A dielectric-enhanced inertial cell sorting device and method

Abstract

The application provides a cell inertia sorting device and method based on dielectric enhancement, and belongs to the technical field of cell filtering and sorting. The device comprises a spiral structure inertia sorting module and a dielectrophoresis enhancement sorting module. Specifically, the device comprises a PDMS cover sheet layer, an Ag-PDMS three-dimensional electrode layer, an ITO wire layer and a glass substrate layer. The spiral structure inertia sorting module can focus cells of different sizes at different balance positions. In the dielectrophoresis enhancement sorting module, a long strip-shaped three-dimensional electrode group and a semicircular three-dimensional electrode are designed to provide strong and stable dielectrophoresis force for cells. Since different cells are subjected to dielectrophoresis forces of different sizes and directions, different directional deviations are generated, so that the separation distance of the two kinds of cells is increased, and the inertia sorting effect is enhanced. The inertia technology and the dielectrophoresis technology are organically combined, the sorting precision is improved, and high-precision and high-throughput separation between different cells is realized.

Description

Technical Field

[0001] This invention relates to the field of cell filtration and sorting technology, and more particularly to a cell inertial sorting device and method based on dielectric enhancement. Background Technology

[0002] Microfluidic technology has received widespread attention since its emergence in the 1990s. Microfluidic chips integrate basic operations such as sample preparation, reaction, sorting, and detection into microscale channels, automating the analysis and processing. They offer advantages such as small sample volume, small size, low cost, high efficiency, portability, and ease of integration, and have been widely applied in various research fields including biology, medicine, chemistry, agriculture, food, and the environment. Due to the excellent scale compatibility between microchannels and cells, they are also widely used in life sciences for cell culture, cell sorting, cell detection, cell microenvironment simulation, and organ-on-a-chip applications.

[0003] Inertial flow sorting technology has become a research hotspot in recent years. Its main feature is that it can achieve focused flow of particles in microchannels by relying on inertia without applying other external forces. The device used in this method is simple, easy to fabricate and manufacture, requires no external mechanical or electronic components, is small in size, and easy to integrate. In a helical flow channel, the focusing equilibrium position of particles strongly depends on particle size, thus enabling high-throughput size-based particle sorting.

[0004] Dielectrophoresis is a technique that describes the movement of electrically neutral particles due to polarization in a non-uniform electric field. The dielectric force acting on a particle depends not only on its size but also on its dielectric properties. Therefore, dielectrophoresis can be used to sort particles of similar size but different dielectric properties. Because of its advantages such as non-contact operation, batch processing capability, ease of control, and low cost, dielectrophoresis is widely used in the sorting of macromolecules such as cells.

[0005] However, each sorting technology has its limitations, such as low sorting purity, low throughput, and complex manufacturing processes. For complex cell sorting applications, using only a single technology is insufficient for effective sorting. Combining multiple technologies can integrate their advantages and is an effective method for solving cell sorting problems under complex conditions. Summary of the Invention

[0006] To address the aforementioned technical problems of low sorting accuracy and difficulty in distinguishing cells of similar size when using only inertial sorting technology, this invention provides a cell inertial sorting device and method based on dielectric enhancement. This invention primarily combines inertial technology and dielectric electrophoresis technology to achieve high-throughput, high-precision cell sorting, overcoming the shortcomings and deficiencies of existing single technologies. By employing multiple technologies in combination, it provides a high-throughput, high-precision cell sorting device, which has significant scientific and practical value for improving the accuracy of inertial sorting and enabling cell sorting applications in complex situations.

[0007] The technical means employed in this invention are as follows:

[0008] A cell inertial sorting device based on dielectric enhancement, comprising:

[0009] The inertial sorting module has an inertial sorting channel, with one end inlet for injecting cell sample solution into the inertial sorting channel and the other end outlet for discharging the sorted cell solution.

