Sheath-free flow filtering and dispersing structure, focusing structure and filtering, dispersing and focusing structure

Abstract

The invention belongs to the technical field of microfluidics, and discloses a sheath-flow-free filtering and dispersing structure, a focusing structure and a filtering, dispersing and focusing structure. According to the invention, a sheath-flow-free filtering and dispersing structure is designed, an inflowing particle flow is divided into two branches through the bifurcated flow channel, each branch realizes primary filtering and separation after passing through the first bent channel, one part of the particle flow flows into the auxiliary filtering branch flow channel, and the other part of the particle flow flows into the second bent channel; the other part of fluid flows into a main filtering branch flow channel and is subjected to secondary filtering separation at a second curve and an array branch, and the two parts of fluid are converged in a first outlet flow channel to realize particle filtering dispersion; then the particles enter the sheath-free flow focusing structure, particle-free branches are led out from the two sides of an inlet of a middle branch respectively and symmetrically converge into a second outlet flow channel from the two sides through side branches, and particle flow centering focusing is achieved through the combined action of particle flow velocity direction correction of the middle branch and convergence of the side branches. The two structure modules can highly uniformly disperse and focus particle flows such as cells, and have good long-term stability and anti-interference performance.

Description

Technical Field

[0001] The invention belongs to the field of microfluidics technology, and relates to a particle flow single dispersion focusing technology, and specifically to a sheathless flow filtering dispersion structure, a focusing structure and a filtering dispersion focusing structure. Background Art

[0002] In single-cell analysis scenarios based on microfluidics, such as single-cell imaging, single-cell sorting, and single-cell encapsulation, it is necessary to control the dispersion and focusing of cells in the fluid into a flow that approximates a line, and passes through the specified flow channel area in sequence. At present, the known single-cell dispersion and focusing technology is achieved by sheath flow. Through multi-layer nested pipelines, the cell suspension is surrounded by sheath fluid, the cell suspension is in the middle as the core flow, and the two sides or a circle around are sheath flow. When the core flow and the sheath flow merge, the cell suspension is dispersed by the symmetrical sheath flow, forming a single arranged cell flow in the middle of the flow channel. However, the flow rate of the sheath flow and the core flow needs to be strictly controlled, and whether the cells can form a single arranged cell flow in the flow channel depends on the ratio of the two. Affected by the properties of the fluid, the cell dispersion and focusing achieved by the sheath flow takes tens of seconds or even longer to achieve stability. At the same time, the cell dispersion and focusing achieved by the sheath flow technology has poor stability. Common phenomena such as gradual blockage of the front-end filtration and rear-end operation will cause the flow resistance of the flow channel to change, thereby destroying the stable flow environment of the sheath flow and the core flow, causing the cell focusing to fluctuate or fail. These problems lead to poor stability and generalization of the single-cell dispersion focusing system achieved by sheath flow technology, making it difficult to work stably for more than a few hours and not conducive to integration with unstable flow structures. In addition, the sheath flow dispersion focusing system requires additional flow channel inlets, pumps and other equipment, which increases cost and complexity.

[0003] Sheath flow technology is a commonly used technology in the field of microfluidics. For example, patent CN110343611A discloses a microfluidic chip, which includes a tightly fitting control layer, an elastic film layer, a fluid channel layer and an electromagnetic microvalve, wherein: the control layer is located at the top, and a plurality of control cavities are arranged inside the control layer, and a blocking magnet is installed inside the control cavity; the elastic film layer is located in the middle; the fluid channel layer is located at the bottom, and a main channel, a sheath flow channel and a screening channel are arranged inside the fluid channel layer, the main channel is used to connect the cell pool, and the middle section of the main channel is used for external equipment detection, the sheath flow channels are arranged on both sides of the main channel to promote cell focusing, and the screening channel is arranged at the rear end of the main channel; the blocking magnet can realize up and down movement under the control of the electromagnetic microvalve, and the up and down movement of the blocking magnet can drive the elastic film layer to block different screening channels to realize cell screening. Figure 2The sheath flow channel allows the sheath flow to enter from the left side and flow into the main channel through two branches, symmetrically flowing into the main channel to achieve focusing. Although focusing can be achieved (walking in the middle of a flow channel wider than the cell), inevitably, the sheath flow input of the sheath flow channel is required, which is equivalent to that the chip structure has at least two inlets, resulting in large equipment and material costs during use. At the same time, the flow rates of the sheath flow and the core flow need to be strictly controlled. Whether the cells can form a single row of cell flow in the flow channel depends on the ratio between the two. Affected by the fluid properties, it takes dozens of seconds or even longer for the cell dispersion and focusing achieved by the sheath flow to reach stability. In this sheath flow mode, if the inlet flow rate at the sheath flow channel is too large, it is very difficult for the cells to flow out at the main channel. That is, if the flow rate at the front end of the main channel fluctuates, the centered focusing will fail.

[0004] The prior art CN103341372A discloses a microfluidic chip structure for a flow cytometer, which mainly includes a sample liquid inlet, a columnar array structure, a conical focusing structure, a microchannel, a detection area, a flow expansion channel, and a waste liquid outlet end. The columnar array structure is located near the sample liquid inlet of the conical focusing structure, and the size interval between adjacent rows of micro-columns is d1, and d1 satisfies: d0 < d1 < d2, where d0 is the maximum cell diameter in the injected sample liquid, and d2 is the cross-sectional width of the microchannel. The focusing effect of the conical focusing structure enables the cell particles to flow into the microchannel individually. The microchannel confines the cells through the channel to make them pass through the detection area individually. The cross-sectional width d2 of the microchannel satisfies: d0 < d2 < 1.5d0. The detection area is the area illuminated by the detection beam of the optical detection system. The flow expansion channel mainly realizes the evacuation of the waste liquid after detection. The waste liquid outlet end is used to connect the catheter leading out of the chip, so that the waste liquid flows out of the entire microfluidic chip. This technology uses a sheathless technology restricted by the flow channel, with filtration at the front and restriction of the cells passing through a narrow flow channel at the back. However, this kind of structure has many problems, resulting in its inability to keep the cells centered for a long time and being difficult to be applied in the actual field. First of all, ordinary filtration is likely to break the cells. In the figure, the filtration position formed by the columnar array structure is perpendicular to the cell flow direction, and the cells will directly hit the cylinders of the columnar array structure. Secondly, as the garbage continuously accumulates at the filtration position, the garbage, like the columnar array structure, will be perpendicular to the cell flow direction and continue to break the cells, damaging the sample. At the same time, the broken cells will block the flow channel. Then, the flow channel restricting the cell flow at the back is relatively narrow, about the size of 2 cells, and is easily blocked by the cell debris gradually washed down from the filtration area. Secondly, the cell dispersion of this structure is not good. Although the flow channel restricts the cells to pass through individually, it cannot make the intervals between each cell as evenly separated as possible. That is, a group of cells that are very close to each other originally will still be very close in the flow channel, without a dispersion function.

[0005] Prior art CN107674820A discloses a microfluidic device for sorting cells, in which a pre-focusing channel with a waveform extension is arranged in the flow path. Through the viscoelastic effect and inertial effect of the microfluid, the cell particles in the sample solution are gradually gathered to the center of the flow path, and then pass through the arc-shaped bifurcated flow path and the sample sorting flow path, so that the cell particles are pushed away from the wall by the combined action of the inertial lift induced by the wall and the elastic force induced by the viscoelastic fluid. Cells of different sizes are subjected to different thrusts, so that cells of different sizes accumulate sufficient distance differences when migrating in the sample sorting flow path, and finally achieve the effect of separation and collection. This technology uses a periodic waveform curve to set up a pre-focusing channel, and uses the viscoelastic effect and inertial effect of the microfluidic to gradually move the cells to the center of the channel where the force is balanced. It can only ensure that the cells are initially centered in the channel, but cannot preserve the linear structure or increase the distance between cells (no dispersion function). In fact, linear dispersion focusing cannot be performed at all. The subsequent arc-shaped bifurcated channel can only complete the function of dividing the cell liquid into two channels, and has no focusing function. In fact, according to the application text, in the arc-shaped bifurcated channel, the cells actually flow along the inner wall of the channel. Therefore, it is not a true dispersion focusing technology.

