WO2022068648A1

WO2022068648A1 - Microfluidic chip cartridge

Info

Publication number: WO2022068648A1
Application number: PCT/CN2021/119609
Authority: WO
Inventors: 林柏均; 娜格拉斯苏妮塔; 陈涛
Original assignee: 苏州莱博睿思生物科技有限公司
Priority date: 2020-09-30
Filing date: 2021-09-22
Publication date: 2022-04-07
Also published as: CN112195094A

Abstract

A microfluidic chip cartridge, comprising: an inner cartridge body (10), a microfluidic chip (20), and an outer cartridge body (30). The inner cartridge body (10) is a housing having an opening top; the microfluidic chip (20) is placed in the inner cartridge body (10); the outer cartridge body (30) is a housing having an open bottom; the outer cartridge body (30) covers the inner cartridge body (10); at least one sample inlet (31) is provided on the top surface of the outer cartridge body (30); at least one microfluidic channel (21) is provided in the microfluidic chip (20); the starting end of the microfluidic channel (21) is connected to the sample inlet (31) by means of an input tube; the tail section of the microfluidic channel (21) comprises a plurality of flow distribution channels (22); flow distribution tubes communicates the tail ends of the plurality of flow distribution channels (22) with at least two flow distribution liquid outlets (11) provided on the inner cartridge body (10). The microfluidic chip cartridge has a simple structure and is easy to operate, implements separation of target cells by means of physical principles, and can achieve the separation effects of high throughput, high recovery rate and high sample purity.

Description

A microfluidic chip box

technical field

The invention relates to the technical field of medical devices, in particular to a microfluidic chip box.

Background technique

Cancer is one of the leading causes of human death. Studies have shown that at least 30 percent of deaths in cancer patients are preventable if cancer patients are diagnosed and treated before metastatic cancer develops. Tumors metastasize when circulating tumor cells (CTCs) flow into the peripheral blood from primary or metastatic tumors. Therefore, the separation of tumor cells in circulating blood by CTC examination is of great significance for the judgment of cancer and the evaluation of the therapeutic effect in the process of cancer treatment.

At present, most of the commonly used CTC separation methods are based on the immunoaffinity method of EpCAM antibody, that is, the EpCAM antigen on the surface of CTCs is identified by the antibody, and the magnetic beads bound to the antibody are combined with an exogenous magnetic field or the surface of a microfluidic chip. Capture CTCs. The immunoaffinity method has been continuously improved in the past ten years, and has been able to increase the capture efficiency of EpCAM-positive CTCs to more than 90%. However, with the development of technology, studies in recent years have shown that the immunoaffinity method has many shortcomings, as follows:

1. Recent studies have shown that nearly half of CTCs do not have EpCAM surface antigens, so the number of CTCs that can actually be captured by immunoaffinity in clinical applications is low.

2. Since the antibody-antigen reaction takes a certain amount of time to stabilize the bond between the two, the sample flow rate needs to be controlled at about 1 ml per hour, so that there is a high probability of collision between the CTC and the added EpCAM antibody and then it can be captured. , which also limits the amount of blood samples that can be processed in clinical applications, resulting in insufficient throughput of the immunoaffinity method.

The two fatal defects of low capture rate and low throughput directly lead to the extremely low number of CTCs that can be extracted for downstream applications. Therefore, its development has always been limited. At present, most of the technologies adopted by various manufacturers belong to the immunoaffinity technology, and there is not much room for breakthrough.

Based on the difference in cell size, label-free technology has gradually emerged in recent years, which utilizes physical properties other than antibody-antigen reaction to separate CTCs. Since the cell size of CTC is generally above 15 μm in diameter, there is at least a 3 μm gap between leukocytes (7-12 μm) and smaller red blood cells (5-6 μm). In addition to this, there are subtle differences between the electric field properties of CTCs and other blood cells. Using cell size characteristics can solve the problem of cell heterogeneity in antibody affinity methods, and cells will not be unable to be captured because of different antigenic expression.

Among the above methods, the most direct method is the filtration method, that is, the use of membrane pores or similar structures with a pore size between normal cells and cancer cells for cell filtration. The filtration method is direct but has considerable problems, such as channel blockage, Cells are susceptible to stress and become inactive, captured cells are not easily removed, and throughput is low. At present, there are many academic and commercial technologies for realizing CTC application by filtration at home and abroad, but the technical limitation cannot be accepted by downstream applications. The most mature technology in the filtration method is CelSee technology from the United States, which uses a microfluidic chip to precisely control cell capture. Due to the limitations of the filtering method itself, the number of CTCs captured in patient samples is too small.

