Microfluidic chip and device for stable capture of CTCs at ultra-high flow rates

Abstract

The application relates to the field of medical diagnosis devices, in particular to a micro-fluidic chip and device capable of stably capturing CTCs at an ultrahigh flow rate. The application increases the upper limit of the flow rate of a single chip by 3 times (about 150 mL / h) through discovery and optimization of a flow rate limiting key structure, and the chip still has excellent capture efficiency and capture purity at a high flow rate. Meanwhile, through optimization of the arrangement mode of the chip, the three-dimensional micro-fluidic chip of the application is more conducive to reducing the flow rate and / or flow of the sample, and the area of a single-layer chip is reduced to 1 / 6-1 / 4 of the original. Through joint use of the above two optimization modes, the upper limit of the flow rate can be multiplied.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnostic devices, and in particular to a microfluidic chip and device that can be used for stably capturing CTCs at ultra-high flow rates. Background Art

[0002] According to the latest data from the International Agency for Research on Cancer, there were approximately 18.19 million new cancer cases and 9.6 million cancer deaths worldwide in 2018. Among these cancer deaths, metastatic cancer is the leading cause of cancer-related deaths. Therefore, early diagnosis and treatment of cancer are crucial to effectively prevent the development of metastatic disease. Circulating tumor cells (CTCs) are cancer cells shed from primary cancer in situ and enter the circulatory system, and are closely linked to cancer metastasis. Research has shown that CTCs are present in the blood of most cancer patients by the time the primary cancer is discovered. Therefore, CTCs play a crucial role in early cancer diagnosis, real-time monitoring, and prognostic assessment, enabling earlier detection of cancer compared to existing diagnostic methods. However, CTCs are rare, occurring in peripheral blood at concentrations ranging from a few to a few hundred per milliliter. Highly sensitive techniques for isolating CTCs from peripheral blood are lacking. Therefore, the development of new technologies that efficiently and efficiently capture CTCs with high purity and ease of use is urgently needed.

[0003] Among existing microfluidic chip designs, one relies on biochemical methods to specifically capture cancer cells through the EpCAM receptor on the surface of cancer cells. This method often requires complex pre-operations, and CTCs in the epithelial-mesenchymal transition (EMT) are difficult to detect. Another method uses the differences in the physical properties of cancer cells and red and white blood cells for separation. However, physical separation methods often cannot take into account both the upper limit of flow rate and robustness to flow rate, and the capture purity is relatively low.

[0004] CN 111909828 A and Lu, C., et al., Lab on a Chip, 2020.20(22): p.4094-4105 both disclose a microfluidic chip suitable for capturing circulating tumor cells (e.g., attached Figure 1 The chip achieved stable capture of over 90% of target particles at flow rates of 5-40 mL / h, with a low leukocyte contamination rate (4 log removal). However, subsequent studies revealed that at higher sample flow rates, the capture efficiency of target particles decreased to a certain extent. Summary of the Invention

[0005] To address the aforementioned technical issues, the present invention investigated the cause of the reduced capture efficiency and discovered that it was due to a jet effect caused by the sudden widening of the sample pathway within the microfluidic chip (particularly at the connection between the converging structure and the diverting channel). Based on this finding, the present invention further optimized the structure of the microfluidic chip, providing a high-efficiency, high-purity, and simple-to-operate microfluidic chip and device for the stable capture of circulating tumor cells at ultra-high flow rates.

[0006] Specifically, the present invention first provides a microfluidic chip, which includes a converging and diverting module, wherein the converging and diverting module includes an inlet, a main channel, one or more converging and diverting units, and an outlet;

[0007] The inlet, one or more converging and diverting units and the outlet are connected through a main channel, and the sample enters the converging and diverting unit from the inlet and is discharged or partially discharged from the outlet;

[0008] The converging and diverting unit converges the target particles in the sample at the center of the liquid flow; at the same time, the side liquid flow that does not contain the target particles is discharged or partially discharged through the outlet, thereby reducing the flow rate and / or flow of the sample without losing the target particles;

[0009] In the sample passage, when the passage along the sample flow direction is widened, the S 宽 / S 窄 ≤8, more preferably S 宽 / S 窄 ≤5; where S 宽 Indicates the maximum cross-sectional area of ​​the channel at the widening point, S 窄 Indicates the minimum cross-sectional area of ​​the channel at the widening point.

[0010] The present invention has found that the above structural improvement can greatly improve the capture efficiency loss of the original chip when the sample flow rate is high.

[0011] Preferably, the converging and diverting unit comprises one or more converging structures and diverting channels arranged in series;

[0012] The converging structure converges the target particles in the sample at the center of the liquid flow, and discharges the separated side liquid flow without the target particles to the outlet through the diversion channel or partially discharges it;

[0013] At the connection channel between the converging structure and the diverting channel, S 宽 / S 窄 ≤8, more preferably S 宽 / S 窄 ≤5; where S 宽 represents the maximum cross-sectional area of ​​the connecting channel, S 窄Indicates the minimum cross-sectional area of ​​the connecting channel.