[0006] In summary, there is no good technology in the existing technology to achieve sheathless flow single-cell focusing and dispersion, so it is necessary to carry out relevant research and development. Summary of the invention

[0007] One of the purposes of the present invention is to provide a sheathless flow filtration dispersion structure based on microfluidics to solve the problems of uneven dispersion of particle flows such as cells and inability to filter large particle impurities in the prior art.

[0008] Another object of the present invention is to provide a sheathless flow focusing structure based on microfluidics, which adopts a brand-new sheathless flow focusing structure to avoid the defects of sheath flow focusing.

[0009] Another object of the present invention is to provide a sheathless flow filtering, dispersing and focusing structure. By combining the sheathless flow filtering, dispersing and focusing structure with the sheathless flow focusing structure, cells and other particles can be dispersed and focused uniformly and with high quality. In the absence of sheath flow, the uniform outflow of cells and other particles to form a highly centered chain particle flow is ensured, providing technical support for single-cell analysis based on microfluidics, such as single-cell imaging, single-cell sorting, and single-cell encapsulation.

[0010] In order to solve the above problems, the present invention adopts the following technical means:

[0011] In one aspect, a microfluidics-based sheathless flow filtration dispersion structure comprises

[0012] A first inlet flow channel is a flow channel for providing inflow;

[0013] A first outlet flow channel is a flow channel for providing outflow;

[0014] A bifurcated flow channel is provided at the end of the first inlet flow channel and is open in a V-shape to divide the fluid into two branches;

[0015] Two main filtering branch flow channels are connected to each branch end of the bifurcated flow channel through first bends that turn inwards relatively, and each main filtering branch flow channel is also provided with a second bend, so that the outlets of the two main filtering branch flow channels merge downstream of the first outlet flow channel;

[0016] The inlets of the two auxiliary filtering branch flow channels are respectively connected to the ends of the corresponding first bends and are arranged inwardly relative to the main filtering branch flow channel, so that after the fluid passes through the first bend, relatively small particles flow into the auxiliary filtering branch flow channel under the action of centrifugal force; the outlets of the two auxiliary filtering branch flow channels merge to form the source of the first outlet flow channel; and

[0017] The array fork is arranged in the outlet of each main filtering branch flow channel and is located downstream of the second bend. The forks in the array fork are arranged along the flow direction of the first outlet flow channel, so that under the combined action of the centrifugal force generated in the second bend and the change of flow resistance in the array fork, the particles in the particle flow are merged into the first outlet flow channel through the array fork at intervals to achieve particle dispersion, and large particle impurities are thrown to the outermost side under the action of centrifugal force, and are filtered and retained by the array fork to achieve filtration.

[0018] Furthermore, the bifurcated flow channel is arranged in alignment with the first inlet flow channel, and the two branches of the bifurcated flow channel are arranged symmetrically.

[0019] Furthermore, the sheathless flow filtration dispersion structure is symmetrically arranged with respect to a center line connecting the first inlet flow channel and the first outlet flow channel.

[0020] Furthermore, the main filtering branch flow channel gradually widens from the inlet to the outlet.

[0021] Furthermore, the inlet width of the auxiliary filter branch flow channel is smaller than the inlet of the main filter branch flow channel, so that the inlet flow of the auxiliary filter branch flow channel is smaller than that of the main filter branch flow channel. At the same time, the auxiliary filter branch flow channel gradually widens from the inlet to the outlet to maintain a suitable flow resistance ratio between the auxiliary filter branch flow channel and the main filter branch flow channel, thereby reducing the flow resistance of the entire structure as much as possible.

[0022] Furthermore, the included angle of the bifurcated flow channel is 60-180 degrees.

[0023] Furthermore, the turning angle of the first curve is 30-90 degrees.

[0024] Furthermore, the turning angle of the second curve is 15-120 degrees.

[0025] Furthermore, a cross section of the first outlet flow channel gradually increases along the flow direction.

[0026] Furthermore, the array bifurcation is a comb-tooth structure formed by arranging a plurality of partitions, and the gap between adjacent comb teeth is a bifurcation for particles to pass through.

[0027] Furthermore, along the flow direction of the main filtering branch flow channel, the internal width of each branch of the comb-tooth structure gradually increases to reduce the flow resistance of the entire structure.

[0028] Furthermore, in the comb-tooth structure, along the flow direction of the first outlet flow channel, the inlet width of each branch gradually decreases, so as to obtain better large impurity filtering performance while ensuring that the flow resistance of the entire structure is small.

[0029] On the other hand, the present invention provides a microfluidic chip with a sheathless flow filtration and dispersion function, the microfluidic chip comprising a chip substrate and a sheathless flow filtration and dispersion flow channel prepared on the chip substrate, the sheathless flow filtration and dispersion flow channel comprising

[0030] A first inlet flow channel is a flow channel for providing inflow;

[0031] A first outlet flow channel is a flow channel for providing outflow;

[0032] A bifurcated flow channel is arranged at the end of the first inlet flow channel and is aligned with the center of the first inlet flow channel, and is arranged to be open in a V-shape, and is used to divide the fluid into two symmetrical branches;

[0033] Two main filtering branch flow channels are connected to each branch end of the bifurcated flow channel through first bends that turn inwards relatively, and each main filtering branch flow channel is also provided with a second bend, so that the outlets of the two main filtering branch flow channels merge downstream of the first outlet flow channel;

[0034] The inlets of the two auxiliary filtering branch flow channels are respectively connected to the ends of the corresponding first bends and are arranged inwardly relative to the main filtering branch flow channel, so that after the fluid passes through the first bend, relatively small particles flow into the auxiliary filtering branch flow channel under the action of centrifugal force; the outlets of the two auxiliary filtering branch flow channels merge to form the source of the first outlet flow channel; and

[0035] The array fork is arranged in the outlet of each main filtering branch flow channel and is located downstream of the second bend. The forks in the array fork are arranged along the flow direction of the first outlet flow channel, so that under the combined effect of the centrifugal force generated in the second bend and the change of flow resistance in the array fork, the particles in the particle flow are merged into the source of the first outlet flow channel formed by the outlet of two symmetrical auxiliary filtering branch flow channels through the array fork at intervals, thereby realizing particle dispersion. Large particle impurities are thrown to the outermost side under the action of centrifugal force and filtered and retained by the array fork to realize filtration.

[0036] On the other hand, the present invention provides a sheathless flow focusing structure based on microfluidics, comprising

[0037] The second inlet flow channel is a flow channel for providing inflow;

[0038] A second outlet flow channel is a flow channel for providing outflow;

[0039] an intermediate branch connected to the end of the second inlet flow channel and the inlet end of the second outlet flow channel, and having a cross-section that decreases along the flow direction; and

[0040] Two side branches are symmetrically arranged on both sides of the middle branch, the inlets of the two side branches guide the flow from the openings on both sides of the inlet of the middle branch, the outlets of the two side branches are connected to the inlet end of the second outlet flow channel, and the inlet width of the side branches is smaller than the inlet width of the middle branch;

[0041] The outlets of the two side branches are arranged on both sides of the outlet of the middle branch to form a confluence at the inlet end of the second outlet flow channel.

[0042] Furthermore, the outlet width of the side branch is greater than the outlet width of the middle branch.

[0043] Furthermore, the width of the second inlet flow channel is smaller than the inlet width of the middle branch.

[0044] Furthermore, the side branch includes an inlet section, a turning section and an outlet section, the inlet section forms an obtuse angle with the flow direction of the fluid in the middle branch, and the outlet section forms an acute angle with the flow direction in the second outlet channel after the confluence.

[0045] Furthermore, the second outlet flow channel includes a focusing section and a reducing section, the focusing section is used to merge the fluids of the two side branches to both sides of the fluid of the middle branch to form a focused confluence, and the reducing section is used to adjust the focused confluence to a desired width.

[0046] Furthermore, the width of the focusing segment is greater than the width of the middle branch outlet.

