A microfluidic chip for sorting sub-micron scale cell adjustable inlet structure

Abstract

The application discloses a micro-fluidic chip for sorting sub-micron scale cells, which is composed of three components of an upper cover plate, a cell sorting micro-channel and a lower bottom plate by layer bonding. The upper cover plate is provided with an outside inlet, an inside inlet, an outside outlet and an inside outlet. The cell sorting micro-channel is composed of three regions of an inlet structure adjusting region, a spiral channel region and an outlet region. The lower bottom plate is provided with a convex wedge-shaped structure, and three kinds of inlet structures of an outside-narrow-inside-wide shape, an outside-inside-equivalent shape and an outside-wide-inside-narrow shape can be obtained by adjusting the position of the wedge-shaped structure. The sub-micron scale cells are sorted by the cooperation of the cell sorting micro-channel and the wedge-shaped structure. The spiral micro-channel is introduced, the cell transverse migration starting point is controlled by adjusting the spiral channel inlet structure and cooperating with suitable flow conditions, and the high-precision sorting of the sub-micron scale cells is finally completed. The cell sorting diversity and adaptability are improved by flexibly adjusting according to specific sorting requirements.

Description

Technical Field

[0001] The present 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 cells with sizes between nanometers and micrometers. Their tiny size and efficient material exchange capabilities facilitate the precise targeting and rapid release of drugs within organisms. As an essential component of living organisms, studying the physiological properties of submicron cells can help discover new therapeutic targets and deepen our understanding of intercellular information transmission and material exchange. Sorting submicron cells from background cells is a prerequisite for studying their physiological properties. However, due to their size, obtaining sufficient quantity and quality of these cells is challenging.

[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 characteristics 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 inertially 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 and Deanli F D When the two interact, 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 them. Therefore, there are few reports on microfluidic chips for sorting submicron-sized cells.

[0004] To promote the development of submicron cell sorting applications, this invention provides a microfluidic chip with an adjustable inlet structure for submicron cell sorting. By utilizing the flow characteristics of Dean vortexes and combining them with a suitable inlet structure, continuous, high-throughput, label-free sorting of submicron-sized cells is achieved. Summary of the Invention

[0005] Aiming at the problem that submicron-sized cells flow in a channel along with Dean vortexes, which makes them difficult to be sorted, the present invention proposes 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-sized 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 symmetrical shape 6, and an outer wide and inner narrow shape 7. The submicron-sized 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 areas of the cover plate 1 are 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 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, thereby achieving 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 interpenetrating with the outer inlet 101 and the inner inlet 102, 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 microscopy platform.

[0010] In a preferred embodiment, the cell sorting microchannel 2 is made of a translucent silicone plate with a thickness of 80 μm. This material provides excellent bonding with the upper cover plate 1 and the lower base plate 3, preventing damage to the synthesis chip 4 due to insufficient bonding strength. Furthermore, the silicone plate is harder than traditional polydimethylsiloxane (PDMS), ensuring that the synthesis chip 4 is not deformed by pressure even under high-throughput flow conditions.

[0011] In a preferred embodiment, the outer inlet 101, inner inlet 102, outer outlet 103, and inner outlet 104 of the upper cover plate 1 all have diameters of 700 μm. Submicron-sized cells are not differentiated by which inlet they enter through; this can be appropriately selected based on specific sorting requirements. Accordingly, the outflow of submicron-sized cells through outer outlet 103 or inner outlet 104 can also be determined based on the lateral migration behavior of the cells within 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 uses a wedge structure 301 to achieve mutual isolation between the outer inlet 101 and the inner inlet 102.

[0013] In a preferred embodiment, the spiral microchannel 202 has an Archimedean spiral shape, with an initial radius of curvature of 3 mm, a spiral pitch of 500 μm, a channel width of 240 μm, and a channel height of 80 μm. A 1 / 2 arc segment is provided at the front end to guide submicron-sized cells and sheath fluid into the spiral microchannel 202. The rear end is connected to the outlet region 203, allowing submicron-sized cells at different lateral positions to flow out through 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, that is, the ratio of the branch channel width to the main channel width, is 1:3 and 2:3, respectively, providing a feasible solution for accurately separating submicron-scale cells and other background cells.

[0015] In a preferred embodiment, the wedge-shaped structure 301 on the lower base plate 3 is 80 μm high 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 within the inlet structure adjustment region 201, it is possible to fabricate a composite chip 4 with three inlet structures: a narrow outer and wide inner shape 5, a symmetrical outer and inner shape 6, and a wide outer and narrow inner shape 7.

[0016] In a preferred embodiment, the inlet width of the synthetic chip with a narrow-outer-wide-inner-5-inlet structure is narrower near the outer channel wall than near the inner channel wall. Correspondingly, the inlet width of the synthetic chip with a wide-outer-narrow-inner-7-inlet structure is the opposite, with the inner and outer inlet widths of the outer and inner equal-sized structure (6) being half the width of the spiral channel, or 120 μm. These three synthetic chips with different inlet structures can handle the vast majority of submicron cell sorting in a wide range of applications.

