Array micro-fluidic chip and separation and purification method of high-concentration leukocytes, platelets and cytokines

Abstract

The invention provides an array micro-fluidic chip and a separation and purification method of high-concentration leukocytes, platelets and cytokines, and relates to the technical field of array micro-fluidic. The micro-column array is respectively connected with the micro-hydrocyclone and the asymmetric curved flow channel, a tangential inlet is formed in the upper part of the micro-hydrocyclone, and an upper overflow port is formed in the top of the micro-hydrocyclone; a plurality of rows of cylinders are arranged in the micro-column array to form a concave-convex array; the asymmetric curved flow channel is composed of a plurality of narrow turns and a plurality of wide turns at intervals, and an outer side outlet allowing red blood cells and a buffer solution to flow and an inner side outlet allowing platelets to flow are formed in the tail end of the asymmetric curved flow channel; the micro-column inlet is communicated with the upper overflow port, the asymmetric bent flow channel is communicated with the second outlet, and the first outlet and the inner side outlet are connected into the collecting pipe. According to the micro-fluidic equipment, a micro-hydrocyclone, a micro-column array and an asymmetric curved runner are combined for use, and a high-concentration autologous leukocyte, platelet and cytokine solution is obtained and kept active in a staged purification mode.

Description

Technical Field

[0001] The present invention relates to the field of array microfluidics technology, in particular to an array microfluidics chip and a method for separating and purifying high-concentration leukocytes, platelets and cytokines. Background Art

[0002] In medical treatment, autologous leukocytes, platelets and other cytokines can be used to treat aseptic inflammatory diseases such as arthritis and tendonitis. It is a relatively new cell therapy with broad application prospects. Compared with the widely used platelet-rich plasma therapy on the market, this solution contains concentrated leukocytes and cytokines. Cytokines provide short-acting anti-inflammatory and analgesic effects, and leukocytes provide long-acting anti-inflammatory factor release, thereby providing long-term inflammation and pain relief, as well as regeneration of injured tissues such as soft tissue or bone tissue.

[0003] In existing technologies, there is no specific separation device for solutions of autologous white blood cells, platelets, and cytokines, making extraction very difficult. While platelets can be obtained clinically by centrifuging platelet-rich plasma, there is no clinical method for extracting an "autologous white blood cell and cytokine solution."

[0004] The primary blood separation methods currently available on the market are density gradient separation methods based on centrifuges and separation gels. These methods have their limitations: 1. Prolonged high-speed centrifugation is detrimental to white blood cell viability; 2. Since the collection of the white blood cell layer inevitably removes the separation gel layer, its toxic side effects in humans when used as a drug are unknown, and a washing step is required to remove the medium; 3. Lymphocyte separation media Ficoll and Histopaque-1077 are the most commonly used methods for separating the white blood cell layer, but their density is insufficient, and they can only separate a portion of white blood cells (mononuclear leukocytes, such as lymphocytes). Separating the entire white blood cell layer and maintaining a stable ratio of various white blood cell components (mononuclear leukocytes and polynuclear leukocytes) are crucial for maintaining the microenvironment in which white blood cells survive, which directly determines the activity of the solution after autologous solution reinfusion. Ficoll and Histopaque-1077 are products of Sigma, and polysucrose and sodium diatrizoate solution have a density of 1.077 g / ml; 4. Sodium diatrizoate solution, as an iodine-containing benzoate contrast agent, cannot be used for therapeutic purposes due to its radioactivity.

