Spiral structure inertial focusing microfluidic cell screening device and screening method thereof

Abstract

The invention provides a spiral structure inertia focusing microfluidic cell screening device and a screening method thereof.The spiral structure inertia focusing microfluidic cell screening device comprises a planar spiral structure, the planar spiral structure is a single-ring planar spiral structure, and a single-ring planar spiral structure channel in the planar spiral structure is provided with at least two inlet channels and at least three outlet channels; the single-ring plane spiral structure channel, the inlet channel and the outlet channel are rectangular cross section channels, the single-ring plane spiral structure is provided with a plurality of stabbing structures, and the injection pump is driven to adjust the flow of a blood sample and sheath flow liquid so as to adjust the starting point Reynolds number of the single-ring plane spiral structure channel, so that stable cell inertial focusing is realized; the device can replace a complex and high-cost multi-separation microfluidic device, is not easy to block, easy to clean, high in stability, not easy to deform, high in experiment accuracy and reliability, beneficial to long-term experiment operation and use, and high in repeated utilization rate.

Description

Technical Field

[0001] The present invention belongs to the field of medical research, specifically to the field of cancer diagnosis, prognosis, treatment and tumor metastasis research sample pretreatment, and more specifically to a spiral structure inertial focusing microfluidic cell screening device and a screening method thereof. Background Art

[0002] Circulating tumor cells (CTCs) are a general term for various types of tumor cells present in peripheral blood. They are shed from solid tumor lesions (primary lesions, metastatic lesions) into peripheral blood due to spontaneous or treatment operations. Although most of them can undergo apoptosis or be phagocytosed, a few become metastatic lesions, increasing the risk of death. Circulating tumor cells have been proven to be independent biomarkers for early diagnosis and prognosis of cancer patients. However, the frequency of circulating tumor cells in the peripheral blood of cancer patients is low (1~10 CTC / mL), and the difficulty of accurate counting and separation has prevented them from being widely used in cancer diagnosis and treatment. Therefore, in order to promote the research of cancer diagnosis, prognosis, treatment and tumor metastasis, it is urgent and critical to introduce new technologies to obtain the detection and separation of circulating tumor cells with high capture efficiency, enrichment, purity and throughput.

[0003] Microfluidics technology has the advantage of precise manipulation of liquids at the microscale. It can accurately control and process smaller volumes of samples according to mechanical and biological properties, making it very popular in cell sorting and separation research. There are two types of efficient and reliable microfluidic platforms. One type is to complete cell separation through the differences in biological properties between cells, which is the affinity strategy. However, specific antigens on different circulating tumor cells (such as Epcam) have different expression levels. Some circulating tumor cells may be lost during separation. At the same time, complex operations and high costs also limit the promotion and use of this method. The other type of technology is to complete cell separation through the differences in physical properties between cells (such as size, density and other physical quantities), which is also called a label-free strategy.

[0004] Label-free strategies include active separation techniques and passive separation techniques. Active separation techniques include acoustophoresis (AP), optical tweezers, dielectrophoresis (DEP), magnetophoresis (MP), etc. However, active separation techniques such as acoustics and optics have low throughput due to the limitation of fluid velocity, and usually require more complex external devices to provide additional complex physical fields. Passive separation techniques include filtration-based methods, deterministic lateral displacement (DLD), and inertial focusing. Among them, deterministic lateral displacement (DLD) and filtration separation techniques face problems such as easy channel clogging, inability to ensure cell viability after treatment, and poor device stability.

[0005] Inertial focusing is a passive separation technology for microfluidic processing. The Reynolds number (re) is a similarity criterion number that characterizes the effect of viscosity in fluid mechanics. It can be used to characterize the dimensionless number of fluid flow conditions. When the fluid flows through the microchannel in a low Reynolds number laminar flow, the particles or cells will be affected by the fluid shear force, wall interaction and inertial force. When these forces are balanced, the motion state of the particles or cells will be stable, causing the particles or cells to focus at a specific position in the microchannel. Inertial focusing technology has excellent separation performance and stability as well as the advantages of easy production and use.

[0006] Secondary flow refers to the fluid movement generated outside the mainstream direction, which usually occurs in channels with curved or disturbed obstacles. Due to the mismatch of fluid momentum in the center of curvature and the near-wall area, a radial pressure gradient will be generated, thus forming a secondary flow. This secondary flow usually manifests itself as two counter-rotating vortices, called Dean vortices. During the inertial focusing process, secondary flows (such as Dean vortices) will affect the equilibrium position of particles or cells. Due to the existence of secondary flows, particles or cells will be subjected to a drag force perpendicular to the mainstream direction. This force will change the equilibrium position of particles or cells, thereby affecting the effect of inertial focusing. On the other hand, inertial focusing itself will also be affected by the channel structure and fluid properties, which will also indirectly affect the generation and distribution of secondary flows.

