Sheathless Flow Filtering and Dispersion Structure, Focusing Structure, and Filtering, Dispersion, and Focusing Structure

Abstract

The present invention belongs to the field of microfluidic technology and discloses a sheathless flow filtration and dispersion structure, a focusing structure, and a filtration, dispersion, and focusing structure. The present invention designs a sheathless flow filtration and dispersion structure, which divides the incoming particle flow into two branches through a bifurcated flow channel. Each branch undergoes the first filtration and separation after passing through the first bend, with a part flowing into the auxiliary filtration branch flow channel and the other part flowing into the main filtration branch flow channel and undergoing the second filtration and separation at the second bend and the array bifurcation. The two parts of the fluid converge in the first outlet flow channel to achieve particle filtration and dispersion. Then, it enters the sheathless flow focusing structure, where a particle-free branch flow is respectively led out from both sides of the entrance of the middle branch and symmetrically converges into the second outlet flow channel from both sides through the side branch. The particle flow is centered and focused through the combined action of the correction of the particle flow velocity direction by the middle branch and the convergence of the side branch. The two structural 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 present invention belongs to the technical field of microfluidics, and relates to a single-cell dispersion focusing technology for particle flow, specifically to a sheathless flow filtering and dispersion structure, a focusing structure, and a filtering, dispersion, and 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 cells to be dispersed and focused into an approximately linear flow in the fluid and pass through the specified flow channel area in sequence. Currently, the well-known single-cell dispersion focusing technology is achieved through sheath flow. By means of a multi-layer nested pipeline, the cell suspension is surrounded by sheath fluid, with the cell suspension serving as the core flow in the middle and the sheath flow on both sides or in a circle around it. When the core flow and the sheath flow converge, under the action of the symmetric sheath flow, the cell suspension is dispersed, and a single-row cell flow is formed in the middle of the flow channel. However, the flow rates of the sheath flow and the core flow need to be strictly controlled, and whether the cells can form a single-row cell flow in the flow channel depends on the ratio between the two. Affected by the properties of the fluid, it takes dozens of seconds or even longer to reach stability for cell dispersion and focusing achieved through sheath flow. At the same time, the stability of cell dispersion and focusing achieved by the sheath flow technology is relatively poor. Common phenomena such as gradual blockage of the front-end filter and back-end operations will cause changes in the flow resistance of the flow channel, thereby destroying the stable flow environment of the sheath flow and the core flow, resulting in cell focusing fluctuations or focusing failures. These problems lead to poor stability and generalization of the single-cell dispersion focusing system achieved by the sheath flow technology, making it difficult to perform stable work for more than several hours and being unfavorable for integration with structures with unstable flows. In addition, the sheath flow dispersion focusing system requires additional equipment such as flow channel inlets and pumps, increasing costs and complexity.

[0003] The sheath flow technology is a commonly used technology in the field of microfluidics. For example, Patent CN110343611A discloses a microfluidic chip, which includes a closely attached control layer, an elastic film layer, a fluid channel layer, and an electromagnetic micro-valve, wherein: the control layer is located at the top, and multiple control cavities are arranged inside the control layer, and a flow-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 channel is arranged on both sides of the main channel to promote cell focusing, and the screening channel is arranged at the back end of the main channel; the flow-blocking magnet can move up and down under the control of the electromagnetic micro-valve, and the up and down movement of the flow-blocking magnet can drive the elastic film layer to block different screening channels to achieve cell screening. Among them Figure 2The sheath flow channel allows the sheath flow to enter from the left side and flow through two branches into the main channel, symmetrically flowing into the main channel to achieve focusing. Although focusing can be achieved (by flowing in the middle of a channel wider than the cell), inevitably, the input of the sheath flow in the sheath flow channel is required, which means that the chip structure has at least two inlets, resulting in high 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-file cell flow in the channel depends on the ratio of 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 method, if the inlet flow rate at the sheath flow channel is too large, it is difficult for the cells to flow out at the main channel. That is, if there are fluctuations in the flow rate at the front end of the main channel, 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, and the cross-sectional width d2 of the microchannel satisfies: d0 < d2 < 1.5d0, and 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 to allow the waste liquid to flow out of the entire microfluidic chip. This technology is a sheathless technology using channel restriction, with filtration at the front and restriction of the cells passing through a narrow channel at the back. However, this type of structure has many problems, resulting in its inability to achieve long-term cell centering and being difficult to apply in practical fields. First, ordinary filtration can easily 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. Second, 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 channel. Then, the 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. Second, the cell dispersion of this structure is not good. Although the 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 originally very close to each other will still be very close in the 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] The first outlet channel, which is a channel for providing outflow;

[0014] The bifurcated channel is arranged at the end of the first inlet channel and is arranged in a V - shaped opening to divide the fluid into two branches;

[0015] Two main filtering branch channels are respectively connected to the end of each branch of the bifurcated channel through a first bend that turns relatively inwards. A second bend is also provided in each main filtering branch channel, so that the outlets of the two main filtering branch channels converge downstream of the first outlet channel;