[0005] The microfluidic method is a new particle separation method that does not require a centrifuge. In the microfluidic system, blood maintains a laminar flow state, reducing physical damage to sensitive biological molecules such as cells or proteins. , such as spiral channel inertial separation technology. However, since current microfluidic technology is mainly based on medical research or detection purposes and can only separate a single type of cell, such as white blood cells, from a small amount of blood, it cannot be used as a therapeutic platform for the following reasons: Since the amount of blood required for cell therapy is much greater than that required for diagnosis, there is an urgent need for ultra-high throughput methods. The current cell diagnostic platform has limited flow, such as 1ul / min of laboratory microarray microfluidic chips [Davis, John A., et al. "Deterministic hydrodynamics: taking blood apart." Proceedings of the National Academy of Sciences 103.40(2006): 14779-14784], and 500μL / min of the fastest commercial inertial microfluidic chip [Lu, Xiaoguang, Mahnoush Tayebi, and Ye Ai. "Alow-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood." Electrophoresis 42.21-22(2021):2281-2292], a single microchannel is not sufficient for commercially relevant flow rates, and when the cell concentration is higher than 10 7 When the number of cells / mL is 1000, the efficiency of the system drops significantly because the number of red blood cells far exceeds the number of white blood cells typically useful in treatment, with a ratio of approximately 1000:1. When whole blood enters the microchannel, the large number of red blood cells tends to stack, squeeze, and fill the channel, causing blockage. This can also cause blockage in such a small hydraulic diameter channel. Moreover, highly diluted blood means longer processing time. Currently, to separate cells from whole blood, most blood pretreatment steps require a lysis step to reduce the volume of red blood cells. However, the lysis buffer is toxic, and non-toxic treatment methods are necessary. In addition, because the blood is heavily diluted in the chip, the active ingredients are also diluted and cannot be used in treatment. Summary of the Invention

[0006] The present invention provides an array microfluidic chip and a method for separating and purifying high-concentration leukocytes, platelets and cytokines, which can separate and purify high-concentration leukocytes and cytokines using a simpler, gentler and continuous high-throughput cell sorting and solution preparation method.

[0007] The specific technical solution is an array microfluidic chip, including: a micro hydrocyclone, a micro pillar array and an asymmetric curved channel. The micro pillar array located between the micro hydrocyclone and the asymmetric curved channel is respectively connected to the micro hydrocyclone and the asymmetric curved channel. The micro hydrocyclone is a device that uses fluid pressure to generate rotation to separate substances. It has a tangential inlet on the top, an overflow port on the top, and an underflow port on the bottom. The interior of the micro pillar array is provided with multiple rows of cylinders to construct a concave-convex array, with a micro pillar inlet at the front end and a first and second outlets at the rear end. The flow channel ratio of the first outlet to the second outlet is 1:5. The asymmetric curved channel is composed of multiple narrow turns and multiple wide turn intervals. The end of the asymmetric curved channel forms an outer outlet for the flow of red blood cells and buffer and an inner outlet for the circulation of platelets. The micro pillar inlet is connected to the overflow port, the asymmetric curved channel is connected to the second outlet, and both the first outlet and the inner outlet are connected to a collection pipe.

[0008] Furthermore, in the micro-pillar array, the columns in the rear row are laterally offset from the front end to the rear end relative to the columns in the front row.

[0009] Furthermore, in the micropillar array, the critical diameter of the column Dc is Dc = 1.4gε 0.48 , g represents the gap width between the cylinders, ε represents the relative displacement fraction; ε = d / λ, the distance between the cylinders is λ, and the relative displacement between adjacent column rows is d.

[0010] Furthermore, in the micropillar array, the critical diameter of the pillar is 5 μm.

[0011] Furthermore, the micro hydrocyclone adopts the classic design of Bradley hydrocyclone.

[0012] Furthermore, in the micro hydrocyclone, a cylinder is used as the main body, and a tangential inlet is manufactured above the cylinder using the SU-8 photolithography process. The width of the tangential inlet channel is 50 μm and the depth is 100 μm. A tangential outlet connected to the underflow port is manufactured below the cylinder using the SU-8 photolithography process. The width of the tangential outlet is 70 μm and the depth is 100 μm. The overflow port coaxial with the cylinder has a diameter of 70 μm and a height of 0.5 mm.

[0013] Furthermore, precision pumps are connected to the tangential inlet, overflow port and underflow port to control the flow rate.