[0007] In inertial focusing technology, spiral microfluidic devices have been widely studied due to their ease of operation, relatively simple channel design, high throughput, high energy efficiency, low cost, and portability. The principle of spiral microfluidic inertial focusing technology is the main factor affecting particle focusing performance. (inertial lift) and The synergistic effect of inertial lift and Dean drag determines the inertial focusing behavior and equilibrium position in a curved channel. The numerical meaning of this ratio is as follows: Inertial lift tends to keep particles at a specific cross-sectional position within the channel. In contrast, Dean drag tends to entrain particles to follow counter-rotating vortex streamlines. > When , the inertial lift force dominates and the particle migrates to the equilibrium position independently of the Dean flow. < When the spiral channel is properly designed, the Dean drag dominates the behavior of the particles, and the particles remain entrained in the Dean flow. When the spiral channel is properly designed, the cells with larger particle sizes will gradually migrate to the equilibrium position in the channel and form a focusing zone due to the dominant inertial lift. Only when the two forces are in the middle range of the same magnitude can the inertial equilibrium position be corrected by the Dean flow. The reasonable introduction of this correction method can have a positive effect on the focusing performance of larger cells. At the same time, smaller cells are entrained in the Dean flow due to the dominant Dean drag. By properly designing the channel length, cell sorting of different particle sizes can be achieved. However, the Archimedean spiral of the spiral inertial separation device has a continuously changing radius of curvature, and the radius of curvature of the spiral channel formed is also constantly changing, which leads to the change of the radius of curvature R during the movement of cells in the channel. (inertial lift) and (Dean resistance) ratio changes, and the instability of this ratio has a negative impact on the focusing performance of the cells. Therefore, the current traditional spiral microfluidic device for separating target cells is highly sensitive to flow rate. Only when Re reaches a certain large value can the cell focusing zone be stable. However, a larger Re brings about a larger device hydraulic pressure, which greatly reduces the stability of the device. Moreover, in the microfluidic devices of the prior art, the two-sorting device can separate a particle solution of a target particle size from particles of different particle sizes. It is a more common microfluidic sorting device, while the multi-sorting device can separate particle solutions of more than two target particle sizes from particles of different particle sizes. It is relatively rare. The sorting device can be used in the separation and screening of circulating tumor cells due to its working principle, and the multi-sorting device has a complex structure and high processing cost. The reasons are as follows: 1. When it meets , the cell can achieve inertial focusing, among which, is the ratio of cell diameter to characteristic length; 2. At the same time, The lift acting on the cell can overcome the Dean drag when The size of describes the strength of the competition between the cell inertial lift and the Dean drag, that is, The larger the value, the greater the inertial lift of the cell is, which is greater than the Dean drag. 3. Other factors, such as if the spacing between cell bands of different sizes is too narrow, will destroy the equilibrium state of the cell bands. In the sorting device, recesses or depressions are often set to enhance the focusing effect, but the retention of fluid particles, bubbles or other impurities in the recessed area structure can easily lead to residue accumulation and even channel blockage, and the local recessed structure in the microfluidics is difficult to clean thoroughly. In addition, the local depression will also bring a sudden increase in pressure to the channel, which may deform the original structure of the channel, affect the accuracy and reliability of subsequent experiments, and is not conducive to long-term experimental operation and use. The device has a low reusability rate. Therefore, a new technology is needed to improve this situation. Summary of the invention

[0008] In view of the above problems in the prior art, the present invention provides a spiral structure inertial focusing microfluidic cell screening device and a screening method thereof, which improves the spiral microfluidic device and overcomes the above problems. (inertial lift) and The problem of negative effect on the focusing performance of target cells caused by the change in the ratio of (Dean resistance) and the problem of target cells being highly sensitive to flow velocity are overcome, so that target cells can achieve a stable motion state at a lower flow rate and achieve stable cell inertial focusing. It can replace complex and expensive multi-sorting microfluidic devices to achieve efficient cell separation effects, is not easy to clog, is easy to clean, and has strong device stability and is not easy to deform. The experiment has high accuracy and reliability, is conducive to long-term experimental operation and use, and has a high reusability rate.

[0009] The present invention is implemented by the following technical scheme: a spiral structure inertial focusing microfluidic cell screening device, comprising a planar spiral structure, wherein the planar spiral structure is a single-ring planar spiral structure, wherein the single-ring planar spiral structure is an Archimedean spiral, wherein the Archimedean spiral is a clockwise rotating spiral, wherein a hollow channel is provided inside the single-ring planar spiral structure, wherein the hollow channel is a single-ring planar spiral structure channel;

[0010] The starting point of the single-ring planar spiral structure channel is connected by branches and is provided with at least two inlet channels, the inlet channels form an arc-shaped structure as a whole, the inlet channel includes an inner wall inlet channel and an outer wall inlet channel, the end point of the single-ring planar spiral structure is connected by branches and is provided with at least three outlet channels, the outlet channels form a straight surface structure as a whole, the single-ring planar spiral structure channel, the inlet channel, and the outlet channel are connected in the inner cavity, the single-ring planar spiral structure channel and the inlet channel and the outlet channel are rectangular cross-section channels, the outlet channel includes an inner wall outlet channel, a middle outlet channel, and an outer wall outlet channel;

[0011] The single-ring planar spiral structure is provided with a plurality of spur structures, wherein the spur structures are specifically of a corner type, and the spur structures are connected to the outside of the single-ring planar spiral structure, and the channel of the single-ring planar spiral structure is connected to the inner cavity of the spur structure; the top of the contour of the outer side of the spur structure is a processing curve, and the processing curve is a partial elliptical contour line A, and the partial elliptical contour line A is symmetrical about the vertex o of the partial elliptical contour line A, and each endpoint a of the partial elliptical contour line is connected to the adjacent endpoint b of the outer cross-section contour of the single-ring planar spiral structure through a rounded arc line B with a radius r, wherein the endpoint a , the endpoint b is connected to the rounded arc line B in a tangent line; the center point x of the partial elliptical contour line A is located on the outer cross-sectional contour line C of the single-ring planar spiral structure, the line connecting the vertex o of the partial elliptical contour line A and the center point x of the partial elliptical contour line A is the major axis of the thorn structure, the length of the major axis of the thorn structure and the height of the cross-sectional area of ​​the single-ring planar spiral structure channel are both α, the intersection point e of the extension line D of the partial elliptical contour line A and the outer cross-sectional contour line C of the single-ring planar spiral structure, the line connecting two adjacent intersection points e is the minor axis of the thorn structure, and the length of the minor axis of the thorn structure is L;