[0014] The present invention further discovered that after the connection channel between the converging structure and the diversion channel is set in the above manner, the capture efficiency of the chip is significantly improved at high sample flow rates.

[0015] More preferably, the connecting channel between the converging structure and the diverting channel is in an arc shape, more preferably an arc-shaped branching structure.

[0016] Preferably, the converging structure includes a central channel and side branch channels;

[0017] Wherein, the central channel is connected to the main channel at both ends and is coaxially arranged;

[0018] The side branch channels intersect with the main channel and the central channel at both ends; further preferably, there are two side branch channels, and further preferably, the two side branch channels are arranged on both sides of the central channel, and more preferably, the two side branch channels are symmetrically arranged on both sides of the central channel and have the same size parameters.

[0019] In the present invention, by controlling the flow resistance (i.e., flow resistance) of each channel, the converging and diverting unit converges the target particles in the sample at the center of the liquid flow; at the same time, the side liquid flow that does not contain the target particles is discharged or partially discharged through the outlet, thereby reducing the flow rate and / or flow of the sample without losing the target particles.

[0020] The proportional relationship of the flow resistance is based on the L(H+W) of the channel. 2 / (HW) 3 The length, width and height of the channel are obtained by the ratio of L, W and H, respectively.

[0021] For details on the principles of flow resistance calculation, please refer to CN 111909828 A.

[0022] In the present invention, the sample is derived from whole blood, plasma, serum, perfusate, urine, tissue fluid, cerebrospinal fluid, cell culture fluid, or a cell mixture. More preferably, the sample is whole blood or perfusate. Preferably, the target particles are tumor cells, more preferably circulating tumor cells (CTCs). Those skilled in the art can set the specific parameters of each sample channel in the microfluidic chip based on the parameters of the above-mentioned sample and target particles.

[0023] As a preferred solution, a main channel narrowing portion is provided at the position where the sample flows from the main channel into the converging and diverting unit; Zrepresents half the width of the main channel, W2 represents the width of the narrowed main channel, d1 represents the distance between the center of mass of the target particle closest to the channel boundary on the side of the narrowed main channel and the boundary of the side channel, R1 is the flow resistance of the central channel of the first converging structure in each converging and diverting unit, and R2 is the flow resistance of a single side branch channel in the first converging structure. The above parameters meet the following conditions:

[0024]

[0025] Among them, the value of d1 is equal to r cell , or slightly smaller than r cell , r cell Indicates the average radius of target particles in the sample;

[0026] As a preferred solution, let the flow resistance of the central channel of the nth converging structure be R1 n , width Ws n The flow resistance of a single lateral branch channel is R2 n , the flow resistance of the central channel of the n+1 converging structure is R1 n+1 The flow resistance of each lateral branch channel is R2 n+1 , which satisfies the following conditions:

[0027]

[0028] Among them, the value of d1 is equal to r cell , or slightly smaller than r cell , r cell It represents the average radius of target particles in the sample, and n is a natural number greater than or equal to 1 and less than the total number of convergent structures.

[0029] For target particles with a certain rigidity (such as cells, especially cancer cells such as CTCs), d1 can be considered to be equal to r cell , because the target particles are difficult to compress. In actual calculations, in order to ensure that the designed channel does not allow all target particles to be mistakenly discharged, d1 can be made slightly smaller than r cell The term “slightly less than” in the present invention is understood by those skilled in the art, that is, as long as the value of d1 is less than r cell , we can ensure that the target particles enter the central channel to a greater extent in the calculation, for example, d1 can be r cell More than 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or the range and value between any two of the above points.

[0030] Preferably, both ends of the diversion channel intersect with the main channel, wherein the upstream intersection is close to the last converging structure in the converging and diversion unit, and the downstream intersection is close to the outlet of the microfluidic chip; preferably, there are two diversion channels, arranged on both sides of the main channel; more preferably, they are symmetrically arranged on both sides of the main channel and have the same size parameters.

[0031] As a preferred solution, the flow resistance of the diversion channel of the mth converging and diverting unit is determined as follows: let the overall flow resistance of the area between the two intersections of the diversion channel of the mth converging and diverting unit and the main channel (or the overall flow resistance of the area inside the diversion channel) be R1 m , the flow resistance of each diversion channel is R2 m , Wherein, k is the proportion of the liquid flow in the diversion channel to the overall liquid flow; m is a natural number greater than or equal to 1 and less than or equal to the total number of converging and diverting units.

[0032] Preferably, the microfluidic chip further comprises a capture module for capturing target particles.

[0033] More preferably, the capture module is located downstream of the converging and diverting unit, and both ends thereof are connected to the main channel.