[0016] Two auxiliary filtering branch channels have their inlets respectively connected to the ends of the corresponding first bends and are arranged closer to the inside relative to the main filtering branch channels. So that after the fluid passes through the first bend, under the action of centrifugal force, relatively small particles flow into the auxiliary filtering branch channels; the outlets of the two auxiliary filtering branch channels converge to form the source of the first outlet channel; and

[0017] The array bifurcation is arranged inside the outlet of each main filtering branch channel and is located downstream of the second bend. The bifurcations in the array bifurcation are arranged along the flow direction of the first outlet channel. So that under the combined action of the centrifugal force generated in the second bend and the change in flow resistance inside the array bifurcation, the particles in the particle flow pass through the array bifurcation at intervals and flow into the first outlet channel, realizing particle dispersion. The large - particle impurities are thrown to the outermost side under the action of centrifugal force and are filtered and intercepted through the array bifurcation to achieve filtration.

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

[0019] Furthermore, the sheathless flow filtering and dispersing structure is symmetrically arranged with respect to the central connection line of the first inlet channel and the first outlet channel.

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

[0021] Furthermore, the inlet width of the auxiliary filtering branch channel is smaller than that of the main filtering branch channel, so that the inlet flow rate of the auxiliary filtering branch channel is less than that of the main filtering branch channel. At the same time, the auxiliary filtering branch channel gradually widens from the inlet to the outlet to maintain an appropriate flow resistance ratio between the auxiliary filtering branch channel and the main filtering branch channel and minimize the flow resistance of the entire structure as much as possible.

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

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

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

[0025] Further, the first outlet flow channel gradually increases in cross-section along the flow direction.

[0026] Further, the array bifurcates into a comb-like structure formed by arranging a plurality of partition plates, and the gap between adjacent comb teeth is a bifurcation for particles to pass through.

[0027] Further, along the flow direction of the main filtration branch flow channel of the comb-like structure, the inner width of each bifurcation gradually increases to reduce the flow resistance of the entire structure.

[0028] Further, along the flow direction of the first outlet flow channel of the comb-like structure, the inlet width of each bifurcation gradually decreases to ensure a relatively small flow resistance of the entire structure while obtaining better large impurity filtration performance.

[0029] On the other hand, the present invention provides a microfluidic chip with a sheathless flow filtration and dispersion function. The microfluidic chip includes 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 includes

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

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

[0032] A bifurcated flow channel, which 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 in a V-shaped opening for dividing the fluid into two symmetric branches;

[0033] Two main filtration branch flow channels, which are respectively connected to the end of each branch of the bifurcated flow channel through a first bend that turns inward relatively. A second bend is also provided in each main filtration branch flow channel, so that the outlets of the two main filtration branch flow channels converge downstream of the first outlet flow channel;

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

[0035] An array bifurcation, which is arranged inside the outlet of each main filtration branch flow channel and is located downstream of the second bend. The bifurcations in the array bifurcation 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 in flow resistance in the array bifurcation, the particles in the particle flow pass through the array bifurcation at intervals and converge into the source of the first outlet flow channel formed by the convergence of the outlets of the two symmetric auxiliary filtration branch flow channels to achieve particle dispersion, and large particle impurities are thrown to the outermost side under the action of centrifugal force and are filtered and intercepted through the array bifurcation to achieve filtration.

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

[0037] a second inlet channel, which is a channel for providing inflow;

[0038] a second outlet channel, which is a channel for providing outflow;

[0039] a middle branch, which is connected to the end of the second inlet channel and the inlet end of the second outlet channel, and whose cross-section shrinks along the flow direction; and

[0040] two side branches, which are symmetrically arranged on both sides of the middle branch. The inlets of the two side branches are diverted from 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 channel, and the inlet width of the side branch is smaller than the inlet width of the middle branch;

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

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

[0043] Furthermore, the width of the second inlet 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 included angle between the inlet section and the flow direction of the fluid in the middle branch is an obtuse angle, and the included angle between the outlet section and the flow direction in the second outlet channel after the confluence port is also an acute angle.

[0045] Furthermore, the second outlet channel includes a focusing section and a variable diameter section. The focusing section is used to converge the fluids of the two side branches to both sides of the fluid in the middle branch to form a focused combined flow, and the variable diameter section is used to adjust the focused combined flow to the required width.

[0046] Furthermore, the width of the focusing section is larger than the outlet width of the middle branch.

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

[0049] a second inlet channel, which is a 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] Configure an intermediate branch with a gradually shrinking cross-section to correct the direction of particle velocity, gradually squeeze the particle flow to reduce the width, and form a particle flow with a direction consistent with the central axis;

[0066] Configure side branches, take a branch without particle flow from the inflowing particle flow respectively, symmetrically introduce the two branches to both sides of the particle flow flowing out of the intermediate branch, form a protective layer, and achieve sheathless focusing of the particle flow.

