Optical tweezer test dielectrophoresis force chip and manufacturing method and test method thereof

Abstract

The application provides a light tweezer test dielectrophoresis force chip and a manufacturing method and a test method thereof. The light tweezer test dielectrophoresis force chip comprises a bottom substrate, an electrode layer on the bottom substrate, a micropore array layer on the electrode layer and an arrayed test positioning layer. The electrode layer comprises a plurality of pairs of oppositely arranged first interdigital electrodes and second interdigital electrodes, and each pair of first interdigital electrodes and second interdigital electrodes has a gap. The micropore array layer comprises a plurality of arrayed micropores. The micropores are located between the first interdigital electrodes and the second interdigital electrodes, and each row of first interdigital electrodes and second interdigital electrodes has a plurality of micropores. Each row of micropores is located between two test positioning layers in the row. The application realizes accurate, efficient and comprehensive analysis of the DEP behavior of a single particle / cell or multiple particles / cells simultaneously by using the light tweezer to assist in analyzing the DEP of the particles / cells in the chip.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of testing dielectrophoresis force by optical tweezers, and particularly relates to a chip for testing dielectrophoresis force by optical tweezers, a manufacturing method thereof and a testing method. BACKGROUND

[0002] Lab on a Chip is based on micro-electro-mechanical technology, and can design and integrate micro-pump, micro-valve, micro-channel, micro-cavity and micro-electrode and other functional units on a small chip platform to realize precise and stable particle / cell manipulation. At present, by integrating manipulation methods based on different principles such as fluid mechanics, optics, electricity, magnetism and acoustics into a microfluidic chip system, a series of particle / cell manipulation platforms suitable for different application scenarios are obtained. The method of electric field manipulation is mainly based on dielectrophoresis (DEP). DEP describes the directional migration movement of dielectric particles due to polarization in a non-uniform electric field. When the polarization performance of the particles is better than that of the buffer medium, the particles will be subjected to positive dielectrophoresis force (p-DEP) and move towards the high electric field, and when the polarization performance of the particles is not as good as that of the buffer medium, the particles will be subjected to negative dielectrophoresis force (n-DEP) and move towards the low electric field. The microfluidic chip based on DEP can realize high-throughput, non-invasive and low-cost particle manipulation, and DEP manipulation of cells has been successfully applied in researches such as separation of dead cells and live cells, separation of circulating tumor cells in whole blood, separation between different cell subpopulations, single cell separation and manipulation, etc. In the research of DEP-based particle / cell manipulation microfluidic chip, analyzing the DEP behavior of particles / cells in the chip is the premise and basis of chip design.

[0003] Analyzing the DEP behavior of particles / cells by formula calculation and analysis is the most commonly used method, but it has the problems of complex process and insufficient accuracy. First, there are many parameters involved in the formula, and the values of the parameters in the formula need to be measured again when the experimental conditions are changed, so the calculation process is obviously complex. The results obtained by such a complex calculation are not accurate enough. Taking cells as an example, when the dielectric constant of cells is measured by techniques such as resonant cavity perturbation method, the measured is often the "average value" of the dielectric constant of a group of cells, and the same is true for the conductivity of cells. Therefore, the Re[K(ω)] and the size of DEP force calculated by such a method are not accurate enough. Therefore, it is very important to simply and accurately analyze the DEP behavior of cells. SUMMARY

[0004] The application aims to provide an optical tweezer test dielectrophoresis force chip, a manufacturing method thereof, and a testing method, so as to realize accurate, efficient, and comprehensive analysis of the DEP behavior of a single microparticle / cell or multiple microparticles / cells.

[0005] The application provides an optical tweezer test dielectrophoresis force chip, which comprises a bottom substrate, an electrode layer on the bottom substrate, a micropore array layer between the first and second interdigital electrodes, and an arrayed test positioning layer; the electrode layer comprises a plurality of pairs of oppositely arranged first and second interdigital electrodes, and each pair of first and second interdigital electrodes has a gap therebetween; the micropore array layer comprises an arrayed plurality of micropores; the micropores are located between the first and second interdigital electrodes, and each row of micropores is located between the two adjacent test positioning layers in the row.