Other uses of cell size properties for separation are Inertial-Based Size Separation. This technology uses a spiral microchannel to separate large and small cells, but due to technical limitations, the CTC capture rate is only about 80%, and the final CTC purity is low (the leukocyte removal rate is too low), making it difficult for this technology to be connected to downstream applications.

SUMMARY OF THE INVENTION

(1) Technical problems to be solved

The purpose of the present invention is to provide a microfluidic chip box that utilizes a physical method to separate target cells and can achieve separation effects of high throughput, high recovery rate and high sample purity, aiming at the above-mentioned deficiencies of the prior art.

(2) Technical solutions

In order to solve the above problems, the present invention provides a microfluidic chip box, comprising: an inner box body, a microfluidic chip and an outer box body; the inner box body is a shell with an open top surface, and the microfluidic control The chip is placed in the inner box body; the outer box body is a shell with an open bottom surface, the outer box body is covered on the inner box body; at least one sample is arranged on the top surface of the outer box body The microfluidic chip is provided with at least one microfluidic channel; the initial end of the microfluidic channel is connected with the sample inlet through an input pipe; the tail section of the microfluidic channel includes a plurality of A shunt channel, a shunt pipe communicates the ends of the plurality of shunt channels with at least two shunt liquid outlets provided on the inner box body.

Optionally, the microfluidic channel includes a plurality of segments and a plurality of corners, and the corners are arranged at the junction of two adjacent segments.

Optionally, the segment is a straight line segment or a circular arc segment with a set radius.

Optionally, the cross section of the microfluidic channel is rectangular.

Optionally, the ratio of the length to the width of the cross section of the microfluidic channel is 1 to 10.

Optionally, the angle of the turning angle is not less than 90°.

Optionally, the initial section of the microfluidic channel includes a plurality of input channels, and the initial end of the input channel is communicated with the sample inlet through the input tube.

Optionally, the microfluidic chip includes a chip bottom plate and a chip body, and the chip bottom plate and a microfluidic groove machined on the bottom surface of the chip body form the microfluidic channel.

Optionally, the chip body is provided with a plurality of inlet channels and a plurality of outlet channels; the lower end of the inlet channel is connected to the starting end of the input channel, and the upper end of the inlet channel is opened at the end of the chip body. outer surface; the upper end of the inlet channel is connected to the sample inlet through the input pipe; the lower end of the outlet channel is connected to the end of the shunt channel, and the upper end of the outlet channel is open to the chip body The outer surface; the upper end of the outlet channel is communicated with at least two shunt liquid outlets provided on the inner box body through the shunt pipe.

Optionally, a limiting groove is provided at the bottom of the inner box, and the limiting groove is used to fix the shunt pipe.

(3) Beneficial effects

The microfluidic chip box provided by the present invention is composed of a microfluidic chip box placed in the inner box and an outer box covered on the inner box to form a microfluidic chip box for separating cells in a sample. Utilize the physical structural properties of the microfluidic channel in the microfluidic chip, such as the shape and size of the cross-section of the microfluidic channel, the lengths and bending radii of the multiple segments that make up the microfluidic channel, and the angle, position and number of turning corners Enables separation of cells of different sizes in a sample. The microfluidic chip box has a simple structure and is easy to use. It can separate a variety of cells at the same time without pre-labeling the cells in the sample. It has high throughput, fast separation speed and large sample volume (50ml of samples can be processed at one time). ), the viability of the isolated cells was not affected. The microfluidic chip of the invention has good separation effect on cells in the sample, the capture rate of cancer cells reaches 95.9%, and the removal rate of white blood cells reaches 99.99%; the detection sensitivity of clinical CTC is high, the breast cancer and pancreatic cancer reach 95%, and the lung cancer reaches 100% , liver cancer reached 88.1% (75% in stage I, 96.2% in stage II-IV).

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without creative effort.

Fig. 1 is the exploded view of the microfluidic chip box in the embodiment of the present invention;

2A and 2B are schematic structural diagrams of an inner box body of a microfluidic chip box in an embodiment of the present invention;

3A and 3B are schematic structural diagrams of an outer box body of a microfluidic chip box in an embodiment of the present invention;

4 is a schematic structural diagram of a microfluidic chip of a microfluidic chip box in an embodiment of the present invention;

5 is a schematic structural diagram of a microfluidic channel in a microfluidic chip according to an embodiment of the present invention;

6 is a schematic diagram of a style of a microfluidic channel in a microfluidic chip according to an embodiment of the present invention;

7A to 7J are schematic diagrams of various styles of microfluidic channels in the microfluidic chip according to an embodiment of the present invention;