[0067] On the other hand, the present invention provides a method for filtering, dispersing and focusing a particle flow without sheath flow, including the following steps:

[0068] Configure a first inlet flow channel for inputting a particle flow, and the particle flow contains large particle impurities;

[0069] Configure a bifurcated flow channel to divide the input particle flow into two branches that open in a V shape;

[0070] Configure a first bend to turn the two branch fluids inward for the first centrifugation;

[0071] Configure a main filtration branch flow channel to receive the particle flow containing large particle impurities on the outside after the first bend;

[0072] Configure an auxiliary filtration branch flow channel to receive the particle flow after the first bend;

[0073] Configure a first outlet flow channel to export the particle flow from which large particle impurities have been filtered;

[0074] Sequentially configure a second bend and an array bifurcation in the main filtration branch flow channel, perform a second centrifugation on the particle flow containing large particle impurities through the second bend and pass through the array bifurcation, so that the particles enter the first outlet flow channel in a dispersed manner, and the large particle impurities accumulate on the outermost side of the array bifurcation;

[0075] Configure a transition flow channel to receive the particle flow from which large particle impurities have been filtered;

[0076] Configure a second inlet flow channel to receive the particle flow conveyed through the transition flow channel;

[0077] Configure an intermediate branch with a gradually shrinking cross-section to correct the direction of particle velocity, gradually squeeze the particle flow to reduce the width, and form a particle flow with a direction consistent with the central axis;

[0078] Configure side branches, take a branch without particle flow from the inflowing particle flow respectively, symmetrically introduce the two branches to both sides of the particle flow flowing out of the intermediate branch, form a protective layer, and achieve sheathless focusing of the particle flow.

[0079] Compared with the prior art, the beneficial effects of the present invention are as follows:

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

[0081] The sheathless flow focusing structure designed by the present invention can achieve the dispersion and focusing effects without using sheath flow, is not restricted by the application scenarios of sheath flow, has good generalization and stability, allows for 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, greatly reducing the consumable costs during the experiment. The chip structure of the present invention is simple, the operation is convenient, no complex equipment control is required, reducing the equipment cost and labor cost.

[0082] After the sheathless flow filtration and dispersion structure and the sheathless flow focusing structure of the present invention are used in combination, while satisfying the focusing obtained by the sheath flow focusing technology, the interval height of the particle flow is also highly uniform, and the dispersion performance is superior to the sheath flow focusing technology; in addition, the present invention has good generalization and stability, allows for 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 filtration and 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 the filtration and dispersion function and the focusing function respectively. In specific applications related to cell analysis, one or both of the above structures can be selected according to the requirements for cells to achieve the purpose. At the same time, in the analysis of particles other than cells, the corresponding topological structures of the above structures can also be used to achieve dispersion and focusing; the operation and use are flexible and can be combined according to different scenarios. Description of the Drawings

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

[0085] Figure 2 It is a schematic diagram of the sheathless flow focusing structure in Embodiment 2 of the present invention.

[0086] Figure 3 It is a schematic diagram of the sheathless flow filtration, dispersion and focusing structure in Embodiment 3 of the present invention.

[0087] Figure 4 It is a schematic diagram of the height control of the cell fluctuating in the vertical direction.

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

[0089] Figure 6 Schematic diagram of the single-cell filtration dispersion chip in Example 5, where the blue dots are cells, the red blocks are debris, and the yellow arrows represent the fluid flow direction.

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

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

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

[0093] Figure 10 Cell image in Example 4. It can be seen that each cell is separated and has good centering and clear edges.

[0094] Figure 11A Result image of the observation area in Example 6, where Figure 11A (c) and (d) in it are images of two consecutive time periods in the observation area. It can be seen from Figure 11A that the cells are uneven, and there are phenomena of a large number of cells passing through in a short time or no cells passing through for a period of time, and the flux is unstable.

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

[0096] Figure 12 Result image in Comparative Example 1. The cells are not centered, and many cells that are close together are easily captured at the same moment.

[0097] Note: Figure 12 Compared with Figure 10 The optical system and camera model used for image acquisition are different, so the texture of the cell images is different, but it does not affect the observation of the outer contour and the position of the cells in the flow channel.

[0098] 100 - sheathless flow filtration and dispersion structure, 110 - first inlet flow channel, 120 - outlet flow channel, 130 - bifurcated flow channel, 131 - flow direction, 140 - main filtration branch flow channel, 141 - front - stage filtration flow channel, 142 - rear - stage 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 - intermediate branch, 230 - second outlet flow channel, 231 - focusing section, 232 - variable - diameter section, 240 - side branch, 241 - inlet section, 242 - turning section, 243 - outlet section;

[0100] 310 - cell; 320 - debris;

