Micro-fluidic chip with adjustable inlet structure for sorting submicron-scale cells

Abstract

The invention discloses a micro-fluidic chip with an adjustable inlet structure for sorting submicron-scale cells. The micro-fluidic chip is synthesized by bonding an upper cover plate, a cell sorting micro-channel and a lower bottom plate layer by layer. The upper cover plate is provided with an outer side inlet, an inner side inlet, an outer side outlet and an inner side outlet. The cell sorting microchannel is composed of an inlet structure adjusting area, a spiral channel area and an outlet area. The lower bottom plate is provided with a wedge-shaped structure protruding upwards, and three inlet structures which are narrow outside and wide inside, equal outside and inside and wide outside and narrow inside can be manufactured by adjusting the position of the wedge-shaped structure. And the submicron-scale cells are sorted by matching the cell sorting microchannels with the wedge-shaped structures. The spiral micro-channel is introduced, the purpose of controlling the transverse migration starting point of the cells is achieved by adjusting the inlet structure of the spiral channel to be matched with a proper flowing working condition, and finally high-precision sorting of the submicron-scale cells is completed. And flexible adjustment can be performed according to specific sorting requirements, so that the diversity and adaptability of cell sorting are improved.

Description

Technical Field

[0001] The invention relates to the field of cell sorting microfluidic chips, and in particular to a microfluidic chip with an adjustable inlet structure for sorting submicron-scale cells. Background Art

[0002] Submicron cells are a type of cells with a particle size between nanometers and microns. Their tiny size and efficient material exchange capabilities facilitate the precise positioning and rapid release of drugs in vivo. As an important part of the organism, studying the physiological characteristics of submicron cells helps to discover new therapeutic targets and deepen the understanding of information transfer and material exchange between cells. Sorting submicron cells from background cells is a prerequisite for studying their physiological characteristics. However, due to their size, it is challenging to obtain sufficient quantity and quality of such cells.

[0003] Inertial microfluidics technology uses the inertial effect of cells in microchannels to achieve precise manipulation of their lateral migration behavior, thereby efficiently sorting cells of different particle sizes. Compared with other cell sorting technologies, inertial microfluidics technology does not require complex external force intervention, and only relies on the fluid properties in the microchannel to achieve accurate and efficient cell sorting. The soundness and practicality of its technological development are particularly prominent in the field of cell sorting. In 2023, Huang Yuxiang and others disclosed a circulating tumor cell sorting chip based on inertial microfluidics, which realized the sorting of tumor cells. Ni Zhonghua and others disclosed a tumor cell sorting and detection instrument and sorting method, which can realize the integration of tumor cell sorting and detection. However, most of the currently disclosed cell inertial sorting methods are aimed at inertial focusable cells, and the blocking ratio λ of such cells is ≥0.07, that is, the ratio of the characteristic size to the hydraulic diameter of the channel is ≥0.07. Such cells are subject to the net inertial lift F L Dean F D When the two are in dynamic equilibrium, the cells are inertially focused near the inner channel wall. However, submicron-scale cells (λ<0.07) are affected by F D The cells are dominant and flow in the channel with the Dean vortex, and cannot be focused into a beam relatively stably, which greatly increases the difficulty of sorting. Therefore, there are few reports on microfluidic chips for sorting submicron-sized cells.

[0004] In order to promote the development of submicron cell sorting applications, the present invention provides a microfluidic chip with an adjustable inlet structure for submicron cell sorting. By utilizing the Dean vortex flow characteristics and combining with a suitable inlet structure, continuous, high-throughput, and label-free sorting of submicron-sized cells can be achieved. Summary of the invention

[0005] Aiming at the problem that submicron-sized cells flow in a channel along with Dean vortex, which makes it difficult to sort them, the present invention provides a microfluidic chip with an adjustable inlet structure for submicron-sized cell sorting.

[0006] The present invention solves the above problems through the following technical solutions:

[0007] A microfluidic chip with an adjustable inlet structure for sorting submicron-scale cells is synthesized by bonding three components layer by layer: an upper cover plate 1, a cell sorting microchannel 2 and a lower base plate 3. The upper cover plate 1 is provided with an outer inlet 101, an inner inlet 102, an outer outlet 103 and an inner outlet 104. The cell sorting microchannel 2 is composed of three regions: an inlet structure adjustment region 201, a spiral channel region 202 and an outlet region 203. An upwardly convex wedge-shaped structure 301 is provided on the lower base plate 3. By adjusting the position of the wedge-shaped structure 301, three inlet structures can be obtained: an outer narrow and inner wide shape 5, an outer and inner equilateral shape 6 and an outer wide and inner narrow shape 7. The submicron-scale cells are sorted by the cell sorting microchannel 2 and the wedge-shaped structure 301.