[0014] The method for separating and purifying high-concentration white blood cells, platelets and cytokines comprises the following steps:

[0015] S1. Rough separation removes most of the red blood cells. Whole blood diluted 1 to 10 times is injected into the micro hydrocyclone. Under the outward radial centrifugal force generated by the tangential velocity, different blood components remain dispersed and are driven by the fluid. Most of the red blood cells are discharged from the overflow port, and white blood cells, platelets and a small amount of red blood cells are discharged from the overflow port.

[0016] S2: Fine separation to separate white blood cells. The white blood cells, platelets and a small amount of red blood cells in step S1 continue to flow into the microcolumn array. The deterministic lateral displacement principle is used to separate white blood cells and red blood cells, and a high-concentration white blood cell and diluted platelet solution is obtained.

[0017] S3, platelet concentration: the diluted platelet solution in step S2 continues to flow into the asymmetric curved channel, and the buffer and red blood cells are removed to obtain concentrated platelets.

[0018] S4. The leukocytes, cytokines and platelets are pooled. The concentrated platelets in step S3 and the high-concentration leukocytes in step S2 are pooled to obtain a concentrated autologous leukocyte, platelet and cytokine solution.

[0019] Furthermore, S1 was diluted with 2-5% dextran buffer.

[0020] Furthermore, in step S1, the whole blood flow rate is 1 ml / min and the blood cell concentration is 10 4 -10 8 / ul.

[0021] The array microfluidic chip of the present invention combines a micro hydrocyclone, a microcolumn array and an asymmetric curved channel for use. Through a staged purification method, whole blood can remove most of the red blood cells by passing through the micro hydrocyclone, and then enter the microcolumn array to separate high-concentration white blood cells. The remaining diluted platelet solution continues to enter the asymmetric curved channel to eliminate the red blood cells to obtain concentrated platelets, which are then combined with the high-concentration white blood cells in the previous stage to finally obtain a high-concentration autologous white blood cell, platelet and cytokine solution while maintaining its activity. Compared with traditional centrifugation and existing microfluidic methods, the present invention can achieve a simpler, gentler and continuous high-throughput cell sorting and solution preparation method, thereby producing a solution with higher concentration, stronger functionality and rich in highly active white blood cells and cytokines for the treatment of diseases such as arthritis. High flow is reflected in a flow rate greater than 10 mL / min, and high concentration is higher than 10 7 cells / mL to meet the needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 is a schematic diagram of the structure of the array microfluidic chip in the present invention,

[0024] Figure 2 This is a schematic diagram of the cyclone process of a micro hydrocyclone.

[0025] Figure 3 This is a schematic diagram of the forces acting on cells in the cyclone.

[0026] Figure 4 This is a schematic diagram of the micropillar array process.

[0027] Figure 5 This is the principle diagram for calculating the critical diameter of the micropillar array process.

[0028] Figure 6 This is a schematic diagram of the separation and purification process of high-concentration white blood cells, platelets and cytokines.

[0029] Figure 7 This is a schematic diagram of the micromachining process of micro hydrocyclone.

[0030] Among them, 1. Micro hydrocyclone, 2. Micro column array, 3. Asymmetric curved flow channel, 4. Collection pipe,

[0031] 101, tangential inlet, 102, overflow outlet, 103, underflow outlet,

[0032] 201, micro column inlet, 202, first outlet, 203, second outlet,

[0033] 301, narrow turn, 302, wide turn, 303, outside exit, 304, inside exit. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and the accompanying drawings. Here, the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but are not intended to limit the present invention.

[0035] The following is a description of the present invention with reference to the accompanying drawings:

[0036] Combine Figure 1The array microfluidic chip comprises: a micro hydrocyclone 1, a micro pillar array 2 and an asymmetric curved channel 3. The micro pillar array 2 located between the micro hydrocyclone 1 and the asymmetric curved channel 3 is respectively connected to the micro hydrocyclone 1 and the asymmetric curved channel 3. The micro hydrocyclone 1 is a device that uses fluid pressure to generate rotation to separate substances. It is provided with a tangential inlet 101 on the top, an overflow port 102 on the top, and a downflow port 103 on the bottom. The interior of the micro pillar array 2 is provided with multiple rows of columns to form a concave-convex array, and a micro pillar inlet 20 is provided at the front end. 1, a first outlet 202 and a second outlet 203 are provided at the rear end, and the flow channel ratio of the first outlet 202 to the second outlet 203 is 1:5; the asymmetric curved flow channel 3 is composed of multiple narrow turns 301 and multiple wide turns 302, and the end of the asymmetric curved flow channel 3 forms an outer outlet 303 for the flow of red blood cells and buffer and an inner outlet 304 for the circulation of platelets; the microcolumn inlet 201 is connected to the overflow port 102, the asymmetric curved flow channel 3 is connected to the second outlet 203, and the first outlet 202 and the inner outlet 304 are both connected to the collection pipe 4.

[0037] In one embodiment, in the micro-pillar array 2 , the columns in the rear row are laterally offset from the front end to the rear end relative to the columns in the front row.

[0038] In one embodiment, in the micropillar array 2, the critical diameter of the pillar Dc is Dc=1.4gε 0.48 , g represents the gap width between the cylinders, ε represents the relative displacement fraction; ε = d / λ, the distance between the cylinders is λ, and the relative displacement between adjacent column rows is d.

[0039] In one embodiment, in the micro-pillar array 2 , the critical diameter of the pillars is 5 μm.

[0040] In one embodiment, the micro hydrocyclone 1 adopts the classic design of Bradley hydrocyclone.

[0041] In one embodiment, in the micro hydrocyclone 1, a cylinder is used as the main body, and a tangential inlet 101 is manufactured on the top of the cylinder using the SU-8 photolithography process. The channel width of the tangential inlet 101 is 50 μm and the depth is 100 μm. A tangential outlet connected to the underflow port 103 is manufactured below the cylinder using the SU-8 photolithography process. The tangential outlet has a width of 70 μm and a depth of 100 μm. The overflow port 102 coaxial with the cylinder has a diameter of 70 μm and a height of 0.5 mm.

[0042] In one embodiment, precision pumps are connected to the tangential inlet 101, the overflow port 102 and the underflow port 103 to control the flow rate.

[0043] The method for separating and purifying high-concentration white blood cells, platelets and cytokines comprises the following steps:

[0044] S1. Rough separation removes most of the red blood cells. Whole blood diluted 1 to 10 times is injected into the micro hydrocyclone 1. Under the outward radial centrifugal force generated by the tangential velocity, different blood components remain dispersed and are driven by the fluid. Most of the red blood cells are discharged from the overflow port 103, and white blood cells, platelets and a small amount of red blood cells are discharged from the overflow port 102.

[0045] S2: Separate the white blood cells. The white blood cells, platelets and a small amount of red blood cells in step S1 continue to flow into the microcolumn array 2. The deterministic lateral displacement principle is used to separate the white blood cells and red blood cells, and a high-concentration white blood cell and diluted platelet solution is obtained.

[0046] S3, platelet concentration: the diluted platelet solution in step S2 continues to flow into the asymmetric curved channel 3, and the buffer and red blood cells are removed to obtain concentrated platelets. The asymmetric curved channel 3 is an inertial focusing pipe with an asymmetric inertial focusing bending separation microchannel structure. The inertial focusing pipe is composed of multiple narrow turns and multiple wide turn intervals. The end of the inertial focusing pipe forms an outer outlet for the flow of plasma, buffer and red blood cells and an inner outlet for the circulation of platelets.

[0047] S4. The leukocytes, cytokines and platelets are pooled. The concentrated platelets in step S3 and the high-concentration leukocytes in step S2 are pooled to obtain a concentrated autologous leukocyte, platelet and cytokine solution.

[0048] In one embodiment, 2-5% dextran buffer is used for dilution in S1.

[0049] In one embodiment, the whole blood flow rate in step S1 is 1 ml / min, and the blood cell concentration is 10 4 -10 8 / ul.