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

[0102] 510 - substrate, 520 - cover plate. Detailed implementation manners

[0103] The following further describes the implementation manners of the present invention in detail in conjunction with the drawings and embodiments. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0104] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is 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 thus cannot be understood as a limitation of the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes 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 "connected" 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 directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0106] Example 1: As 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 bifurcated flow channel 130 is arranged to open in a V-shaped manner, dividing the particle flow to be filtered (a fluid formed by particles suspended in a liquid environment, where the particles include cells, polyethylene spheres, hydrogel spheres, etc.) into two branches. The two main filtering branch channels 140 are connected to the end of each branch of the bifurcated flow channel 130 through the first bend 170 that turns relatively inwards. At the same time, an auxiliary filtering branch channel 150 is arranged inside. When the particle flow passes through the first bend 170, a rotating fluid is formed, generating a centrifugal force. Under the action of the centrifugal force, large-particle particles are thrown to the right and enter the main filtering branch channel 140 together with the particle flow. A small part of the particle flow of small particles enters the auxiliary filtering branch channel 150 under the action of the swirling flow, realizing the first filtering separation; the two auxiliary filtering branch channels 150 converge to form the source of the first outlet flow channel 120. Due to the symmetric design, the fluids in the two auxiliary filtering branch channels 150 basically form a forward-propagating flow in the first outlet flow channel 120 after converging; larger-particle particles and large-particle impurities enter the main filtering branch channel 140 together and form a swirling flow again under the action of the second bend 180, generating a centrifugal force, causing the particle flow to disperse according to size and enter the array bifurcation 160. The large-particle impurities accumulate on the right side of the array bifurcation 160 ( Figure 1 in the figure, the red part is the garbage 320 formed by the accumulation of large-particle impurities), which does not affect most of the functions of the array bifurcation 160 itself. After the particles of different sizes are dispersed at the entrance of the array bifurcation 160, they pass through each bifurcation respectively and enter the first outlet flow channel 120 in a staggered manner. The large-particle impurities are filtered and intercepted by the array bifurcation 160, realizing the second filtering separation; the particles flow into the first outlet flow channel 120 in a decentralized manner under the combined action of the array bifurcation 160 and the liquid flow converging downstream of the auxiliary filtering branch channel 140, realizing sheathless flow decentralized filtration.

[0114] When there is a particle passing through a certain bifurcation in the array bifurcation 160, the flow resistance in that bifurcation increases rapidly and the flow velocity slows down. When a new particle reaches the entrance of that bifurcation, it will flow with the fluid to a nearby bifurcation where no cell is passing, thus automatically avoiding the situation where multiple particles continuously enter a single bifurcation. Therefore, the particle flow in the main filtering branch channel 140 enters the first outlet flow channel 120 one by one at intervals after passing through the array bifurcation 160, making the particle dispersion in the first outlet flow channel 120 very good.

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

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

[0117] In some embodiments, the sheathless flow filtration and dispersion structure 100 is symmetrically arranged with respect to the central connection line of the first inlet channel 110 and the first outlet channel 120. By the symmetric arrangement, it is ensured that the fluid in the two auxiliary filtration branch channels 150 can be centered and dispersed after entering the main filtration branch channel 140, avoiding the occurrence of situations such as vortex and turbulence that cause the particles to refocus after dispersion; by the symmetric arrangement, it can also ensure that the kinetic energies of the two fluid flows at the confluence of the outlets of the two main filtration branch channels 140 are almost the same, and the particles can be kept in a dispersed state after confluence, avoiding the occurrence of unidirectional swirl and thus refocusing when the flow rates are inconsistent or the confluence angles are inconsistent.

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

[0119] See Figure 1, in some embodiments, the main filtration branch flow channel 140 gradually widens from the first inlet flow channel 110 to the outlet; the fluid velocity is reduced by the gradually changing main filtration branch flow channel 140 to prevent particles from hitting the array bifurcation 160 too fast, while providing sufficient space for a sufficient number of bifurcations on the array bifurcation 160, thereby improving the dispersion efficiency and preventing a large number of particles from concentrating at a certain bifurcation and causing blockage.

[0120] It should be noted that the width at the array bifurcation 160 in the main filtration branch flow channel 140 can be estimated based on the particle concentration. Generally, the number of cells reaching the array bifurcation simultaneously estimated from the particle concentration does not exceed twice the number of array voids 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 good dispersion effect and prevent uneven spacing of the particles flowing through the first outlet flow channel 120 after dispersion.

[0121] It should be noted that the distance between the second bend 180 and the array bifurcation 160 should not be too large or too small. A too large distance will result in insufficient space provided for the array bifurcation, and a too small distance will cause the velocity of the particles not to decrease sufficiently after turning, affecting the dispersion effect.

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

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

[0124] See Figure 1 , the included angle between the two branches of the bifurcated flow channel 130 provides a necessary guarantee for the subsequent turning of the particle flow to form a centrifugal force. If the included angle is too large, it will cause too large a flow resistance in the bifurcated flow channel 130, and at the same time, the particle flow will hit the side wall of the bifurcated flow channel 130 almost head-on, causing particles such as cells to break and affecting the experimental quality. If the included angle is too small, there will not be enough turning angle for the subsequent first bend 170 and second bend 180 to form sufficient centrifugal force; therefore, the included 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 filtration branch channel 140 when the main filtration branch channel 140 and the auxiliary filtration branch channel 150 bifurcate. The larger the turning angle, the better the centrifugal effect of the particle flow. However, an overly large turning angle will leave no margin for the second bend 180. Therefore, the turning angle B of the first bend 170 is appropriately in the range of 30 - 90 degrees, and the best choice is 60 - 80 degrees.