[0034] Further preferably, the capture module includes multiple arrays, each array being composed of small blocks of arbitrary shapes (e.g., cubes, triangular prisms, cylinders, etc., or having square, rectangular, triangular, circular, etc. cross-sections). There is a gap d between adjacent blocks, where the size of the gap d is defined as the distance between the two closest points on the surfaces of two adjacent blocks. The gap d between blocks in each array is the same, and the gap d between blocks in different arrays decreases step by step along the direction of sample inflow. For example, when the number of arrays is four, the sizes of the gap d in each array along the direction of sample inflow are 12 μm, 10 μm, 8 μm, and 6 μm, respectively.

[0035] Preferably, a filtering unit is provided between the inlet and the connecting channel of the first converging and diverting unit to prevent the chip from being blocked.

[0036] In a preferred embodiment of the present invention, the material of the microfluidic chip is polydimethylsiloxane (PDMS).

[0037] Those skilled in the art can combine the above solutions to obtain a preferred embodiment of the microfluidic chip of the present invention.

[0038] Furthermore, the present invention also provides a three-dimensional microfluidic chip, which includes a stacked convergence and diversion layer chip and a capture layer chip.

[0039] Among them, a converging and diverting module is provided on the converging and diverting layer chip, and the structure of the converging and diverting module is the same as described above;

[0040] The converging and diverting unit on the converging and diverting layer chip is provided with a first outlet and a second outlet downstream, the first outlet is used to discharge the liquid flow containing the target particles, and the second outlet is used to discharge the side liquid flow not containing the target particles;

[0041] A sample inlet and a sample outlet are provided on the capture layer chip. The sample inlet is communicated with the first outlet; the sample outlet is communicated with the second outlet.

[0042] The present invention also found that the above-mentioned stacking arrangement is more conducive to reducing the flow rate and / or flow of the sample, thereby increasing the upper limit of the sample flow rate while reducing the area of ​​the single-layer chip, which is also conducive to controlling costs.

[0043] More preferably, one or more arrays, and more preferably multiple arrays, are provided between the connecting passages between the sample inlet and the sample outlet of the capture layer chip. The arrays are composed of small blocks of any shape (e.g., cubes, triangular prisms, cylinders, etc., or with square, rectangular, triangular, circular, etc. cross-sections). A gap d exists between adjacent small blocks, where the size of the gap d is defined as the distance between the two closest points on the surfaces of two adjacent small blocks. The gap d between small blocks in each array is the same, and the gap d between small blocks in different arrays decreases step by step along the direction of sample inflow. For example, when there are four arrays, the sizes of the gap d in each array along the direction of sample inflow are, respectively, 12 μm, 10 μm, 8 μm, and 6 μm.

[0044] In the capture module or capture layer chip of the present invention, the upper limit of the flow rate can be further increased by refining the small block structure in the array, and / or increasing the area of ​​the array, and / or increasing the number of arrays (the gap d of which decreases step by step along the direction of sample inflow), which all fall within the scope of protection claimed by the present invention.

[0045] In a preferred embodiment of the present invention, the microfluidic chip and the capture layer chip are made of polydimethylsiloxane (PDMS).

[0046] Preferably, a stacked converging and diverting layer chip and a capture layer chip are formed into a group, and the three-dimensional microfluidic chip comprises multiple groups of stacked converging and diverting layer chips and capture layer chips; the inlets of the converging and diverting layer chips in different groups are connected, and at the same time, the second outlets of the converging and diverting layer chips in different groups and the sample outlets of the capture layer chips are connected.

[0047] Those skilled in the art can combine the above solutions to obtain a preferred embodiment of the three-dimensional microfluidic chip of the present invention.

[0048] The present invention also provides a device for enriching and / or capturing target particles in a sample, which comprises: the microfluidic chip and / or the three-dimensional microfluidic chip.

[0049] Preferably, the device further comprises a detection unit for detecting the liquid flow containing the target particles and / or the side liquid flow not containing the target particles.

[0050] The present invention also provides applications of the microfluidic chip, the three-dimensional microfluidic chip, or the device in any of the following aspects:

[0051] (i) enriching and / or capturing target particles in a sample;

[0052] (ii) removing target particles from the sample solution;

[0053] (iii) Reduce the sample flow rate and / or flow rate.

[0054] Preferably, the sample is derived from whole blood, plasma, serum, perfusate, urine, tissue fluid, cerebrospinal fluid, cell culture fluid or cell mixture, more preferably, the sample is whole blood or perfusate;

[0055] Preferably, the target particles are tumor cells, more preferably circulating tumor cells (CTCs).

[0056] Preferably, the flow rate of the sample in the microfluidic chip or three-dimensional microfluidic chip is within 150 mL / h, for example, 5-150 mL / h.

[0057] The present invention further provides a method for enriching and / or capturing target particles, comprising:

[0058] A sample solution containing target particles is injected into the microfluidic chip, the three-dimensional microfluidic chip, or the device, and the liquid flow containing the target particles is collected.

[0059] The present invention further provides a method for removing target particles from a sample solution, comprising:

[0060] A sample solution containing target particles is injected into the microfluidic chip, the three-dimensional microfluidic chip, or the device, and a side flow without target particles is collected.