[0010] The dielectrophoresis-enhanced sorting module is connected to an external power supply and has electrodes distributed on both sides of the inertial sorting channel to provide dielectrophoretic forces in different directions, causing cells to shift in different directions under the action of dielectrophoretic forces, thereby enhancing the effect of inertial sorting.

[0011] A glass substrate is used to fix the inertial sorting module and the dielectric-enhanced sorting module.

[0012] Furthermore, the inertial sorting module employs a PDMS cover layer, which is tightly bonded to the glass substrate layer, encapsulating the dielectrophoresis-enhanced sorting module within it;

[0013] The PDMS cover layer includes a spiral inertial sorting channel, a sample inlet, cell outlet I, cell outlet II, electrode groove I, and multiple electrode grooves II. The spiral inertial sorting channel is in a multi-turn spiral shape. The sample inlet is located at the beginning of the innermost spiral inertial sorting channel and at the center of the spiral inertial sorting channel. The outermost spiral inertial sorting channel has two branch channels at its end, which are connected to cell outlet I and cell outlet II, respectively. The electrode groove I and multiple electrode grooves II are located on the inner and outer sides of the outermost spiral inertial sorting channel.

[0014] Furthermore, the initial radius of the spiral inertial sorting channel is 2000 μm, the channel width is 100 μm, and the number of turns is 3; the radius of the sample inlet and the two cell outlets is 1000 μm, the channel height is 38 μm, and the angle between cell outlet I and cell outlet II is 120°; the size of the spiral inertial sorting channel is adjusted according to the size of the cells to be separated.

[0015] Furthermore, the electrode groove I is provided in a semi-circular shape, with a width of 500 μm, a length of 6000 μm, and a height of 38 μm; the size of the electrode groove I is adjusted according to the actual situation of the cells to be separated.

[0016] Furthermore, the electrode groove II is elongated and arranged in three groups at intervals, with each group containing four elongated electrode grooves II. The end of the electrode groove II near the spiral inertial sorting channel has a sharp corner. Each elongated electrode groove II has a width of 200 μm, a length of 1400 μm, a height of 100 μm for the sharp corner, a sharp corner angle of 45°, and a height of 38 μm. The distance between two adjacent elongated electrode grooves II is 200 μm. The size of the electrode groove II is adjusted according to the actual situation of the cells to be separated.

[0017] Furthermore, the dielectric electrophoresis-enhanced sorting module employs an Ag-PDMS three-dimensional electrode layer. This Ag-PDMS three-dimensional electrode layer is a three-dimensional electrode made of Ag-PDMS material, comprising a semi-circular Ag-PDMS three-dimensional electrode and multiple elongated three-dimensional electrodes, which are respectively embedded in electrode groove I and multiple electrode grooves II. The semi-circular Ag-PDMS three-dimensional electrode generates a negative dielectric electrophoretic force, while the elongated three-dimensional electrodes generate a positive dielectric electrophoretic force. The Ag-PDMS three-dimensional electrode has the same size and number as the electrode groove I, and the elongated three-dimensional electrodes have the same size and number as the electrode grooves II.

[0018] Furthermore, it also includes an ITO conductive layer, on which the Ag-PDMS 3D electrode layer is fabricated. The ITO conductive layer is deposited on a glass substrate and connected to an external power source.

[0019] Furthermore, the ITO conductive layer is fabricated on the glass substrate using a wet etching method; the ITO conductive layer includes an upper ITO conductive layer and a lower ITO conductive layer, which serve as conductive layers connecting the semi-circular Ag-PDMS 3D electrode and the elongated Ag-PDMS 3D electrode to the external power supply, respectively.

[0020] Furthermore, the PDMS cover layer is formed by using soft photolithography to create a silicon-based mold, which is then sequentially cleaned, pre-baked, spin-coated, intermediate-baked, exposed, post-baked, and developed, and finally cast onto the silicon-based mold using PDMS.

