Inertial micro-fluidic chip integrating cell focusing and sorting and preparation method thereof

Abstract

The invention discloses an inertial micro-fluidic chip integrating cell focusing and sorting and a preparation method thereof. The inertial micro-fluidic chip comprises a cover plate layer, a runner layer and a substrate layer, a chip inlet, a first chip outlet and a second chip outlet are formed in the cover plate layer; the flow channel layer is provided with a sample liquid flow channel inlet, a first flow channel outlet and a second flow channel outlet, the chip inlet is communicated with the sample liquid flow channel inlet, the first chip outlet is communicated with the first flow channel outlet, and the second chip outlet is communicated with the second flow channel outlet. The flow channel layer is provided with a spiral cell sorting flow channel and a cell focusing flow channel, the front end of the spiral cell sorting flow channel is communicated with a sample liquid flow channel inlet, the tail end of the spiral cell sorting flow channel is communicated with the cell focusing flow channel and a first flow channel outlet, and the tail end of the cell focusing flow channel is communicated with a second flow channel outlet. Through a microfluid vortex focusing technology and a spiral flow channel inertia sorting technology, accurate sorting and focusing of cells are realized, and the problems that the focusing state of the sorted cells is influenced and the cell counting detection precision is low are effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological particle manipulation, and in particular to an inertial microfluidic chip integrating cell focusing and sorting and a preparation method thereof. Background Art

[0002] Microfluidic cell sorting technology selectively manipulates and sorts cells or other biological particles within a specifically designed microstructure based on their physical properties, such as size, shape, density, or dielectric properties. Depending on the operating principle, manipulation of cells using external fields such as electric, magnetic, acoustic, or optical fields is generally referred to as microfluidic field dynamic sorting, while manipulation of cells solely using the fluid properties within a microchannel is referred to as microfluidic hydrodynamic sorting.

[0003] Microfluidic vortexes are generated by modifying periodic contraction-expansion structures on both sides of the direct current channel. The working principle is that when the fluid enters the expansion area from the contraction area, local vortices are induced by changes in flow velocity and streamline path. Then, under the coupling of local vortex-induced lift and inertial lift, cells of different sizes are migrated to a specific equilibrium position to achieve sorting.

[0004] Inertial microfluidics utilizes fluid dynamics at finite Reynolds numbers to precisely manipulate cells of varying sizes. The flow channel structure in inertial microfluidics is a key factor in determining chip functionality and performance. Reported flow channel structures include linear, sinusoidal, spiral, and other modified forms. Inertial migration refers to the combined effects of laminar shear-induced lift and wall-induced lift on cells in a fluid, resulting in relative displacement of cells relative to the wall and eventual stabilization at an equilibrium position. Secondary flow occurs in curved or perturbed straight channels, where the fluid at the center of the channel has higher momentum than the fluid near the channel walls. This outward flow drives the relatively stagnant fluid near the channel walls inward along the circumference, forming a pair of vortices in opposite directions across the channel cross section, known as Dean vortices. Cell extraction using inertial microfluidics utilizes these inertial lift and Dean vortex drag forces to adjust the position of cells within the channel.

[0005] In various body fluids such as blood, urine, and pleural effusion, there are some rare cells that are present in extremely low levels but are of great clinical significance. Their extremely low abundance makes counting and detecting these cells significantly difficult. Although the rare cell detection methods commonly used in clinical laboratories, such as histochemical staining, immunohistochemistry, and flow cytometry, are relatively mature technologies, they still face limitations such as insufficient sensitivity, complex operating procedures, and expensive equipment. Currently, many microfluidic chips that use passive control have emerged. For example, cells are sorted based on size characteristics, and then the sorted cells are directly counted and detected. However, after the sorted cells enter the new flow channel, they may affect the original focusing state of the cells, thereby affecting the detection accuracy. In addition, when counting and detecting cells, it is difficult for the cells to maintain focus on a single position, and problems such as cells passing side by side or multiple cells passing through the counting and detection area at the same time may occur, reducing the accuracy of cell counting detection. Summary of the Invention

[0006] Purpose of the invention: The present invention proposes an inertial microfluidic chip and preparation method for integrated cell focusing and sorting, which improves the accuracy in the field of cell sorting and detection through microfluidic vortex focusing technology and spiral flow channel inertial sorting technology.

[0007] Technical solution: The present invention proposes an inertial microfluidic chip with integrated cell focusing and sorting, comprising a cover layer, a flow channel layer and a base layer from top to bottom; the base layer supports the cover layer and the flow channel layer; the cover layer is provided with a plurality of inlets and outlets adapted to the flow channel layer, the cover layer is provided with a chip inlet, a first chip outlet and a second chip outlet, the flow channel layer is provided with a sample liquid flow channel inlet, a first flow channel outlet and a second flow channel outlet, the chip inlet is connected to the sample liquid flow channel inlet, the first chip outlet is connected to the first flow channel outlet, and the second chip outlet is connected to the second flow channel outlet; the flow channel layer is also provided with a spiral cell sorting flow channel and a cell focusing flow channel connected to the spiral cell sorting flow channel, the front end of the spiral cell sorting flow channel is connected to the sample liquid flow channel inlet, the end of the spiral cell sorting flow channel is connected to the cell focusing flow channel and the first flow channel outlet, and the end of the cell focusing flow channel is connected to the second flow channel outlet.