[0006] Preferably, the micropores have a pore diameter of 10-20 microns; the center distance between the micropores in two adjacent rows is 50-200 microns; the height of the first interdigital electrode and the height of the second interdigital electrode are both 100-300 nanometers; the spacing between each pair of first and second interdigital electrodes is 4-8 microns; and the width of the first interdigital electrode and the width of the second interdigital electrode are both 15-25 microns.

[0007] Preferably, the lower surface or the upper surface of the test positioning layer is on the same straight line as the center line of the corresponding row of micropores.

[0008] The application further provides a manufacturing method of the optical tweezer test dielectrophoresis force chip, which comprises the following steps:

[0009] S1: cleaning the bottom substrate;

[0010] S2: forming an electrode protection layer on the bottom substrate;

[0011] S3: forming the first and second interdigital electrodes of the electrode layer;

[0012] S4: forming the micropore array layer between the first and second interdigital electrodes;

[0013] S5: forming the arrayed test positioning layer, and each row of micropores is located between the two test positioning layers in the row.

[0014] Preferably, the resistance of a pair of first and second interdigital electrodes needs to be tested after step S4.

[0015] The application further provides a testing method of the optical tweezer test dielectrophoresis force chip, which is fixed in a device, and comprises the following steps:

[0016] S1: a solution with microspheres is put into the device;

[0017] S2: the optical tweezer captures at least one microsphere so that the microsphere corresponds to a microwell, and the center of the microsphere and the lower surface or upper surface of the test positioning layer corresponding to the microwell are in the same focal plane;

[0018] S3: an electric signal is applied to the optical tweezer test dielectrophoresis force chip to generate dielectrophoresis force, and the microsphere moves laterally in the optical tweezer under the action of the dielectrophoresis force;

[0019] S4: the direction of the dielectrophoresis force is observed, and the size of the dielectrophoresis force is obtained by testing and calculating the lateral optical tweezer force.

[0020] Preferably, for step S2, whether the center of the microsphere and the lower surface or upper surface of the test positioning layer corresponding to the microwell are in the same focal plane is observed under a microscope.

[0021] Preferably, the specific method of step S2 is that after the microsphere is put into the device, the optical tweezer captures the microsphere and moves the microsphere to the vicinity of the test positioning layer corresponding to the microwell, and under observation under a microscope, the optical tweezer moves the microsphere up and down, and when the center of the microsphere and the lower surface or upper surface of the test positioning layer are both focused, it is determined that the microsphere is in the vicinity of the microwell directly above.

[0022] Preferably, step S2 uses a laser to capture the microsphere.

[0023] Preferably, the specific method of step S3 is that when the electrode layer of the optical tweezer test dielectrophoresis force chip is electrified, the microsphere moves under the action of the dielectrophoresis force, and when the focal plane of the microsphere is observed to be unchanged, it is determined that the microsphere is closer to the microwell directly above, and at this time, a one-dimensional direction of dielectrophoresis force is generated.

[0024] The optical tweezer assisted analysis of the chip realizes accurate, efficient and comprehensive analysis of the DEP behavior of a single micro-particle / cell or multiple micro-particles / cells at the same time. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0026] Figure 1 is the front view of the optical tweezer test dielectrophoresis force chip of the embodiment of the present application;

[0027] Figure 2 is a top view of an optical tweezer test dielectrophoresis force chip which is an embodiment of the present application;

[0028] Figure 3 is a perspective view of an optical tweezer test dielectrophoresis force chip which is an embodiment of the present application;

[0029] Figure 4 is a perspective view of a device for testing dielectrophoresis force which is an embodiment of the present application. DETAILED DESCRIPTION

[0030] Embodiments of the present application are described in detail below with reference to the attached drawings, which show by way of example, embodiments in which like reference numerals refer to like elements or elements having similar functions. The embodiments described below are exemplary and are intended to be illustrative of the present application, and are not to be construed as limiting the present application.

[0031] In the description of the present application, it is to be understood that the orientations or positional relationships indicated by the terms "length", "width", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like are based on the orientations or positional relationships shown in the drawings, and are merely for the purpose of facilitating the description of the present application and simplifying the description, and are not intended to indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be construed as limiting the present application. In addition, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise explicitly and specifically limited.

[0032] Figures 1 to 3 A structural schematic diagram of an optical tweezer test dielectrophoresis force chip is shown, the optical tweezer test dielectrophoresis force chip 10 comprising a bottom substrate 11, an electrode layer 12 on the bottom substrate 11, a microwell array layer 13 on the electrode layer 12, and a test positioning layer 14 arranged in an array.