8 is a schematic diagram of multiple input channels of a microfluidic channel in an embodiment of the present invention;

9 is a schematic structural diagram of a bottom plate of a microfluidic chip box in an embodiment of the present invention;

10A and 10B are schematic diagrams of the overall structure of the microfluidic chip box in the embodiment of the present invention;

FIG. 11 is a schematic diagram of the force of cells in the sample in the microfluidic channel according to the embodiment of the present invention;

12A and 12B are schematic diagrams of the equilibrium positions of cells in a sample in a microfluidic channel in an embodiment of the present invention;

13A and 13B are schematic diagrams of Dean flow of samples in corners of different radii in a microfluidic channel according to an embodiment of the present invention;

14A is a schematic diagram of a microfluidic channel in an embodiment of the present invention;

14B to 14D are schematic diagrams of cell separation in a sample during the flow of the sample in the microfluidic channel shown in FIG. 14A .

The reference signs in the accompanying drawings are:

10. Inner box, 11. Outlet of shunt liquid, 12, Limit slot, 20, Microfluidic chip, 21, Microfluidic channel, 211, Section, 212, Corner, 22, Shunt channel, 23, Input channel, 24. Chip bottom plate, 25, Chip body, 251, Inlet channel, 252, Outlet channel, 30, Outer box, 31, Sample inlet, 32, Syringe head, 40, Bottom plate, 41, Outlet hole, 42, Positioning protrusion .

Detailed ways

The specific embodiments of the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. Here, the following examples of the present invention are used to illustrate the present invention, but not to limit the scope of the present invention.

As shown in FIGS. 1 to 4 , an embodiment of the present invention provides a microfluidic chip box, including: an inner box body 10 , a microfluidic chip 20 and an outer box body 30 . The inner box body 10 is a shell with an open top surface, and the microfluidic chip 20 is placed in the inner box body 10 ; the outer box body 30 is a shell with an open bottom surface, which covers the inner box body 10 . In this embodiment, the inner box body 10 and the outer box body 30 are both cubes (cuboids or cubes), and the outer box body 30 covers the inner box body 10 to form a closed box body. In practical applications, the shapes of the inner box body 1 and the outer box body 30 can be set as required, as long as the outer box body 30 can cover the inner box body 10 to form a closed box body. Specific restrictions.

As shown in FIG. 3a and FIG. 3b, at least one sample inlet 31 is provided on the top surface of the outer box body 30 for inputting the sample into the microfluidic chip 20 in the microfluidic chip box. The number of the sample inlets 31 and the positions of the sample inlets 31 on the top surface of the outer box body 30 may be set as required, which are not specifically limited in the embodiment of the present invention. As shown in FIG. 4 , the microfluidic chip 20 is provided with at least one microfluidic channel 21 , and the initial end of the microfluidic channel 21 is connected to the sample inlet 31 through an input tube (not shown in the figure). The tail section of the microfluidic channel 21 includes a plurality of shunt channels 22 . The ends of the shunt channels 22 communicate with one end of the shunt tube, and the other end of the shunt tube communicates with at least two shunt liquid outlets 11 provided on the inner box 10 . In this embodiment, the microfluidic channel 21 is divided into four shunt channels 22 at its tail section, and each shunt channel 22 corresponds to a type of cell separated from the sample. The ends of each shunt channel 22 are respectively connected with a shunt tube (not shown in the figure), which transports the separated cells out of the microfluidic chip box.

As shown in FIG. 4 , the microfluidic chip 20 includes a chip bottom plate 24 and a chip body 25 . The chip bottom plate 24 and the microfluidic grooves machined on the bottom surface of the chip body 25 form a microfluidic channel 21 . The microfluidic chip 20 can be made of metal, plastic, polymer, inorganic compound, glass, silicon (eg -Si-Si-), silicone resin (eg -Si-O-Si- or PDMS), epoxy resin, semiconductor or Combinations of them; alternatively, from different siloxane polymers, organic polymers such as polyethylene terephthalate (PET), polyimide, polyether ether ketone (PEEK) and/or their Combination made. The microfluidic chip 20 may be fabricated using techniques known in the art, including molding, photolithography, electroforming, machining, chemical vapor deposition, and the like. In this embodiment, the chip base plate 24 is made of glass; the chip body 25 is made of polydimethylsiloxane (PDMS, polydimethylsiloxane), and microfluidic control is etched on the bottom surface of the chip body 25 by soft lithography technology. Then, the chip bottom plate 24 and the bottom surface of the chip body 25 are fixedly connected by surface plasma treatment, that is, the chip bottom plate 24 serves as the bottom surface of the microfluidic channel 21 and is combined with the chip body 25 to form the microfluidic channel 21.