[0008] Preferably, the cover layer, the flow channel layer and the base layer are each provided with three positioning holes.

[0009] Preferably, the cover plate layer and the flow channel layer are aligned and assembled on the base layer through positioning holes.

[0010] Preferably, the curvature radius of the spiral cell sorting flow channel is 8-12 mm, and the width and height of the rectangular cross-section of the spiral cell sorting flow channel are 200 μm and 90 μm, respectively.

[0011] Preferably, the cell focusing channel is a contraction-expansion array embedded in a cuboid.

[0012] Preferably, the cell focusing flow channel has a length of 24-26 mm and a width of 220-420 μm.

[0013] Preferably, the cover layer and the base layer are made of colorless and transparent PET silicone film with a thickness of 100 microns.

[0014] Preferably, the flow channel layer is made of a translucent silicone plate with a thickness of 90 microns.

[0015] A method for preparing an inertial microfluidic chip integrating cell focusing and sorting comprises the following steps:

[0016] S1: Make the cover layer, flow channel layer and base layer respectively;

[0017] S2: Place the base layer in the tooling, and bond the flow channel layer to the base layer through the corresponding positioning holes. At this time, the flow channel layer and the base layer together form an open flow channel.

[0018] S3: Pass the cover layer through the corresponding positioning holes and the flow channel layer to complete the chip packaging.

[0019] Preferably, the cover layer, the flow channel layer and the base layer in S1 are prepared by laser cutting or soft lithography.

[0020] Beneficial effects: The present invention proposes an inertial microfluidic chip and preparation method for integrated cell focusing and sorting. Due to the inertial effect and Dean flow in the spiral flow channel, the fluid gathers rare cells and other cells at different equilibrium positions. The rare cells are then introduced into the cell focusing channel through the bifurcated flow channel, while the other cells are guided out. The cell focusing channel is a contraction-expansion array structure embedded in a rectangular parallelepiped, which can generate precise cross-sectional vortices. Under the coupling effect of local vortex-induced lift and inertial lift, rare cells are focused on one side of the flow channel. This avoids the possibility that rare cells entering the new flow channel will affect the original focusing state of the cells, and solves problems such as cells passing side by side or multiple cells passing through the counting and detection area at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the top view of the inertial microfluidic chip integrating cell focusing and sorting;

[0022] Figure 2 It is a structural diagram of the cover layer;

[0023] Figure 3 It is a structural diagram of the flow channel layer;

[0024] Figure 4 It is a schematic diagram of the structure of the basal layer;

[0025] Figure 5 is a schematic diagram of the structure of the cell focusing flow channel;

[0026] Figure 6 This is an exploded diagram of the inertial microfluidic chip for cell focusing and sorting; DETAILED DESCRIPTION

[0027] Example 1

[0028] like Figure 1-6 As shown, the present invention proposes an inertial microfluidic chip with integrated cell focusing and sorting, comprising a cover layer 1, a flow channel layer 2 and a base layer 3 arranged in sequence from top to bottom; the cover layer 1 is provided with a chip inlet 4, a first chip outlet 5, a second chip outlet 6 and three positioning holes; the flow channel layer 2 is provided with a sample liquid inlet 7, a first flow channel outlet 8, a second flow channel outlet 9 and three positioning holes; the base layer 3 is provided with three positioning holes, and the cover layer 1 and the flow channel layer 2 are aligned and assembled on the base layer 3 through the positioning holes; the base layer 3 supports the cover layer 1 and the flow channel layer 2.

[0029] The inertial microfluidic chip with integrated cell focusing and sorting is aligned and assembled from bottom to top through the positioning holes set on each layer, with the flow channel layer 2 between the base layer 3 and the cover layer 1; the chip inlet 4 of the inertial microfluidic chip with integrated cell focusing and sorting is aligned with the sample liquid inlet 7, the first chip outlet 5 is aligned with the first flow channel outlet 8, and the second chip outlet 6 is aligned with the second flow channel outlet 9.

[0030] The cover layer 1 and base layer 3 are made of a colorless, transparent PET silicone film, both 100 microns thick. The flow channel layer 2 is a translucent silicone sheet, 90 microns thick. These two materials provide excellent bonding and are relatively hard, protecting the flow channel structure from pressure deformation.

[0031] A cell focusing flow channel 10 and a spiral cell sorting flow channel 11 are provided on the flow channel layer 2; the front end of the spiral cell sorting flow channel 11 is connected to the sample liquid flow channel inlet 7, the end of the spiral cell sorting flow channel 11 is connected to the cell focusing flow channel 10 and the first flow channel outlet 8, and the end of the cell focusing flow channel 10 is connected to the second flow channel outlet 9.