[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] The present invention has the following advantages over existing cell sorting chips:

[0022] 1. High-precision sorting of submicron cells. The introduction of spiral microchannels makes it possible to regulate the lateral migration of cells. However, submicron cells flow within the channels with Dean vortices, making them difficult to separate uniformly. By adjusting the spiral channel entrance structure and combining it with appropriate flow conditions, the initial position of submicron cells entering the spiral channel can be controlled, thereby achieving the goal of controlling the starting point of cell lateral migration and ultimately achieving high-precision sorting of submicron cells.

[0023] 2. Wide application and strong operability. This microfluidic chip can be widely used not only for sorting submicron cells, but also for sorting nanoparticles and biomolecules, with a wide range of applications. In addition, different inlet structures and sorting modes can be flexibly adjusted according to specific sorting needs, improving the diversity and adaptability of cell sorting. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 Top views and local magnified views of the components of the microfluidic chip; (a) is the upper cover plate, (b) is the cell sorting microchannel, (c) is the lower base plate, (d) is a local magnified plan view of the spiral channel, (e) is a local three-dimensional magnified view of the inlet structure adjustment area, and (f) is a local magnified plan 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 outer surface and a wide inner surface;

[0028] Figure 1 Middle, upper cover plate 1, cell sorting microchannel 2, lower base plate 3 and synthesis chip 4; Figure 2Middle, upper cover: outer inlet 101, inner inlet 102, outer outlet 103 and inner outlet 104, cell sorting microchannel: inlet structure adjustment area 201, spiral channel 202 and outlet area 203, lower bottom plate: wedge-shaped structure 301; Figure 3 Among them, there are entrance structures of outer narrow and inner wide type 5, outer and inner equal type 6 and outer wide and inner narrow type 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 specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0030] Example: A microfluidic chip 4 with an adjustable inlet structure for sorting submicron-scale cells is constructed from 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 comprises an inlet structure adjustment region 201, a cell sorting microchannel 202, and an outlet region 203. The lower base plate 3 is provided with an upwardly convex wedge-shaped structure 301. Adjusting its position allows the chip 4 to produce three inlet structures: a narrow outer and wide inner shape 5, a shape with equal outer and inner shapes 6, and a wide outer and narrow inner shape 7.

[0031] Using a diluted red blood cell solution 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 Figure A, the red blood cell dilution solution 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 rate 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 main flow direction of the spiral channel, the red blood cells gradually migrate toward the center of the channel. Figure 4 Figure B shows the state when the red blood cells are at a transverse position of 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. Afterwards, 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 to the inner channel wall and then back to the outer channel wall. This demonstrates that the outer narrow, inner wide 5-shaped synthetic chip effectively defines the initial position of red blood cells upon entry into the spiral microchannel. This allows for the lateral position of red blood cells to be manipulated during subsequent flow, facilitating the differential separation of red blood cells from other background cells.

[0032] The above figures and texts illustrate the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip with an adjustable inlet structure for sorting submicron-scale cells, comprising an upper cover plate (1), a cell sorting microchannel (2), and a lower base plate (3) bonded 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); and is characterized in that: 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); the position of the wedge-shaped structure (301) is adjusted to obtain 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); 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 (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 upper cover (1) is in contact with the spiral channel area (202), thereby achieving a single-sided closure of the cell sorting microchannel (2); the communication between the inlet structure adjustment area (201) and the outlet area (203) in the cell sorting microchannel (2) is achieved through the spiral channel area (20 2) connected; the lower bottom plate (3) contacts the spiral channel area (202) and the outlet area (203), thereby realizing a 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), thereby dividing the inlet structure adjustment area (201) into two mutually isolated spaces, and respectively connected with the outer inlet (101) and the inner inlet (102), thereby ensuring that the two solutions flowing in from the outer inlet (101) and the inner inlet (102) do not mix with each other; The curve shape of the spiral channel region (202) is an Archimedean spiral, wherein the initial curvature radius 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 its front end to introduce submicron-scale cells and sheath fluid into the spiral channel region (202), and the rear end is connected to the outlet region (203), and submicron-scale cells at different lateral positions will flow out from different outlets.

2. The microfluidic chip with an adjustable inlet structure for sorting submicron-sized 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 translucent 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 utilizes a wedge-shaped structure (301) 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 outlet area (203) branches into two outlets, an outer outlet (103) and an inner outlet (104), and the ratio of the width of the occupied channels, 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.

7. 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 base 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 inner wide shape (5), an outer and inner equal shape (6), and an outer wide inner narrow shape (7), is manufactured.

8. 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, that is, 120 μm.

9. 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 cell sorting involves the following specific steps: S1: Fabricate the upper cover plate (1), cell sorting microchannel (2) and 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 based on 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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