[0033] The electrode layer 12 comprises a plurality of pairs of first interdigital electrodes 121 and second interdigital electrodes 122 arranged oppositely, each pair of first interdigital electrodes 121 and second interdigital electrodes 122 having a gap therebetween. The microwell array layer 13 comprises a plurality of microwells 131 arranged in an array, the microwells 131 being located between the first interdigital electrodes 121 and the second interdigital electrodes 122, and there being a plurality of microwells 131 between each row of first interdigital electrodes 121 and second interdigital electrodes 122.

[0034] The pore size of the micropores 131 is 10-20 microns, the height of the first interdigital electrode 121 and the height of the second interdigital electrode 122 are both 100-300 nanometers, the spacing between each pair of first interdigital electrode 121 and second interdigital electrode 122 is 4-8 microns, and the width of the first interdigital electrode 121 and the width of the second interdigital electrode 122 are both 15-25 microns. The micropore array layer 13 is formed by negative photoresist lithography on the electrode layer 12.

[0035] By loading a voltage on the electrode layer 12, a dielectrophoresis force can be formed in the micropores 131.

[0036] The test positioning layer 14 is used to position the functional layer of the microsphere when the optical tweezer test force is tested.

[0037] The plurality of micropores 131 in each row are located between the two adjacent test positioning layers 14 of the row, and the lower surface or the upper surface of the test positioning layer 14 is on the same straight line as the center line of the corresponding row of micropores 131.

[0038] The center distance of the micropores 131 of the adjacent two rows is 50-200um.

[0039] The application also discloses a manufacturing method of the optical tweezer test dielectrophoresis force chip, comprising the following steps:

[0040] S1: cleaning the bottom substrate 11;

[0041] The bottom substrate 11 is an ITO glass substrate. First, the ITO glass substrate is surface cleaned to ensure the cleanliness and adhesion of the ITO glass substrate surface. Specifically, a square ITO glass substrate with a square resistance of 20Ω and a side length of 8cm is placed in a cleaning rack and soaked in a mixed solution of hydrogen peroxide, ammonia and deionized water in a ratio of 1:1:6. After 40 minutes of 70℃ water bath heating, the ITO glass substrate is taken out. After the surface of the sample is sprayed by a water gun, the water is blown dry by nitrogen, and the ITO glass substrate is placed in a silicon wafer box for storage. The purpose of surface cleaning is to remove various particles and impurities on the surface of the ITO glass substrate and increase the surface energy to ensure that the subsequent spin-coated photoresist film is flat and has sufficient adhesion.

[0042] After cleaning, the surface of the ITO glass substrate is subjected to ultraviolet (UV) irradiation for 30 minutes to remove the impurity dangling bonds on the surface of the ITO glass substrate and further increase the surface energy of the ITO glass substrate, so that the photoresist has sufficient adhesion to adhere to the surface of the ITO glass substrate.

[0043] S2: performing positive photoresist lithography on the bottom substrate 11 by using a hard contact lithography process to form an electrode protection layer;

[0044] Before spin-coating the positive photoresist, the ITO glass substrate is baked on a hot plate at 180°C for 20 minutes to remove the moisture remaining on the surface of the ITO glass substrate, and then the positive photoresist is spin-coated in a spin coater according to the spin-coating parameters in Table 1:

[0045] Table 1 Spin-coating parameters of the positive photoresist

[0046]

[0047]

[0048] After spin-coating, the ITO glass substrate with the spin-coated positive photoresist is pre-baked on a hot plate at 95°C for 2 minutes. Then the ITO glass substrate with the spin-coated positive photoresist is fixed on an alignment sheet, and then placed in a photoetching machine together with a mask plate to adjust to a fixed position for exposure, with an exposure dose of 40 mJ / cm2. After exposure, the ITO glass substrate is placed in an appropriate amount of developing solution for 30 seconds, and then washed with deionized water and dried with nitrogen. Finally, the ITO glass substrate with the spin-coated positive photoresist is heated on a hot plate at 150°C for 2 minutes to harden the film, and the electrode protection layer is completed.