[0032] The length and width of the cell focusing channel 10 are 24-26 mm and 220-420 μm. The cell focusing channel 10 is a contraction-expansion array structure embedded in a rectangular parallelepiped. It can focus particles into beams at a specific flow rate, thereby realizing particle recovery and facilitating counting. The rectangular cross-section width and height of the spiral cell sorting channel 11 are 200 μm and 90 μm, respectively. The spiral cell sorting channel 11 is a counterclockwise spiral with a radius of 8-12 mm from the inside to the outside. After the sheath fluid and sample fluid at a specific flow rate are introduced, particles of different sizes can be sorted. A fork is set at the end of the channel to realize the recovery of particles of different sizes.

[0033] Example 2

[0034] like Figure 1-6 As shown, the preparation method of an inertial microfluidic chip integrated with cell focusing and sorting proposed by the present invention comprises the following steps:

[0035] S1: Fabricate the cover layer 1, flow channel layer 2, and base layer 3 by laser cutting or soft lithography.

[0036] S2: Place the base layer 3 in the tooling, and bond the flow channel layer 2 to the base layer 3 through the corresponding positioning holes. At this time, the flow channel layer 2 and the base layer 3 together form an open flow channel;

[0037] S3: Place the cover layer 1 through the corresponding positioning holes and the flow channel layer 2 to complete the chip packaging.

Claims

1. An inertial microfluidic chip integrating cell focusing and sorting, characterized in that: The invention comprises a cover layer (1), a flow channel layer (2) and a base layer (3) from top to bottom; the base layer (3) supports the cover layer (1) and the flow channel layer (2); the cover layer (1) is provided with a plurality of inlets and outlets adapted to the flow channel layer (2); the cover layer (1) is provided with a chip inlet (4), a first chip outlet (5) and a second chip outlet (6); the flow channel layer (2) is provided with a sample liquid flow channel inlet (7), a first flow channel outlet (8) and a second flow channel outlet (9); the chip inlet (4) is connected to the sample liquid flow channel inlet (7); the first chip outlet (5) is provided with a second chip outlet (6); the sample liquid flow channel inlet (7) is connected to the first chip outlet (8) and the second chip outlet (9); (5) is connected to the first flow channel outlet (8), and the second chip outlet (6) is connected to the second flow channel outlet (9); the flow channel layer (2) is also provided with a spiral cell sorting flow channel (11) and a cell focusing flow channel (10) connected to the spiral cell sorting flow channel (11), the front end of the spiral cell sorting flow channel (11) is connected to the sample liquid flow channel inlet (7), the end of the spiral cell sorting flow channel (11) is connected to the cell focusing flow channel (10) and the first flow channel outlet (8), and the end of the cell focusing flow channel (10) is connected to the second flow channel outlet (9).

2. The inertial microfluidic chip with integrated cell focusing and sorting according to claim 1, characterized in that: The cover plate layer (1), the flow channel layer (2) and the base layer (3) are each provided with three positioning holes.

3. The inertial microfluidic chip with integrated cell focusing and sorting according to claim 2, characterized in that: The cover plate layer (1) and the flow channel layer (2) are aligned and assembled on the base layer (3) through positioning holes.

4. The inertial microfluidic chip with integrated cell focusing and sorting according to claim 1, characterized in that: The curvature radius of the spiral cell sorting flow channel (11) is 8-12 mm, and the width and height of the rectangular cross section of the spiral cell sorting flow channel (11) are 200 μm and 90 μm respectively.

5. The inertial microfluidic chip integrated with cell focusing and sorting according to claim 1, characterized in that: The cell focusing flow channel (10) is a contraction-expansion array embedded in a rectangular parallelepiped.

6. The inertial microfluidic chip integrated with cell focusing and sorting according to claim 1, characterized in that: The cell focusing channel (10) has a length of 24-26 mm and a width of 220-420 μm.

7. The inertial microfluidic chip integrated with cell focusing and sorting according to claim 1, characterized in that: The cover layer (1) and the base layer (3) are made of a colorless and transparent PET silicone film with a thickness of 100 micrometers.

8. The inertial microfluidic chip integrated with cell focusing and sorting according to claim 1, characterized in that: The flow channel layer (2) is made of a translucent silica gel plate with a thickness of 90 microns.

9. A method for preparing an inertial microfluidic chip integrated with cell focusing and sorting according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1: preparing the cover layer (1), the flow channel layer (2) and the base layer (3) respectively; S2: placing the base layer (3) in the tooling, and bonding the flow channel layer (2) to the base layer (3) through the corresponding positioning holes, so that the flow channel layer (2) and the base layer (3) together form an open flow channel; S3: The cover layer (1) is passed through the corresponding positioning holes and the flow channel layer (2) to complete the chip packaging.

10. The method for preparing an inertial microfluidic chip integrated with cell focusing and sorting according to claim 9, characterized in that: The cover layer (1), flow channel layer (2) and base layer (3) in S1 are prepared by laser cutting or soft lithography.

Images