[0061] Preferably, before injecting the sample, the sample passage is wetted and pretreated with 1× phosphate buffered saline (PBS) and 0.1% bovine serum albumin solution (BSA). More preferably, the wetting and pretreatment time is 1.5 to 3 hours.

[0062] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0063] By discovering and optimizing key flow rate limiting structures, the present invention increases the upper limit of a single chip flow rate by three times (approximately 150 mL / h), while maintaining excellent capture efficiency and purity at high flow rates. Simultaneously, by optimizing the chip layout, the three-dimensional microfluidic chip of the present invention reduces the area of ​​a single-layer chip to 1 / 6-1 / 4 of its original size, while further facilitating the reduction of sample flow rate and / or flow rate. By combining these two optimization methods, an exponential increase in the upper limit of flow rate can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The structural diagram and physical picture of the original designed chip (Lu, C., et al., Lab on a Chip, 2020.20(22): p.4094-4105); among them, B is the physical picture, while A and CG are both structural diagrams.

[0065] Figure 2 Schematic diagram of the structure of a three-dimensional microfluidic chip provided by an embodiment of the present invention; Figure 2 Among them, 1. Converging and diverting layer chip; 11. Filtering unit; 111. Square filtering structure; 112. S-shaped filtering structure; 12. Converging and diverting unit; 121. Converging structure; 122. Diverting channel; 13. Outlet; 131. First outlet; 132. Second outlet; 2. Capturing layer chip; 21. Triangular capturing area; 22. Sample inlet and outlet; 221. Sample inlet; 222. Sample outlet.

[0066] Figure 3 1 is a flow chart of a method for manufacturing a three-dimensional microfluidic chip structure provided by an embodiment of the present invention;

[0067] Figure 4 Schematic diagram of a method for fabricating the structure of a three-dimensional microfluidic chip provided by an embodiment of the present invention;

[0068] Figure 5 1 is a flow chart of a method for using the structure of a three-dimensional microfluidic chip provided by an embodiment of the present invention;

[0069] Figure 6 Schematic diagram and scanning electron microscope image of the gap size of the triangular capture area (array) of the structure of the three-dimensional microfluidic chip provided by an embodiment of the present invention;

[0070] Figure 7 This is a physical diagram of the structure of a three-dimensional microfluidic chip provided by an embodiment of the present invention;

[0071] Figure 8A comparison of (i) a streamline simulation diagram of the first diversion unit of the original design and (ii) a streamline simulation diagram of the first diversion unit of the structure of the three-dimensional microfluidic chip provided by an embodiment of the present invention;

[0072] Figure 9 Comparison of (i) the measured streamline diagram of green fluorescent E. coli in the first diversion unit of the original design and (ii) the measured streamline diagram of green fluorescent E. coli in the first diversion unit of the three-dimensional microfluidic chip structure provided by an embodiment of the present invention;

[0073] Figure 10 It is a comparison of the inlet flow rate and capture efficiency of the structure of the three-dimensional microfluidic chip provided by the original design and the embodiment of the present invention. DETAILED DESCRIPTION

[0074] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0075] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0076] Example 1

[0077] This embodiment provides a three-dimensional microfluidic chip for detecting CTCs. Figure 2 As shown, it is a double-layer chip, including: a converging and diverting layer chip 1 and a capturing layer chip 2.

[0078] The converging and diverting layer chip 1 is provided with a converging and diverting module, which includes an inlet, a filtering unit 11, six converging and diverting units 12 connected in series, and an outlet 13, which are sequentially connected by a main channel.

[0079] The filter unit 11 includes a square filter structure 111 with a height of 60 microns and an S-shaped filter structure 112 with a height of 30 microns to prevent chip clogging.

[0080] The converging and diverting unit 12 includes a collection port, a narrowed main channel, five converging structures 121, and two diverting channels 122 on either side, each 60 microns high. Flow resistance calculations show that while retaining cancer cells, some white blood cells and red blood cells are diverted, achieving a concentrated and decelerated flow.

[0081] Specifically, the cross-sectional diameter W1 of the widest part of the collecting port is 200 μm, and the whole structure is funnel-shaped. The cross-sectional diameter W2 of the narrowed part of the main channel is 30 μm, and the cross-sectional diameter of the main channel is 90 μm. For each converging and diverting unit 12, the widths of the side diverting channels 122 of the five converging structures 121 are 30, 40, 40, 40, 40 μm, respectively, and the lengths are 1290, 1210, 830, 650, 490 μm (single-side length), respectively. The width Ws of the central channel is 30 μm, and the width of the main channel is 2W. Z The six groups of converging and diverting units 12 have the same number and size of converging structures 121, and the diverting channels 122 have widths of 100, 100, 80, 80, 70 and 60 μm from top to bottom, and lengths of 29,000, 30,000, 20,500, 21,500, 18,000 and 15,000 μm (total length of one side). The S of the connecting channel between the converging structure 121 and the diverting channel 122 is 100, 100, 80, 80, 70 and 60 μm from top to bottom, and the ... 宽 / S 窄 is 3.3, which effectively weakens the jet effect; at the same time, the diversion site is changed to an arc-shaped branching structure to reduce the effective width of the diversion site. Specifically, the widths of the three branches from left to right are 50 microns (connecting the diversion channel 122), 80 microns (connecting the main channel) and 50 microns (connecting the diversion channel 122).

