A spiral structure inertial focusing microfluidic cell screening device and its screening method

Abstract

The present invention provides a spiral structure inertial focusing microfluidic cell screening device and a screening method thereof, including a planar spiral structure. The planar spiral structure is a single-ring planar spiral structure, and at least two inlet channels and at least three outlet channels are provided in the single-ring planar spiral structure channel inside it. The single-ring planar spiral structure channel, the inlet channels and the outlet channels are rectangular cross-section channels. The single-ring planar spiral structure is provided with a plurality of spike structures. By driving an injection pump to adjust the flow rates of the blood sample and the sheath fluid, the Reynolds number at the starting point of the single-ring planar spiral structure channel is adjusted to achieve stable cell inertial focusing and an efficient cell separation effect. It can replace complex and costly multi-sorting microfluidic devices, is not easily blocked, is easy to clean, has strong stability and is not easily deformed, has high experimental accuracy and reliability, is conducive to long-term experimental operation and use, and has a high repeat utilization rate.

Description

Technical Field

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

[0002] Circulating tumor cells (CTCs) are a collective term for various types of tumor cells present in peripheral blood. They shed from solid tumor lesions (primary lesions, metastatic lesions) into peripheral blood spontaneously or due to medical procedures. Although most of them can undergo apoptosis or be phagocytosed, a small number become metastatic foci, 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 relatively low (1 - 10 CTC / mL), and the difficulties in accurate counting and separation have prevented their widespread use in cancer diagnosis and treatment. Therefore, in order to promote cancer diagnosis, prognosis, treatment and tumor metastasis research, it is urgent and crucial to introduce new technologies to achieve high capture efficiency, enrichment, purity and throughput for circulating tumor cell detection and separation.

[0003] Microfluidic technology is popular in cell sorting and separation research due to its advantage of precise manipulation of liquids at the microscale, enabling precise control and processing of smaller volumes of samples based on mechanical and biological properties. It includes two types of highly efficient and reliable microfluidic platforms. One type is to complete cell separation based on different biological properties between cells, namely the affinity strategy. However, different circulating tumor cells have different expression levels of specific antigens (such as Epcam), and some circulating tumor cells may be lost during separation. At the same time, complex operations and high costs also limit the popularization and use of this method. The other type of technology is to complete cell separation based on different physical properties between cells (such as physical quantities like size, density, etc.), also known as the label-free strategy.

[0004] The label-free strategy includes active separation techniques and passive separation techniques. Active separation techniques include, for example, acoustic trapping (AP), optical tweezers technology, dielectrophoresis (DEP), and magnetic trapping (MP). However, in active separation techniques such as acoustic and optical techniques, the throughput is relatively low due to the limitation of fluid velocity magnitude, and usually, more complex external devices are required to provide additional complex physical fields. Passive separation techniques include filtration-based methods, deterministic lateral displacement (DLD), and inertial focusing. Among them, separation techniques such as deterministic lateral displacement (DLD) and filtration separation face problems such as easy channel clogging, inability to ensure cell viability after processing, and poor device stability.

[0005] Inertial focusing belongs to a passive separation technology for microfluidic processing. The Reynolds number (Re) is a similarity criterion number in fluid mechanics that characterizes the influence of viscosity and is a dimensionless number used to characterize fluid flow conditions. When a fluid flows through a microchannel in a low-Reynolds-number laminar flow, particles or cells are subjected to fluid shear forces, wall interactions, and inertial forces. Under the condition that the actions of these forces reach equilibrium, the motion state of the particles or cells will reach stability, resulting in the focusing of the particles or cells at a specific position within the microchannel. Inertial focusing technology has excellent separation performance, stability, and the advantages of being easy to produce and use.

[0006] Secondary flow refers to the fluid motion generated outside the main flow direction, which usually appears in channels with curved or perturbed obstacles. Due to the fluid momentum mismatch between the center of curvature and the near-wall region, a radial pressure gradient is generated, which in turn forms secondary flow. This secondary flow usually manifests as two counter-rotating vortices, called Dean vortices. During inertial focusing, secondary flow (such as Dean vortices) will affect the equilibrium position of particles or cells. Due to the existence of secondary flow, particles or cells are subjected to a drag force perpendicular to the main flow direction, and this force will change the equilibrium position of the particles or cells, thus affecting the effect of inertial focusing. On the other hand, inertial focusing itself is also affected by the channel structure and fluid properties, and these factors will also indirectly affect the generation and distribution of secondary flow.