[0021] The Ag-PDMS 3D electrode layer is manufactured by using photolithography dry film method, which involves sequentially cleaning, pressing, exposing, and developing to process an electrode mold on a dry film, then filling it with Ag PDMS material, and finally baking, curing, and demolding.

[0022] The present invention also provides a dielectric-enhanced cell inertial sorting method, which is used in the above-mentioned dielectric-enhanced cell inertial sorting device and includes the following steps:

[0023] Step 1: Prepare a mixed solution of the two cell samples to be separated, and load the prepared sample solution onto the syringe pump;

[0024] Step 2: After plasma cleaning, fix the sorting device on the microscope stage and connect the electrodes and external power supply.

[0025] Step 3: Use a syringe pump to inject the sample solution into the sample inlet at a certain speed;

[0026] Step 4: After passing through the spiral structure inertial sorting channel, under the combined action of inertial lift and Dean force, the two types of cells of different sizes will focus at different equilibrium positions.

[0027] Step 5: When the two types of cells flow through the dielectrophoretic sorting area, the separation distance between the two types of cells will be further increased under the action of the dielectrophoretic force because the directions of the dielectrophoretic forces on the two types of cells are different, effectively enhancing the sorting effect;

[0028] Step 6: Under the combined action of inertial sorting and dielectrophoretic enhanced sorting in the spiral structure channel, cells subjected to positive dielectrophoretic force will flow out from cell outlet II, and cells subjected to negative dielectrophoretic force will flow out from cell outlet I, realizing high-throughput and high-precision inertial sorting of cells based on dielectro-enhanced technology.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] 1. The cell inertial sorting device and method based on dielectric enhancement provided by the present invention achieves high-throughput and high-precision cell sorting by combining inertial technology and dielectric electrophoresis technology.

[0031] 2. The cell inertial sorting device and method based on dielectric enhancement provided by the present invention, through a set of strip electrodes and a semi-circular electrode, causes two types of cells subjected to dielectric forces in different directions to shift in different directions, thereby enhancing the effect of inertial sorting and improving sorting accuracy.

[0032] 3. The cell inertial sorting device and method based on dielectric enhancement provided by the present invention have the advantages of simple operation and high degree of automation.

[0033] In summary, the technical solution of this invention can solve the problems of low sorting accuracy and difficulty in distinguishing cells of similar size caused by using inertial sorting technology alone. This invention overcomes the shortcomings of low sorting accuracy when using inertial sorting technology alone and the defects of low throughput when using dielectrophoresis technology alone. By organically combining the two technologies, high-throughput and high-precision sorting is achieved.

[0034] Based on the above reasons, this invention can be widely applied in fields such as cell sorting. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram of the overall structure of the cell inertial sorting device based on dielectric enhancement according to the present invention.

[0037] Figure 2 This is a schematic diagram of the structure of the PDMS cover layer of the present invention.

[0038] Figure 3 This is a schematic diagram of the structure of the Ag-PDMS three-dimensional electrode layer of the present invention.

[0039] Figure 4 This is a schematic diagram of the structure of the ITO conductive layer of the present invention.

[0040] In the figure: 1. PDMS cover sheet layer; 2. Ag-PDMS 3D electrode layer; 3. ITO lead wire layer; 4. Glass substrate layer; 1-1. Spiral structure inertial sorting channel; 1-2. Sample inlet; 1-3. Cell outlet I; 1-4. Cell outlet II; 1-5. Electrode groove I; 1-6. Electrode groove II; 2-1. Ag-PDMS 3D electrode; 2-2. Strip-shaped 3D electrode; 3-1. Upper ITO lead wire; 3-2. Lower ITO lead wire. Detailed Implementation

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0044] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following figures denote similar items; therefore, once an item is defined in one figure, it need not be further discussed in subsequent figures.

[0045] In the description of this invention, it should be understood that the orientation or positional relationship indicated by directional terms such as "front, back, up, down, left, right", "horizontal, vertical, horizontal" and "top, bottom" is generally based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this invention and simplifying the description. Unless otherwise stated, these directional terms do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the scope of protection of this invention. The directional terms "inner" and "outer" refer to the inner and outer contours relative to the outline of each component itself.