[0050] Example 1: Combination Figure 1The array microfluidic chip comprises: a micro hydrocyclone 1, a micro pillar array 2 and an asymmetric curved channel 3. The micro pillar array 2 located between the micro hydrocyclone 1 and the asymmetric curved channel 3 is respectively connected to the micro hydrocyclone 1 and the asymmetric curved channel 3; the micro hydrocyclone 1 is a device that uses fluid pressure to generate rotation to separate substances, and is provided with a tangential inlet 101 on the top, an overflow port 102 on the top, and an underflow port 103 on the bottom; the interior of the micro pillar array 2 is provided with multiple rows of columns to form a concave-convex array, with a micro pillar inlet 201 at the front end, a first outlet 202 at the rear end and a The flow channel ratio of the second outlet 203, the first outlet 202 and the second outlet 203 is 1:5; the asymmetric curved channel 3 is composed of multiple narrow turns 301 and multiple wide turns 302 at intervals, and the ends of the asymmetric curved channel 3 form an outer outlet 303 for the flow of red blood cells and buffer and an inner outlet 304 for the circulation of platelets; the micropillar array 2 is connected to the overflow port 102 of the micro hydrocyclone 1, and the asymmetric curved channel 3 is connected to the second outlet 203 of the micropillar array 2. The first outlet 202 of the micropillar array 2 and the inner outlet 304 of the asymmetric curved channel 3 are both connected to the collecting pipe 4.

[0051] The method for separating and purifying high-concentration white blood cells, platelets and cytokines comprises the following steps:

[0052] S1. Rough separation removes most of the red blood cells. A micro hydrocyclone 1 is used to separate the particles in the blood treatment. A single flow rate of 1 ml / min is used to inject 2-5% dextran buffer diluted 1-10 times of whole blood. Due to the outward radial centrifugal force generated by the tangential velocity, the particles are pushed to the wall according to their size, shape and density. Different blood components remain dispersed and are driven by the fluid and discharged through different outlets. Among them, after the red blood cells are mixed with glucose, their density and force area increase. They follow the outer downward vortex and are discharged through the overflow port 103, while white blood cells, platelets and a small amount of red blood cells are discharged through the internal upward vortex and the overflow port 102. Figure 2 As shown; in the micro hydrocyclone 1 cell is subjected to centrifugal force and drag force, the force analysis is shown in Figure 3 ,

[0053] Working principle of cyclone: The fluid velocity moves in a spiral pattern, so the fluid velocity can be decomposed into two component velocities: tangential component v t and the radial velocity component v r If the diameter is D p And the volume is v p The particles are rotating in the upper cylindrical component of the cyclone with a diameter of Dc and a rotation radius of r from the central axis of the cyclone. ris the drift velocity when dynamic equilibrium is reached, the drag friction generated by the particle moving in the fluid balances the centrifugal force of rotation, and the particle has no radial acceleration and travels at a constant speed. p is the particle density, ρ f is the fluid density,

[0054]

[0055] When the cyclone size and fluid properties are given, the cutoff diameter Dp of the particles that can be separated by the cyclone, i.e. the filtration radius, can be obtained. Particles exceeding this radius will be separated.

[0056] S2, fine separation to separate white blood cells. The white blood cells, platelets and a small amount of red blood cells in step S1 continue to flow into the microcolumn array 2. White blood cells are separated according to the size of different blood components. The separation of white blood cells and red blood cells is achieved by the column array in the microfluidics. The diameter of white blood cells is 10-20 μm, and the thickness of red blood cells is 2 μm and the diameter is 9 μm. Using the deterministic lateral displacement principle, the geometric shape of the cylindrical microcolumn is designed to have a critical diameter of 5 μm, which deflects white blood cell particles. The ratio of the two outlet flow channels is 1:5, and a high concentration of white blood cells and diluted platelet solution can be obtained. Figure 4 It is understood that the principle formula of microarray separation follows the literature: [Davis, John A., et al. "Deterministic hydrodynamics: taking blood apart." Proceedings of the National Academy of Sciences 103.40 (2006): 14779-14784.]. The fluid flows through a microcolumn concave-convex array, in which each row of columns is designed to be slightly laterally offset relative to the row in front of it. Particles below the critical fluid dynamics diameter Dc circulate along the streamlines through the gaps, moving in an average rightward flow direction. Particles above the critical fluid dynamics diameter do not fit into the first streamline, but are moved to sequential streamlines at each column by the fluid dynamics lateral resistance. Therefore, their movement is not parallel to the fluid flow, but at an angle determined by the ratio of the column offset to the row spacing. The size of the critical diameter Dc is related to the offset angle and the microcolumn gap, and the critical diameter calculation formula is Dc = 1.4gε 0.48 , g represents the gap width between the cylinders, ε represents the relative displacement fraction; ε = d / λ = tanθ, the distance between the cylinders is λ, and the relative displacement between adjacent cylinder rows is d. The critical diameter of the cylinder is 5μm. This angle θ is the direction of movement of the impacting object when passing through the array, combined with Figure 5 understand.