[0012] The Archimedean spiral is drawn by a Cartesian coordinate system, and the position of one point of the Archimedean spiral in the Cartesian coordinate system is assigned as θ, and the angle formed by the vector formed by one point of the Archimedean spiral and the origin of the Cartesian coordinate system and the positive direction of the horizontal axis of the Cartesian coordinate system is 2πθ; the position of one of the starting points of the stab structure in the Cartesian coordinate system is assigned as θs, and the position of one of the ending points of the stab structure in the Cartesian coordinate system is assigned as θe, θe-θs=β, and the geometric parameters of a single stab structure are: α=300μm, r=0.2mm, β=0.05, wherein the floating value of α is ±10μm, the floating value of β is ±0.001, and the floating value of r is ±0.03mm, and the numerical relationship between β and the short axis length L of the stab structure is as follows:

[0013] .

[0014] Furthermore, the coordinate equation of the starting point θs of one of the spur structures in the Cartesian coordinate system is:

[0015]

[0016]

[0017] Wherein, the x and y are the horizontal coordinate value and the vertical coordinate value corresponding to a point in the Cartesian coordinate system.

[0018] Furthermore, there are five stab structures, and the positions of the five stab structures from the entrance to the exit are θ1, θ2, θ3, θ4, and θ5 respectively. The absolute position of a single stab structure unit is: θ1=0.3, θ2=0.35, θ3=0.4, θ4=0.49, θ5=0.53; wherein the floating values ​​of θ1, θ2, θ3, θ4, and θ5 are ±0.01.

[0019] Furthermore, the Archimedean spiral expression (equation driven curve):

[0020] Inner wall:

[0021] ;

[0022] Outer wall:

[0023] ; The range of θ: 0<=θ<=1.

[0024] Furthermore, the rectangular cross-sectional height of the single-ring planar spiral structure channel is 450um, with a floating value of ±10um, and the rectangular cross-sectional heights of the inlet channel and the outlet channel are both 180um, with a floating value of ±3um.

[0025] The present invention also includes a screening method of a spiral structure inertial focusing microfluidic cell screening device, which specifically includes the following steps:

[0026] S1: respectively loading syringes containing sheath fluid and mixed particle solutions with different particle sizes into syringe pumps, injecting sheath fluid into the inner wall inlet channel and injecting mixed particle solutions with different particle sizes into the outer wall inlet channel, wherein the mixed particle solutions with different particle sizes are blood samples, and the sheath fluid and the blood sample merge into a fluid solution at the starting point of the single-ring planar spiral structure channel, wherein the blood sample contains one or more of the target sorting cells, human breast cancer cells or lung cancer human alveolar basal epithelial cells;

[0027] S2: driving the syringe pump to make the fluid solution flow, the laminar flow at the inlet of the inner wall channel flows evenly at a flow rate of 0.95 times the total flow rate Q, the laminar flow at the inlet of the outer wall channel flows evenly at a flow rate of 0.05 times the total flow rate Q, and the static pressure P of the outlet channel is 0;

[0028] S3: The Reynolds number at the starting point of the single-ring planar spiral structure channel is adjusted by adjusting the flow rate of the blood sample and the sheath fluid to make the Reynolds number below 45. The distribution and focusing state of various cells in the channel is used to determine that this stage is the chaotic period.

[0029] When the Reynolds number is between 45 and 60, the focusing trend of the target cells, human breast cancer cells and human alveolar basal epithelial cells of lung cancer, is used to determine that this stage is transition stage 1;

[0030] When the Reynolds number is between 60 and 70, the stage is judged as sorting stage 1 by the formation of the target cell human breast cancer cells, lung cancer human alveolar basal epithelial cells and the unsorted cells in the outlet channel;

[0031] When the Reynolds number is between 70 and 90, the moving direction of the unsorted cells in the outlet channel is used to determine that this stage is transition period 2;

[0032] When the Reynolds number is between 90 and 100, the distribution of unsorted cells and lung cancer human alveolar basal epithelial cells in the outlet channel determines that this stage is sorting stage 2;

[0033] S4: Post-processing by flow cytometry to count the sorted target cells human breast cancer cells and lung cancer human alveolar basal epithelial cells.

[0034] Furthermore, the optimal Reynolds number range for inertial aggregation sorting of the blood sample is 65-68.

[0035] Beneficial Effects

[0036] The device is provided with a spiral microfluidic device with a rectangular cross-section. When the fluid flows in the spiral microfluidic channel, due to the centrifugal acceleration in the radial direction outward from the center of curvature, two counter-rotating vortices (Dean vortices) are formed at the upper and lower parts of the channel, and the cells in the channel are subjected to inertial lift. and Dean resistance Influence, during the flow in the channel, the inertial lift caused by the shear flow ( ) causes larger cells to move toward the inner wall, while the Dean drag ( ) pushes the small cells toward the outer wall, and Under the influence of Under the leading role of the , medium-sized cells (such as lung cancer human alveolar basal epithelial cells) formed a cell band in the middle part of the channel and flowed out from the middle outlet, while small red blood cells and white blood cells Under the leading role of the spiking structure, the cells gradually migrated to the outer wall and formed a cell belt. At the same time, the introduction of the spike structure sequence in the spiral microfluidic device channel produced a The effect is similar to lift. When the fluid passes through the sequence of spur structures, and The ratio of can be locally amplified, and the overall force and movement state of the target cell are positively regulated, thereby enhancing the focusing performance of the target cell and forming a narrower cell focusing band, while overcoming the (inertial lift) and The problem of negative effect on the focusing performance of target cells caused by the change in the ratio of (Dean resistance) and the problem of target cells being highly sensitive to flow velocity are overcome, so that target cells can achieve a stable motion state at a lower flow rate and achieve stable cell inertial focusing. It can replace complex and expensive multi-division microfluidic devices, and ultimately achieve a more efficient cell separation effect. It is not easy to be clogged and easy to clean, and the device is stable and not easy to deform. The experiment has high accuracy and reliability, which is conducive to long-term experimental operation and use and has a high reusability rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 A perspective view of an embodiment of the present invention;