[0007] In inertial focusing technology, spiral microfluidic devices have been widely studied due to their advantages such as simple 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 (Dean drag). 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 the counter-rotating vortex streamlines. When > , inertial lift dominates, and particles migrate to the equilibrium position independently of the Dean flow. When < When Dean drag dominates the behavior of the particles, the particles remain entrained within the Dean flow. When the spiral channel is reasonably designed, for larger-sized cells, due to the dominant inertial lift force, the particles will gradually migrate to the equilibrium position within the channel and form a focusing band. Only within the intermediate range where the two forces are of the same order of magnitude can the inertial equilibrium position be corrected by the Dean flow. Reasonably introducing this correction method can have a positive effect on the focusing performance of larger cells. At the same time, for smaller cells, since the Dean drag they experience dominates, they are entrained in the Dean flow. By reasonably designing the channel length, cell sorting of different sizes can be achieved. However, for the Archimedean spiral of the spiral inertial separation device, since the radius of curvature continuously changes, the radius of curvature of the formed spiral channel also changes continuously. This leads to a change in the ratio of (inertial lift force) and (Dean drag) during the movement of the cells within the channel. 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 the flow rate. Only when Re reaches a relatively large value can a stable cell focusing band be formed. However, a larger Re also brings a greater hydraulic pressure to the device, which greatly reduces the stability of the device. And in the microfluidic devices of the existing technology, the binary sorting device can separate a particle solution of a target particle size from particles of different sizes, which is a relatively common microfluidic sorting device, while the multi-sorting device can separate a particle solution of two or more target particle sizes from particles of different sizes, which is relatively rare. The sorting device can be applied to 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 for the following reasons: 1. When is satisfied, inertial focusing of the cells can be achieved, where is the ratio of the cell diameter to the characteristic length; 2. At the same time, when is satisfied, the lift force acting on the cells can overcome the Dean drag, where describes the strength relationship of the competition between the inertial lift force and the Dean drag of the cells, that is, The larger it is, the more it indicates that the inertial lift force on the cells is greater than the Dean drag force. 3. Other factors, such as if the spacing between different-sized cell bands is too narrow, it will disrupt the balance state of the cell bands. And in the sorting device, concave cavities or recessed areas are often set to enhance the focusing effect. However, fluid particles, bubbles, or other impurities are likely to remain in the structure of the concave cavities or recessed areas, leading to the accumulation of residues and even channel blockage. Moreover, it is difficult to thoroughly clean the local structure of the recesses in microfluidics. Additionally, the local recesses will bring a sudden increase in pressure to the channel, and this pressure may deform the original structure of the channel, affecting the accuracy and reliability of subsequent experiments, being not conducive to long-term experimental operations and use, and having a low repeat utilization rate of the device. Therefore, a new technology is needed to improve this situation. Summary of the Invention

[0008] Aiming at the problems in the above-mentioned prior art, the present invention provides a spiral structure inertial focusing microfluidic cell screening device and its screening method. By improving the spiral microfluidic device, it simultaneously overcomes (inertial lift force) and (Dean drag force) ratio changes that have a negative effect on the focusing performance of target cells, and overcomes the problem that target cells are more sensitive to the flow rate, enabling target cells to achieve a stable motion state at a lower flow rate, realizing stable cell inertial focusing, which can replace complex and costly multi-sorting microfluidic devices, achieving efficient cell separation, being not easily blocked, easy to clean, having strong device stability and not being easily deformed, high accuracy and reliability of experiments, being conducive to long-term experimental operations and use, and having a high repeat utilization rate.