[0082] The outlet 13 includes a first outlet 131 for entering the capture layer chip 2 and a second outlet 132 for discharging a side liquid flow that does not contain target particles after capture, and has a height of 130 microns.

[0083] Capture layer chip 2 (gap size diagram and SEM image see Figure 6 ) includes several branching channel structures, a triangular capture region 21, and two sample inlets and outlets 22, each 60 microns in height. The triangular capture region 21 comprises four layers of triangular structures with varying gaps of 12, 10, 8, and 6 microns, respectively. The two sample inlets and outlets 22 include a sample inlet 221 connected to the first outlet 131 and a sample outlet 222 connected to the second outlet 132.

[0084] The material of the converging and diverting layer chip 1 and the capturing layer chip 2 is polydimethylsiloxane, that is, the chip processing material is PDMS (polydimethylsiloxane).

[0085] After functional partitioning of the chip, the chip area can be significantly reduced. The actual chip picture is as follows: Figure 7 Obviously, the area of ​​a single-row chip is more than four times smaller than that of the original chip, and the area of ​​the 4× parallel chip is smaller than that of the original chip. Therefore, this design can allow parallel connection within a limited area to achieve an increase in the overall flow rate limit.

[0086] It should be mentioned that the size and height of the channel in the present invention can be adjusted based on experiments. For details on the principle, please refer to CN111909828 A.

[0087] Example 2

[0088] This embodiment provides a method for manufacturing the three-dimensional microfluidic chip in Example 1, such as Figure 3 As shown, it mainly includes the following steps:

[0089] Step S1: preparing molds for the convergence and diversion layer chip and the capture layer chip respectively.

[0090] Step S2: coating the prepared material on the mold and performing a curing process.

[0091] Step S3: Align and bake the connection sites of the convergence and diversion layer chip and the capture layer chip.

[0092] Step S4: Plasma treatment is performed on the chip structure, and the chip is bonded to the glass slide and baked.

[0093] In a specific embodiment, Figure 4 As shown, the steps for making the structure of a three-dimensional microfluidic chip are as follows:

[0094] (1) Preparation of mold. Use L-Edit to draw and print it on a plastic film or optical mask as an exposure mask for subsequent experiments. A total of one silicon wafer mold is required to make the convergence and diversion layer chip, and one chrome plate is used for back exposure to make the capture layer chip. The preparation process of the two molds is basically the same, except that the mask is different. Use SU8 photoresist, pre-spin at a speed of 500 rpm for 10 seconds when spreading the glue, and then set different speeds for 60 seconds according to different height requirements. After baking on a 95-degree hot plate for 30 minutes, put it into the exposure machine for exposure. The exposure time needs to be determined by referring to the help document of the exposure machine and the actual light intensity. Then bake it on a 95-degree hot plate for 15 minutes, and develop it to get the mold.

[0095] (2) Chip production. The cell injection layer and the culture layer are both obtained by PDMS injection molding. The convergence and diversion layer chip and the capture layer chip require a ratio of A glue (monomer) to B glue (cross-linking agent) of 8:1. Mix them evenly, vacuum to remove bubbles, and then spin-coat PDMS on the convergence and diversion layer chip mold through a glue spreader (1000rpm, 30s). A PDMS film with a thickness of about 100um can be obtained. The capture layer chip thickness is more than 3mm. When curing, first put the two molds into a 70-degree oven and bake for 40 minutes, then take out the two layers of chips. At this time, the PDMS has been preliminarily cured. Use a microscope to align the through-hole structure and put it in an oven to bake overnight. The PDMS will continue to cure and the two layers of chips will be firmly bonded together.

[0096] (3) Chip packaging and modification. The chip and the glass slide are plasma treated and then baked overnight. Before using the chip, it is necessary to vacuum it for 20 minutes to facilitate the discharge of gas inside the chip when the liquid is added. It should be noted that after the vacuum treatment, the internal pressure of the chip will gradually balance with the external air pressure, so the pretreatment operation needs to be completed within 30 minutes after the treatment.

[0097] Example 3

[0098] This embodiment provides a method for using the three-dimensional microfluidic chip in Example 1, such as Figure 5 As shown, the method mainly includes the following steps:

[0099] Step S1: 1×PBS and 0.1% BSA were passed through the chip to wet and pretreat it, and the chip was left to stand for 2 hours.

[0100] Step S2: The test sample is passed into the chip for CTC capture, and the outflowing liquid is collected using a well plate.

[0101] Step S3: Microscopically photograph and count the triangular capture area of ​​the chip and the well plate.