[0047] On the other hand, the present invention provides a sheathless flow filtering dispersion focusing structure, comprising the above-mentioned sheathless flow filtering dispersion structure and the sheathless flow focusing structure, wherein the first outlet flow channel of the sheathless flow filtering dispersion structure is connected to the second inlet flow channel of the sheathless flow focusing structure through a transition flow channel.

[0048] On the other hand, the present invention provides a microfluidic chip with a sheathless flow focusing function, comprising a chip substrate and a sheathless flow focusing channel prepared on the chip substrate, wherein the sheathless flow focusing channel comprises

[0049] The second inlet flow channel is a flow channel for providing inflow;

[0050] A second outlet flow channel is a flow channel for providing outflow;

[0051] an intermediate branch connected to the end of the second inlet flow channel and the inlet end of the second outlet flow channel, and having a cross-section that decreases along the flow direction; and

[0052] Two side branches are symmetrically arranged on both sides of the middle branch, the inlets of the two side branches guide the flow from the openings on both sides of the inlet of the middle branch, the outlets of the two side branches are connected to the inlet end of the second outlet flow channel, and the inlet width of the side branches is smaller than the inlet width of the middle branch;

[0053] The outlets of the two side branches are arranged on both sides of the outlet of the middle branch to form a confluence at the inlet end of the second outlet flow channel.

[0054] On the other hand, the present invention provides a microfluidic chip with sheathless flow filtration dispersion focusing function, including a chip substrate and a sheathless flow filtration dispersion focusing flow channel prepared on the chip substrate, wherein the sheathless flow filtration dispersion focusing flow channel includes the above-mentioned sheathless flow filtration dispersion flow channel and sheathless flow focusing flow channel.

[0055] In another aspect, the present invention provides a particle flow filtration dispersion method without sheath flow, comprising the following steps:

[0056] A first inlet flow channel is configured for inputting a particle flow, wherein the particle flow contains large impurities;

[0057] A bifurcated flow channel is configured to divide the input particle flow into two branches opening in a V shape;

[0058] The first bend is configured to turn the two branch fluids inwards for the first centrifugation;

[0059] A main filtering branch flow channel is configured to receive the particle flow containing large impurities on the outside after the first bend;

[0060] An auxiliary filtering branch flow channel is configured to receive the particle flow after the first bend;

[0061] A first outlet flow channel is configured to guide out the particle flow from which large particles of impurities have been filtered out;

[0062] A second bend and an array fork are sequentially arranged in the main filtering branch flow channel, and the particle flow containing large particle impurities is subjected to a second centrifugation through the second bend and the array fork, so that the particles enter the first outlet flow channel in a dispersed manner, and the large particle impurities are accumulated on the outermost side of the array fork.

[0063] In another aspect, the present invention provides a sheathless particle flow focusing method, comprising the following steps:

[0064] A second inlet flow channel is configured for inflow of particle flow;

[0065] An intermediate branch with a gradually decreasing cross section is configured to correct the direction of particle velocity, gradually squeeze the particle flow and reduce its width to form a particle flow with the direction consistent with the central axis;

[0066] Configure side branches, take a branch without particle flow from the incoming particle flow, and symmetrically introduce the two branches into the two sides of the particle flow outflowing from the middle branch to form a protective layer, so as to achieve sheath-free flow focusing of the particle flow.

[0067] In another aspect, the present invention provides a sheathless particle flow filtration dispersion focusing method, comprising the following steps:

[0068] A first inlet flow channel is configured for inputting a particle flow, wherein the particle flow contains large impurities;

[0069] A bifurcated flow channel is configured to divide the input particle flow into two branches opening in a V shape;

[0070] The first bend is configured to turn the two branch fluids inwards for the first centrifugation;

[0071] A main filtering branch flow channel is configured to receive the particle flow containing large impurities on the outside after the first bend;

[0072] An auxiliary filtering branch flow channel is configured to receive the particle flow after the first bend;

[0073] A first outlet flow channel is configured to guide out the particle flow from which large particles of impurities have been filtered out;

[0074] A second bend and an array fork are sequentially arranged in the main filtering branch flow channel, and the particle flow containing large particle impurities is subjected to a second centrifugation through the second bend and the array fork, so that the particles enter the first outlet flow channel in a dispersed manner, and the large particle impurities are accumulated on the outermost side of the array fork;

[0075] A transition channel is configured to receive the particle flow that filters out large particles of impurities;

[0076] A second inlet flow channel is configured to receive the particle flow delivered through the transition flow channel;

[0077] An intermediate branch with a gradually decreasing cross section is configured to correct the direction of particle velocity, gradually squeeze the particle flow and reduce its width to form a particle flow with the direction consistent with the central axis;

[0078] Configure side branches, take a branch without particle flow from the incoming particle flow, and symmetrically introduce the two branches into the two sides of the particle flow outflowing from the middle branch to form a protective layer, so as to achieve sheath-free flow focusing of the particle flow.

[0079] Compared with the prior art, the present invention has the following beneficial effects:

[0080] The sheathless flow filtering and dispersion structure designed in the present invention can automatically intercept large particle impurities in the particle flow while dispersing without the need for additional filtering structures and sheath flow structures, thereby achieving filtering and dispersion. The present invention has a good dispersion effect. During the dispersion process, the array bifurcation naturally has the property of allowing particles to tend to enter intervals with smaller flow resistance, so that after the dispersion of the present invention, the particle flow hardly aggregates or there is no particle outflow for a long time.

[0081] The sheathless flow focusing structure designed in the present invention can achieve a dispersed focusing effect without using a sheath flow, is not limited by the application scenarios of the sheath flow, has good generalization and stability, allows long-term stable operation, is insensitive to external interference, and is not easily disturbed by problems such as flow channel blockage. No additional reagents are required except for the particle flow suspension sample, which greatly reduces the cost of consumables during the experiment. The chip of the present invention has a simple structure and is easy to operate. It does not require complex equipment control, which reduces equipment costs and labor costs.

[0082] The present invention combines the sheathless flow filtering dispersion structure and the sheathless flow focusing structure to achieve the focusing required by the sheath flow focusing technology while also making the particle flow intervals highly uniform, with a dispersion performance superior to that of the sheath flow focusing technology. In addition, the present invention has good chemical resistance and stability, allows long-term stable operation, is insensitive to external interference, and is not easily disturbed by problems such as flow channel blockage.

[0083] The sheathless flow filtering dispersion structure and the sheathless flow focusing structure of the present invention can be used in combination to achieve single-particle dispersion and focusing of the particle flow, or can be used separately to achieve filtering dispersion function and focusing function respectively. In specific applications related to cell analysis, one or two of the above structures can be selected to achieve the purpose according to the requirements for cells. At the same time, in the analysis of particles other than cells, the topological structure corresponding to the above structure can also be used to achieve dispersion and focusing; the operation and use are flexible, and can be used in combination according to different scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is a schematic diagram of the sheathless flow filtration dispersion structure in Example 1 of the present invention.

[0085] Figure 2 Schematic diagram of the sheathless flow focusing structure in Example 2 of the present invention.

[0086] Figure 3 Schematic diagram of the sheathless flow filtration dispersion focusing structure in Example 3 of the present invention.

[0087] Figure 4 Schematic diagram of highly controlled cell fluctuations in the vertical direction.

[0088] Figure 5Schematic diagram of the sheathless flow single-cell dispersion focusing chip in Example 4, wherein the blue dots are cells, the red blocks are garbage, and the yellow arrows represent the direction of fluid flow.

[0089] Figure 6 This is a schematic diagram of the single cell filtration dispersion chip in Example 5, wherein blue dots are cells, red blocks are garbage, and yellow arrows represent fluid flow directions.

[0090] Figure 7 Schematic diagram of the sheathless flow single-cell focusing chip in Example 6, wherein blue dots are cells, red blocks are garbage, and yellow arrows represent the direction of fluid flow.

[0091] Figure 8 Schematic diagram of the variable cross-section flow channel focusing chip in Comparative Example 1, where blue dots are cells, red blocks are garbage, and yellow arrows represent fluid flow direction.

[0092] Fig. 9 Schematic diagram of the sheath flow single-cell focusing chip in Comparative Example 1, where blue dots are cells, red blocks are garbage, and yellow arrows represent the direction of fluid flow.