[0057] S3, platelet concentration: the diluted platelet solution in step S2 continues to flow into the asymmetric curved channel 3, and the buffer and red blood cells are removed to obtain concentrated platelets.

[0058] The asymmetric curved channel 3 is an inertial focusing pipe having an asymmetric inertial focusing curved separation microchannel structure, which realizes platelet enrichment.

[0059] The asymmetric curved channel 3 is composed of multiple narrow turns 301 and multiple wide turns 302. The width of the narrow turn 301 is 350 μm, and the width of the wide turn 302 is 650 μm. The average curvature radius of the narrow turn 301 and the wide turn 302 are 325 μm and 890 μm, respectively. This geometric shape causes the Dean resistance of the asymmetric system to be 8 times larger in small radius turns than in large turns. The end of the asymmetric curved channel 3 forms an outer outlet 303 for the flow of plasma and buffer and an inner outlet 304 for the circulation of platelets.

[0060] Regarding the design description of the channel width and curvature radius at the narrow turn 301 and the wide turn 302 in the asymmetric curved channel 3, there is a correlation, and the formula is as follows:

[0061]

[0062] Where r is the average curvature radius of the channel, r1 is the curvature radius of the narrow turn 301, r2 is the curvature radius of the wide turn 302, and D h is the hydraulic diameter of the channel, a c is the cutoff diameter of the particle, that is, the particle diameter that the channel can separate.

[0063] In the experiment, we obtained a set of feasible parameters: In this application we set a c The width of the narrow bend is 325μm, the height is 890μm, the width at the bend with a small curvature radius is 350μm, and the width at the bend with a large curvature radius is 650μm. The average curvature radii of narrow bends and wide bends are 325μm and 890μm respectively.

[0064] There are two main fabrication processes for the chips in steps S2 and S3: Micropillar arrays below 100 mm are fabricated using PBMS soft lithography, which is low-cost. Specifically, a photolithography method is used, such as SU-8, to generate a master structure in a photoresist, and then the SU-8 master structure is replicated in a soft elastomeric polymer material, usually PDMS.

[0065] S4. The leukocytes, cytokines and platelets are pooled. The concentrated platelets in step S3 and the high-concentration leukocytes in step S2 are pooled to obtain a concentrated autologous leukocyte, platelet and cytokine solution.

[0066] The array microfluidic chip of the present invention combines a micro hydrocyclone, a microcolumn array and an asymmetric curved channel for use. Through a staged purification method, whole blood can remove most of the red blood cells by passing through the micro hydrocyclone, and then enter the microcolumn array to separate high-concentration white blood cells. The remaining diluted platelet solution continues to enter the asymmetric curved channel to eliminate the red blood cells to obtain concentrated platelets, which are then combined with the high-concentration white blood cells in the previous stage to finally obtain a high-concentration autologous white blood cell, platelet and cytokine solution while maintaining its activity. Compared with traditional centrifugation and existing microfluidic methods, the present invention can achieve a simpler, gentler and continuous high-throughput cell sorting and solution preparation method, thereby producing a solution with higher concentration, stronger functionality and rich in highly active white blood cells and cytokines for the treatment of diseases such as arthritis. High flow is reflected in a flow rate greater than 10 mL / min, and high concentration is higher than 10 7 cells / mL to meet the needs.