[0046] For ease of description, spatial relative terms such as "above," "over," "on the upper surface of," "above," etc., are used herein to describe the spatial positional relationship of a device or feature as shown in the figures to other devices or features. It should be understood that spatial relative terms are intended to encompass different orientations in use or operation besides the orientation of the device as described in the figures. For example, if the device in the figures is inverted, a device described as "above" or "above" other devices or structures would subsequently be positioned as "below" or "under" other devices or structures. Thus, the exemplary term "above" can include both "above" and "below." The device may also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatial relative descriptions used herein will be interpreted accordingly.

[0047] Furthermore, it should be noted that the use of terms such as "first" and "second" to define components is merely for the purpose of distinguishing the corresponding components. Unless otherwise stated, the above terms have no special meaning and therefore should not be construed as limiting the scope of protection of this invention.

[0048] The purpose of this invention is to overcome the shortcomings and deficiencies of existing single technologies by combining multiple technologies to provide a high-throughput, high-precision cell sorting device. This invention overcomes the problems of low sorting accuracy and difficulty in distinguishing cells of similar size that arise from using only inertial sorting technology. This will have significant scientific and practical value for improving the accuracy of inertial sorting and enabling cell sorting applications in complex situations.

[0049] As shown in the figure, the present invention provides a cell inertial sorting device based on dielectric enhancement, including a helical inertial sorting module and a dielectric-enhanced sorting module.

[0050] The spiral inertial sorting module has one sample inlet, two cell collection outlets, and a three-turn spiral inertial sorting channel. This module can focus cells of different sizes at different equilibrium positions. The dielectrophoresis-enhanced sorting module includes a semi-circular three-dimensional Ag-PDMS electrode and three sets of elongated three-dimensional Ag-PDMS electrodes. The two electrode groups are connected to the positive and negative terminals of an external power supply via ITO conductive layers. In the dielectrophoresis-enhanced sorting module, the designed elongated three-dimensional electrode groups and semi-circular three-dimensional electrodes provide strong and stable dielectrophoretic forces to the cells. Because different cells experience different magnitudes and directions of dielectrophoretic forces, they deflect in different directions, thereby increasing the separation distance between the two cell types and enhancing the inertial sorting effect.

[0051] Specifically, when sample cells enter the spiral inertial sorting channel at a certain speed, under the action of inertial lift and Dean's drag, cells of different sizes will focus to different equilibrium positions. However, when the size difference between cells is small, the equilibrium positions of the focused cells will be relatively close, making separation difficult. Dielectrophoresis technology can sort cells based on their size and dielectric properties, thus enabling the separation of cells of the same size but different dielectric properties. When two types of cells to be sorted flow through the dielectrophoresis-enhanced sorting module, due to the different magnitudes and directions of the dielectrophoretic forces acting on the two types of cells, they will be deflected in different directions under the action of the dielectrophoretic forces, thereby increasing the sorting distance and enhancing the sorting effect.

[0052] This invention organically combines inertial technology and dielectrophoresis technology, improving sorting accuracy and achieving high-precision, high-throughput separation between different cells.

[0053] The present invention also provides a sorting method for a dielectric-enhanced cell inertial sorting device, the method comprising the following steps:

[0054] (1) Prepare the cell sample solution to be sorted.

[0055] (2) Load the prepared sample solution onto the syringe pump.

[0056] (3) After plasma cleaning, the sorting device is fixed on the microscope stage.

[0057] (4) Connect the electrodes of the dielectric electrophoresis enhanced sorting module to the external power supply.

[0058] (5) Adjust the flow rate of the syringe pump so that the sample solution enters the DLD sorting module at a suitable flow rate.

[0059] (6) Turn on the signal generator, adjust the voltage and frequency, and achieve cell sorting under the combined action of inertial force and dielectrophoretic force.