[0038] Figure 2 A diagram of an inlet channel and an outlet channel according to an embodiment of the present invention;

[0039] Figure 3 A three-dimensional diagram of a stab structure according to an embodiment of the present invention;

[0040] Figure 4 A cross-sectional view of a stab structure according to an embodiment of the present invention;

[0041] Figure 5 It is a plan view of the absolute positions of five spur structures θ1, θ2, θ3, θ4, and θ5 according to an embodiment of the present invention;

[0042] Figure 6 A finite element analysis cloud diagram of a spur structure according to an embodiment of the present invention;

[0043] Figure 7 Finite element analysis cloud diagrams of sections 1, 2, 3, 4 and 5 of the spur structure according to an embodiment of the present invention;

[0044] Figure 8 This is a flow chart of a screening method of a spiral structure inertial focusing microfluidic cell screening device for sorting target cells, which are human breast cancer cells and lung cancer human alveolar basal epithelial cells, according to an embodiment of the present invention;

[0045] Fig. 9 It is a diagram showing the movement of human breast cancer cells (MCF-7), lung cancer human alveolar basal epithelial cells (A549), white blood cells (WBCS), and red blood cells (RBCS) in a channel near an outlet as the normalized distance from the inner wall changes with the Reynolds number Re in an embodiment of the present invention;

[0046] Fig.10 This is a diagram showing the distribution status of various types of cells in the outlet channel according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0048] This embodiment is obtained by using particles of 24um, 15um, 10um, and 8um sizes to replace human breast cancer cells (MCF-7), lung cancer human alveolar basal epithelial cells (A549), white blood cells (WBCS), and red blood cells (RBCS) in blood samples to conduct COMSOL simulation experiments.

[0049] This embodiment is implemented by the following technical solutions: Figure 1 As shown, a spiral structure inertial focusing microfluidic cell screening device includes a planar spiral structure, the planar spiral structure is a single-ring planar spiral structure, the single-ring planar spiral structure is an Archimedean spiral, the Archimedean spiral is a clockwise rotating spiral, and a hollow channel is provided inside the single-ring planar spiral structure, and the hollow channel is a single-ring planar spiral structure channel;

[0050] like Figure 2 As shown, the starting point of the single-loop planar spiral structure channel is connected by branches with two entrance channels, the entrance channels form an arc-shaped structure as a whole, the entrance channels include an inner wall entrance channel and an outer wall entrance channel, the end point of the single-loop planar spiral structure is connected by branches with three exit channels, the exit channels form a straight-face structure as a whole, the single-loop planar spiral structure channel, the entrance channel, and the exit channel are connected in the inner cavity, the single-loop planar spiral structure channel and the entrance channel and the exit channel are rectangular cross-section channels, and the exit channel includes an inner wall exit channel, a middle exit channel, and an outer wall exit channel.

[0051] like Figure 3-Figure 5As shown, the single-ring planar spiral structure is provided with 5 spur structures, which are specifically of corner type. The spur structures are connected to the outside of the single-ring planar spiral structure, and the channel of the single-ring planar spiral structure is connected with the inner cavity of the spur structure; the top of the contour of the outer side of the spur structure is a processing curve, and the processing curve is a partial elliptical contour line A, and the partial elliptical contour line A is symmetrical about the vertex o of the partial elliptical contour line A. The end point a of each partial elliptical contour line is connected with the end point b of the outer cross-section contour of the adjacent single-ring planar spiral structure through a rounded arc line B with a radius r, wherein the end point Point a and end point b are connected to the rounded arc line B in a tangent line; the center point x of the partial ellipse contour line A is located on the outer cross-sectional contour line C of the single-ring planar spiral structure; the line connecting the vertex o of the partial ellipse contour line A and the center point x of the partial ellipse contour line A is the long axis of the spur structure; the length of the long axis of the spur structure and the height of the cross-sectional area of ​​the single-ring planar spiral structure channel are both α; the intersection point e of the extension line D of the partial ellipse contour line A and the outer cross-sectional contour line C of the single-ring planar spiral structure, the line connecting the two adjacent intersection points e is the short axis of the spur structure, and the length of the short axis of the spur structure is L;

[0052] The Archimedean spiral is drawn in a Cartesian coordinate system. The position of one point of the Archimedean spiral in the Cartesian coordinate system is assigned as θ. The angle formed by the vector formed by one point of the Archimedean spiral and the origin of the Cartesian coordinate system and the positive direction of the horizontal axis of the Cartesian coordinate system is 2πθ; the position of the starting point of one of the spur structures in the Cartesian coordinate system is assigned as θs, and the position of the ending point of one of the spur structures in the Cartesian coordinate system is assigned as θe, θe-θs=β, and the geometric parameters of a single spur structure are: α=300μm, r=0.2mm, β=0.05, wherein the floating value of α is ±10μm, the floating value of β is ±0.001, and the floating value of r is ±0.03mm. The numerical relationship between β and the short axis length L of the spur structure is as follows:

[0053] .