[0067] The preparation process of the autologous leukocyte and cytokine solution is as follows: 20 ml of the patient's autologous blood is drawn; the blood and buffer are pumped into the array microfluidic chip of this application to obtain approximately 3 ml of a mixed solution of autologous leukocytes and cytokines; the mixed solution is passed through a concentration tube or 8KDa filter paper to obtain 1.5 ml of a concentrated autologous leukocyte and cytokine solution; this solution can be directly injected into the joint cavity or ligament injury site for the treatment of chronic traumatic aseptic inflammatory diseases, such as arthritis and tendonitis.

[0068] Example 2: Combination Figure 7 Understand that the main body of the micro hydrocyclone 1 is a cylinder with a diameter of 350μm and a height of 0.5mm, namely PMMA2 in the figure; the tangential inlet 101 is formed on the PMMA2 substrate by the SU-8 photolithography process through photoresist 1, and the tangential outlet is formed on the PMMA3 substrate by the SU-8 photolithography process through photoresist 2. The overflow port 102 is located at the top of the main body, namely PMMA1 in the figure, and PMMA1, PMMA2 and PMMA3 are bonded together. For the separation of particles, the classic design of the Bradley hydrocyclone is used. The tangential inlet channel at the top of the cylinder has a width of 50μm, a thickness / depth of 100μm, a flow rate of 1ml / min, and a blood cell concentration of 10 4 -10 8 / ul, manufactured on a wafer using SU-8 photolithography. The cyclone has a conical bottom, connecting the cylinder and the overflow port, and is manufactured on a PMMA substrate using micron milling technology. The outlet is divided into an overflow port 102 and an overflow port 103. The tangential outlet at the bottom of the cylindrical hole is connected to the overflow port 103. The tangential outlet has a width of 70μm, a thickness of 100μm, and a flow rate of 600μl / min. -1 The overflow port 102 has a diameter of 70 μm along the cylindrical axis, a height of 0.5 mm, and a flow rate of 400 μl min-1 Precision pumps are connected to the inlet and outlet to control the flow rate. In micropillar array 2, the rear row of pillars is laterally offset from the front to the back relative to the front row. The critical diameter of the pillars is 5 μm.

[0069] During use, the array microfluidic chip is connected to a controller to form a separation and purification system with real-time closed-loop feedback control. By parallelizing the microchannels, 2 to 5 microchannels can be used in parallel to increase the total flow rate to 10 ml / min.

[0070] Example 3: A small cyclone for rapidly separating plasma from whole blood can be used in battlefield situations, far from hospitals and without access to centrifuges, to treat patients in hemorrhagic shock who require rapid plasma transfusions, as well as patients who require platelet-rich plasma for wound healing. The cyclone flow rate is modified to 100 ml / min due to its high throughput, low cost, and low maintenance requirements. First, 450 ml of PBS buffer containing 5% glucose is introduced at a rate of 100 ml / min and circulated through the cyclone. Then, 50 ml of undiluted whole blood is slowly injected at a rate of 10 ml / min. High centrifugal forces cannot guarantee cell viability. However, they are suitable for rapid solid-liquid separation of plasma, platelets, and blood cell-free components. The cyclone can be directly manufactured from 3D printing, and the cyclone design follows the formula in this application. Example dimensions: 1.5 mm overflow port, 1 mm underflow port and tangential inlet port, and 3 mm cyclone diameter.

[0071] To increase the throughput to more than 100 ml, the micropillar arrays were realized on silicon wafers using deep reactive ion etching and the Bosch process in silicon after an initial photolithography step, resulting in deeply etched and high-aspect-ratio microstructures.