In an optional embodiment, the microfluidic chip 20 may be transparent, or approximately transparent, so that the state of the sample in the microfluidic channel 21 can be observed from the top and/or bottom and/or side.

As shown in FIG. 5 , the microfluidic channel 21 includes a plurality of segments 211 and a plurality of corners 212 . The segment 211 may be a straight line segment or a circular arc segment with a set radius. The corner 212 is provided at the junction of two adjacent segments 211 . The angle of the corner 212 is not less than 90°. The cross section of the microfluidic channel 21 is a rectangle; preferably, the cross section is a rectangle, and the horizontal side is the long side, and the vertical side is the short side. The shape and size of the cross section of the microfluidic channel 21, the shape and length of each segment 211 in the microfluidic channel 21, the number and position of the corners 212 in the microfluidic channel 21, and the angle of each corner 212 can be determined as needed , and the pattern of the microfluidic channel 21 formed by the plurality of segments 211 and the plurality of corners 212 , etc., these factors will affect the separation effect of the microfluidic channel 21 on different types of cells in the sample. The microfluidic channel 21 may be a labyrinth pattern composed of straight line segments and circular arc segments, as shown in FIG. 5 , or a pattern composed of all straight line segments, as shown in FIG. 6 . 7A to 7J show schematic diagrams of various types of microfluidic channels 21, but the microfluidic channels 21 are not limited to these. The embodiment of the present invention does not specifically limit the style of the microfluidic channel 21 .

As shown in FIG. 4 , a plurality of inlet channels 251 and a plurality of outlet channels 252 are provided in the chip body 25 . The lower end of the inlet channel 251 is connected to the starting end of the microfluidic channel 21, and the upper end of the inlet channel 251 is opened on the outer surface of the chip body 25, for example, on the top surface of the chip body 25; One end of the tube is connected to the sample inlet 31 , and the other end is inserted into the inlet channel 251 and connected to the starting end of the microfluidic channel 21 , so that the sample is injected into the microfluidic channel 21 . The lower end of the outlet channel 252 is connected to the end of the shunt channel 22, and the upper end of the outlet channel 252 is opened on the outer surface of the chip body 25, for example, on the top surface of the chip body 25; thus, by inserting one end of the shunt pipe into the outlet The inside of the channel 252 is connected with the end of the shunt channel 22, so that the cells separated in the shunt channel 22 can be transported to the outside of the microfluidic chip box. An outlet channel 252 is provided at the end of each shunt channel 22 and corresponds to a shunt pipe. As shown in FIG. 4 , in this embodiment, the tail section of the microfluidic channel 21 is divided into four shunt channels 22 ; four outlet channels 252 are arranged in the chip body 25 ; the four shunt pipes are respectively inserted into the four outlet channels 252 , It is connected with the four shunt channels 22, and the corresponding cells separated in the four shunt channels 22 are transported out of the microfluidic chip box.

In an optional embodiment, the initial section of the microfluidic channel 21 includes a plurality of input channels 23, as shown in FIG. 8 . When the initial section of the microfluidic channel 21 includes a plurality of input channels 23 , the initial end of each input channel 23 is provided with an inlet channel 251 connected to it, and a corresponding input pipe and is provided on the top surface of the outer box body 30 on the sample inlet 31.

As shown in FIGS. 4 to 8 , for different styles of microfluidic channels 21 , and the situation where different numbers of input channels 23 and shunt channels 22 are respectively set at the beginning and end of the microfluidic channel 21 , The starting end of each input channel 23 is provided with an inlet channel 251, a corresponding input tube and a sample inlet 31; the end of each split channel 22 is provided with an outlet channel 252, and a corresponding split Tube. The number of the sample inlets 31 and the positions on the top surface of the outer box body 30 can be determined as required. The same applies to the case where a plurality of microfluidic channels 21 are provided in the microfluidic chip 20 .

