves from the smaller cells, and therefore quickly separate from the mixed cells and enter the sample outlet. Although the smaller cells have also begun to separate, the period of their streamlines is much greater than that of the larger cells, so that they cannot make sufficient lateral displacement before approaching the wall. They are still in the microchannel, waiting to enter the second-stage deterministic lateral displacement sorting module for further separation. S4. After the cells enter the second-level deterministic lateral displacement sorting module, the deflection angle of the elliptical microcolumns changes to a negative value opposite to that of the first-level sorting microcolumn array, causing the actual movement period of the remaining two sizes of smaller cells in the flow channel to be greatly reduced compared to the first-level sorting flow channel, making them more susceptible to lateral displacement and being quickly sorted into their respective corresponding sample outlet channels.

[0044] It should be noted that the above content merely illustrates the technical idea of the present invention and cannot be used to limit the scope of protection of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications all fall within the scope of protection of the claims of the present invention.

Claims

1. A DLD chip based on high-flow composite flow channel multi-target sorting, characterized by: It includes a sample inlet, a sheath liquid inlet, an inlet rectification module, and a two-stage deterministic lateral displacement sorting module; The inlet rectification module is composed of a circular micro-column array, which is respectively arranged in the sample inlet channel and the sheath liquid inlet channel; The two-stage deterministic lateral displacement sorting module includes a main channel, a first-stage deterministic lateral displacement sorting module and a second-stage deterministic lateral displacement sorting module; The first-stage deterministic lateral displacement sorting module includes a first-stage sorting microcolumn array, a sample outlet channel I, and a sample outlet I; The second-stage deterministic lateral displacement sorting module includes a second-stage sorting micro-pillar array, a sample outlet channel II, a sample outlet II, a sample outlet channel III, and a sample outlet III; The first-stage sorting micro-pillar array and the second-stage sorting micro-pillar array are arranged in the main channel, and a partition is provided in the main channel to separate the first-stage sorting micro-pillar array from the second-stage sorting micro-pillar array; The sample inlet and the sheath fluid inlet merge into the inlet of the first-stage sorting microcolumn array. The upper end of the first-stage deterministic lateral displacement sorting module is connected to the sample outlet channel I, and the lower end is connected to the second-stage deterministic lateral displacement sorting module. The second-level deterministic lateral displacement sorting module is connected to the sample outlet channel II and the sample outlet channel III.

2. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: All microchannels of the chip are made of PDMS material and are manufactured through a soft photolithography casting process.

3. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The rectifying modules at the inlet are all composed of circular microcolumn arrays with a diameter of 60 μm and a spacing of 80 μm. The angle between the connecting line of each row of microcolumn array and the central axis corresponding to the deterministic lateral displacement microchannel connected to the sample inlet and the sheath liquid inlet is 15°, and the critical size is 30 μm.

4. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The sheath liquid inlet adopts a gradually expanding micro-channel, and the sample inlet adopts a contracting micro-channel.

5. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The sample outlet I and the sample outlet II in the deterministic lateral displacement sorting module use slow closing.

6. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The first-level sorting micropillar array consists of several elliptical micropillars with a major axis length of 60 μm, a minor axis length of 30 μm, a spacing of 68.8 μm, an angle of -45° between the major axis of the micropillar and the central axis of the microfluidic channel, an angle of 11° between the line connecting each row of the micropillar array and the central axis of the deterministic lateral displacement microfluidic channel, and a critical size of 27 μm; except for the angle of 45° between the major axis of the micropillar and the central axis of the microfluidic channel, the other parameters of the second-level deterministic lateral displacement sorting module micropillar array are consistent with those of the first-level deterministic lateral displacement sorting module micropillar array.

7. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The angle between the sample inlet and the sheath liquid inlet is 46°, the angle between the sample outlet I and the sample outlet II is 47°, and the angle between the sample outlet II and the sample outlet III is 47°.

8. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The second-stage deterministic lateral displacement sorting module is directly connected to the first-stage lateral displacement sorting module, and the flow channel size is narrower than that of the first-stage lateral displacement sorting module, and the flow channel length is longer than that of the first-stage lateral displacement sorting module.

9. The DLD chip based on high-flow composite flow channel multi-target sorting according to claim 1, characterized in that: The deterministic lateral displacement sorting module is designed to facilitate structural design, and the micro-pillar array migrates downward by one row when each cycle is met.

10. A method for sorting cells in a high-flow environment using the DLD chip for multi-target sorting based on high-flow composite flow channels according to claim 1, characterized in that: The following steps are involved: S1. Connect the syringe pump to the sheath fluid inlet on the DLD chip through a hose. Turn on the syringe pump. When the buffer passes through the rectifying microcolumn array, impurities and bubbles are filtered out. Wait for the buffer to completely expel the bubbles in the chip to maintain a laminar flow state. S2. Mix the cells evenly and draw them into the syringe, which is then connected to the sample inlet via a flexible tube. S3. The sample is pushed into the chip. After the cells pass through the narrowed sample inlet channel, the flow rate increases and focuses at the bottom of the inlet of the first-stage deterministic lateral displacement sorting module, and then enters the first-stage deterministic lateral displacement sorting module. At this time, the deflection angle of the elliptical microcolumns is negative 45°. The reaction force of the microcolumns on the fluid makes it easier for the fluid to flow over the microcolumns. Smaller cells are more likely to make zigzag movements, reducing blockage, while larger cells always collide with the microcolumns, quickly distance themselves from the smaller cells, and therefore quickly separate from the mixed cells and enter the sample outlet. Although the smaller cells also begin to separate, the period of their streamlines is much larger than that of the larger cells, so that they cannot make sufficient lateral displacement before approaching the wall and remain in the microchannel, waiting to enter the second-stage deterministic lateral displacement sorting module for further separation. S4. After the cells enter the second-level deterministic lateral displacement sorting module, the deflection angle of the elliptical microcolumns changes to 45°, which is opposite to the first-level sorting microcolumn array. As a result, the actual movement period of the remaining two sizes of smaller cells in the flow channel is greatly reduced compared to the first-level sorting flow channel, making them more susceptible to lateral displacement and being quickly sorted into their respective corresponding sample outlet channels.

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