[0049] S3: forming the first interdigital electrode 121 and the second interdigital electrode 122 of the electrode layer 12;

[0050] After the electrode protection layer is made, wet etching is performed using an ITO etching solution to completely etch the ITO layer in the areas not protected by the photoresist, leaving the array pattern of the first interdigital electrode 121 and the second interdigital electrode 122 required; then the photoresist protection layer is washed away with acetone to obtain the ITO electrode layer 12; the ITO etching solution is heated to 35°C in a water bath, and then the ITO glass substrate is placed in the etching solution for 180 seconds, and then quickly transferred to a beaker containing deionized water to dilute the etching solution, and finally washed with water and dried.

[0051] After etching, the positive photoresist protection layer needs to be removed to obtain the bare electrode layer 12: only the ITO glass substrate needs to be placed in an acetone solution for ultrasonic treatment for 1 minute, and then directly dried to remove the positive photoresist protection layer (i.e. the electrode protection layer). After obtaining the electrode layer 12, the resistance of the integrated pair of first interdigital electrode 121 and second interdigital electrode 122 is tested using a multimeter. If the first interdigital electrode 121 and the second interdigital electrode 122 are conductive, it indicates that the electrode layer has failed, and if they are not conductive, they can be used for the next step of manufacturing. Before the next photoetching, the ITO glass substrate still needs to be subjected to ultraviolet irradiation for 30 minutes and baking at 180°C for 20 minutes to ensure that the adhesion between the photoresist and the ITO glass substrate is sufficient.

[0052] S4: forming the micropore array layer 13;

[0053] A microporous layer 13 with an array of holes was fabricated on the ITO electrode layer using hard contact photolithography to perform negative photoresist lithography. First, the negative photoresist was spin-coated onto the ITO glass substrate according to Table 2, as detailed in Table 2:

[0054] Table 2 Spin-coating parameters for negative photoresists

[0055]

[0056] Then, a pre-baking process was performed on a hot plate at 95°C for approximately 2 minutes. After pre-baking, the ITO glass substrate was left to stand for 20 minutes to allow the photoresist to fully cure. Because the exposure window of the negative photoresist is relatively small, a layer of black wallpaper was adhered to the back of the ITO glass substrate to absorb light and prevent unnecessary overexposure caused by UV light reflection within the substrate. Next, the mask with a microporous structure and the ITO glass substrate were placed sequentially into the lithography machine. Alignment was performed using alignment marks on the mask and electrode layer, followed by hard-contact exposure at an exposure dose of 100 mJ / cm². When UV light irradiated the unshielded negative photoresist, the photoresist produced acid that caused polymer cross-linking, which then remained in the developer. After exposure, the ITO glass substrate was placed on a hot plate at 95°C for 2 minutes for post-baking, and then allowed to cool to room temperature. After cooling, the ITO glass substrate can be developed. Immerse the ITO glass substrate in the developer solution, shaking the container evenly to allow the developer solution to flow. After 60 seconds, remove the ITO glass substrate, fix it with isopropanol, and then dry the sample with nitrogen. Finally, heat the ITO glass substrate at 150°C for 2 minutes to harden the photoresist, ensuring it is fully set and stable.

[0057] After the above steps are completed, a microporous layer 13 with an array thickness of approximately 4 μm can be obtained.

[0058] S5: Forming the test positioning layer 14 of the array setup.

[0059] The test positioning layer 14 is fabricated by performing negative photoresist lithography again on the microporous layer 13 of the array using hard contact photolithography.

[0060] The negative photoresist was spin-coated onto the ITO glass substrate according to Table 3, as detailed in Table 3:

[0061] Table 3 Spin Coating Parameters for Negative Photoresist

[0062]

[0063] Then, the ITO glass substrate is placed on a hot plate at 65 °C for 1 min for pre-baking, and then transferred to a hot plate at 95 °C for 5 min for post-baking. After pre-baking, the ITO glass substrate is allowed to stand for 20 min for complete drying of the photoresist. Similarly, a black wallpaper is pasted on the back of the ITO glass substrate for light absorption. Then, the mask plate with the micro-trap structure and the ITO glass substrate are placed in a photoetching machine in sequence, and aligned by using the alignment marks on the mask plate and the electrode layer 12, and then exposed by hard contact exposure with an exposure dose of 180 mJ / cm2. After exposure, the ITO glass substrate is placed on a hot plate at 65 °C for 1 min for post-baking, and then transferred to a hot plate at 95 °C for 6 min for post-baking. After standing and cooling to room temperature, the ITO glass substrate is developed, and the ITO glass substrate is immersed in a developing solution, taken out after 60 s, fixed with isopropyl alcohol, and then dried with nitrogen. Finally, the ITO glass substrate is heated on a hot plate at 150 °C for 2 min for hardening, so that the photoresist is completely shaped and stable. After the above steps are completed, a test positioning layer 14 with a thickness of about 50 μm is obtained. After verifying that the size of the micro-trap meets the expected value under a microscope, the manufacture of the optical tweezer test dielectrophoresis force chip 10 is completed.