[0009] The present invention is implemented through the following technical solutions: 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, and the Archimedean spiral is a clockwise rotating spiral. The inside of the single-ring planar spiral structure is provided with a hollow channel, and the hollow channel is a single-ring planar spiral structure channel;

[0010] At least two inlet channels are connected through branches at the starting point of the single-ring planar spiral structure channel. The overall of the inlet channels forms an arc structure. The inlet channels include an inner wall inlet channel and an outer wall inlet channel. At least three outlet channels are connected through branches at the end point of the single-ring planar spiral structure. The overall of the outlet channels forms a straight surface structure. The inner cavities of the single-ring planar spiral structure channel, the inlet channels, and the outlet channels are interconnected. The single-ring planar spiral structure channel, the inlet channels, and the outlet channels are rectangular cross-section channels. The outlet channels include 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 spike structures. The spike structures are specifically corner types. The spike structures are connected to the outside of the single-ring planar spiral structure, and the channel of the single-ring planar spiral structure communicates with the inner cavity of the spike structures. The top of the outer side surface contour of the spike structure is a machining curve, and the machining curve is a partial elliptical contour line A. The partial elliptical contour line A is symmetric about the vertex o of the partial elliptical contour line A. The end point a of each partial elliptical contour line is connected to the end point b of the outer side cross-section contour of the adjacent single-ring planar spiral structure by a fillet arc B with a radius of r. Among them, the end point a, the end point b and the fillet arc B are tangentially connected. The center point x of the partial elliptical contour line A is located on the outer side cross-section contour line C of the single-ring planar spiral structure. The connection line between the vertex o and the center point x of the partial elliptical contour line A is the major axis of the spike structure. The length of the major axis of the spike structure and the cross-section height of the channel of the single-ring planar spiral structure are both α. The intersection point e of the extension line D of the partial elliptical contour line A and the outer side cross-section contour line C of the single-ring planar spiral structure. The connection line of two adjacent intersection points e is the minor axis of the spike structure, and the length of the minor axis of the spike structure is L.

[0012] The Archimedean spiral is drawn through the Cartesian coordinate system. The position of one point of the Archimedean spiral in the Cartesian coordinate system is assigned as θ. The included 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 starting point of one of the spike structures is assigned as θs in the Cartesian coordinate system, and the ending point of one of the spike structures is assigned as θe in the Cartesian coordinate system. θe - θs = β. The geometric parameters of a single spike structure: α = 300μm, r = 0.2mm, β = 0.05. Among them, the floating value of α is ±10μm, the floating value of β is ±0.001, and the floating value of r is ±0.03mm. There is the following relationship between β and the length L of the minor axis of the spike structure:

[0013] 。

[0014] Further, the starting point θs of one of the spike structures has the following coordinate equation in the Cartesian coordinate system:

[0015]

[0016]

[0017] Among them, x and y are the abscissa value and ordinate value corresponding to one point in the Cartesian coordinate system.

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

[0019] Further, the Archimedes spiral expression (equation-driven curve):

[0020] Inner wall:

[0021] ;

[0022] Outer wall:

[0023] ; The range of θ: 0 <= θ <= 1.

[0024] Further, the height of the rectangular cross-section of the single-ring planar spiral structure channel is 450um, and the floating value is ±10um. The height of the rectangular cross-section of the entrance channel and the height of the rectangular cross-section of the exit channel are both 180um, and the floating value is ±3um.

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

[0026] S1: Syringes containing sheath fluid and a mixed particle solution with different particle sizes are respectively loaded into the injection pumps. The sheath fluid is injected into the inner wall entrance channel, and the mixed particle solution with different particle sizes is injected into the outer wall entrance channel. The mixed particle solution with different particle sizes is a blood sample. The sheath fluid and the blood sample converge into a fluid solution at the starting point of the single-ring planar spiral structure channel. Among them, the blood sample contains one or more of the target sorted cells, human breast cancer cells or human alveolar basal epithelial cells of lung cancer;

[0027] S2: Drive the injection pump to make the fluid solution flow. The laminar flow at the entrance of the inner wall channel flows uniformly at 0.95 times the total flow rate Q, and the laminar flow at the entrance of the outer wall channel flows uniformly at 0.05 times the total flow rate Q. The static pressure P at the exit channel is 0;

[0028] S3: Adjust the Reynolds number at the starting point of the single-ring planar spiral structure channel by adjusting the flow rates of the blood sample and the sheath fluid, so that the Reynolds number is below 45. Determine this stage as the chaotic period by the state of the distribution and focusing of various cells in the channel;

[0029] When the Reynolds number is between 45 and 60, this stage is determined as transition period 1 by judging the focusing trend of target cells, human breast cancer cells and human alveolar basal epithelial cells of lung cancer;

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

[0031] When the Reynolds number is between 70 and 90, this stage is determined as transition period 2 by judging the moving direction of unsorted cells in the outlet channel;

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

[0033] S4: Perform post-processing through a flow cytometer to count the sorted target cells, human breast cancer cells and human alveolar basal epithelial cells of lung cancer.