In an optional embodiment, the bottom of the inner box body 10 is provided with a limiting groove 12 , as shown in FIGS. 2 a and 2 b , one end of the limiting groove 12 is connected to the shunt liquid outlet 11 provided at the bottom of the inner box body 10 It is used to fix the pipe body of the shunt pipe in the limiting groove 12 . One end of the shunt pipe is communicated with the end of the shunt channel 22, and the other end can pass through the bottom of the inner box body 10, and then the shunt pipe is placed in the limit groove 12 on the bottom of the inner box body 10, and along the limit The groove 12 reaches the shunt liquid outlet 11 provided at the bottom of the inner box body 10, and the cells separated from the sample are transported out of the microfluidic chip box. The number, length and arrangement of the limiting grooves 12 may be set as required, which are not specifically limited in the embodiment of the present invention. The number and position of the shunt liquid outlets 11 provided at the bottom of the inner box body 10 can be determined as required. For example, to separate a type of CTC cells from a blood sample, at least two shunt outlets 11 need to be provided, and the shunt tube for delivering CTC cells is placed in the limit groove 12 connected to the shunt outlet 11 for outputting CTC cells The rest of the shunt pipes are placed in the limiting groove 12 connected to the other shunt liquid outlet 11 .

In an optional embodiment, the microfluidic chip box further includes a bottom plate 40 , as shown in FIG. 9 , the bottom plate 40 is disposed at the bottom of the inner box body 10 . The bottom plate 40 is provided with an outlet hole 41 corresponding to the shunt liquid outlet 11 at the bottom of the inner box 10 . The correspondence here means that the number of the outlet holes 41 is the same as that of the shunt liquid outlet 11, and the positions are opposite, so that the separated cells and other cells are output from the microfluidic chip box. Four corners of the bottom plate 40 are provided with positioning protrusions 42 for positioning the inner box body 10 when the bottom plate 40 is connected to the inner box body 10 . The bottom plate 10 can be fixedly connected with the inner box body 10 through a detachable structure, for example, a snap-fit structure. Similarly, the inner box body 10 and the outer box body 30 can also be fixedly connected by a detachable structure. This embodiment of the present invention does not specifically limit this. The bottom plate 40 , the inner box 10 , the microfluidic chip 20 and the outer box 30 are assembled together to form a microfluidic chip box, as shown in FIGS. 10A and 10B .

In an optional embodiment, as shown in FIG. 1 and FIG. 10A, the microfluidic chip box further includes a syringe head 32, which is placed in the sample inlet 31 on the outer box body 30, and whose output end is connected to the input tube connected. Each inlet channel 251 corresponds to an input tube, a sample inlet 31 and a syringe head 32 placed in the sample inlet 31 .

When using the microfluidic chip box of the embodiment of the present invention to separate cells in a sample, for example, to separate rare cells in the sample, the syringe head 32 is taken out from the sample inlet 31 on the outer box body 30, and the syringe head 32 is removed from the sample inlet 31. The end is connected to a container containing a sample, such as a test tube, and the sample is injected through the syringe head 32 and the input tube, and enters the starting end of the microfluidic channel 21 through the inlet channel 251 . Then, the sample flows through the microfluidic channel 21 at a speed such that cells of the same size (different types of cells have different sizes) in the sample converge into a flow, and the flows of cells of different sizes are separated from each other. Finally, at the end of the microfluidic channel 21 , the flow of cells of different sizes flows out from the corresponding shunt channel 22 , enters the shunt pipe through the outlet channel 252 , and is transported to the shunt liquid outlet 11 at the bottom of the inner box 30 through the shunt pipe , the microfluidic chip box is discharged from the outlet hole 41 provided on the bottom plate 40 . The samples in the embodiments of the present invention may be various liquids such as blood samples and body fluid samples. The embodiment of the present invention does not specifically limit the types of samples.

When the sample enters the microfluidic channel 21 and flows in the microfluidic channel 21 including multiple segments 211 and multiple corners 212 at a certain flow rate, the cells in the sample are not only affected by the inertial lift force F _Z , but also by the inertial lift force F Z. Dean's force F _D. Under the action of these two forces, cells of the same size converge into a flow at a certain equilibrium position in the microfluidic channel 21 , and the flow converged by cells of different sizes are separated from each other in different microfluidic channels 21 Equilibrium position, whereby target cells in a sample can be separated from other cells, for example, rare cells of a blood sample can be separated from other cells.