[0054] In this embodiment, the coordinate equation of a starting point θs of one of the spur structures in the Cartesian coordinate system is:

[0055]

[0056]

[0057] Wherein, x, y are the horizontal and vertical coordinate values ​​corresponding to a point in the Cartesian coordinate system.

[0058] In this embodiment, there are five stab structures, and the position θs of the starting point of each stab structure in the Cartesian coordinate system is taken as the position of the stab structure. The positions of the five stab structures from the entrance to the exit are θ1, θ2, θ3, θ4, and θ5, respectively. The absolute positions of the five stab structures θ1, θ2, θ3, θ4, and θ5 are shown in Table 1:

[0059] Spike structural unit θ1 θ2 θ3 θ4 θ5 Absolute Position 0.3 0.35 0.4 0.49 0.53

[0060] Table 1: Absolute position parameter table of the stab structure

[0061] The spacing between adjacent spurs θ1-θ2, θ2-θ3, θ3-θ4, and θ4-θ5 is shown in Table 2:

[0062] Adjacent spur structures θ1-θ2 θ2-θ3 θ3-θ4 θ4-θ5 spacing 2.8 (mm) 2.8 (mm) 5.1 (mm) 2.3 (mm)

[0063] Table 2: Parameters of spacing between adjacent spur structures

[0064] It can be seen that the absolute position of a single stab structure unit is: θ1=0.3, θ2=0.35, θ3=0.4, θ4=0.49, θ5=0.53, among which the floating values ​​of θ1, θ2, θ3, θ4, and θ5 are ±0.01; the spacing between a single stab structure unit: the distance from θ1 to θ2 is 2.8mm, the distance from θ2 to θ3 is 2.8mm, the distance from θ3 to θ4 is 5.1mm, and the distance from θ4 to θ5 is 2.3mm.

[0065] In this embodiment, the Archimedean spiral expression (equation driven curve):

[0066] Inner wall:

[0067] ;

[0068] Outer wall:

[0069] ; The range of θ: 0<=θ<=1, when θ=0, it is the starting point of the Archimedean spiral, and when θ=1, it is the ending point of the Archimedean spiral;

[0070] The inlet channel is formed as a semicircular arc structure with a radius of R1 = 4 mm;

[0071] The outlet channel formed an overall length of 1.4 mm.

[0072] In this embodiment, the rectangular cross-section width of the single-ring planar spiral structure channel is 180um, and the height is 450±10um. The rectangular cross-section height of the inlet channel and the rectangular cross-section height of the outlet channel are both 180um, and the floating value is ±3um. The rectangular cross-section width of the inner wall inlet channel is 315um, and the rectangular cross-section width of the outer wall inlet channel is 135um. The rectangular cross-section width of the inner wall outlet channel is 105um, the rectangular cross-section width of the middle outlet channel is 300um, and the rectangular cross-section width of the outer wall outlet channel is 243um.

[0073] like Figure 6-Figure 7 As shown, by performing finite element analysis on the velocity field of the fluid solution passing through the stab structure and the five cross-sectional locations of the stab structure, namely, section 1, section 2, section 3, section 4, and section 5, it can be seen that in the process of the fluid solution passing through the stab structure, the velocity of the fluid solution is a process of first decreasing and then increasing. More specifically, a specific velocity field characterization is performed on the cross-sections at five different positions of the stab structure. The velocity field characterization includes the magnitude and direction of the velocity. The background color (blue) of the cross-sectional area indicates the distribution of the magnitude of the velocity field, and the arrow indicates the direction of the velocity vector. In the process of the fluid solution passing through the stab structure, it moves from section 1 to section 5. In addition to the above-mentioned laws occurring at the level of flow velocity magnitude, the direction of the velocity field inside the fluid solution also changes. The reason for the change in the magnitude of the velocity in the velocity field is that the introduction of the stab structure causes a change in the cross-sectional area of ​​the channel, and the reason for the change in the direction of the velocity vector is that the stab structure generates a lift force similar to the FL effect.

[0074] The present invention also includes a screening method of a spiral structure inertial focusing microfluidic cell screening device, such as Figure 8 As shown, the specific steps include:

[0075] S1: The syringes containing sheath fluid and mixed particle solutions with different particle sizes are respectively loaded into the syringe pump, the sheath fluid is injected into the inner wall inlet channel, and the mixed particle solutions with different particle sizes are injected into the outer wall inlet channel, the mixed particle solutions with different particle sizes are blood samples, the sheath fluid and the blood sample are merged into a fluid solution at the starting point of the single-ring planar spiral structure channel, wherein the blood sample contains one or more of the target sorting cells human breast cancer cells or lung cancer human alveolar basal epithelial cells;

[0076] S2: Drive the injection pump to make the fluid solution flow. The laminar flow at the inlet of the inner wall channel flows evenly at a flow rate of 0.95 times the total flow rate Q, and the laminar flow at the inlet of the outer wall channel flows evenly at a flow rate of 0.05 times the total flow rate Q. Specifically, the inner wall inlet flow rate of the single-ring spiral microfluidic device is Q1=0.95*Q=1167.07ul / min, the outer wall inlet flow rate is Q2=0.05*Q=61.42ulmin, the total flow rate Q is 1228.50ul / min, and the static pressure P of the outlet channel is 0.