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

[0035] Beneficial effects

[0036] By setting a rectangular cross-section spiral microfluidic device in this apparatus, when the fluid flows in the spiral microfluidic channel, due to experiencing centrifugal acceleration in the radial direction outward from the center of curvature, two counter-rotating vortices (Dean vortices) are formed in the upper and lower parts of the channel. The cells in the channel are affected by inertial lift and Dean drag During the flow in the channel, the inertial lift caused by the shear flow ( ) causes the larger cells to move towards the inner wall, while the Dean drag caused by the curvature of the spiral channel ( ) pushes the small cells towards the outer wall. Under the influence of and , the large cells (such as human breast cancer cells) gather into a cell band close to the inner wall under the dominant role of . The medium-sized cells (such as human alveolar basal epithelial cells of lung cancer) form a cell band in the middle part of the channel and flow out from the middle outlet, while the small red blood cells and white blood cells gradually migrate to the outer wall and form a cell band under the dominant role of . Meanwhile, the introduction of the spike structure sequence in the spiral microfluidic device channel generates a lift similar to the effect. When the fluid passes through the spike structure sequence, and The ratio can be locally amplified, and the overall force and motion state of the target cells are positively regulated, so that the focusing performance of the target cells is enhanced, and a narrower cell focusing band is formed. At the same time, it overcomes (inertial lift) and (Dean drag) the problem that the change in the ratio has a negative effect on the focusing performance of the target cells, and overcomes the problem that the target cells are more sensitive to the flow rate, enabling the target cells to achieve a stable motion state at a lower flow rate, realizing stable cell inertial focusing, which can replace complex and costly multi-splitting microfluidic devices. Finally, a more efficient cell separation effect state is achieved, which is not easy to block, easy to clean, and the device has strong stability and is not easy to deform. The accuracy and reliability of the experiment are high, which is conducive to long-term experimental operation and use, and has a high repeat utilization rate. Brief Description of the Drawings

[0037] Figure 1 It is a three-dimensional view of an embodiment of the present invention;

[0038] Figure 2 It is a diagram of the inlet channel and outlet channel of an embodiment of the present invention;

[0039] Figure 3 It is a three-dimensional view of the spike structure of an embodiment of the present invention;

[0040] Figure 4 It is a cross-sectional view of the spike structure of an embodiment of the present invention;

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

[0042] Figure 6 It is a finite element analysis nephogram of the spike structure of an embodiment of the present invention;

[0043] Figure 7 It is a finite element analysis nephogram of cross-sections 1, 2, 3, 4, and 5 of the spike structure of an embodiment of the present invention;

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

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

[0046] Figure 10 This is a diagram showing the distribution state of various types of cells at the exit channel in an embodiment of the present invention. Detailed implementation manners

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

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

[0049] This embodiment is implemented through the following technical solutions: As Figure 1 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, and the Archimedean spiral is a clockwise rotating spiral. 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] As Figure 2 shown, two inlet channels are provided through branch connections at the starting point of the single-ring planar spiral structure channel. The overall shape formed by the inlet channels is an arc structure. The inlet channels include an inner wall inlet channel and an outer wall inlet channel. Three outlet channels are provided through branch connections at the end point of the single-ring planar spiral structure. The overall shape formed by the outlet channels is a straight surface structure. The inner cavities of the single-ring planar spiral structure channel, the inlet channels, and the outlet channels are connected through. The single-ring planar spiral structure channel, the inlet channels, and the outlet channels are rectangular cross-section channels. The outlet channels include an inner wall outlet channel, a middle outlet channel, and an outer wall outlet channel.