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. An array microfluidic chip, characterized in that: include: A micro hydrocyclone (1), a micro-pillar array (2) and an asymmetric curved flow channel (3); the micro-pillar array (2) located between the micro hydrocyclone (1) and the asymmetric curved flow channel (3) is connected to the micro hydrocyclone (1) and the asymmetric curved flow channel (3) respectively. The micro hydrocyclone (1) is a device that uses fluid pressure to generate rotation to separate substances. It is provided with a tangential inlet (101) on the top, an overflow port (102) on the top, and an underflow port (103) on the bottom. The micro-column array (2) is internally provided with multiple rows of columns to form a concave-convex array. The front end is provided with a micro-column inlet (201), and the rear end is provided with a first outlet (202) and a second outlet (203). The flow channel ratio of the first outlet (202) to the second outlet (203) is 1:

5. The asymmetric curved channel (3) is composed of a plurality of narrow turns (301) and a plurality of wide turns (302) at intervals. The ends of the asymmetric curved channel (3) form an outer outlet (303) for the flow of red blood cells and buffer and an inner outlet (304) for the flow of platelets. The microcolumn inlet (201) is connected to the overflow port (102), the asymmetric curved channel (3) is connected to the second outlet (203), and the first outlet (202) and the inner outlet (304) are both connected to the collecting pipe (4).

2. The array microfluidic chip according to claim 1, characterized in that: In the microcolumn array (2), the columns in the rear row are laterally offset from the front end to the rear end relative to the columns in the front row.

3. The array microfluidic chip according to claim 2, characterized in that: In the micropillar array (2), the critical diameter of the cylinder is Dc, Dc = 1.4gε 0.48 , g represents the gap width between the cylinders, ε represents the relative displacement fraction; ε = d / λ, the distance between the cylinders is λ, and the relative displacement between adjacent column rows is d.

4. The array microfluidic chip according to claim 3, characterized in that: In the micropillar array (2), the critical diameter of the pillar is 5 μm.

5. The array microfluidic chip according to claim 1, characterized in that: The micro hydrocyclone (1) adopts the classic design of Bradley hydrocyclone.

6. The array microfluidic chip according to any one of claims 1 to 5, characterized in that: In the micro hydrocyclone (1), a cylinder is used as the main body, and a tangential inlet (101) is manufactured on the cylinder using SU-8 photolithography technology. The channel width of the tangential inlet (101) is 50 μm and the depth is 100 μm. A tangential outlet connected to an underflow port (103) is manufactured below the cylinder using SU-8 photolithography technology, wherein the width of the tangential outlet is 70 μm and the depth is 100 μm; The overflow port (102) coaxial with the cylinder has a diameter of 70 μm and a height of 0.5 mm.

7. The array microfluidic chip according to claim 6, characterized in that: The tangential inlet (101), the overflow port (102) and the underflow port (103) are connected to precision pumps to control the flow rate.

8. A method for separating and purifying high-concentration leukocytes, platelets and cytokines, characterized in that: Here are the steps: S1. Rough separation removes most of the red blood cells. Whole blood diluted 1 to 10 times is injected into the micro hydrocyclone (1). Under the outward radial centrifugal force generated by the tangential velocity, different blood components remain dispersed and are driven by the fluid. Most of the red blood cells are discharged from the overflow port (103), and white blood cells, platelets and a small amount of red blood cells are discharged from the overflow port (102). S2, fine separation to separate the white blood cells, the white blood cells, platelets and a small amount of red blood cells in step S1 continue to flow into the microcolumn array (2), and the deterministic lateral displacement principle is used to achieve the separation of white blood cells and red blood cells, and obtain a high-concentration white blood cell and diluted platelet solution. S3, platelet concentration, the diluted platelet solution in step S2 continues to flow into the asymmetric curved channel (3), and the buffer and red blood cells are removed to obtain concentrated platelets. S4. The leukocytes, cytokines and platelets are pooled. The concentrated platelets in step S3 and the high-concentration leukocytes in step S2 are pooled to obtain a concentrated autologous leukocyte, platelet and cytokine solution.

9. The method for separating and purifying high-concentration leukocytes, platelets and cytokines according to claim 8, characterized in that: S1 was diluted with 2-5% dextran buffer.

10. The method for separating and purifying high-concentration leukocytes, platelets and cytokines according to claim 8, characterized in that: In step S1, the flow rate of whole blood injected is 1 ml / min, and the blood cell concentration is 10 4 -10 8 / ul.

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