Specifically, when the sample flows in the straight section in the microfluidic channel 21, it will be subjected to the inertial lift force F _Z generated by the combination of the shear gradient lift force (Shear Gradient) and the wall lift force (Wall Effect); wherein, The shear gradient lift force pushes the cells in the sample to the side wall of the microfluidic channel 21, and the wall lift force pushes the cells to the center of the microfluidic channel 21. As shown in Figure 11, the inertial lift force F _Z combines the The wall lift effect of the cells pushing to the wall of the microfluidic channel and the wall lift effect of pushing the cells to the center of the microfluidic channel, the action of the two forces makes the cells in the sample maintain a specific equilibrium position in the microfluidic channel, as shown in the figure 12A and 12B. In Fig. 12A, there are four equilibrium positions (E1, E2, E3, E4) in the microfluidic channel 21 with a square cross-section; in Fig. 12B, there are two in the microfluidic channel 21 with a rectangular cross-section Equilibrium position (E5, E6). The inertial lift force F _Z tends to confine cells in a straight channel to several equilibrium positions, the number of which is related to the geometry and size of the cross-section of the microfluidic channel 21 . Although the size of the cell does not affect the equilibrium position of the cell when it is acted on by the inertial lift force F _Z alone, it affects whether cells of different sizes will converge on the equilibrium position under specific fluid conditions. When the inertial lift force F _Z received by the cell exceeds the critical value, the cells will converge in the equilibrium position, and the inertial lift force F _Z is quadratically related to the size of the cell, that is, the larger the cell, the easier it is to exceed the inertial lift force F _Z. Physical conditions for critical values. For example, in a linear segment 211, cells of all sizes will appear in the same equilibrium position under certain physical conditions such as the cross-sectional geometry of the microfluidic channel and the flow rate, fluid viscosity, fluid density, etc.; but at the same time Larger cells may have satisfied the condition to reach the equilibrium position while smaller cells have not, so the larger cells are concentrated in the equilibrium position, while the smaller cells are also randomly scattered in the microfluidic channel. everywhere in the cross section.

The formula for calculating inertial lift F _Z is as follows:

Among them, Re _p is the cell Reynolds number, Re _c is the Reynolds number of the microfluidic channel, and x _c is the position of the cell in the microfluidic channel 21 .

When the sample flows through the arc segment 211 and the corner 212 in the microfluidic channel 21 , Dean Flow will occur. When the sample flows through the above-mentioned arc segment 211 and the corner 212, it will first be affected by centrifugal force, and the Dean flow is usually a secondary flow, which is mainly manifested as a vortex that causes the sample to rotate in reverse, so that the Cells migrate passively along eddies; where flow at the middle of arc segment 211 or corner 212 is directed outward around the axis of both, and flow at the top and bottom of arc segment 211 or corner 212 is Guided inwardly around the axis of both. Usually, the fluid close to the inner wall of the arc segment 211 or the corner 212 is thrown towards the outer wall by centrifugal force, so as to form a compensating flow near the top and bottom of the arc segment 211 or the corner 212 . Two eddies appear at the top and bottom.

The Dean flow creates a drag force F _D on the cells in the sample, which pushes the cells in the sample inward (towards the center of the corner bend radius) away from the equilibrium position previously acted by the inertial lift force F _Z , and move to a new equilibrium position. Figures 13A and 13B show the Dean flow for corners 212 of different radii. The size of the drag force (represented by _FD ) on the cells in the sample produced by the Dean flow is related to the size of the cells in the sample, and the radius of the arc segment 211 or the radius of the corner 212, that is, the strength of the Dean flow This varies with the radius of the arcuate segment 211 or the radius of the corner 212; the smaller the radius, the stronger the Dean flow. Therefore, in the arc segment 211 or the corner 212 with a smaller radius, the cells in the sample are farther away from the center of the microfluidic channel 21 .

The drag force _FD generated by the Dean flow and the inertial lift force act on the cells in the sample together, so that the cells are in a new equilibrium position in the microfluidic channel 21 . Since the inertial lift force F _Z and drag force F _D acting on cells of different sizes in the sample are different, the cells of different sizes are in different equilibrium positions in the microfluidic channel 21 . Therefore, cells of different sizes in the fluid can be placed in different equilibrium positions. separate. That is, the cells are separated based on the size of the different cells in the sample using the inertial lift force F _Z and the drag force F _D generated by the Dean flow.

In the presence of an inertial lift force F _Z that holds the cell in a resting position, the drag force F _D is usually expressed as F _D ~ U _m ² aD _h ² r ^-1 , where U _m is the maximum channel velocity and a is the cell , D _h is the hydraulic diameter, and r is the radius of the arc segment 211 or the corner 212 . The inertial lift force F _Z tends to keep cells stably positioned on the cross-sectional centerline of the microfluidic channel 21 , while the Dean flow drags the cells to circulate across the cross-sectional surface of the microfluidic channel 21 . The new equilibrium position of the cell is related to the ratio of F _Z to F _D as a function of radius (δ) and cell size (a). Therefore, cells of various sizes can be separated with appropriate radii. Then, the new equilibrium position can be estimated from the ratio of _Fz to FD as follows:

where δ is the radius ratio. δ=D _h /2r.