[0051] As Figures 3 - 5As shown, the single-ring planar spiral structure is provided with 5 spike structures. The spike structures are specifically corner types and are connected to the outside of the single-ring planar spiral structure. The channel of the single-ring planar spiral structure is in communication with the inner cavity of the spike structure. The top of the contour on the outer side of the spike structure is a machining curve, and the machining curve is a partial elliptical contour line A. The partial elliptical contour line A is symmetric about the vertex o of the partial elliptical contour line A. The end point a of each partial elliptical contour line is connected to the end point b of the outer cross-section contour of the adjacent single-ring planar spiral structure by a fillet arc B with a radius of r. Among them, the end point a, the end point b and the fillet arc B are tangentially connected. The center point x of the partial elliptical contour line A is located on the outer cross-section contour line C of the single-ring planar spiral structure. The connection line between the vertex o and the center point x of the partial elliptical contour line A is the major axis of the spike structure. The length of the major axis of the spike structure and the height of the cross-section of the channel of the single-ring planar spiral structure are both α. The intersection point e of the extension line D of the partial elliptical contour line A and the outer cross-section contour line C of the single-ring planar spiral structure. The connection line of two adjacent intersection points e is the minor axis of the spike structure, and the length of the minor axis of the spike structure is L;

[0052] The Archimedean spiral is drawn through the 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 starting point of one of the spike structures is assigned as θs in the Cartesian coordinate system. The ending point of one of the spike structures is assigned as θe in the Cartesian coordinate system, and θe - θs = β. The geometric parameters of a single spike structure: α = 300μm, r = 0.2mm, β = 0.05. Among them, 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 relationship between β and the length L of the minor axis of the spike structure is as follows:

[0053] 。

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

[0055]

[0056]

[0057] Among them, x and y are the abscissa value and ordinate value corresponding to one point in the Cartesian coordinate system.

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

[0059] Thrust structure 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 spike structures

[0061] The position spacings between adjacent spikes θ1-θ2, θ2-θ3, θ3-θ4, and θ4-θ5 are shown in Table 2:

[0062] Adjacent thrust 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: Spacing parameter table between adjacent spike structures

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

[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 position of the Archimedean spiral, and when θ = 1, it is the ending point position of the Archimedean spiral;

[0070] The overall shape formed by the inlet channel is a semi-circular arc structure with a radius R1 = 4 mm;

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

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

[0073] As Figures 6 - 7 shown, by performing a finite element analysis on the velocity fields of the fluid solution passing through the five cross-section parts of the spike structure and the cross-sections 1, 2, 3, 4, and 5 of the spike structure, it can be seen that during the process of the fluid solution passing through the spike structure, the magnitude of the velocity of the fluid solution is a process of first decreasing and then increasing. More specifically, the velocity fields of 5 different positions of the spike structure are specifically characterized. The velocity field characterization includes the magnitude and direction of the velocity. The cross-section background color (blue) represents the distribution of the velocity field magnitude, and the arrow represents the direction of the velocity vector. During the process of the fluid solution passing through the spike structure, it moves from cross-section 1 to cross-section 5. In addition to the above law occurring at the flow velocity magnitude level, the direction of the velocity field inside the fluid solution also undergoes a changing process. Among them, the reason for the change in the velocity magnitude in the velocity field is that the introduction of the spike structure causes a change in the cross-sectional area of the channel, and the reason for the change in the velocity vector direction is a lift force generated by the spike structure similar to the FL effect.

[0074] The present invention also includes a screening method for a spiral structure inertial focusing microfluidic cell sorting device. As Figure 8 shown, it specifically includes the following steps:

[0075] S1: Load syringes containing sheath fluid and a mixed particle solution with different particle sizes into the injection pumps respectively. Inject the sheath fluid into the inner wall inlet channel and inject the mixed particle solution with different particle sizes into the outer wall inlet channel. The mixed particle solution with different particle sizes is a blood sample. The sheath fluid and the blood sample converge into a fluid solution at the starting point of the single-loop planar spiral structure channel. Among them, the blood sample contains one or more of the target sorted cells, human breast cancer cells or human alveolar basal epithelial cells of lung cancer;

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

[0077] S3: Adjust the Reynolds number at the starting point of the single-loop planar spiral structure channel by regulating the flow rates of the blood sample and the sheath fluid, so that the Reynolds number is below 45, and various types of cells in the channel are in a chaotic state, and various types of cells 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 show a tendency to focus. This stage is the transition period 1;