[0126] See Figure 1 , since the second bend 180 is provided in the main filtration branch channel 140, the main filtration branch channel 140 can be divided into a front - segment filtration channel 141 and a rear - segment filtration 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 - segment filtration channel 141 relative to the center line of the corresponding bifurcated channel 130.

[0127] The front - segment filtration channel 141 has a certain length, that is, a certain aspect ratio, such as an aspect ratio of 3:1 - 15:1. By setting an appropriate aspect ratio (since the main filtration branch channel 140 is a variable - cross - section channel, so it is actually an equivalent ratio), the end of the front - segment filtration 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 Figure 1 , the turning angle of the second bend 180 affects the arrangement of the particle flow and impurities according to size on the array bifurcation 160. Therefore, an appropriate turning angle needs to be set to achieve a better dispersion effect. If the turning angle is too small, the generated centrifugal force is insufficient to separate large - particle impurities, and the particle flow itself is also difficult to be effectively dispersed. If the turning angle is too large, it may cause the flow direction of the rear - segment filtration channel 142 and the flow direction in the first outlet channel 120 to have too small an angle. When the particles enter the first outlet channel 120, the lateral fluctuation is too large, and even a reverse flow may be formed, seriously affecting the dispersion of the particles passing through the array bifurcation 160 in the first outlet channel 120. Therefore, the turning angle C of the second bend 180 is generally appropriately in the range of 15 - 120 degrees, and the best choice is 30 - 60 degrees. Since the main filtration 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 - segment filtration channel 142 relative to the center line of the front - segment filtration channel 141.

[0129] See Figure 1, It should be noted that the velocity component of the flow velocity in the rear-stage filtration channel 142 in the flowing direction in the first outlet channel 120 should be the same as the flowing direction in the first outlet channel 120. Otherwise, it will cause the particles entering the first outlet channel 120 through the array bifurcation 160 to flow back, resulting in the re-focusing of the particles. In this case, it is only required that the angle D between the rear-stage filtration channel 142 and the first outlet channel 120 is an acute angle. Generally speaking, the smaller the angle D between the rear-stage filtration channel 142 and the first outlet channel 120, the smaller the lateral fluctuation when converging into the first outlet channel 120 through the array bifurcation 160, and the better the converging effect. However, if the angle D is too small, there will not be enough turning angle margin for the first bend 170 and the second bend 180. Generally, the angle D of 30-80 degrees is more appropriate.

[0130] In some embodiments, the cross-section of the first outlet channel 120 gradually becomes larger along the flowing direction, and the flow velocity slows down during the process of the width increasing. At the same time, it provides enough lateral movement margin for the particles to converge through the array bifurcation 160, preventing the particle flow from impacting the side wall of the first outlet channel 120 after converging into the first outlet channel 120.

[0131] It should be noted that in this embodiment, the size of each bifurcation of the array bifurcation 160 should match the size of the particles to be dispersed, so that the particles pass through each bifurcation individually. For example, the size of each bifurcation 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 the particles pass through each bifurcation individually; preferably, it should also be smaller than the size of the large particle impurities. The array bifurcation 160 of the present invention can be either an array of holes arranged in an array or a comb structure formed by several partition plates. No matter which structure, it is sufficient to satisfy that the particles pass through the corresponding channels individually. See Figure 1 , in this embodiment, the surface of the array bifurcation 160 close to the main filtration branch channel 140 is defined as the dispersion surface, and the dispersion surface is substantially parallel to the flowing direction (center line) of the first outlet channel 120. In this way, the rear-stage filtration channel 142 and the dispersion surface are arranged at an angle. 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 in a certain bifurcation, the flow resistance in this bifurcation is much greater than that in the bifurcations without particles. Therefore, under the action of this velocity component, the particles will move to the next bifurcation with a smaller flow resistance and pass through, so as to realize the single-particle intermittent passing through each bifurcation, and the shortest interval time is the time required for the particles to pass through this bifurcation.

[0132] In some embodiments, the array bifurcation 160 is a comb structure formed by arranging several partition plates, and the gap between adjacent combs is a bifurcation for the particles to pass through. Of course, the array bifurcation 160 is not limited to the comb structure, and it can also be other pore arrays with similar sizes.

[0133] In some embodiments, along the flow direction of the main filtration branch flow channel, the inner width of each channel of the comb structure gradually increases to reduce the flow resistance of the particle flow passing through the comb structure.

[0134] In some embodiments, along the flow direction of the first outlet flow channel, the inlet width of each bifurcation gradually decreases to ensure a relatively small overall flow resistance while achieving better large impurity filtration performance. Since there is a centrifugal force acting on the particle flow after passing through the second bend 180, it is almost impossible for large particle impurities to enter the left side of the array bifurcation 160. Therefore, the bifurcation size on the left side of the array bifurcation 160 can be set relatively larger to reduce the flow resistance, while the bifurcation size on the right side is set smaller to ensure the efficiency of filtering large particle impurities. Exemplarily, taking the passage of cells as an example, the bifurcation width of the rightmost part of the array bifurcation 160 is about 10 micrometers to filter large particle impurities, and the bifurcation width of the leftmost part of the array bifurcation 160 is approximately 15 - 20 micrometers to reduce the resistance for cells to pass through.