[0077] S3: The Reynolds number at the starting point of the single-ring planar spiral structure channel is adjusted by adjusting the flow rate of the blood sample and the sheath fluid to make the Reynolds number below 45. The various cells in the channel are in a chaotic state and cannot achieve inertial focusing. This stage is the chaotic period.

[0078] When the Reynolds number is between 45 and 60, the target cells gradually begin to focus, and this stage is transition period 1;

[0079] When the Reynolds number is between 60 and 70, the target cells human breast cancer cells and lung cancer human alveolar basal epithelial cells begin to form a single tight cell focusing zone, among which human breast cancer cells form a cell focusing zone in the inner wall outlet channel, lung cancer human alveolar basal epithelial cells form a cell focusing zone in the middle outlet channel, and unsorted cells form a cell focusing zone in the outer wall outlet channel. At this time, the three classifications of human breast cancer cells, lung cancer human alveolar basal epithelial cells and unsorted cells can be achieved. This stage is sorting stage 1;

[0080] When the Reynolds number is between 70 and 90, red blood cells and white blood cells move toward the inner wall outlet channel. This stage is transition period 2.

[0081] When the Reynolds number is between 90 and 100, red blood cells, white blood cells, and lung cancer human alveolar basal epithelial cells are only distributed in the middle outlet channel. At this time, human breast cancer cells and unsorted cells can be classified into two categories. This stage is sorting stage 2.

[0082] like Fig. 9 As shown in the figure, according to the normalized distance of human breast cancer cells (MCF-7), lung cancer human alveolar basal epithelial cells (A549), white blood cells (WBCS), and red blood cells (RBCS) from the inner wall and the movement of them near the outlet channel as the Reynolds number Re changes, when the entrance channel Reynolds number gradually increases from 30 to 60, human breast cancer cells (MCF-7) gradually form a tight focusing zone at the inner wall of the channel, and lung cancer human alveolar basal epithelial cells (A549) also gradually form a focusing zone in the middle of the channel. When Re continues to increase, MCF-7 and A549 show better focusing performance, and the focusing equilibrium position tends to be stable;

[0083] However, only when the channel entrance Reynolds number is in the range of 60 to 70, the white blood cell (WBCS) and red blood cell (RBCS) focusing bands are close to the side of the channel outer wall;

[0084] When Re gradually increases from 70 to 90, the cell bands of the two gradually migrate to the center of the channel. When Re gradually increases from 90 to 100, the white blood cells (WBCS) and red blood cells (RBCS) focus bands are located in the middle of the channel.

[0085] S4: Post-processing by flow cytometry to count the sorted target cells human breast cancer cells and lung cancer human alveolar basal epithelial cells

[0086] In this embodiment, the optimal Reynolds number range for achieving inertial aggregation sorting of blood samples is 65-68.

[0087] The working principle of this embodiment is as follows:

[0088] 1. Through the rectangular cross-section spiral microfluidic device, when the fluid flows in the spiral microfluidic channel, due to the centrifugal acceleration in the radial direction outward from the center of curvature, it forms two counter-rotating vortices (Dean vortices) at the upper and lower parts of the channel. The rectangular channel can satisfy the following relationship:

[0089]

[0090] in is the channel width, is the channel height, is the hydraulic diameter.

[0091] 2. Due to the introduction of Dean flow, the cells in the channel are mainly affected by two forces: one is inertial lift , one is Dean resistance . The shear gradient lift force pushes the cells toward the channel wall. and wall lift The inertial lift force formed can guide cells with larger particle size to move to the inner wall. The inertial lift force can be described by the following equation:

[0092]

[0093] in is the lift coefficient, which is the cell position and the channel Reynolds number Re, is the diameter of the cell, and are the maximum values ​​of fluid density and fluid velocity, respectively, where =1.5 , is the average flow velocity;

[0094] (inertial lift) and The ratio of (Dean's resistance) can be described by the following equation: , where R is the radius of curvature in the Archimedean spiral.

[0095] 3. Such as Fig.10 As shown in the figure, the movement of cells of different particle sizes in the channel near the outlet with different Reynolds numbers. For a rectangular channel, when Re < 100, the lift coefficient is equal to 0.5, and the Dean drag caused by the curvature of the spiral channel ( ) is used to push the small cells toward the outer wall of the spiral channel. The Dean resistance can be described by the following equation: ,in , , They represent dynamic viscosity, fluid velocity, and cell velocity, respectively;

[0096] When 1 < Re < 100, the Dean resistance can be calculated as , It is used to characterize the Dean velocity and can be described as the following equation:

[0097] in, It represents the Dean number, which can be used to measure the size of the secondary flow.

[0098] 4. The synergistic effect of inertial lift and Dean drag. Dean drag entrains cells to follow the counter-rotating vortex streamline. When the two forces are at the same magnitude, the inertial equilibrium position can be corrected by the Dean flow, and only then can the cells be effectively separated. and Under the influence of Under the leading role of the medium-sized cells, they gathered into a cell belt near the inner wall. The medium-sized cells formed a cell belt in the middle part of the channel and flowed out from the middle outlet, while the small cells Under the leading role of the The size of describes the strength of the competition between the cell inertial lift and the Dean drag, that is, The larger it is, the stronger the inertial lift force on the cell is than the Dean drag. Whether it is greater than 0.07 is the index of particle size. Can cells with a characteristic length of The spiral microfluidic channel is a necessary condition for achieving excellent inertial focusing. Whether the index is greater than 0.08 indicates that the particle size in the spiral microfluidic channel is Whether the inertial lift of the cells can overcome the Dean drag, and the strength of the inertial lift of the cells to overcome the Dean drag also reflects the equilibrium position of the corresponding cell band on the channel cross section. as well as The value of can effectively help explain the movement state of cells of various sizes in the channel and their distribution on the cross section of the outlet channel. Diameter of human breast cancer cells, lung cancer human alveolar basal epithelial cells, white blood cells, and red blood cells , cell diameter With characteristic length Ratio , The data is shown in Table 3:

[0099]

[0100] Table 3: Cell diameter , cell diameter With characteristic length Ratio , Data Sheet

[0101] For human breast cancer cells: the ratio of the diameter to the characteristic length of human breast cancer cells =0.093>0.07, the strength relationship between the inertial lift and Dean drag of human breast cancer cells =0.542>0.08, so human breast cancer cells can achieve inertial focusing on the inner wall of the channel;

[0102] For lung cancer human alveolar basal epithelial cells, although the ratio of the diameter to the characteristic length of lung cancer human alveolar basal epithelial cells =0.058<0.07, but the strength of the competition between the inertial lift and Dean drag of human alveolar basal epithelial cells in lung cancer =0.212>0.08, so although the lung cancer human alveolar basal epithelial cells cannot be focused on the inner wall, the lift force can effectively overcome the Dean drag, so they can form a stable cell belt in the middle of the channel;

[0103] For white blood cells, the ratio of the white blood cell diameter to the characteristic length =0.039<0.07, the strength relationship between the competition between the inertial lift of white blood cells and the Dean drag =0.095>0.08, but considering that The value is close to the critical value of the index, so the effect of lift overcoming resistance is not obvious. The cell band formed by white blood cells is more biased to the outside of the channel than the cell band of human alveolar basal epithelial cells of lung cancer. The difference is large, and the cell bands of the two will not overlap;

[0104] For red blood cells, the ratio of the red blood cell diameter to the characteristic length =0.031<0.07, the strength of the competition between the inertial lift of red blood cells and the Dean drag =0.06<0.08, at this time, the cell band formed by the red blood cells is closest to the outer wall of the channel compared with the previous types of cell bands. In summary, the equilibrium position of the cell bands formed by the four types of cells changes from the inner wall of the channel to the outer wall of the channel in descending order with the cell size, and satisfies the following relationship:

[0105]

[0106]

[0107] On this basis, three-step sorting of target cells was achieved.

[0108] The advantages of this embodiment are as follows:

[0109] 1. Due to the introduction of the spur structure, the inertial lift of the target cells is adjusted by increasing the cross-sectional area of ​​the channel to cause additional lift, thereby promoting cell focusing. Compared with the similar concave structure used in the prior art, the physical structure is just the opposite, which improves the original problem of easy blockage and easy change of the channel. In addition, the chip only needs to use a syringe to quickly inject clean water into the channel to flush out impurities, and the channel can be cleaned. The cleaning step is very simple. At the same time, although the existing similar concave structure and the proposed spur structure both have the function of regulating the movement of target cells, the regulation effect of the concave structure is more radical, mainly regulating cell movement by enhancing the Dean speed, while the regulation effect of the proposed spur structure is more gentle, and in comparison, it causes less damage to cells in practical applications;

[0110] 2. By introducing the spike sequence, the sensitivity of the target sorted cells to the flow rate was reduced. The focused human breast cancer cells achieved a stable cell focusing zone after Re=60 and were no longer affected by the flow rate.

[0111] 3. The regulatory effect induced by the spiking structure makes the force on the cells in the channel more stable during movement, overcoming the (Dean resistance) and The problem of negative effect on the focusing performance of target cells caused by the change in the ratio of (inertial lift) is solved, and the problem of target cells being more sensitive to flow velocity is overcome, so that target cells can achieve a stable motion state at a lower flow rate. In this way, target cells can focus and migrate to the equilibrium position faster, effectively shortening the focusing length.

[0112] The above is only one of the preferred embodiments of the present invention and is not a formal limitation of the present invention. It should be understood that the techniques, methods and devices known to ordinary technicians in the relevant fields may not be discussed in detail, but in appropriate cases, the techniques, methods and devices should be regarded as part of the authorization specification and should be included in the protection scope of the present invention within the scope of the features defined in the claims.