When the sample flows through the microfluidic channel 21 containing a plurality of segments 211 and corners 212 at a certain flow rate, the inertial lift force F _Z acts as the driving force for the cells in the sample to converge, and the drag force F _D generated by the Dean flow Acts as a force to deflect the converging cells away from the center of the microfluidic channel 21, thereby enabling the separation of different cells in the sample based on their size. Among the inertial lift force F _Z and drag force F _D acting on the cell, when the drag force F _D is dominant (F _D > F _Z ), the cells may not converge due to insufficient inertial lift force Fz, or due to the strong drag force F _D , so that cells of different sizes may be dragged to the same convergence position; when the inertial lift force Fz is dominant (F _Z > F _D ), due to the lack of the drag force F _D , cells of different sizes are kept in the microfluidic The same balance position in the control channel 21 cannot be separated. The ratio of the inertial lift force _Fz to the drag force FD can be adjusted by using the flow rate of the sample and the radius of the arc segment 211 and the corner 212 in the microfluidic channel 21 to achieve the desired cell separation effect.

The value of the ratio of the inertial lift force _Fz to the drag force FD is preferably 1 to 10. It can be understood that, for the sample flowing in the arc segment 211 with a relatively large radius in the microfluidic channel 21, the ratio of the inertial lift force F _Z to the drag force F _D of the larger cells and the smaller cells ( F _Z /F _D ) are all greater than 1, which means that the equilibrium position of the cells is dominated by the inertial lift force F _Z , which is similar to the situation when the sample flows within the segment 211 of the straight line, regardless of the size, the cells tend to concentrate closer to the at the center of the microfluidic channel 21 . For a sample flowing in a circular arc segment 211 or a corner 212 with a smaller radius in the microfluidic channel 21, the ratio of the inertial lift force F _Z to the drag force F _D (F _Z of the larger cells and the smaller cells) /F _D ) are all close to 1, which means that the equilibrium position of the cell is dominated by the drag force F _D generated by the Dean flow. In this case, the cells may be pushed to the inner wall of the microfluidic channel 21 by the strong drag force _FD , where the large-sized cells and the small-sized cells usually converge at the same location. Therefore, the desired state is that the positions of cells of different sizes in the sample are in between, where the _FZ /F _D ratio of the larger sized cells is greater than the _FZ /F _D ratio of the smaller sized cells, which allows Separate cells of different sizes well.

In one embodiment, the microfluidic channel 21 includes an arc segment with a relatively small radius, and/or the angle of the corner 212 is not less than 90°, so that the sample flows through the corner 212 and/or the arc with a relatively small radius There is a sharp turn in the flow direction during the period. This large change in the direction of sample flow facilitates the convergence of smaller-sized cells (eg, red blood cells (RBCs) and white blood cells (WBCs)) generated by the Dean flow-generated drag force _FD . Because the inertial lift force Fz is not strong enough (at least in part), it is more difficult for cells of smaller size to converge. When the direction of flow of the sample changes greatly somewhere, the resulting strong Dean flow causes smaller cells at corners 212 and/or arc segments with smaller radii to follow the vortices caused by the Dean flow Spin passive migration. The sample undergoes multiple such flow direction changes, causing the smaller cells in the sample to continuously change their positions, eventually moving to an equilibrium position and converging into a flow.

The physical properties of the microfluidic channel 21, for example, the shape and size of the cross-section of the microfluidic channel 21, the length and number of straight segments 211 in the microfluidic channel 21, the radius, length and number of arc segments 211, and the corners The angle, number, and position of 212 will affect the separation of cells in the sample.

FIG. 14A shows an example of a microfluidic channel, and FIGS. 14B to 14D show the process of separating cells in the blood sample after the blood sample is input into the microfluidic channel 21 . At A near the inlet of the microfluidic channel 21, various cells in the blood sample are mixed together, as shown in Figure 14B. During the flow of the blood sample in the microfluidic channel 21, under the action of inertial lift force F _Z and drag force F _D , cells of the same size converge into a flow, and cells of different sizes (rare cells, white blood cells and cells) flow. separated from each other (in their respective equilibrium positions) as shown in Figure 14C. Finally, at the end of the microfluidic channel 21, the separated cells of different types are respectively discharged from the microfluidic channel 21 through the corresponding shunt channel, as shown in FIG. 14D. The separation between the streams of rare cells, leukocytes and erythrocytes is 50 to 150 μm.