[0093] Fig.10 This is the cell image in Example 4. It can be seen that each cell is separated, well centered, and has clear edges.

[0094] Fig.11A is the result image of the observation area in Example 6, wherein: Fig.11A (c) and (d) are images of the observation area in two consecutive time periods. Fig.11A It can be seen that the cells are uneven, with a large number of cells passing through in a short period of time or no cells passing through for a period of time, and the flux is unstable.

[0095] Fig. 11B This is the overall area result image of the sheathless flow focusing structure in Example 6. It can be seen that in the second outlet flow channel 230, the cell centering performance is very good, but there is local aggregation, a large number of cells pass through in a short period of time or no cells pass through for a period of time, and the flux is unstable.

[0096] Fig.12 This is the result image of Comparative Example 1. The cells are not centered, and many cells close to each other are easily photographed at the same time.

[0097] Note: Fig.12 and Fig.10 The cell image textures are different due to the different optical systems and camera models used for image acquisition, but this does not affect the observation of the outer contour and the position of the cells in the flow channel.

[0098] 100-sheathless flow filtration dispersion structure, 110-first inlet flow channel, 120-outlet flow channel, 130-forked flow channel, 131-flow direction, 140-main filtration branch flow channel, 141-front section filtration flow channel, 142-rear section filtration flow channel, 150-auxiliary filtration branch flow channel, 160-array bifurcation, 170-first bend, 180-second bend;

[0099] 200-sheathless flow focusing structure; 210-second inlet flow channel, 220-middle branch, 230-second outlet flow channel, 231-focusing section, 232-diameter reducing section, 240-side branch, 241-inlet section, 242-turning section, 243-outlet section;

[0100] 310-cells; 320-garbage;

[0101] 410 - feed inlet, 420 - transition channel, 430 - discharge channel, 440 - straight channel, 450 - outlet, 460 - variable cross-section channel, 470 - cell fluid feed inlet, 480 - sheath fluid feed inlet;

[0102] 510-substrate, 520-cover plate. DETAILED DESCRIPTION

[0103] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0104] In the description of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0105] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0106] Example 1: Figure 1As shown, this embodiment provides a microfluidics-based sheathless flow filtration dispersion structure 100, comprising

[0107] The first inlet flow channel 110 is a flow channel for providing inflow;

[0108] The first outlet flow channel 120 is a flow channel for providing outflow;

[0109] The bifurcated flow channel 130 is disposed at the end of the first inlet flow channel and is aligned with the center of the first inlet flow channel, and is arranged to be open in a V-shape, and is used to divide the fluid into two symmetrical branches;

[0110] Two main filtering branch flow channels 140 are connected to each branch end of the bifurcated flow channel 130 through first bends 170 that turn inwards relatively to each other. A second bend 180 is also provided in each main filtering branch flow channel 140, so that the outlets of the two main filtering branch flow channels 140 merge at the downstream of the first outlet flow channel 120;

[0111] The inlets of the two auxiliary filtering branch flow channels 150 are respectively connected to the ends of the corresponding first bends 170 and are arranged inwardly relative to the main filtering branch flow channel 140, so that after the fluid (particle flow or suspension) passes through the first bend 170, under the action of centrifugal force, relatively small particles flow into the auxiliary filtering branch flow channels 150; the outlets of the two auxiliary filtering branch flow channels 150 merge to form the source of the first outlet flow channel 120; and

[0112] The array fork 160 is disposed in the outlet of each main filtering branch flow channel 140 and is located downstream of the second bend 180. The forks in the array fork 160 are arranged along the flow direction of the first outlet flow channel 120, so that under the combined effect of the centrifugal force generated in the second bend 180 and the change in flow resistance in the array fork 160, the particles in the particle flow pass through the array fork 160 at intervals and merge into the first outlet flow channel 120, thereby achieving particle dispersion, and large particle impurities are thrown to the outermost side under the action of the centrifugal force ( Figure 1 ), filtering is achieved by bifurcating the array and filtering and intercepting.

[0113] In this embodiment, the particle flow to be filtered (a fluid formed by particles suspended in a liquid environment, the particles include cells, polyethylene balls, hydrogel balls, etc.) is divided into two branches through a bifurcated flow channel 130 that is opened in a V-shape. The two main filtering branch flow channels 140 are connected to each branch end of the bifurcated flow channel 130 through a first bend 170 that turns relatively inward. At the same time, an auxiliary filtering branch flow channel 150 is arranged on the inner side. When the particle flow passes through the first bend 170, a rotating fluid is formed to generate centrifugal force. Under the action of the centrifugal force, large particles are thrown to the right and enter the main filtering branch flow channel 140 together with the particle flow, and a small number of small particles are The sub-flow enters the auxiliary filtering branch flow channel 150 under the action of the swirl flow, and realizes the first filtering separation; the two auxiliary filtering branch flow channels 150 merge to form the source of the first outlet flow channel 120. Due to the symmetrical design, the fluids in the two auxiliary filtering branch flow channels 150 merge to form a forward propulsion flow in the first outlet flow channel 120; the larger particles and the large particle impurities enter the main filtering branch flow channel 140 together, and a swirl flow is formed again under the action of the second bend 180, generating centrifugal force, so that the particle flow is dispersed according to size and enters the array fork 160, and the large particle impurities accumulate on the right side of the array fork 160 ( Figure 1 The middle red color represents garbage 320 formed by the accumulation of large particles of impurities, which does not affect most of the functions of the array fork 160 itself. After being dispersed at the entrance of the array fork 160, particles of different sizes pass through each fork and enter the first outlet channel 120 in an alternating manner. The large particles of impurities are filtered and retained by the array fork 160, realizing a second filtration separation; the particles flow into the first outlet channel 120 in a dispersed manner under the joint action of the liquid flow converged downstream of the array fork 160 and the auxiliary filtration branch channel 140, realizing sheathless flow dispersed filtration.

[0114] When a particle is passing through a fork in the array fork 160, the flow resistance in the fork increases rapidly and the flow rate slows down. When a new particle reaches the entrance of the fork, it will flow with the fluid to a fork where no cells are passing nearby, thereby automatically avoiding the situation where multiple particles enter one fork continuously. As a result, the particle flow in the main filter branch channel 140 passes through the array fork 160 and enters the first outlet channel 120 one by one at intervals, so that the particle dispersion in the first outlet channel 120 is very good.

[0115] In this embodiment, by setting the inlet of the auxiliary filter branch flow channel 150 on the inner side relative to the main filter branch flow channel 140, when the particle flow passes through the first bend 170, due to the centrifugal force, large particle impurities enter the main filter branch flow channel 140 with most of the particle flow, and a small part of the particle flow without large particle impurities follows the action of the fluid to enter the auxiliary filter branch flow channel 150, thereby realizing the first filtering and separation of the particle flow, so that a small part of the smaller particle flow without large particle impurities enters the first outlet flow channel 120 through the auxiliary filter branch flow channel 150 and is automatically dispersed.

[0116] Since the two auxiliary filtering branch flow channels 150 directly merge to form the source of the first outlet flow channel 120 after merging, the smaller particle flow entering the auxiliary filtering branch flow channel 150 can quickly enter the first outlet flow channel 120, and since the flow resistance in the first outlet flow channel 120 is small, the smaller particles will reach the outlet end of the first outlet flow channel 120 as quickly as possible, thereby being dispersed from the particle flow containing large particle impurities entering the main filtering branch flow channel 140. After passing through the sheathless flow filtering dispersion structure 100 of the present invention, the particle flow can filter out large particle impurities, and the particles are individually distributed in the first outlet flow channel 120 along the flow direction of the flow channel.

[0117] In some embodiments, the sheathless flow filtering dispersion structure 100 is symmetrically arranged with respect to the center line of the first inlet flow channel 110 and the first outlet flow channel 120. The symmetrical arrangement ensures that the fluids of the two auxiliary filtering branch flow channels 150 can be dispersed in the center after entering the main filtering branch flow channel 140, thereby avoiding the formation of eddy currents, turbulence, etc., which may cause the particles to re-focus after dispersion. The symmetrical arrangement can also ensure that the kinetic energy of the two fluids at the confluence of the outlets of the two main filtering branch flow channels 140 is almost the same, and the particles can remain dispersed after confluence, thereby avoiding unidirectional vortex flow caused by inconsistent flow rates or inconsistent confluence angles, thereby causing re-focusing.