[0060] The above sorting method specifically includes the following steps:

[0061] A. Prepare a mixed solution of cell A and cell B samples to be separated;

[0062] B. Connect the electrodes, external power supply, and signal generator;

[0063] C. Use a syringe pump to inject the sample solution into sample inlet 1-2 at a certain speed;

[0064] D. After passing through the spiral structure inertial sorting channel 1-1, under the combined action of inertial lift and Dean force, cells A and B of different sizes will focus at different equilibrium positions.

[0065] E. When cells A and B flow through the dielectrophoretic sorting area, the separation distance between the two cells will be further increased under the action of the dielectrophoretic force because the directions of the dielectrophoretic forces on the two types of cells are different, effectively enhancing the sorting effect;

[0066] F. Under the combined action of inertial sorting and dielectrophoretic-enhanced sorting in the spiral flow channel, cells subjected to positive dielectrophoretic force will flow out from cell outlet II1-4, and cells subjected to negative dielectrophoretic force will flow out from cell outlet I1-3, realizing high-throughput and high-precision inertial sorting of cells based on dielectro-enhanced sorting.

[0067] Example 1

[0068] like Figure 1 As shown, a cell inertial sorting device based on dielectric enhancement includes a PDMS cover layer 1, an Ag-PDMS 3D electrode layer 2, an ITO conductive layer 3, and a glass substrate layer 4. The Ag-PDMS 3D electrode layer 2 is fabricated on the ITO conductive layer 3 using Ag-PDMS material; the ITO conductive layer 3 is deposited on the glass substrate layer 4 and connected to an external power source; the PDMS cover layer 1 is tightly bonded to the glass substrate layer 4, encapsulating the Ag-PDMS 3D electrode layer 2 within it.

[0069] like Figure 2As shown, the PDMS cover layer 1 of this invention includes a spiral inertial sorting channel 1-1 (spiral inertial sorting channel), a sample inlet 1-2, two cell outlets, a semi-circular electrode groove I1-5, and three sets of elongated electrode grooves II1-6. The two cell outlets are cell outlet I1-3 and cell outlet II1-4. The spiral inertial sorting channel 1-1 is in a multi-turn spiral shape. The sample inlet 1-2 is located at the beginning of the innermost circle of the spiral inertial sorting channel 1-1 and at the center of the spiral inertial sorting channel 1-1. The outermost circle of the spiral inertial sorting channel 1-1 has two branch channels at its end, which are connected to cell outlet I1-3 and cell outlet II1-4, respectively. The electrode groove I1-5 and multiple electrode grooves II1-6 are located on the inner and outer sides of the outermost circle of the spiral inertial sorting channel 1-1. The spiral inertial sorting channel 1-1 has an initial radius of 2000 μm and a channel width of 100 μm; it has 3 turns. The sample inlet 1-2 and the two cell outlets each have a radius of 1000 μm and a channel height of 38 μm. The angle between cell outlet I 1-3 and cell outlet II 1-4 is 120°. These dimensions can be adjusted according to the size of the cells to be separated. The semi-circular electrode groove I has a width of 500 μm, a height of 38 μm, and a length of 6000 μm. These dimensions can also be adjusted according to the size of the cells to be separated. The elongated electrode groove II consists of 3 groups, equally spaced, with 4 elongated electrode grooves II in each group. Each elongated electrode groove II has a width of 200μm, a length of 1400μm, a height of 100μm at the pointed corner, a pointed corner angle of 45°, and a height of 38μm. The distance between two adjacent elongated electrode grooves II is 200μm, and there are a total of 3 sets. The dimensions of the elongated electrode grooves II can be adjusted according to the actual situation of the cells to be separated.