[0079] When the Reynolds number is between 60 and 70, the target cells, human breast cancer cells and human lung alveolar basal epithelial cells of lung cancer, begin to form a single and tight cell focusing band. Among them, human breast cancer cells form a cell focusing band at the inner wall outlet channel, human lung alveolar basal epithelial cells of lung cancer form a cell focusing band at the middle outlet channel, and unsorted cells form a cell focusing band at the outer wall outlet channel. At this time, the three-way classification of human breast cancer cells, human lung alveolar basal epithelial cells of lung cancer and unsorted cells can be achieved. This stage is the sorting period 1;

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

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

[0082] As Figure 9 shown, according to the movement conditions of human breast cancer cells (MCF-7), human lung alveolar basal epithelial cells of lung cancer (A549), white blood cells (WBCS), and red blood cells (RBCS) at the normalized distance from the inner wall with the change of the Reynolds number Re near the outlet channel, when the Reynolds number at the inlet channel gradually increases from 30 to 60, human breast cancer cells (MCF-7) gradually form a tight focusing band at the channel inner wall, and human lung alveolar basal epithelial cells of lung cancer (A549) also gradually form a focusing band in the middle of the channel. When Re continues to increase, MCF-7 and A549 show better focusing performance, and the focusing equilibrium positions tend to be stable;

[0083] However, only when the Reynolds number at the channel entrance is in the range of 60 to 70, the bands of white blood cells (WBCs) and red blood cells (RBCs) are close to the outer wall of the channel;

[0084] When Re gradually increases from 70 to 90, the bands of both types of cells gradually migrate towards the center of the channel. When Re gradually increases from 90 to 100, the bands of white blood cells (WBCs) and red blood cells (RBCs) are located in the middle of the channel.

[0085] S4: Perform post-processing through a flow cytometer to count the sorted target cells, human breast cancer cells and human alveolar basal epithelial cells of lung cancer.

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

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

[0088] 1. Through a rectangular cross-section spiral microfluidic device, when the fluid flows in the spiral microfluidic channel, due to experiencing 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. The rectangular channel satisfies the following relationship:

[0089]

[0090] Where 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 the inertial lift , and the other is the Dean drag . is the inertial lift composed of the shear-gradient lift that pushes the cells towards the channel wall and the wall lift . It can guide the cells with larger particle sizes to move towards the inner wall. The inertial lift can be described by the following equation:

[0092]

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

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

[0095] 3. As Figure 10 shown, for cells of different particle sizes moving in the channel near the outlet with different Reynolds numbers. For a channel with a rectangular cross-section, 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 small cells towards the outer wall of the spiral channel. The Dean drag can be described by the following equation: , where , , respectively represent the dynamic viscosity, the velocity of the fluid, and the velocity of the cell;

[0096] When 1 < Re < 100, the Dean drag can be calculated as , used to characterize the magnitude of the Dean velocity and can be described by the following equation:

[0097] where, represents the Dean number, and the magnitude of the secondary flow can be measured through the Dean number.

[0098] 4. The synergistic effect of inertial lift and Dean drag. The Dean drag entrains cells to follow the reverse-rotating vortex streamlines. When the two forces are of the same order of magnitude, the inertial equilibrium position can be corrected by the Dean flow, and only then can cells be effectively separated. Under the influence of and , large cells aggregate into a cell band near the inner wall under the dominant effect of , medium-sized cells form a cell band in the middle part of the channel and flow out from the middle outlet, while small cells gradually migrate to the outer wall and form a cell band under the dominant effect of . The magnitude of describes the strength relationship of the competition between the inertial lift and Dean drag of cells, that is, the larger it is, the stronger the inertial lift on the cell is compared to the effect of the Dean drag. Whether it is greater than 0.07 is an indicator for whether cells with a particle size of can achieve excellent inertial focusing in a spiral microfluidic channel with a characteristic length of . Whether it is greater than 0.08 indicates that for particles with a size of in the spiral microfluidic channel. Whether the inertial lift force received by the cells can overcome the Dean drag, and the intensity of the inertial lift force overcoming the Dean drag by the cells also reflects the equilibrium position of the corresponding cell band on the channel cross-section. By calculating and The values can effectively help to illustrate the motion states of various particle-size cells in the channel and their distribution on the outlet channel cross-section. The diameters of human breast cancer cells, human lung alveolar basal epithelial cells, white blood cells, and red blood cells , cell diameter and the characteristic length The ratio , The data are shown in Table 3:

[0099]