[0118] In some embodiments, the bifurcated flow channel 130 has a certain length, that is, a certain aspect ratio, such as an aspect ratio of 5:1-20:1, with 10:1 being the best. By setting a suitable aspect ratio, the end of the bifurcated flow channel 130 can maintain a sufficient flow velocity in the required flow direction, providing a basis for the centrifugal separation of the first bend 170. Similarly, the flow channel between the first bend and the second bend also needs to ensure that the particles have a sufficient flow velocity in the flow direction when entering the second bend.

[0119] See also Figure 1In some embodiments, the main filter branch flow channel 140 gradually widens from the first inlet flow channel 110 to the outlet; the fluid flow rate is reduced by the gradual main filter branch flow channel 140 to prevent particles from flowing too fast and hitting the array fork 160, while providing space for setting up enough forks on the array fork 160, thereby improving the dispersion efficiency and avoiding a large number of particles from concentrating at a certain fork to cause blockage.

[0120] It should be noted that the width of the array bifurcation 160 in the main filtering branch flow channel 140 can be estimated by the particle concentration. Generally, the number of cells reaching the array bifurcation at the same time estimated by the particle concentration shall not exceed twice the number of array gaps to avoid blockage of the array bifurcation 160. The optimal value is 1-2 times, which can not only avoid blockage but also achieve a better dispersion effect and prevent uneven spacing of particles in the first outlet flow channel 120 after dispersion.

[0121] It should be noted that the distance between the second bend 180 and the array fork 160 should not be too large or too small. If the distance is too large, it will not provide sufficient space for the array fork, and if the distance is too small, the speed of the particles will not drop sufficiently after passing the bend, affecting the dispersion effect.

[0122] It should be noted that the inlet width of the auxiliary filter branch flow channel 150 is smaller than the inlet of the main filter branch flow channel 140, so that the inlet flow of the auxiliary filter branch flow channel 150 is smaller than that of the main filter branch flow channel 140. At the same time, the auxiliary filter branch flow channel 150 gradually widens from the inlet to the outlet to maintain a suitable flow resistance ratio between the auxiliary filter branch flow channel and the main filter branch flow channel, thereby reducing the flow resistance of the entire structure as much as possible.

[0123] In some embodiments, the inlet width of the auxiliary filtering branch channel 150 can be set to be smaller than the diameter of the large particles, so that the filtering and separation effect at the inlet of the auxiliary filtering branch channel 150 is better.

[0124] See also Figure 1 The angle between the two branches of the bifurcated flow channel 130 is necessary to ensure that the particle flow turns to form centrifugal force. If the angle is too large, the flow resistance in the bifurcated flow channel 130 will be too large, and the particle flow will hit the side wall of the bifurcated flow channel 130 head-on, causing the cells and other particles to break, affecting the quality of the experiment. If the angle is too small, the subsequent first bend 170 and the second bend 180 will not have enough turning angles to form sufficient centrifugal force; therefore, the angle A between the two branches of the bifurcated flow channel 130 is 60-180 degrees, and 120-160 degrees has a better effect.

[0125] The turning angle of the first bend 170 greatly affects the efficiency of the particle flow entering the main filter branch flow channel 140 when the main filter branch flow channel 140 and the auxiliary filter branch flow channel 150 diverge. The larger the turning angle, the better the centrifugal effect of the particle flow. However, too large a turning angle will leave no margin for the second bend 180. Therefore, the turning angle B of the first bend 170 is in the appropriate range of 30-90 degrees, among which 60-80 degrees is the best.

[0126] See also Figure 1 Since a second bend 180 is provided in the main filtering branch channel 140, the main filtering branch channel 140 can be divided into a front filtering channel 141 and a rear filtering channel 142 with the second bend 180 as the boundary. Then the turning angle B of the first bend 170 is the deflection angle of the center line of the front filtering channel 141 relative to the center line of the corresponding forked channel 130.

[0127] The front-section filter channel 141 has a certain length, that is, a certain aspect ratio, such as a length-to-width ratio of 3:1-15:1; by setting a suitable aspect ratio (since the main filter branch channel 140 is a variable cross-section channel, it is actually an equivalent ratio), the end of the front-section filter channel 141 can maintain a sufficient flow velocity in the required flow direction, providing a basis for the centrifugal separation of the second bend 180.

[0128] See also Figure 1 The turning angle of the second bend 180 affects the particle flow and the arrangement of impurities on the array fork 160 according to their size. Therefore, a suitable turning angle needs to be set to achieve a better dispersion effect. If the turning angle is too small, the centrifugal force generated is insufficient, and large impurities cannot be separated, and the particle flow itself is difficult to disperse effectively. If the turning angle is too large, the flow direction of the rear filter channel 142 and the flow direction in the first outlet channel 120 may be too small. When the particles enter the first outlet channel 120, the lateral fluctuation is too large, and even a backflow is formed, which seriously affects the dispersion of the particles passing through the array fork 160 in the first outlet channel 120. Therefore, the turning angle C of the second bend 180 is generally 15-120 degrees, which is the appropriate range, and 30-60 degrees is the best. Since the main filter branch channel 140 gradually widens from the first inlet channel 110 to the outlet, the second turning angle is the deflection angle of the center line of the rear filter channel 142 relative to the center line of the front filter channel 141.

[0129] See also Figure 1It should be noted that the velocity component of the flow velocity in the rear-stage filtering channel 142 in the flow direction inside the first outlet channel 120 should be the same as the flow direction in the first outlet channel 120, otherwise it will cause the particles to flow back through the array bifurcation 160 into the first outlet channel 120, causing the particles to focus again. In this case, the angle D between the rear-stage filtering channel 142 and the first outlet channel 120 is required to be an acute angle. Generally speaking, the smaller the angle D between the rear-stage filtering channel 142 and the first outlet channel 120, the smaller the lateral fluctuation when merging into the first outlet channel 120 through the array bifurcation 160, and the better the merging effect. However, if the angle D is too small, the first bend 170 and the second bend 180 will not have sufficient turning angle margin. Generally, an angle D of 30-80 degrees is more appropriate.

[0130] In some embodiments, the first outlet channel 120 gradually increases in cross-section along the flow direction, and the flow velocity slows down as the width increases. At the same time, sufficient lateral movement margin is provided for particles to merge through the array fork 160 to prevent the particle flow from impacting the side wall of the first outlet channel 120 after merging into the first outlet channel 120.

[0131] It should be noted that in this embodiment, the size of each fork of the array fork 160 should match the size of the particles to be dispersed, so that a single particle can pass through each fork. For example, the size of each fork is larger than the largest single particle to be dispersed, and smaller than twice the largest single particle, so as to meet the requirement that a single particle can pass through each fork; preferably, it should also be smaller than the size of large impurities. The array fork 160 of the present invention can be either an array of holes or a comb-tooth structure formed by a number of partitions. Regardless of the structure, it is sufficient to meet the requirement that a single particle can pass through the corresponding channel. Figure 1 In this embodiment, the side of the array fork 160 close to the main filtering branch channel 140 is defined as a dispersion surface, and the dispersion surface is roughly parallel to the flow direction (center line) of the first outlet channel 120, so that the rear-stage filtering channel 142 is set at an angle to the dispersion surface. After the particle flow contacts the dispersion surface, in addition to the velocity component entering the first outlet channel 120, there is also a velocity component along the dispersion surface downstream. When there are particles that have not completely passed through a fork, the flow resistance in the fork is much greater than the fork without particles. Therefore, under the action of this velocity component, the particles will move to the next fork with a smaller flow resistance to pass through, thereby realizing a single particle passing through each fork at intervals, and the shortest interval time is the time required for the particle to pass through the fork.

[0132] In some embodiments, the array fork 160 is a comb tooth structure formed by setting a plurality of partitions, and the gap between adjacent comb teeth is a fork for particles to pass through. Of course, the array fork 160 is not limited to the comb tooth structure, and can also be other channel arrays of similar size.