[0070] like Figure 3 As shown, the Ag-PDMS three-dimensional electrode layer 2 of the present invention is a three-dimensional electrode made of Ag-PDMS material, including a semi-circular Ag-PDMS three-dimensional electrode 2-1 and three sets of elongated three-dimensional electrodes 2-2. The dimensions are the same as the semi-circular electrode groove I1-5 and the elongated electrode groove II1-6 in the PDMS cover layer, respectively. It is fabricated on the ITO conductive layer 3 and embedded in the corresponding electrode grooves. The ITO conductive layer 3 supplies power to each electrode.

[0071] like Figure 4As shown, the ITO conductor layer 3 of this invention comprises two parts: an upper ITO conductor 3-1 and a lower ITO conductor 3-2. The upper ITO conductor 3-1 supplies power to the semi-circular Ag-PDMS 3D electrode 2-1, while the lower ITO conductor 3-2 supplies power to the three sets of elongated 3D electrodes 2-2. In other words, the upper ITO conductor 3-1 and the lower ITO conductor 3-2 serve as conductors connecting the semi-circular Ag-PDMS 3D electrode 2-1 and the elongated Ag-PDMS 3D electrode 2-2 to the external power source, respectively.

[0072] The PDMS cover layer 1 of this invention is fabricated using soft photolithography to create a silicon-based mold, which involves several steps: cleaning, pre-baking, spin coating, intermediate baking, exposure, post-baking, and development. Then, PDMS is cast onto the silicon-based mold.

[0073] like Figure 3 As shown, the Ag-PDMS 3D electrode layer 2 of the present invention is fabricated by photolithography dry film method. Through the operation methods of cleaning, pressing, exposure and development, an electrode mold is processed on the dry film, and then Ag PDMS material is filled in. Then, the Ag-PDMS 3D electrode is fabricated by baking, curing and demolding.

[0074] like Figure 4 As shown, the ITO conductive layer 3 of the present invention is fabricated by wet etching, and the ITO conductive layer is deposited on the glass substrate layer 4.

[0075] The PDMS capping layer 1 and the glass substrate layer 4 with the etched ITO electrode layer 3 described in this invention are tightly bonded together, tightly encapsulating the Ag-PDMS 3D electrode layer 2 therein.

[0076] An embodiment of the sorting method of the cell inertial sorting device based on dielectric enhancement described in this invention includes the following steps:

[0077] 1. Place 15 mL of the sample solution of cells to be sorted (taking Chlorella and Crescentella cells as examples) into a centrifuge, centrifuge at 8000 rpm for 10 min at room temperature, and pour out the supernatant.

[0078] 2. Remove the cells obtained from centrifugation and place them in a 1.5 mL test tube. Add PBS buffer with a conductivity of 3000 μS / cm and vortex until well mixed.

[0079] 3. Mix the two types of microalgae cells in a certain proportion, shake well, and obtain Chlorella cell and Crescentella cell sample solutions (hereinafter referred to as sample solutions).

[0080] 4. Draw the prepared sample solution into the syringe and load it onto the syringe pump, adjusting the speed to 400 μL / min.

[0081] 5. Place the fabricated dielectric-enhanced cell inertial sorting device chip (hereinafter referred to as the chip) into a plasma cleaner and clean it for 2 minutes to ensure the hydrophilicity of the channel.

[0082] 6. Place the cleaned chip under the microscope, adjust the focus, and connect the external power supply and signal generator.

[0083] 7. Slowly inject the prepared PBS buffer into the chip to remove air from the chip and prevent air bubbles from forming in the microchannels. This also prevents cells from adhering to the channel walls.

[0084] 8. Connect the sample solution to the sample inlet 1-2 of the chip via a conduit.

[0085] 9. Turn on the power supply and signal generator, and adjust the voltage and frequency of the signal generator.

[0086] 10. Turn on the syringe pump, and the sample solution flows into the chip at a stable flow rate. The sample cells focus under the action of inertial force. The focusing position of Chlorella is close to the inner wall of the channel, while the focusing position of Crescentella is in the middle of the channel, closer to the outer wall.