[0135] Due to the setting of the second bend 180, the particle flow undergoes centrifugal action, causing large particle impurities to be thrown to the right side. Therefore, large particle impurities are concentrated on the right side of the dispersion surface ( Figure 1 in the garbage 320), which can maximally avoid the blockage of the array bifurcation 160 by large particle impurities, thereby extending the service life and long - term stability of the array bifurcation 160.

[0136] After passing through the sheath - free flow filtration and dispersion structure 100 as described above, the particle flow is filtered out of large particle impurities and the particles are dispersed, enabling the particles to be arranged in the front - to - back direction along with the fluid flow, greatly reducing the difficulty of sheath - flow or sheath - free flow focusing.

[0137] Embodiment 2: This embodiment provides a sheath - free flow focusing structure 200 based on microfluidics, including

[0138] A second inlet flow channel 210, which is a flow channel for providing inflow;

[0139] A second outlet flow channel 230, which is a flow channel for providing outflow;

[0140] An intermediate branch 220, connected to the end of the second inlet flow channel 210 and the inlet end of the second outlet flow channel 230, and having a reduced cross - section along the flow direction; and

[0141] Two side branches 240, symmetrically arranged on both sides of the intermediate branch 220. The inlets of the two side branches 240 are opened for diversion from both sides of the inlet of the intermediate branch 220. 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 branch 240 is smaller than the inlet width of the intermediate branch 220;

[0142] Among them, 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 port at the inlet end of the second outlet flow channel 230.

[0143] In this embodiment, a fluid is respectively drawn from both sides of the inlet of the middle branch 220 through the side branches 240. By setting the inlet width of the side branches 240, the fluid basically does not contain particles. Since the initial size of the middle branch 220 is large, the particle flow all enters the middle branch 220. The width of the second half of the middle branch 220 gradually decreases, and only a single particle can pass through the outlet end, correcting the speed and direction of the particles, realizing that the particles enter the second outlet flow channel 230 in a single and centered arrangement. The fluid in the side branches 240 symmetrically converges from both sides of the inlet of the second outlet flow channel 230, wrapping the particle flow in the middle. Under the combined action of the fluid convergence of the middle branch 220 and the two side branches 240, particle flow focusing is completed. Since the inlet width of the second outlet flow channel 230 is greater than the outlet width of the middle branch 220, better particle focusing can be achieved after the fluid of the two side branches 240 is injected. It should be noted that if a large number of particles gather in the second inlet flow channel 210, the subsequent focusing effect will be 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 to keep the flow direction of the fluid therein stable, preventing turbulence from causing lateral movement of the particles to block the inlet of the side branch 240 and affecting the focusing effect.

[0145] In some embodiments, the width of the second inlet flow channel 210 is uniform to maintain better particle flow stability, enabling the particle flow to mainly move forward without generating turbulence and reducing lateral fluctuations; and the width of the second inlet flow channel 210 is less than the inlet width of the middle branch 220, enabling the particle flow to smoothly enter the middle branch 220. Even in the case of particle flow backflow caused by the diameter change of the middle branch 220, the inlet 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 (the angle between the centerlines of the two flow channels) between the inlet section 241 and the fluid flow direction in the intermediate branch 220 is an obtuse angle, which can ensure the smooth introduction of the fluid in the side branch 240 while avoiding particle blockage at the inlet of the side branch 240. If the angle E is too large, it will cause difficulty in draining the side branch 240. Therefore, the general design range is 90 - 120 degrees. The angle F (the angle between the centerlines of the two flow channels) between the outlet section 243 and the flow direction in the second outlet flow channel 230 is also an acute angle. By setting the angle F as an acute angle, when the fluid in the side branch 240 flows into the second outlet flow channel 230, a sheath flow effect can be well formed from both sides, avoiding 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; if it is set too small, it will cause the side branch 240 to occupy too much space. Therefore, in general, the design range of the angle F is 20 - 89 degrees, and the range of 30 - 60 degrees is the best.

[0147] In some embodiments, the second outlet flow channel 230 includes a focusing section 231 and a diameter-changing section 232. The focusing section 231 is used to converge the fluids from the two side branches 240 to both sides of the outlet fluid of the intermediate branch 220 to form a focused combined flow. The diameter-changing section 232 is used to adjust the focused combined flow to the required width. The width of the diameter-changing section 232 can be increased or decreased, specifically determined according to the use of the particle flow after focusing. For example, in this embodiment, the width of the diameter-changing section 232 is decreased to facilitate the observation of the experiment.