[0133] In some embodiments, the comb-tooth structure is arranged along the flow direction of the main filtering branch flow channel, and the internal width of each channel gradually increases to reduce the flow resistance of the particle flow through the comb-tooth structure.

[0134] In some embodiments, the comb-tooth structure gradually decreases the inlet width of each fork along the flow direction of the first outlet flow channel to obtain better large impurity filtering performance while ensuring that the flow resistance of the entire structure is small. Since the particle flow is subjected to centrifugal force after passing through the second bend 180, it is basically impossible for large particle impurities to enter the left side of the array fork 160. Therefore, the fork size on the left side of the array fork 160 can be relatively larger to reduce flow resistance, while the fork size on the right side is set smaller to ensure the efficiency of filtering large particle impurities. For example, taking cell passage as an example, the rightmost fork width of the array fork 160 is about 10 microns to filter large particle impurities, and the leftmost fork width of the array fork 160 is about 15-20 microns to reduce cell passage resistance.

[0135] Due to the setting of the second bend 180, the particle flow is centrifuged to cause large particles to be thrown to the right, so that large impurities are concentrated on the right side of the dispersion surface ( Figure 1 The medium garbage 320) can avoid the large particles of impurities clogging the array fork 160 to the greatest extent, thereby increasing the service life and long-term stability of the array fork 160.

[0136] After passing through the sheathless flow filtering and dispersing structure 100, large impurities are filtered out of the particle flow, and particle dispersion is achieved, so that the particles are arranged in the front and back directions and flow with the fluid, which greatly reduces the difficulty of focusing the sheath flow or sheathless flow.

[0137] Embodiment 2: This embodiment provides a sheathless flow focusing structure 200 based on microfluidics, comprising

[0138] The second inlet flow channel 210 is a flow channel for providing inflow;

[0139] The second outlet flow channel 230 is a flow channel for providing outflow;

[0140] The middle branch 220 is connected to the end of the second inlet flow channel 210 and the inlet end of the second outlet flow channel 230, and has a cross-section that decreases along the flow direction; and

[0141] Two side branches 240 are symmetrically arranged on both sides of the middle branch 220. The inlets of the two side branches 240 are opened on both sides of the inlet of the middle branch 220 to guide the flow. The outlets of the two side branches 240 are connected to the inlet end of the second outlet flow channel 230, and the inlet width of the side branches 240 is smaller than the inlet width of the middle branch 220.

[0142] The outlets of the two side branches 240 are arranged on both sides of the outlet of the middle branch 220 to form a confluence at the inlet end of the second outlet flow channel 230 .

[0143] In this embodiment, a stream of fluid is drawn out from both sides of the inlet of the middle branch 220 through the side branch 240. By setting the inlet width of the side branch 240, the fluid is basically free of particles. Since the middle branch 220 is initially large in size, all particle flows enter the middle branch 220. The width of the second half of the middle branch 220 is gradually reduced, and only a single particle can pass through the outlet end, which corrects the speed and direction of the particle, and realizes that a single particle is arranged in the center and enters the second outlet channel 230. The fluid in the side branch 240 is symmetrically merged from both sides of the inlet of the second outlet channel 230, wrapping the particle flow in the middle. Under the joint action of the fluids from the middle branch 220 and the two side branches 240, the particle flow is focused. Since the inlet width of the second outlet channel 230 is greater than the outlet width of the middle branch 220, the fluids from the two side branches 240 can achieve better particle focusing after being injected. It should be noted that if a large number of particles gather in the second inlet flow channel 210 , the subsequent focusing effect is very limited. Therefore, inputting the filtered and dispersed particle flow into the second inlet flow channel 210 of this embodiment can have a better effect.

[0144] In this embodiment, the second inlet flow channel 210 needs to maintain a certain length so that the flow direction of the fluid therein remains stable to prevent turbulence from causing lateral movement of particles to block the inlet of the side branch 240 and affect the focusing effect.

[0145] In some embodiments, the width of the second inlet flow channel 210 is uniform to maintain good particle flow stability, so that the particle flow mainly moves forward without generating turbulence and reducing lateral fluctuations; and the width of the second inlet flow channel 210 is smaller than the inlet width of the middle branch 220, so that the particle flow can smoothly enter the middle branch 220, and even if the change in diameter of the middle branch 220 causes the particle flow to flow back, the entrance of the side branch 240 will not be blocked.

[0146] In some embodiments, the side branch 240 includes an inlet section 241, a turning section 242 and an outlet section 243. The angle E between the inlet section 241 and the flow direction of the fluid in the middle branch 220 (the angle between the center lines of the two flow channels) is an obtuse angle, which can ensure the smooth introduction of the fluid in the side branch 240 while avoiding particles blocking the entrance of the side branch 240; if the angle E is too large, it will make it difficult to drain the side branch 240, so the general design range is 90-120 degrees. The angle F between the outlet section 243 and the flow direction in the second outlet channel 230 (the angle between the center lines of the two channels) is also an acute angle. By setting the angle F to an acute angle, when the fluid in the side branch 240 merges into the second outlet channel 230, it can form a sheath flow-like effect from both sides to avoid deflecting the particle flow. Generally speaking, the angle F needs to be set within a suitable range. If the angle F is set too large, it is easy to disperse the focused particle flow, and if it is set too small, the side branch 240 will occupy too much space. Therefore, in general, the design range of the angle F is 20-89 degrees, among which 30-60 degrees is optimal.

[0147] In some embodiments, the second outlet flow channel 230 includes a focusing section 231 and a diameter-reducing section 232. The focusing section 231 is used to merge the fluids of the two side branches 240 to the two sides of the outlet fluid of the middle branch 220 to form a focused confluence. The diameter-reducing section 232 is used to adjust the focused confluence to a desired width. The width of the diameter-reducing section 232 can be increased or decreased, depending on the purpose of the focused particle flow. For example, in this embodiment, the width of the diameter-reducing section 232 is reduced to facilitate observation experiments.

[0148] The ratio of the width of the focusing section 231 to the outlet width of the middle branch 220 determines the width of the protective fluid (sheath flow) on both sides of the middle particle flow in the second outlet flow channel 230. The width ratio needs to be appropriate. If the width ratio is too small, the relative width of the protective fluid on both sides of the particle flow will be smaller, and the centering degree of the particle flow in the second outlet flow channel 230 will be poor; if the width ratio is too large, it is easy to cause a low flow rate in the middle branch, which is not conducive to focusing. Generally, the width ratio range is 1-3, and 2 times is the best.

[0149] Example 3: Figure 3As shown, this embodiment provides a sheathless flow filtering dispersion focusing structure, including the sheathless flow filtering dispersion structure 100 described in Example 1 and the sheathless flow focusing structure 200 described in Example 2, a feed port 410 is set at the inlet of the sheathless flow filtering dispersion structure 100, the first outlet flow channel 120 of the sheathless flow filtering dispersion structure 100 is connected to the second inlet flow channel 210 of the sheathless flow focusing structure 200 through a transition flow channel 420, and a discharge flow channel 430 is set at the outlet of the sheathless flow focusing structure 200 to transfer the focused particles Flow is exported; after the particle flow is input into the first inlet flow channel 110 of the sheathless flow filtering and dispersing structure 100 through the feed port 430, the large particle impurities are filtered out and the particle flow is dispersed by the sheathless flow filtering and dispersing structure 100 to achieve preliminary focusing of the particle flow; then, it is transported to the second inlet flow channel 210 of the sheathless flow focusing structure 200 through the transition flow channel 420 with a reduced diameter, and the sheathless flow focusing structure 200 performs sheathless flow focusing on the filtered and dispersed particle flow, and after focusing, a single arranged and highly centered particle flow is output through the discharge flow channel 430.

[0150] The above-mentioned flow channel structure can be made by laser engraving, molding, casting, etching and other means in the existing technology to form a channel on a substrate made of PDMS, acrylic and other materials, and then covered with a cover plate (such as a glass plate, acrylic plate, etc.) to form a microfluidic chip. Since the present application does not make any changes to the flow channel preparation process and materials, the flow channel preparation process and materials have no significant impact on the technical problem solved by the present invention, and therefore the present application will not elaborate on this.