[0087] 11. At a certain frequency (e.g., 20MHz), *Chlorella* cells are repelled by negative mesophoretic forces and moved away from the elongated three-dimensional electrode, while *Crescentia* cells are attracted by positive mesophoretic forces and moved closer to the electrode. This further increases the separation distance between the two cell types, enhancing the sorting effect. Ultimately, *Chlorella* cells flow out from cell outlet I1-3, and *Crescentia* cells flow out from cell outlet II1-4, thus achieving high-throughput and high-precision sorting of the two cell types.

[0088] This invention overcomes the shortcomings of low sorting accuracy when using inertial sorting technology alone and low throughput when using dielectrophoresis technology alone. By organically combining the two technologies, high throughput and high precision sorting are achieved.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dielectrophoretic-based inertial cell sorting device, comprising: The application relates to a microfluidic chip for cell sorting, which comprises the following parts: an inertial sorting module with an inertial sorting flow channel, one end of which is used for injecting a cell sample solution into the inertial sorting flow channel, and the other end of which is used for discharging a sorted cell solution; a dielectrophoresis enhanced sorting module connected with an external power supply and provided with electrodes distributed on both sides of the inertial sorting flow channel, which are used for providing dielectrophoresis forces in different directions so that cells produce different directional deviations under the action of the dielectrophoresis forces, thereby enhancing the inertial sorting effect; a glass substrate layer (4) used for fixing the inertial sorting module and the dielectrophoresis enhanced sorting module; the inertial sorting module adopts a PDMS cover layer (1), the PDMS cover layer (1) is tightly bonded with the glass substrate layer (4), and the dielectrophoresis enhanced sorting module is wrapped in the PDMS cover layer (1); the PDMS cover layer (1) comprises a spiral structure inertial sorting flow channel (1-1), a sample inlet (1-2), a cell outlet I (1-3), a cell outlet II (1-4), an electrode groove I (1-5) and a plurality of electrode grooves II (1-6), the spiral structure inertial sorting flow channel (1-1) is in a multi-turn spiral shape, the sample inlet (1-2) is arranged at the starting end of the innermost turn of the spiral structure inertial sorting flow channel (1-1) and located at the center of the spiral structure inertial sorting flow channel (1-1); the tail end of the outermost turn of the spiral structure inertial sorting flow channel (1-1) has two branch flow channels, and the two branch flow channels are connected with the cell outlet I (1-3) and the cell outlet II (1-4) respectively; the electrode groove I (1-5) and the plurality of electrode grooves II (1-6) are arranged on the inner side and the outer side of the outermost turn of the spiral structure inertial sorting flow channel (1-1); the initial radius of the spiral structure inertial sorting flow channel (1-1) is 2000 mu m, the flow channel width is 100 mu m, the number of turns is 3, the flow channel height is 38 mu m, and the included angle between the cell outlet I (1-3) and the cell outlet II (1-4) is 120 DEG; the electrode groove I (1-5) is in a semicircular shape, the width is 500 mu m, and the length is 6000 mu m; the electrode groove II (1-6) is in a strip shape, is arranged in a spaced mode, has three groups, each group contains four strip-shaped electrode grooves II (1-6), the width of each strip-shaped electrode groove II (1-6) is 200 mu m, the length is 1400 mu m, the height of a sharp corner part is 100 mu m, the sharp corner angle is 45 DEG, the electrode height is 38 mu m, and the spacing between two adjacent strip-shaped electrode grooves II (1-6) is 200 mu m; the dielectrophoresis enhanced sorting module adopts an Ag-PDMS three-dimensional electrode layer (2) and an ITO wire layer (3), the Ag-PDMS three-dimensional electrode layer (2) comprises a semicircular Ag-PDMS three-dimensional electrode (2-1) and a plurality of strip-shaped three-dimensional electrodes (2-2), and the ITO wire layer (3) is connected with an external power supply; the electrode groove I (1-5) is provided with one semicircular electrode groove I (1-5) with a height of 38 mu m; and the size of the electrode groove I (1-5) is adjusted according to the actual situation of the cells to be separated. The Ag-PDMS three-dimensional electrode layer (2) is a three-dimensional electrode made of Ag-PDMS material. The semi-circular Ag-PDMS three-dimensional electrode (2-1) and multiple elongated three-dimensional electrodes (2-2) are respectively embedded in electrode groove I (1-5) and multiple electrode grooves II (1-6). The semi-circular Ag-PDMS three-dimensional electrode (2-1) generates negative dielectric force, and the elongated three-dimensional electrode (2-2) generates positive dielectric force. The Ag-PDMS three-dimensional electrode (2-1) has the same size and number as the electrode groove I (1-5), and the elongated three-dimensional electrode (2-2) has the same size and number as the electrode groove II (1-6).