[0008] The outer inlet 101 and the inner inlet 102 in the upper cover plate 1 are interconnected with the inlet structure adjustment area 201, the outer outlet 103 and the inner outlet 104 are interconnected with the outlet area 203, and the remaining unopened area of ​​the cover plate 1 is in contact with the spiral sorting channel 202, so as to achieve a single-sided closure of the cell sorting microchannel 2. The connection between the inlet structure adjustment area 201 and the outlet area 203 in the cell sorting microchannel 2 is connected through the spiral channel area 202. The lower base plate 3 is in contact with the spiral channel area 202 and the outlet area 203, so as to achieve a single-sided closure of the cell sorting microchannel 2. The wedge-shaped structure 301 passes through the inlet structure adjustment area 201, and then contacts the upper cover plate 1, dividing the inlet structure adjustment area 201 into two isolated spaces, and respectively interpenetrates with the outer inlet 101 and the inner inlet 102, so as to ensure that the two solutions flowing in from the outer inlet 101 and the inner inlet 102 do not mix with each other.

[0009] In a preferred embodiment, the upper cover plate 1 and the lower base plate 3 are made of a colorless and transparent PET silicone film with a thickness of 5 mm, which can facilitate light to penetrate the cell sorting microchannel 2, ensuring that the submicron scale cell migration movement behavior therein can be observed on a high-speed microscopic photography platform.

[0010] In a preferred embodiment, the cell sorting microchannel 2 is made of a semi-transparent silicone plate material with a thickness of 80 μm, which has an excellent bonding effect with the upper cover plate 1 and the lower base plate 3, avoiding the chip damage problem caused by insufficient bonding strength of the synthesis chip 4. In addition, the hardness of the silicone plate material is higher than that of the traditional polydimethylsiloxane PDMS material, which can ensure that the synthesis chip 4 is not deformed under pressure under high-throughput flow conditions.

[0011] In a preferred embodiment, the diameters of the outer inlet 101, the inner inlet 102, the outer outlet 103 and the inner outlet 104 in the upper cover plate 1 are all 700 μm. It is not distinguished which inlet the submicron-sized cells flow in from, and a reasonable selection can be made according to specific sorting requirements. Accordingly, the outflow of the submicron-sized cells from the outer outlet 103 or the inner outlet 104 can also be determined according to the lateral migration behavior of the cells in the spiral microchannel.

[0012] In a preferred embodiment, the shape of the inlet structure adjustment area 201 is a combination of a rectangle and an isosceles right triangle; the inlet structure adjustment area 201 is connected to the outer inlet 101 and the inner inlet 102 at the top, and is connected to the lower bottom plate 3 at the bottom, and a wedge-shaped structure 301 is used to isolate the outer inlet 101 and the inner inlet 102 from each other.

[0013] In a preferred embodiment, the spiral microchannel 202 has an Archimedean spiral shape, wherein the initial radius of curvature is 3 mm, the spiral pitch is 500 μm, the channel width is 240 μm, and the channel height is 80 μm. A 1 / 2 arc is provided at the front end thereof, which serves to introduce submicron-scale cells and sheath fluid into the spiral microchannel 202, and the rear end is connected to the outlet area 203, and submicron-scale cells at different lateral positions will flow out from different outlets.

[0014] In a preferred embodiment, the outlet region 203 branches into two outlets, an outer outlet 103 and an inner outlet 104, and the ratio of the occupied channel widths, i.e., the ratio of the branch channel width to the main channel width, is 1:3 and 2:3 respectively, which provides a feasible solution for accurately separating submicron-scale cells and other background cells.

[0015] In a preferred embodiment, the height of the wedge-shaped structure 301 on the lower bottom plate 3 is 80 μm, and after penetrating the inlet structure adjustment area 201, it contacts the upper cover plate 1. By adjusting the position of the wedge-shaped structure 301 in the inlet structure adjustment area 201, the synthesis chip 4 of three inlet structures, namely, the outer narrow inner wide shape 5, the outer inner equal shape 6, and the outer wide inner narrow shape 7, can be manufactured.

[0016] In the preferred embodiment, the inlet width of the synthetic chip with the outer narrow and inner wide shape 5 is narrower near the outer channel wall than the inlet width near the inner channel wall. Correspondingly, the inlet width of the synthetic chip with the outer wide and inner narrow shape 7 is the opposite, and the inner and outer inlet widths of the outer and inner equal shape 6 are 1 / 2 of the spiral channel width, that is, 120μm. The synthetic chips with three different inlet structures can solve the sorting of most submicron-scale cells in a large range.

[0017] The manufacturing of the above-mentioned microfluidic chip for submicron scale cell sorting includes the following specific steps:

[0018] S1: The upper cover plate 1, the cell sorting microchannel 2 and the lower base plate 3 are manufactured by laser cutting or soft lithography.