[0100] Table 3: Cell diameter , cell diameter and the characteristic length The ratio , The data table

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

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

[0103] For white blood cells, the ratio of the diameter of white blood cells to the characteristic length = 0.039 < 0.07, and the strength relationship between the inertial lift force and the Dean drag of white blood cells = 0.095 > 0.08. However, considering that at this time is close to the index critical value, the effect of the lift force overcoming the drag is not obvious. The cell band formed by white blood cells is more biased towards the outer side of the channel than the cell band of human lung alveolar basal epithelial cells of lung cancer, and because The difference is relatively large, and there will be no overlapping phenomenon between the two cell bands;

[0104] For red blood cells, the ratio of the red blood cell diameter to the characteristic length = 0.031 < 0.07, the strength relationship between the inertial lift and Dean drag of red blood cells = 0.06 < 0.08. At this time, the cell band formed by red blood cells is closest to the outer wall of the channel among the previous types of cell bands. In summary, the equilibrium positions of the cell bands formed by the four types of cells change from the inner wall to the outer wall of the channel in order of decreasing cell size, and satisfy the following relationship:

[0105]

[0106]

[0107] On this basis, the three-way sorting of target cells is realized.

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

[0109] 1. Due to the introduction of the spike structure, the inertial lift force on the target cells is adjusted by increasing the cross-sectional area of the channel to cause an additional lift force, thereby promoting cell focusing. Compared with the existing technology using a similar concave structure, it is exactly the opposite in physical structure, improving the problems of easy blockage and easy deformation of the original channel. Moreover, the chip only needs to quickly inject clean water into the channel with a syringe to flush out impurities, and the cleaning step is very simple. At the same time, although the existing similar concave structure and the proposed spike structure both have the effect of adjusting the movement of target cells, the adjustment effect of the concave structure is more radical, mainly by enhancing the Dean velocity to adjust cell movement, while the adjustment effect of the proposed spike structure is more gentle. Comparatively, 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 is reduced, and the focused human breast cancer cells achieve a stable cell focusing band after Re = 60 and are no longer affected by the flow rate;

[0111] 3. The regulatory effect induced by the spike structure makes the force received by the cells in the channel during movement more stable, overcoming (Dean drag) and (inertial lift force) the problem of the negative effect on the focusing performance of target cells caused by the change in the ratio, and overcoming the problem that the target cells are more sensitive to the flow rate, enabling the target cells to achieve a stable movement state at a lower flow rate. In this regard, the 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 does not impose a formal limitation on the present invention. It should be understood that technologies, methods, and devices known to those of ordinary skill in the relevant field may not be discussed in detail. However, under appropriate circumstances, the technologies, methods, and devices should be regarded as part of the authorized specification. Within the scope of the features defined by the claims, they should all be included within the protection scope of the present invention.

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; Due to the introduction of the spiking structure, the inertial lift of the target cells is adjusted by increasing the channel cross-sectional area to cause additional lift, thereby promoting cell focusing; 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: The coordinate equation of the starting point θs of one of the spur structures in the Cartesian coordinate system is: x=(8.255+2θ)cos(2πθ) y=(8.255+2θ)sin(2πθ) Wherein, x and y are the abscissa and ordinate values ​​corresponding to a point in the Cartesian coordinate system; By introducing the spike sequence, the sensitivity of the target sorted cells to the flow rate was reduced.

2. 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.

3. The spiral structure inertial focusing microfluidic cell screening device according to claim 1, characterized in that: The Archimedean screw expression is: Inner wall: x=(7.775+2θ)cos(2πθ) y = (7.775 + 2θ) sin (2πθ); Outer wall: x=(8.225+2θcos(2πθ) y=(8.225+2θ)sin(2πθ); the range of θ is: 0<=θ<=1.

4. 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.

5. A screening method of a spiral structure inertial focusing microfluidic cell screening device, using the spiral structure inertial focusing microfluidic cell screening device according to any one of claims 1 to 4, 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 stage 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 to count the sorted target cells human breast cancer cells and lung cancer human alveolar basal epithelial cells.

6. The screening method of the spiral structure inertial focusing microfluidic cell screening device according to claim 5, characterized in that: In step S1, the optimal Reynolds number range for inertial aggregation sorting of the blood sample is 65-68.

Images