2. The dielectrophoretic cell inertial sorting device of claim 1, wherein, The radius of the sample inlet (1-2) and the two cell outlets is 1000 μm, and the size of the spiral inertial sorting channel (1-1) is adjusted according to the size of the cells to be separated.

3. The dielectrophoretic cell inertial sorting device of claim 1, wherein, The electrode groove II (1-6) has a sharp corner at the end near the spiral inertial sorting channel (1-1). The size of each long strip electrode groove II (1-6) is adjusted according to the actual situation of the cells to be separated.

4. The dielectrophoretic inertial cell sorting device of claim 1, wherein, The Ag-PDMS 3D electrode layer (2) is fabricated on the ITO conductive layer (3), which is deposited on the glass substrate layer (4).

5. The dielectrophoretic cell inertial sorting device of claim 4, wherein, The ITO conductive layer (3) is fabricated on the glass substrate layer (4) using a wet etching method. The ITO conductive layer (3) includes an upper ITO conductive layer (3-1) and a lower ITO conductive layer (3-2), which serve as conductive lines connecting the semi-circular Ag-PDMS 3D electrode (2-1) and the elongated Ag-PDMS 3D electrode (2-2) to the external power supply, respectively.

6. The dielectrophoretic inertial cell sorting device of claim 1, wherein, The PDMS cover layer (1) is made by using soft photolithography to create a silicon-based mold, which is then cleaned, pre-baked, spin-coated, mid-baked, exposed, post-baked, and developed in sequence, and then cast onto the silicon-based mold using PDMS. The Ag-PDMS three-dimensional electrode layer (2) is made by using photolithography dry film method, sequentially cleaning, pressing, exposing and developing, processing the dry film to form an electrode mold, filling it with Ag-PDMS material, and then baking, curing and demolding.

7. A dielectric-enhanced inertial cell sorting method, comprising: This sorting method is used in the dielectric-enhanced cell inertial sorting device according to any one of claims 1-6, and includes the following steps: Step 1: Prepare a mixed solution of the two cell samples to be separated, and load the prepared sample solution onto the syringe pump; Step 2: After plasma cleaning, fix the sorting device on the microscope stage and connect the electrodes and external power supply. Step 3: Use a syringe pump to inject the sample solution into the sample inlet (1-2) at a certain speed; Step 4: After passing through the spiral structure inertial sorting channel (1-1), under the combined action of inertial lift and Dean force, the two types of cells of different sizes will focus at different equilibrium positions. Step 5: When the two types of cells flow through the dielectrophoretic sorting area, the separation distance between the two types of cells will be further increased under the action of the dielectrophoretic force because the directions of the dielectrophoretic forces on the two types of cells are different, effectively enhancing the sorting effect; Step 6: Under the combined action of inertial sorting and dielectrophoretic enhanced sorting in the spiral structure channel, cells subjected to positive dielectrophoretic force will flow out from cell outlet II (1-4), and cells subjected to negative dielectrophoretic force will flow out from cell outlet I (1-3), thus realizing high-throughput and high-precision inertial sorting of cells based on dielectro-enhanced sorting.

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