Claims

1. A spiral structure inertial focusing microfluidic cell screening device, characterized in that: It comprises a planar spiral structure, wherein the planar spiral structure is a single-ring planar spiral structure, wherein the single-ring planar spiral structure is an Archimedean spiral, wherein the Archimedean spiral is a clockwise rotating spiral, wherein a hollow channel is provided inside the single-ring planar spiral structure, and wherein the hollow channel is a channel of the single-ring planar spiral structure; The starting point of the single-ring planar spiral structure channel is connected by branches and is provided with at least two inlet channels, the inlet channels form an arc-shaped structure as a whole, the inlet channel includes an inner wall inlet channel and an outer wall inlet channel, the end point of the single-ring planar spiral structure is connected by branches and is provided with at least three outlet channels, the outlet channels form a straight surface structure as a whole, the single-ring planar spiral structure channel, the inlet channel, and the outlet channel are connected in the inner cavity, the single-ring planar spiral structure channel and the inlet channel and the outlet channel are rectangular cross-section channels, the outlet channel includes an inner wall outlet channel, a middle outlet channel, and an outer wall outlet channel; The single-ring planar spiral structure is provided with a plurality of spur structures, wherein the spur structures are specifically of a corner type, and the spur structures are connected to the outside of the single-ring planar spiral structure, and the channel of the single-ring planar spiral structure is connected to the inner cavity of the spur structure; the top of the contour of the outer side of the spur structure is a processing curve, and the processing curve is a partial elliptical contour line A, and the partial elliptical contour line A is symmetrical about the vertex o of the partial elliptical contour line A, and each endpoint a of the partial elliptical contour line is connected to the adjacent endpoint b of the outer cross-section contour of the single-ring planar spiral structure through a rounded arc line B with a radius r, wherein each endpoint a , each of the endpoints b is connected to the rounded arc line B in a tangent line; the center point x of the partial elliptical contour line A is located on the outer cross-sectional contour line C of the single-ring planar spiral structure, the line connecting the vertex o of the partial elliptical contour line A and the center point x of the partial elliptical contour line A is the major axis of the thorn structure, the length of the major axis of the thorn structure and the height of the cross-sectional area of ​​the single-ring planar spiral structure channel are both α, the intersection point e of the extension line D of the partial elliptical contour line A and the outer cross-sectional contour line C of the single-ring planar spiral structure, the line connecting two adjacent intersection points e is the minor axis of the thorn structure, and the length of the minor axis of the thorn structure is L; The Archimedean spiral is drawn by a Cartesian coordinate system, and the position of one point of the Archimedean spiral in the Cartesian coordinate system is assigned as θ, and the angle formed by the vector formed by one point of the Archimedean spiral and the origin of the Cartesian coordinate system and the positive direction of the horizontal axis of the Cartesian coordinate system is 2πθ; the position of one of the starting points of the stab structure in the Cartesian coordinate system is assigned as θs, and the position of one of the ending points of the stab structure in the Cartesian coordinate system is assigned as θe, θe-θs=β, and the geometric parameters of a single stab structure are: α=300μm, r=0.2mm, β=0.05, wherein the floating value of α is ±10μm, the floating value of β is ±0.001, and the floating value of r is ±0.03mm, and the numerical relationship between β and the short axis length L of the stab structure is as follows: 。 2. The spiral structure inertial focusing microfluidic cell screening device according to claim 1, characterized in that: The coordinate equation of the starting point θs of one of the spur structures in the Cartesian coordinate system is: Wherein, the x and y are the horizontal coordinate value and the vertical coordinate value corresponding to a point in the Cartesian coordinate system.

3. The spiral structure inertial focusing microfluidic cell screening device according to claim 1, characterized in that: There are five stab structures, and the positions of the five stab structures from the entrance to the exit are θ1, θ2, θ3, θ4, and θ5 respectively. The absolute position of a single stab structure unit is: θ1=0.3, θ2=0.35, θ3=0.4, θ4=0.49, θ5=0.53, wherein the floating values ​​of θ1, θ2, θ3, θ4, and θ5 are ±0.

01.

4. The spiral structure inertial focusing microfluidic cell screening device according to claim 1, characterized in that: The Archimedean screw expression is: Inner wall: ; Outer wall: ; The range of θ: 0<=θ<=1.

5. The spiral structure inertial focusing microfluidic cell screening device according to claim 1, characterized in that: The rectangular cross-sectional height of the single-ring planar spiral structure channel is 450um, and the floating value is ±10um. The rectangular cross-sectional height of the inlet channel and the rectangular cross-sectional height of the outlet channel are both 180um, and the floating value is ±3um.

6. A screening method of a spiral structure inertial focusing microfluidic cell screening device, characterized in that: The specific steps include: S1: respectively loading syringes containing sheath fluid and mixed particle solutions with different particle sizes into syringe pumps, injecting sheath fluid into the inner wall inlet channel and injecting mixed particle solutions with different particle sizes into the outer wall inlet channel, wherein the mixed particle solutions with different particle sizes are blood samples, and the sheath fluid and the blood sample merge into a fluid solution at the starting point of the single-ring planar spiral structure channel, wherein the blood sample contains one or more of the target sorting cells, human breast cancer cells or lung cancer human alveolar basal epithelial cells; S2: driving the syringe pump to make the fluid solution flow, the laminar flow at the inlet of the inner wall channel flows evenly at a flow rate of 0.95 times the total flow rate Q, the laminar flow at the inlet of the outer wall channel flows evenly at a flow rate of 0.05 times the total flow rate Q, and the static pressure P of the outlet channel is 0; S3: The Reynolds number at the starting point of the single-ring planar spiral structure channel is adjusted by adjusting the flow rate of the blood sample and the sheath fluid to make the Reynolds number below 45. The distribution and focusing state of various cells in the channel is used to determine that this stage is the chaotic period. When the Reynolds number is between 45 and 60, the focusing trend of the target cells, human breast cancer cells and human alveolar basal epithelial cells of lung cancer, is used to determine that this stage is transition period 1; When the Reynolds number is between 60 and 70, the stage is judged as sorting stage 1 by the formation of the target cell human breast cancer cells, lung cancer human alveolar basal epithelial cells and the unsorted cells in the outlet channel; When the Reynolds number is between 70 and 90, the moving direction of the unsorted cells in the outlet channel is used to determine that this stage is transition period 2; When the Reynolds number is between 90 and 100, the distribution of unsorted cells and lung cancer human alveolar basal epithelial cells in the outlet channel determines that this stage is sorting stage 2; S4: Post-processing by flow cytometry was performed to count the sorted target cells human breast cancer cells and lung cancer human alveolar basal epithelial cells.

7. The screening method of the spiral structure inertial focusing microfluidic cell screening device according to claim 6, characterized in that: The optimal Reynolds number range for achieving the inertial aggregation sorting of the blood sample is 65~68.

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