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

[0149] Embodiment 3: As Figure 3As shown in the figure, this embodiment provides a sheathless flow filtration and dispersion focusing structure, which includes the sheathless flow filtration and dispersion structure 100 described in Embodiment 1 and the sheathless flow focusing structure 200 described in Embodiment 2. A feed port 410 is arranged at the inlet of the sheathless flow filtration and dispersion structure 100. The first outlet channel 120 of the sheathless flow filtration and dispersion structure 100 is connected to the second inlet channel 210 of the sheathless flow focusing structure 200 through a transition channel 420. A discharge channel 430 is arranged at the outlet of the sheathless flow focusing structure 200 to export the focused particle flow. After the particle flow is input into the first inlet channel 110 of the sheathless flow filtration and dispersion structure 100 through the feed port 430, large particle impurities are filtered out by the sheathless flow filtration and dispersion structure 100 and the particle flow is dispersed to achieve preliminary focusing of the particle flow. Then, it is transported to the second inlet channel 210 of the sheathless flow focusing structure 200 through the variable-diameter transition channel 420. 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 channel 430.

[0150] The above channel structure can be fabricated on a substrate made of materials such as PDMS and acrylic by means of laser engraving, die stamping, casting, etching, etc. in the prior art to form channels, and then covered with a cover plate (such as a glass plate, an acrylic plate, etc.) to form a microfluidic chip. Since this application does not modify the channel preparation process and materials, the channel preparation process and materials do not have a significant impact on solving the technical problems of the present invention. Therefore, this application will not elaborate on this further.

[0151] In addition, it should be noted that as Figure 4 shown, the channels and bifurcations in the present invention are all formed by processing channels on the substrate 510 and covering the cover plate 520. Therefore, the channel size generally refers to the channel width, and the channel height H is not limited. Generally, in order to ensure that all particles pass through, the channel height should be greater than the diameter of the largest particle. If the channel is too high, the particle flow is likely to fluctuate in the height direction. Therefore, generally, it should be less than twice the diameter of the largest particle. If the particles are elastic such as cells, the height can be slightly less than the particle diameter.

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

[0153] (1) Cell preparation: A 6 cm culture dish and adherent Hela cells at a density of 80% are used. After trypsin digestion and centrifugation, a 6 ml cell suspension is obtained. After filtering through a filter with a pore size of about 30 μm and slightly standing, the upper layer liquid is taken and mixed with a density regulator to keep the cells from sinking to the bottom for a long time, serving as the cell suspension for the experiment.

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

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

[0156] (4) Optical path and image acquisition: Select a 20x microscope lens for bright-field observation, use a red light source of about 560 nm, and import the images formed by multiple groups of optical path components into a high-speed camera respectively. The high-speed camera operates with an exposure time of 10 μs and a frame rate of 1000 fps for acquisition. Using the middle part of the straight flow channel 440 ( Figure 5 the dashed box in) after the sheathless flow focusing structure 200 as the observation area, observation experiments are carried out.

[0157] (5) Start collecting cell images after the straight flow channel 440 is filled with liquid, record the centering of the cells on the horizontal plane of the straight flow channel 440, and whether the flux is uniform (the cells come at intervals, and there won't be a situation where many cells crowd together at one time and few cells come at another time).

[0158] The recorded results are as Figure 10 shown, Figure 10 From left to right are 5 different cells continuously photographed by the camera. The shooting parameters are 1000 fps. The numbers in the figure are the frame numbers. The adjacent frame interval is 1 millisecond, that is Figure 10 the cell intervals from left to right are 3 milliseconds, 4 milliseconds, 3 milliseconds, and 4 milliseconds respectively. The cells are basically centered in the flow channel, with clear edges. The cell dispersion is good. During the experiment, the flux is stable, and there are rarely phenomena of a large number of cells passing through in a short time or no cells passing through for a period of time, and it can operate stably for several hours.

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

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

[0161] Steps (2)-(5) are the same as those in Example 4.

[0162] The recorded results are as follows: There are few phenomena of a large number of cells passing through in a short time or no cells passing through for a period of time. The flux is stable, but the cell centering is not good.

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

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

[0165] Steps (2)-(5) are the same as those in Example 4.

[0166] The recorded results are as follows: As Figure 11A and Figure 11B shown in the figure, Figure 11A in (c) and (d) are images at different cell fluxes respectively. (c) shows that there are no cells passing through the flow channel, and (d) shows that there are multiple cells passing through. In actual observation, the cells are uneven, and there are phenomena of a large number of cells passing through in a short time or no cells passing through for a period of time. The flux is unstable. Because the flow channel in the observation area of this example is relatively narrow, the centering cannot be reflected. In addition, the overall part of the sheathless flow focusing structure 200 is imaged, as Figure 11BAs shown, it can be seen that in the second outlet channel 230, the cell centering performance is very good, but there is local aggregation, and there are phenomena such as a large number of cells passing through in a short time or no cells passing through for a period of time, and the flux is unstable.

[0167] Comparative Example 1: This embodiment provides a method for focusing by varying the cross-section of the channel

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

[0169] Steps (2) - (5) are the same as those in Example 4.

[0170] The recorded results are as Figure 12 shown, with unstable cell flux (a large number of cells passing through in a short time or no cells passing through for a period of time) and the phenomenon of cells not being centered (moving close to the channel wall).