[0102] It should be noted that when testing blood samples, the chip needs to be moistened with heparin during the pretreatment stage, and red blood cell lysis solution can be passed through before microscopic counting to facilitate imaging.

[0103] Test example

[0104] The principle explanation and effect difference of the microfluidic chip before and after improvement are explained as follows:

[0105] When the fluid flows in the pipe, there are two main forces in opposite directions: the shear force F caused by the velocity distribution and the LS and the wall force F caused by the viscosity between the fluid and the wall LW ; The shear force is directed toward the wall and its magnitude is determined by Determined by; the direction of the wall force points to the center of the flow channel, and the magnitude is determined by decision; where f L is the lift coefficient, ρ is the fluid density, U m is the maximum velocity of the fluid, a is the particle diameter, W c is the channel width. When the channel size suddenly increases, the wall force acting on particles in the fluid suddenly weakens, and the particles are pushed away from the center of the channel by shear forces, causing a vortex, known as the jet effect. Research has shown that when this jet effect occurs, the Reynolds number (Re) remains within the laminar flow range (Re < 1000), not reaching the size required to form a turbulent vortex. Therefore, this phenomenon is also called a laminar vortex.

[0106] There are two conditions for the occurrence of laminar vortexes: one is that the channel structure has a narrowing-widening structure. In the more universal three-dimensional case, the present invention makes a cross section along the direction of fluid flow and considers the cross-sectional area as the evaluation criterion. Studies have shown that this laminar vortex phenomenon exists not only in two-dimensional narrowing-widening structures, but also in three-dimensional situations where a sudden increase in cross-sectional area leads to a sudden decrease in wall force. The second condition is that the Reynolds number Re reaches a certain value. The Reynolds number is defined as Re = ρU m D h / μ, where ρ is the liquid density, μ is the fluid viscosity, D h =2W C H / (W C (H) is the hydraulic diameter, which is closely related to the maximum linear velocity of the main channel. Research has shown that when the main channel Reynolds number (Re) exceeds 100, the constriction-widening structure begins to exhibit laminar vortexes. When the main channel Reynolds number (Re) exceeds 130, the structure can capture particles larger than 10 microns through vortexes.

[0107] In the original design, Figure 1 Middle C and Figure 8 As shown in (i), the width of the main channel of the last converging unit is 30 microns. When entering the diversion channel after passing the last converging unit, the channel width is 200-300 microns, and the buffer zone is relatively short. After the main channel widens, it quickly enters the diversion structure. According to the calculation, the fluid viscosity μ = 0.8949 mPa·s and the density ρ = 1×10 3 kg / m 3 When the inlet flow rate is 30 mL / h, when entering the first diversion structure, the Reynolds number of the main channel at 30 microns is Re=206, and Re∝U m , that is, the greater the speed, the greater the Reynolds number, and this Re value is consistent with the range of laminar vortex occurrence. After the fluid passes through the 30-micron main channel and enters the diversion unit, the channel width suddenly widens to 200-300 microns, causing the wall force to suddenly weaken. The cells will be pushed away from the center of the channel by the shear force, and due to Particles with larger diameters are more likely to leave the center of the channel. According to Stokes' law, the shear force will be given by the Stokes force Fd =3πμav t equilibrium, so the particle lateral migration speed As the fluid velocity U m Increase, shear force F LS Increase, when U m When a certain value is reached, the narrowing-widening structure of 30 microns to 300 microns in the original design will experience a jet phenomenon, forming a vortex, causing the CTC to be separated by the diversion structure through the vortex, and the capture efficiency will decrease.

[0108] COMSOL streamline simulation of the culture medium system Figure 8 As shown in (i), the parameters used are fluid viscosity μ = 0.8949 mPa·s, density ρ = 1×10 3 kg / m 3 Simulation results show that at an inlet flow rate of 30 mL / h, the first diversion unit exhibits streamline instability. As the inlet flow rate increases, vortexes in the first diversion unit become increasingly apparent. This simulation result is consistent with experimental results. In previous experiments, the capture efficiency of the culture medium system reached over 90% at an inlet flow rate of 5-20 mL / h, dropping to around 80% at 30 mL / h. As the flow rate continues to increase, the capture efficiency drops to 50%-60%.

[0109] From the conditions for the generation of laminar vortex phenomenon, it can be seen that in order to reduce the impact of this phenomenon on chip function, the channel narrowing-widening structure needs to be changed without reducing the inlet flow velocity or the Reynolds number Re. In the narrowing-widening structure, when the ratio of the cross-sectional area S 宽 / S 窄 When S > 5, at high flow rate or high Reynolds number, the structure will have jet phenomenon; 宽 / S 窄 When the S is greater than 8, the jet phenomenon is significant, and the vortex can carry CTC away from the main channel and trap it inside the vortex. In the original design, the 30-300 micron structure S 宽 / S 窄 >8, so the jet effect is obvious, causing the CTC to be captured by the vortex and then separated by the diversion structure.