The microfluidic chip box provided by the present invention is composed of a microfluidic chip box placed in the inner box and an outer box covered on the inner box to form a microfluidic chip box for separating cells in a sample. Utilize the physical structural properties of the microfluidic channel in the microfluidic chip, such as the shape and size of the cross-section of the microfluidic channel, the lengths and bending radii of the multiple segments that make up the microfluidic channel, and the angle, position and number of turning corners Enables separation of cells of different sizes in a sample. The microfluidic chip box has a simple structure and is easy to use. It can separate a variety of cells at the same time without pre-labeling the cells in the sample. It has high throughput, fast separation speed and large sample volume (50ml of samples can be processed at one time). ), the viability of the isolated cells was not affected. The microfluidic chip of the invention has good separation effect on cells in the sample, the capture rate of cancer cells reaches 95.9%, and the removal rate of leukocytes reaches 99.99%; the clinical CTC detection sensitivity is high, the breast cancer and pancreatic cancer reach 95%, and the lung cancer reaches 100% , liver cancer reached 88.1% (75% in stage I, 96.2% in stage II-IV).

In the present invention, terms such as "upper", "lower", "left", "right", "front", "rear", "vertical", "horizontal", "side", "bottom", etc. The orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings, and is only a relational word determined for the convenience of describing the structural relationship of each component or element of the present invention, and does not specifically refer to any component or element in the present invention, and should not be construed as a reference to the present invention. Invention limitations.

In the description of the present invention, it should be noted that the terms "installed", "connected" and "connected" should be understood in a broad sense, unless otherwise expressly specified and limited, for example, it may be a fixed connection or a detachable connection Connection, or integral connection; can be mechanical connection, can also be electrical connection; can be directly connected, can also be indirectly connected through an intermediate medium, can be internal communication between two elements. For those of ordinary skill in the art, they can understand the specific meanings of the above terms in the present invention under specific circumstances, and should not be construed as limiting the present invention.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that various combinations, modifications or equivalent replacements to the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and should cover within the scope of the claims of the present invention.

Claims

A microfluidic chip box, characterized by comprising: an inner box body (10), a microfluidic chip (20) and an outer box body (30);

The inner box body (10) is a shell with an open top surface, the microfluidic chip (20) is placed in the inner box body (10); the outer box body (30) is a shell with an open bottom surface body, the outer box body (30) is covered on the inner box body (10);

At least one sample inlet (31) is provided on the top surface of the outer box body (30);

The microfluidic chip (20) is provided with at least one microfluidic channel (21); the initial end of the microfluidic channel (21) is connected with the sample inlet (31) through an input pipe; the The tail section of the microfluidic channel (21) includes a plurality of shunt channels (22), and a shunt pipe divides the ends of the plurality of shunt channels (22) and the at least two channels arranged on the inner box body (10). The liquid outlet (11) is communicated.
The microfluidic chip box according to claim 1, characterized in that, the microfluidic channel (21) comprises a plurality of segments (211) and a plurality of corners (212), and the corners (212) are arranged in the phase The junction of two adjacent segments (211).
The microfluidic chip box according to claim 2, wherein the segment (211) is a straight segment or a circular arc segment with a set radius.
The microfluidic chip box according to claim 1, wherein the cross section of the microfluidic channel (21) is rectangular.
The microfluidic chip box according to claim 4, wherein the ratio of the length to the width of the cross section of the microfluidic channel (21) is 1 to 10.
The microfluidic chip box according to claim 2, wherein the angle of the corner (212) is not less than 90°.
The microfluidic chip box according to claim 1, wherein the initial section of the microfluidic channel (21) includes a plurality of input channels (23), and the initial end of the input channel (23) passes through The input tube communicates with the sample inlet (31).
The microfluidic chip box according to claim 7, wherein the microfluidic chip (20) comprises a chip bottom plate (24) and a chip body (25), the chip bottom plate (24) and the The microfluidic channel (21) is formed by the microfluidic groove machined on the bottom surface of the chip body (25).
The microfluidic chip box according to claim 8, wherein a plurality of inlet channels (251) and a plurality of outlet channels (252) are provided in the chip body (25);

The lower end of the inlet channel (251) is connected to the starting end of the input channel (23), and the upper end of the inlet channel (251) is opened on the outer surface of the chip body (25); the inlet channel (251) ) is connected with the sample inlet (31) through the input pipe;

The lower end of the outlet channel (252) is connected with the end of the shunt channel (22), and the upper end of the outlet channel (252) is opened on the outer surface of the chip body (25); the outlet channel (252) The upper end of the shunt is communicated with at least two shunt liquid outlets (11) provided on the inner box body (10) through the shunt pipe.
The microfluidic chip box according to claim 9, characterized in that a limiting groove (12) is provided at the bottom of the inner box body (10), and the limiting groove (12) is used for fixing the shunt pipe .