[0151] In addition, it should be noted that Figure 4 As shown, the flow channel and bifurcation in the present invention are formed by machining a channel on the substrate 510 and covering it with a cover plate 520, so the flow channel size generally refers to the flow channel width, and the flow channel height H is not limited. In general, in order to ensure that all particles pass through, the flow channel height should be greater than the diameter of the largest particle. If the flow channel is too high, the particle flow is prone to fluctuate in the height direction. Therefore, in general, it should be less than the two largest particle diameters. If the particles are elastic such as cells, the height can be slightly smaller than the particle diameter.

[0152] Embodiment 4: This embodiment provides a sheathless flow single cell dispersion focusing method, comprising the following steps:

[0153] (1) Cell preparation: 6 cm culture dish, 80% density adherent Hela cells, digested with trypsin, centrifuged to obtain 6 ml of cell suspension, filtered through a 30 um filter, allowed to stand for a while, and the upper layer of liquid was collected and mixed with a density regulator to keep the cells from sinking to the bottom for a long time, which was used as the cell suspension for the experiment.

[0154] (2) Runner preparation: Figure 5As shown, a transfer process is used to prepare channels on the lower surface of the PDMS material (substrate 510) to form a sheathless flow filtering dispersion structure 100 and a sheathless flow focusing structure 200. A hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the inlet of the sheathless flow filtering dispersion structure 100 as a feed inlet 410, a flat flow channel 440 is prepared at the outlet of the sheathless flow focusing structure 200 for observation, and a hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the downstream outlet of the flat flow channel 440 as an outlet 450; Figure 4 As shown, a glass cover plate is then bonded (the glass cover plate is bonded to the lower surface of the substrate 510 to form a cover plate 520) to form a sheathless flow single-cell dispersion focusing chip, and the width of the flat channel 440 is about 30um and the height is about 15um.

[0155] (3) Use an air pressure pump with a pressure of about 500-1000 mbar to pump the cell suspension from the feed port into the feed port 410 of the sheathless flow single cell dispersion and focusing chip.

[0156] (4) Optical path and image acquisition: A 20x microscope lens was used for bright field observation, and a red light source of about 560nm was used. Multiple sets of optical path components were used to introduce the images into the high-speed camera. The high-speed camera had an exposure time of 10us and a frame rate of 1000fps. The middle part of the flat channel 440 connected to the sheathless flow focusing structure 200 ( Figure 5 The dotted box in the middle is used as the observation area for observation experiments.

[0157] (5) After the flat flow channel 440 is filled with liquid, cell images are collected to record the centering of the cells in the horizontal plane of the flat flow channel 440 and whether the flux is uniform (cells are spaced apart, and there will not be many cells crowded together at one time and few cells at another time).

[0158] Record the results as Fig.10 As shown, Fig.10 From left to right, there are 5 different cells captured by the camera in succession. The shooting parameter is 1000fps. The numbers in the figure are frame numbers, and the interval between adjacent frames is 1 millisecond, that is, Fig.10 The cell intervals from left to right are 3 milliseconds, 4 milliseconds, 3 milliseconds, and 4 milliseconds. The cells are basically centered in the flow channel with clear edges and good cell dispersion. The flux is stable during the experiment. There is rarely a large number of cells passing through in a short period of time or no cells passing through for a period of time. It can run stably for several hours.

[0159] Example 5: This example provides a single cell filtration dispersion method

[0160] (1) Runner preparation: Figure 6As shown, a transfer process is used to prepare channels on the lower surface of the PDMS material (substrate 510) to form a sheathless flow filtration dispersion structure 100. A hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the inlet of the sheathless flow filtration dispersion structure 100 as a feed inlet 410, a flat flow channel 440 is prepared at the outlet of the sheathless flow filtration dispersion structure 100 for observation, and a hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the downstream outlet of the flat flow channel 440 as an outlet 450; Figure 4 As shown, a glass cover plate is then bonded (the glass cover plate is bonded to the lower surface of the substrate 510 to form a cover plate 520) to form a single cell filtration dispersion chip, and the width of the flat channel 440 is about 30um and the height is about 15um.

[0161] Step (2) to step (5) are the same as in Example 4.

[0162] The recorded results are: there are rarely a large number of cells passing through in a short period of time or no cells passing through for a period of time. The flux is stable, but the cell centrality is not good.

[0163] Example 6: This example provides a sheathless flow single cell focusing method

[0164] (1) Runner preparation: Figure 7 As shown, a transfer process is used to prepare a channel on the lower surface of the PDMS material (substrate 510) to form a sheathless flow focusing structure 200. A hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the inlet of the sheathless flow focusing structure 200 as a feed inlet 410, a flat flow channel 440 is prepared at the outlet of the sheathless flow focusing structure 200 for observation, and a hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the downstream outlet of the flat flow channel 440 as an outlet 450; Figure 4 As shown, a glass cover plate is then bonded (the glass cover plate is bonded to the lower surface of the substrate 510 to form a cover plate 520) to form a sheathless flow single-cell focusing chip, and the width of the flat channel 440 is about 20um and the height is about 15um.

[0165] Step (2) to step (5) are the same as in Example 4.

[0166] The recorded results are: Fig.11A and Fig. 11B As shown, Fig.11A (c) and (d) are images at different cell fluxes, respectively. (c) indicates that no cells pass through the flow channel, and (d) indicates that multiple cells pass through. In actual observation, the cells appear uneven, with a large number of cells passing through in a short period of time or no cells passing through for a period of time. The flux is unstable. Because the flow channel in the observation area in this embodiment is narrow and cannot reflect the centrality, the overall partial image of the sheathless flow focusing structure 200 is taken, as shown in FIG. Fig. 11BAs shown, it can be seen that in the second outlet flow channel 230, the cell centering performance is very good, but there is local aggregation, a large number of cells pass through in a short period of time or no cells pass through for a period of time, and the flux is unstable.

[0167] Comparative Example 1: This embodiment provides a focusing method based on variable cross-section flow channel

[0168] (1) Runner preparation: Figure 8 As shown, a variable cross-section flow channel 460 with a gradually decreasing width is prepared on the lower surface of the PDMS material (substrate 510) by a transfer process, a hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the inlet of the variable cross-section flow channel 460 as a feed inlet 410, a flat flow channel 440 is prepared at the outlet of the variable cross-section flow channel 460 for observation, and a hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the downstream outlet of the flat flow channel 440 as an outlet 450; Figure 4 As shown, a glass cover plate is then bonded (the glass cover plate is bonded to the lower surface of the substrate 510 to form a cover plate 520) to form a variable cross-section flow channel focusing chip, and the width of the flat flow channel 440 is about 30um and the height is about 15um.

[0169] Step (2) to step (5) are the same as in Example 4.

[0170] Record the results as Fig.12 As shown, the cell flux is unstable (a large number of cells pass through in a short period of time or no cells pass through for a period of time) and the cells are not centered (moving close to the channel wall).

[0171] Comparative Example 2: Known single cell dispersion focusing method based on sheath flow

[0172] (1) Runner preparation: Fig. 9 As shown, a second outlet flow channel based on sheath flow is prepared on the lower surface of the PDMS material (substrate 510) by a transfer process, two holes are prepared at the inlet of the second outlet flow channel based on sheath flow, which serve as a sheath liquid feed port 480 and a cell liquid feed port 470 respectively, a flat flow channel 440 is prepared at the outlet of the second outlet flow channel based on sheath flow for observation, and a hole penetrating into the flow channel is prepared on the upper surface of the substrate 510 at the downstream outlet of the flat flow channel 440 as an outlet 450; Figure 4 As shown, a glass cover plate is then bonded (the glass cover plate is bonded to the lower surface of the substrate 510 to form a cover plate 520) to form a sheath flow single-cell focusing chip, and the width of the flat channel 440 is about 30um and the height is about 15um.

[0173] Step (2) to step (5) are the same as in Example 4.

[0174] The recorded results are basically the same as those of Comparative Example 1.