[0019] S2: Bond the cell sorting microchannel 2 to the lower base plate 3. This step requires precise alignment of the bonding position based on the final synthesized chip type.

[0020] S3: Bonding the cell sorting microchannel bonded to the lower base plate in step S2 to the upper cover plate to form a three-in-one synthetic chip.

[0021] Compared with the existing cell sorting chip, the present invention has the following advantages:

[0022] 1. High-precision sorting of submicron cells. The introduction of spiral microchannels makes it possible to regulate the lateral migration of cells, but submicron cells flow in the channel with Dean vortex and are not easy to be separated uniformly. By adjusting the entrance structure of the spiral channel and matching appropriate flow conditions, the initial position of submicron cells entering the spiral channel can be regulated, thereby achieving the purpose of controlling the starting point of cell lateral migration and finally completing the high-precision sorting of submicron cells.

[0023] 2. Wide application and strong operability. The microfluidic chip can be widely used not only in the sorting of submicron cells, but also in the sorting of nanoparticles and biomolecules, and has a broad application space. In addition, relying on different inlet structures and sorting modes, it can be flexibly adjusted according to specific sorting needs, which improves the diversity and adaptability of cell sorting. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The microfluidic chip components and their synthesis diagrams;

[0025] Figure 2 The top view and local magnified view of each component of the microfluidic chip; (a) is the upper cover plate, (b) is the cell sorting microchannel, (c) is the lower base plate, (d) is the local plane magnified view of the spiral channel, (e) is the local three-dimensional magnified view of the inlet structure adjustment area, and (f) is the local plane magnified view of the outlet area.

[0026] Figure 3 This is an enlarged view of the three inlet structures of the spiral microchannel;

[0027] Figure 4 The lateral migration process of red blood cells in a synthetic chip with a narrow outside and wide inside;

[0028] Figure 1 In the middle, an upper cover plate 1, a cell sorting microchannel 2, a lower bottom plate 3 and a synthesis chip 4; Figure 2In the middle, the upper cover plate includes an outer inlet 101, an inner inlet 102, an outer outlet 103 and an inner outlet 104, the cell sorting microchannel includes an inlet structure adjustment area 201, a spiral channel 202 and an outlet area 203, and the lower bottom plate includes a wedge-shaped structure 301; Figure 3 Among them, there are entrance structures of outer narrow and inner wide shape 5, outer and inner equal shape 6 and outer wide and inner narrow shape 7. DETAILED DESCRIPTION

[0029] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0030] Embodiment: A microfluidic chip 4 with an adjustable inlet structure for sorting submicron-scale cells is synthesized by three components: an upper cover plate 1, a cell sorting microchannel 2, and a lower base plate 3. The upper cover plate 1 is provided with an outer inlet 101, an inner inlet 102, an outer outlet 103, and an inner outlet 104. The cell sorting microchannel is composed of three parts: an inlet structure adjustment area 201, a cell sorting microchannel 202, and an outlet area 203. An upwardly convex wedge-shaped structure 301 is provided on the lower base plate 3, and its position can be adjusted to obtain a synthetic chip 4 with three inlet structures: an outer narrow and inner wide shape 5, an outer and inner equal shape 6, and an outer wide and inner narrow shape 7.

[0031] Using red blood cell dilution with a particle size of 7 μm as a sample experiment, the lateral migration behavior of red blood cells in the 5-shaped synthetic chip with a narrow outer surface and a wide inner surface was observed to verify the feasibility of the synthetic chip in submicron scale cell sorting applications. Figure 4 As shown in A, the red blood cell diluent enters the spiral channel from the outer inlet 101, and the phosphate buffered saline solution PBS flows in from the inner inlet 102. The flow ratio of the two is 40μL / min:140μL / min, so the red blood cells are well confined near the outer channel wall. As the red blood cells flow along the mainstream direction of the spiral channel, the red blood cells gradually migrate to the center of the channel. Figure 4 B shows the state when the lateral position of the red blood cells is about 1 / 2 of the channel width. In the subsequent flow process, the red blood cells migrate laterally toward the inner channel wall. Figure 4 C shows the state of the cell near the inner channel. After that, under the action of Dean vortex, the red blood cell migrates laterally from the inner channel wall to the outer channel wall. Until the red blood cell returns to the outer channel wall, as shown in Figure 4 D, completing a lateral migration from the outer channel wall → the inner channel wall → the outer channel wall. It can be seen that the outer narrow and inner wide shape 5 synthetic chip well defines the initial position of the red blood cells when they enter the spiral microchannel, so that the lateral position of the red blood cells in the later flow process can be artificially controlled, which will help to differentiate the red blood cells from other background cells.