[0171] Comparative Example 2: A well-known single-cell dispersion focusing method based on sheath flow

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

[0173] Steps (2) - (5) are the same as those in Example 4.

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

[0175] From the above recorded results, it can be seen that the sheathless flow filtration and dispersion structure 100 of the present invention can disperse cells (or particles in the particle flow) well, enabling the cells to pass through the flow channel at almost uniform intervals; through the sheathless flow focusing structure 200, the cells can be centered in the middle of the flow channel to achieve cell focusing; when the filtration and dispersion structure is used in combination with the sheathless flow focusing structure 200, the cell suspension can be dispersed and focused to form a cell flow with uniform intervals and centered, so as to meet the experimental requirements of single-cell imaging, single-cell sorting, single-cell encapsulation, etc.

[0176] The above embodiments are only used to illustrate the present invention, rather than limiting the present invention. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that various combinations, modifications or equivalent replacements 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 all be covered within the scope of the claims of the present invention.

Claims

1. A sheathless flow filtration and dispersion structure based on microfluidics, characterized in that, Comprising A first inlet channel, which is a channel for providing inflow; A first outlet channel, which is a channel for providing outflow; A bifurcated channel, arranged at the end of the first inlet channel, opening in a V-shaped manner, and used for dividing the fluid into two branches; Two main filtration branch channels, respectively connected to the end of each branch of the bifurcated channel through a first bend that turns relatively inwards. A second bend is also provided in each main filtration branch channel, such that the outlets of the two main filtration branch channels converge downstream of the first outlet channel; Two auxiliary filtration branch channels, the inlets of which are respectively connected to the ends of the corresponding first bends and are arranged closer to the inside relative to the main filtration branch channels; the outlets of the two auxiliary filtration branch channels converge to form the source of the first outlet channel; And An array bifurcation, arranged inside the outlet of each main filtration branch channel and downstream of the second bend. The bifurcations in the array bifurcation are arranged along the flow direction of the first outlet channel, and the particles in the particle flow pass through the array bifurcation at intervals and flow into the first outlet channel.

2. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, wherein The bifurcated channel is arranged in alignment with the first inlet channel, and the two branches of the bifurcated channel are symmetrically arranged.

3. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, wherein The sheathless flow filtration and dispersion structure is arranged symmetrically with respect to the central connection line of the first inlet channel and the first outlet channel.

4. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, wherein The main filtration branch channel gradually widens from the inlet to the outlet.

5. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, wherein The inlet width of the auxiliary filtration branch channel is smaller than the inlet of the main filtration branch channel, such that the inlet flow rate of the auxiliary filtration branch channel is less than that of the main filtration branch channel. At the same time, the auxiliary filtration branch channel gradually widens from the inlet to the outlet.

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

7. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, characterized in that, The turning angle of the first bend is 30 - 90 degrees.

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

9. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, characterized in that The first outlet channel gradually increases in cross-sectional area along the flow direction.

10. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 1, characterized in that, The array bifurcation is a comb structure formed by arranging a number of partition plates, and the gap between adjacent teeth is a bifurcation for particles to pass through.

11. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 10, characterized in that, Along the flow direction of the main filtration branch channel, the inner width of each bifurcation in the comb structure gradually increases.

12. The sheathless flow filtration and dispersion structure based on microfluidics according to claim 10, wherein Along the flow direction of the first outlet channel, the inlet width of each bifurcation gradually decreases.

13. A sheathless flow focusing structure based on microfluidics, characterized in that, Comprising A second inlet channel, which is a channel for providing inflow; A second outlet channel, which is a channel for providing outflow; An intermediate branch, connected to the end of the second inlet channel and the inlet end of the second outlet channel, and the cross-sectional area shrinks along the flow direction; And Two side branches, symmetrically arranged on both sides of the intermediate branch. The inlets of the two side branches are diverted from both sides of the inlet of the intermediate branch, the outlets of the two side branches are connected to the inlet end of the second outlet channel, and the inlet width of the side branch is smaller than the inlet width of the intermediate branch; Wherein, the outlets of the two side branches are arranged on both sides of the outlet of the intermediate branch to form a confluence port at the inlet end of the second outlet channel.

14. The sheathless flow focusing structure according to claim 13, wherein, The outlet width of the side branch is greater than the outlet width of the intermediate branch.

15. The sheathless flow focusing structure according to claim 13, wherein The width of the second inlet channel is smaller than the inlet width of the intermediate branch.

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

17. The sheathless flow focusing structure according to claim 13, wherein, 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, wherein, The width of the focusing segment is greater than the width of the middle branch exit.

19. A sheathless flow filtration dispersion focusing structure, characterized in that, It comprises the sheathless flow filtering dispersion structure as described in any one of claims 1 to 12 and the sheathless flow focusing structure as described in any one of claims 13 to 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 via a transition flow channel.

20. A sheathless particle flow filtration and 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 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 stream 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 filtering, dispersing and 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 inflowing particle flow, symmetrically introduce the two branches to both sides of the particle flow flowing out of the middle branch to form a protective layer, and achieve sheathless focusing of the particle flow.

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