[0175] It can be seen from the above-mentioned recording results that the sheathless flow filtering dispersion structure 100 of the present invention can better disperse cells (or particles in a particle flow) so that the cells can pass through the flow channel at almost uniform intervals; the sheathless flow focusing structure 200 can better center the cells in the middle of the flow channel to achieve cell focusing; when the filtering dispersion structure is used in conjunction with the sheathless flow focusing structure 200, the cell suspension can be dispersed and focused to form a uniformly spaced and centered cell flow to meet the experimental needs of single cell imaging, single cell sorting, single cell encapsulation, etc.

[0176] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalent substitutions of the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and should be included in the scope of the claims of the present invention.

Claims

1. A microfluidics-based sheathless flow filtration dispersion structure, characterized in that: include A first inlet flow channel is a flow channel for providing inflow; A first outlet flow channel is a flow channel for providing outflow; A bifurcated flow channel is provided at the end of the first inlet flow channel and is open in a V-shape to divide the fluid into two branches; Two main filtering branch flow channels are connected to each branch end of the bifurcated flow channel through first bends that turn inwards relatively, and each main filtering branch flow channel is also provided with a second bend, so that the outlets of the two main filtering branch flow channels merge downstream of the first outlet flow channel; The inlets of the two auxiliary filtering branch flow channels are respectively connected to the ends of the corresponding first bends and are arranged inwardly relative to the main filtering branch flow channel; the outlets of the two auxiliary filtering branch flow channels merge to form the source of the first outlet flow channel; as well as The array fork is arranged in the outlet of each main filtering branch flow channel and is located downstream of the second bend. The forks in the array fork are arranged along the flow direction of the first outlet flow channel, and the particles in the particle flow are intermittently merged into the first outlet flow channel through the array fork.

2. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The bifurcated flow channel is arranged in alignment with the first inlet flow channel, and the two branches of the bifurcated flow channel are arranged symmetrically.

3. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The sheathless flow filtering dispersion structure is symmetrically arranged with respect to a center line connecting the first inlet flow channel and the first outlet flow channel.

4. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The main filtering branch flow channel gradually widens from the inlet to the outlet.

5. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The inlet width of the auxiliary filtering branch flow channel is smaller than the inlet of the main filtering branch flow channel, so that the inlet flow of the auxiliary filtering branch flow channel is smaller than that of the main filtering branch flow channel. At the same time, the auxiliary filtering branch flow channel gradually widens from the inlet to the outlet.

6. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The included angle of the bifurcated flow channel is 60-180 degrees.

7. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The turning angle of the first curve is 30-90 degrees.

8. The microfluidics-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The turning angle of the second curve is 15-120 degrees.

9. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The cross section of the first outlet flow channel gradually increases along the flow direction.

10. The microfluidic-based sheathless flow filtration dispersion structure according to claim 1, characterized in that: The array bifurcation is a comb-tooth structure formed by arranging a plurality of partitions, and the gap between adjacent comb teeth is a bifurcation for particles to pass through.

11. The microfluidics-based sheathless flow filtration dispersion structure according to claim 10, characterized in that: The comb-tooth structure is arranged along the flow direction of the main filtering branch flow channel, and the internal width of each branch gradually increases.

12. The microfluidics-based sheathless flow filtration dispersion structure according to claim 10, characterized in that: The comb-tooth structure has a gradually decreasing inlet width of each fork along the flow direction of the first outlet flow channel.

13. A sheathless flow focusing structure based on microfluidics, characterized in that: include The second inlet flow channel is a flow channel for providing inflow; A second outlet flow channel is a flow channel for providing outflow; The middle branch is connected to the end of the second inlet flow channel and the inlet end of the second outlet flow channel, and has a cross-section that decreases along the flow direction; as well as Two side branches are symmetrically arranged on both sides of the middle branch, the inlets of the two side branches guide the flow from the openings on both sides of the inlet of the middle branch, the outlets of the two side branches are connected to the inlet end of the second outlet flow channel, and the inlet width of the side branches is smaller than the inlet width of the middle branch; The outlets of the two side branches are arranged on both sides of the outlet of the middle branch to form a confluence at the inlet end of the second outlet flow channel.

14. The sheathless flow focusing structure according to claim 13, characterized in that: The outlet width of the side branch is greater than the outlet width of the middle branch.

15. The sheathless flow focusing structure according to claim 13, characterized in that: The width of the second inlet flow channel is smaller than the inlet width of the middle branch.

16. The sheathless flow focusing structure according to claim 13, characterized in that: The side branch includes an inlet section, a turning section and an outlet section. The angle between the inlet section and the flow direction of the fluid in the middle branch is an obtuse angle, and the angle between the outlet section and the flow direction in the second outlet flow channel after the confluence is an acute angle.

17. The sheathless flow focusing structure according to claim 13, characterized in that: The second outlet flow channel includes a focusing section and a diameter-reducing section. The focusing section is used to merge the fluids of the two side branches to both sides of the fluid of the middle branch to form a focused confluence. The diameter-reducing section is used to adjust the focused confluence to a desired width.

18. The sheathless flow focusing structure according to claim 17, characterized in that: The width of the focusing segment is greater than the width of the middle branch exit.

19. A sheathless filtering dispersion focusing structure, characterized in that: It comprises the sheathless flow filtering dispersion structure as described in any one of claims 1-12 and the sheathless flow focusing structure as described in any one of claims 13-18, wherein the first outlet flow channel of the sheathless flow filtering dispersion structure is connected to the second inlet flow channel of the sheathless flow focusing structure through a transition flow channel.

20. A sheathless particle flow filtration dispersion method, characterized in that: The following steps are involved: A first inlet flow channel is configured for inputting a particle flow, wherein the particle flow contains large impurities; A bifurcated flow channel is configured to divide the input particle flow into two branches opening in a V shape; The first bend is configured to turn the two branch fluids inwards for the first centrifugation; A main filtering branch flow channel is configured to receive the particle flow containing large impurities on the outside after the first bend; An auxiliary filtering branch flow channel is configured to receive the particle flow after the first bend; A first outlet flow channel is configured to guide out the particle flow from which large particles of impurities have been filtered out; A second bend and an array fork are sequentially arranged in the main filtering branch flow channel, and the particle flow containing large particle impurities is subjected to a second centrifugation through the second bend and passes through the array fork, so that the particles enter the first outlet flow channel in a dispersed manner, and the large particle impurities are accumulated on the outermost side of the array fork.

21. A sheathless particle flow focusing method, characterized in that: The following steps are involved: A second inlet flow channel is configured for inflow of particle flow; An intermediate branch with a gradually decreasing cross section is configured to correct the direction of particle velocity, gradually squeeze the particle flow and reduce its width to form a particle flow with the direction consistent with the central axis; Configure side branches, take a branch without particle flow from the incoming particle flow, and symmetrically introduce the two branches into the two sides of the particle flow outflowing from the middle branch to form a protective layer, so as to achieve sheath-free flow focusing of the particle flow.

22. A sheathless particle flow filtration dispersion focusing method, characterized in that: The following steps are involved: A first inlet flow channel is configured for inputting a particle flow, wherein the particle flow contains large impurities; A bifurcated flow channel is configured to divide the input particle flow into two branches opening in a V shape; The first bend is configured to turn the two branch fluids inwards for the first centrifugation; A main filtering branch flow channel is configured to receive the particle flow containing large impurities on the outside after the first bend; An auxiliary filtering branch flow channel is configured to receive the particle flow after the first bend; A first outlet flow channel is configured to guide out the particle flow from which large particles of impurities have been filtered out; A second bend and an array fork are sequentially arranged in the main filtering branch flow channel, and the particle flow containing large particle impurities is subjected to a second centrifugation through the second bend and the array fork, so that the particles enter the first outlet flow channel in a dispersed manner, and the large particle impurities are accumulated on the outermost side of the array fork; A transition channel is configured to receive the particle flow that filters out large particles of impurities; A second inlet flow channel is configured to receive the particle flow delivered through the transition flow channel; An intermediate branch with a gradually decreasing cross section is configured to correct the direction of particle velocity, gradually squeeze the particle flow and reduce its width to form a particle flow with the direction consistent with the central axis; Configure side branches, take a branch without particle flow from the incoming particle flow, and symmetrically introduce the two branches into the two sides of the particle flow outflowing from the middle branch to form a protective layer, so as to achieve sheath-free flow focusing of the particle flow.

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