[0110] Therefore, the present invention improves the diversion structure and significantly reduces the jet effect by narrowing the flow, thereby increasing the upper limit of the single-chip flow rate. The specific structure and COMSOL simulation results are shown in Figure 2. Figure 8 As shown in (ii), the present invention changes the original design of the diversion structure with a width of 200-300 microns to 100 microns. 宽 / S 窄<4, effectively reducing the jet effect. At the same time, the diversion site was changed to a branching structure, reducing the effective width of the diversion site. Specifically, the original design's 300-micron diversion channel was changed to a three-branch structure: 50 microns + 80 microns + 50 microns. The goal is to prevent the liquid from completely losing its wall force after reaching the diversion structure. Instead, the flow resistance determines the balance between wall force and shear force after entering different branches, thereby significantly reducing the jet effect. COMSOL simulations show that the improvement in the diversion site can effectively increase the upper limit of the single-chip flow rate by approximately 3 times (from 30-40 mL / h to 100 mL / h).

[0111] In one embodiment, the upper limit of the flow rate of the chip is detected by green fluorescent Escherichia coli, such as Figure 9 shown. Figure 9 (i) is the measured streamline diagram of the first shunt structure in the original design chip. Figure 9 (ii) is the measured streamline diagram of the first shunt structure of the quasi-three-dimensional high-throughput detection chip provided by the present invention. In the original design, at a flow rate of 20mL / h, the streamline distribution of the first shunt structure is normal, and at 40mL / h, obvious streamline instability has appeared. When the inlet flow rate is increased to 60mL / h, the generation of vortices can be clearly seen; and in the chip design provided by the present invention, within the flow rate range of 40-100mL / h, the first shunt structure does not show the phenomenon of streamline instability. It can be seen that the improvement of the shunt site can greatly increase the flow rate upper limit of the chip.

[0112] In a specific experimental example, a certain number of cancer cells were injected into the culture medium system, and the samples were pushed into the original design chip and the three-dimensional microfluidic chip provided by the present invention. The capture efficiency was as follows: Figure 10 As shown, the capture efficiency is defined as: capture efficiency = number of cancer cells captured in the triangular area / (number of cancer cells captured in the triangular area + number of cancer cells in the waste liquid flowing out of the chip). It can be seen that in the original design, within the flow rate range of 5-20mL / h, the capture efficiency of the chip can be stabilized at more than 90%. When the inlet flow rate is increased to 30mL / h, the capture efficiency drops significantly to about 80%. In the three-dimensional microfluidic chip provided by the present invention, within the inlet flow rate range of 5-40mL / h, the chip capture efficiency can reach more than 90%. When the inlet flow rate is increased to 60mL / h, the chip capture efficiency can still be stabilized at about 85%. When the inlet flow rate reaches 80mL / h, the chip capture efficiency drops to 80%. By comparison, it can be seen that the three-dimensional microfluidic chip structure provided by the present invention can increase the upper limit of the flow rate by about 3 times.

[0113] It should be noted that the flow rates discussed in the above specific test examples are all within the culture medium system. Due to the different viscosities of the culture medium system and the blood sample, the upper limit of the flow rate is different from the 100-150 mL / h in the actual test (the sample is generally a blood sample). Here we focus on the increase in the upper limit of the flow rate.

[0114] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A microfluidic chip, characterized in that: It comprises a converging and diverting module, which comprises an inlet, a main channel, one or more converging and diverting units, and an outlet; The inlet, one or more converging and diverting units and the outlet are connected through a main channel, and the sample enters the converging and diverting unit from the inlet and is discharged or partially discharged from the outlet; The converging and diverting unit converges the target particles in the sample at the center of the liquid flow; at the same time, the side liquid flow that does not contain the target particles is discharged or partially discharged through the outlet, thereby reducing the flow rate and / or flow of the sample without losing the target particles; The converging and diverting unit includes one or more converging structures and diverting channels arranged in series; the connecting channel between the converging structure and the diverting channel is an arc-shaped branching structure; In the sample passage, when the passage along the sample flow direction is widened, the , which weakens the jet effect; among them, Indicates the maximum cross-sectional area of ​​the channel at the widening point, Indicates the minimum cross-sectional area of ​​the channel at the widening point; The flow rate of the inlet is 40 mL / h to 100 mL / h.

2. The microfluidic chip according to claim 1, characterized in that In the sample passage, when the passage along the sample flow direction is widened, the .

3. The microfluidic chip according to claim 1, characterized in that The converging structure converges the target particles in the sample at the center of the liquid flow, and discharges the separated side liquid flow without the target particles to the outlet through the diversion channel or partially discharges it; at the connection channel between the converging structure and the diversion channel, ;in, represents the maximum cross-sectional area of ​​the connecting channel, Indicates the minimum cross-sectional area of ​​the connecting channel.

4. The microfluidic chip according to claim 3, characterized in that At the connection channel between the converging structure and the diverting channel, .