[0032] The above figures and texts describe the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A microfluidic chip with an adjustable inlet structure for sorting submicron-scale cells, which is synthesized by bonding three components, namely, an upper cover plate (1), a cell sorting microchannel (2) and a lower base plate (3), layer by layer; the upper cover plate (1) is provided with an outer inlet (101), an inner inlet (102), an outer outlet (103) and an inner outlet (104); characterized in that: The cell sorting microchannel (2) is composed of three regions, namely, an inlet structure adjustment region (201), a spiral channel region (202), and an outlet region (203); an upwardly convex wedge-shaped structure (301) is provided on the lower bottom plate (3); the position of the wedge-shaped structure (301) is adjusted to obtain three inlet structures, namely, an outer narrow and inner wide shape (5), an outer and inner equal shape (6), and an outer wide and inner narrow shape (7); the submicron-scale cells are sorted by the cell sorting microchannel (2) and the wedge-shaped structure (301); The outer inlet (101) and the inner inlet (102) in the upper cover plate (1) are interconnected with the inlet structure adjustment region (201), the outer outlet (103) and the inner outlet (104) are interconnected with the outlet region (203), and the remaining unopened region of the upper cover plate (1) is in contact with the spiral sorting channel (202), thereby achieving a single-sided closure of the cell sorting microchannel (2); the communication between the inlet structure adjustment region (201) and the outlet region (203) in the cell sorting microchannel (2) is achieved through the spiral channel region (20 2) connected; the lower bottom plate (3) contacts the spiral channel area (202) and the outlet area (203) to achieve single-sided closure of the cell sorting microchannel (2); the wedge-shaped structure (301) penetrates the inlet structure adjustment area (201) and then contacts the upper cover plate (1), dividing the inlet structure adjustment area (201) into two mutually isolated spaces, and respectively penetrates the outer inlet (101) and the inner inlet (102), ensuring that the two solutions flowing in from the outer inlet (101) and the inner inlet (102) do not mix with each other.

2. A microfluidic chip with an adjustable inlet structure for sorting submicron-scale cells according to claim 1, characterized in that: The upper cover plate (1) and the lower base plate (3) are made of a colorless and transparent PET silicone film with a thickness of 5 mm.

3. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The cell sorting microchannel (2) is made of a semi-transparent silica gel plate material with a thickness of 80 μm.

4. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The diameters of the outer inlet (101), the inner inlet (102), the outer outlet (103) and the inner outlet (104) in the upper cover plate (1) are all 700 μm.

5. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The shape of the inlet structure adjustment area (201) is a combination of a rectangle and an isosceles right triangle; the inlet structure adjustment area 201 is connected to the outer inlet (101) and the inner inlet (102) at the top and to the lower bottom plate (3) at the bottom, and a wedge-shaped structure (301) is used to achieve mutual isolation between the outer inlet (101) and the inner inlet (102).

6. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The spiral microchannel (202) has a curve shape of an Archimedean spiral, wherein the initial radius of curvature is 3 mm, the spiral pitch is 500 μm, the channel width is 240 μm, and the channel height is 80 μm; a 1 / 2 arc is provided at the front end to introduce submicron-scale cells and sheath fluid into the spiral microchannel (202), and the rear end is connected to the outlet area (203), and submicron-scale cells at different lateral positions will flow out from different outlets.

7. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The outlet area (203) branches into two outlets, an outer outlet (103) and an inner outlet (104), and the ratio of the occupied channel widths, that is, the ratio of the width of the branch channel to the width of the main channel, is 1:3 and 2:3 respectively.

8. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The wedge-shaped structure (301) on the lower bottom plate (3) has a height of 80 μm and contacts the upper cover plate (1) after penetrating the inlet structure adjustment region (201); by adjusting the position of the wedge-shaped structure (301) in the inlet structure adjustment region (201), the synthesis chip (4) of three inlet structures, namely, an outer narrow and inner wide shape (5), an outer and inner equal shape (6), and an outer wide and inner narrow shape (7), is manufactured.

9. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: The inlet structure synthesis chip of the outer narrow and inner wide shape (5) has an inlet width close to the outer channel wall that is narrower than the inlet width close to the inner channel wall; the inlet structure synthesis chip of the outer wide and inner narrow shape (7) is the opposite, and the inner and outer inlet widths of the outer and inner equal shape (6) are 1 / 2 of the spiral channel width, i.e. 120 μm.

10. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized cells according to claim 1, characterized in that: Fabrication of a microfluidic chip for submicron-scale cell sorting includes the following specific steps: S1: manufacturing an upper cover plate (1), a cell sorting microchannel (2) and a lower base plate (3) by laser cutting or soft lithography; S2: Bonding the cell sorting microchannel (2) to the lower base plate (3). This step requires precise alignment of the bonding position according to the final synthesized chip type; S3: Bonding the cell sorting microchannel bonded to the lower base plate in step S2 to the upper cover plate to form a three-in-one synthetic chip.

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