5. The microfluidic chip according to claim 3, characterized in that: The converging structure includes a central channel and side branch channels; Wherein, the central channel is connected to the main channel at both ends and is coaxially arranged; The side branch channels intersect with the main channel and the central channel at both ends.

6. The microfluidic chip according to claim 5, characterized in that: There are two side branch channels.

7. The microfluidic chip according to claim 6, characterized in that: Two side tributary channels are arranged on both sides of the central channel.

8. The microfluidic chip according to claim 7, characterized in that: The two side branch channels are symmetrically arranged on both sides of the central channel and have the same size parameters.

9. The microfluidic chip according to claim 2 or 5, characterized in that: Both ends of the diversion channel intersect with the main channel, wherein the upstream intersection is close to the last converging structure in the converging and diversion unit, and the downstream intersection is close to the outlet of the microfluidic chip.

10. The microfluidic chip according to claim 9, characterized in that: There are two diversion channels, arranged on both sides of the main channel.

11. The microfluidic chip according to claim 10, characterized in that: There are two branch channels, which are symmetrically arranged on both sides of the main channel and have the same size parameters.

12. The microfluidic chip according to claim 11, characterized in that: The flow resistance of the diversion channel of the mth converging and diverting unit is determined as follows: Let the overall flow resistance of the area between the two intersections of the diversion channel of the mth converging and diverting unit and the main channel be R1 m , the flow resistance of each diversion channel is R2 m , , where k is the proportion of the liquid flow in the diversion channel to the overall liquid flow; m is a natural number greater than or equal to 1 and less than or equal to the total number of converging and diverting units.

13. The microfluidic chip according to any one of claims 1 to 12, characterized in that: The microfluidic chip further includes a capture module for capturing target particles.

14. The microfluidic chip according to claim 13, characterized in that: The capture module is located downstream of the converging and diverting module, and both ends are connected to the main channel.

15. The microfluidic chip according to claim 14, characterized in that: The capture module includes multiple arrays, which are composed of small blocks of arbitrary shapes. There is a gap d between adjacent small blocks, and the size of the gap d is defined as the distance between the two closest points on the surfaces of two adjacent small blocks; the gap d between the small blocks in each array is the same, and the gap d between the small blocks in different arrays decreases step by step along the direction of sample inflow.

16. A three-dimensional microfluidic chip, characterized in that: It includes a stacked convergence and diversion layer chip and a capture layer chip. Wherein, a converging and diverting module is provided on the converging and diverting layer chip, and the structure of the converging and diverting module is the same as that described in any one of claims 1 to 15; The converging and diverting unit on the converging and diverting layer chip is provided with a first outlet and a second outlet downstream, the first outlet is used to discharge the liquid flow containing the target particles, and the second outlet is used to discharge the side liquid flow not containing the target particles; The capture layer chip is provided with a sample inlet and a sample outlet. The sample inlet is communicated with the first outlet; the sample outlet is communicated with the second outlet.

17. The three-dimensional microfluidic chip according to claim 16, characterized in that: Multiple arrays are provided between the connecting passages of the sample inlet and the sample outlet of the capture layer chip. The arrays are composed of small blocks of arbitrary shapes. There is a gap d between adjacent small blocks, and the size of the gap d is defined as the distance between the two closest points on the surfaces of two adjacent small blocks; the gap d between the small blocks in each array is the same, and the gap d between the small blocks in different arrays decreases step by step along the direction of sample inflow.

18. The three-dimensional microfluidic chip according to claim 16, characterized in that: A stacked converging and diverting layer chip and a capture layer chip form a group, and the three-dimensional microfluidic chip includes multiple groups of stacked converging and diverting layer chips and capture layer chips; the inlets of the converging and diverting layer chips in different groups are connected, and at the same time, the second outlets of the converging and diverting layer chips in different groups and the sample outlets of the capture layer chips are connected.

19. A device for enriching and / or capturing target particles in a sample, characterized in that: It includes: The microfluidic chip according to any one of claims 1 to 15, and / or the three-dimensional microfluidic chip according to any one of claims 16 to 18.

20. The device for enriching and / or capturing target particles in a sample according to claim 19, characterized in that: The device further comprises a detection unit for detecting the liquid flow containing the target particles and / or the side liquid flow not containing the target particles.

21. Use of the microfluidic chip according to any one of claims 1 to 15, or the three-dimensional microfluidic chip according to any one of claims 16 to 18, or the device according to claim 19 or 20 in any of the following aspects: (i) Enriching and / or capturing target particles in the sample; (ii) removing target particles from the sample solution; (iii) Reduce the sample flow rate and / or flow velocity.

22. The use according to claim 21, characterized in that The sample is derived from whole blood, plasma, serum, perfusate, urine, tissue fluid, cerebrospinal fluid, cell culture fluid or cell mixture.

23. The use according to claim 22, characterized in that The sample is whole blood or perfusate.

24. The use according to claim 23, characterized in that The target particles are tumor cells.

25. The use according to claim 24, characterized in that The target particles are circulating tumor cells.

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