[0064] As shown in Figure 4 The present application also discloses a device for testing dielectrophoresis force, which comprises the optical tweezer test dielectrophoresis force chip 10, a 3D printing clamp 20 for vertically fixing the optical tweezer test dielectrophoresis force chip 10, and a glass slide 30 for bearing the 3D printing clamp 20 and the optical tweezer test dielectrophoresis force chip 10.

[0065] The 3D printing clamp 20 comprises a first clamp 21 and a second clamp 22 with a gap between the first clamp 21 and the second clamp 22, and the optical tweezer test dielectrophoresis force chip 10 is fixed in the gap between the first clamp 21 and the second clamp 22.

[0066] The first clamp 21 is quadrilateral, comprising a first side 211, a second side 212, a third side 213 and a fourth side 214 connected in sequence, wherein the first side 211 and the third side 213 are oppositely arranged, the length of the first side 211 is greater than the length of the third side 213, and the second side 212 and the fourth side 214 are respectively extended from both ends of the first side 211; the second side 212 and the fourth side 214 are oppositely arranged and have the same length.

[0067] The second clamp 22 has three sides, comprising a first extension side 221, a second extension side 222 and a third extension side 223 connected in sequence, wherein the first extension side 221 and the third extension side 223 are oppositely arranged and have the same length, and the second extension side 222 is connected between the first extension side 221 and the third extension side 223.

[0068] The heights of the first and third extending edges 221 and 223 are the same as the heights of the second and fourth edges 212 and 214 respectively.

[0069] The bottom substrate 11 of the optical tweezer test dielectrophoresis force chip 10 is fixed on the first clamp 21 and positioned by the second clamp 22, i.e. the bottom substrate 11 of the optical tweezer test dielectrophoresis force chip 10 is fixed on the first edge 211 of the first clamp 21, and the bottom substrate 11 of the optical tweezer test dielectrophoresis force chip 10 extends out of the second and fourth edges 212 and 214 at both ends; the first and third extending edges 221 and 223 of the second clamp 22 abut at both ends of the bottom substrate 11 of the optical tweezer test dielectrophoresis force chip 10, so that the optical tweezer test dielectrophoresis force chip 10 is fixed between the first clamp 21 and the second clamp 22.

[0070] The electrode layer 12 and the test positioning layer 14 of the optical tweezer test dielectrophoresis force chip 10 are arranged towards the second clamp 22.

[0071] The thickness of the glass slide 30 is 160-180 microns; the optical tweezer test dielectrophoresis force chip 10 is packaged with silica gel, and will not leak liquid during the test.

[0072] After the device for testing dielectrophoresis force is prepared, a row of microwells 131 within a distance of 100 microns or less from the bottom of the glass slide 30 is obtained by randomly cutting a fracture from the device for testing dielectrophoresis force, because the optical tweezer can only pull the microparticles / cells to a height of 100 microns or less, so this is mainly to obtain the microwells 131 within 100 microns from the bottom when cutting along a cross section.

[0073] The method for testing dielectrophoresis force by using the optical tweezer test dielectrophoresis force chip comprises the following steps:

[0074] S1: a solution (not shown in the figure) with microspheres is placed in the device for testing dielectrophoresis force;

[0075] S2: at least one microsphere is captured by the optical tweezer (specifically, the microsphere is captured by laser) so that the microsphere is outside the corresponding microwell 131, and the center of the microsphere and the lower or upper surface of the test positioning layer 14 corresponding to the microwell are on the same focal plane, i.e. whether the center of the microsphere and the lower or upper surface of the test positioning layer are on the same focal plane is observed under a microscope (with a 60x oil lens);

[0076] For step S2, after the microsphere is placed in the device for testing dielectrophoresis force, the optical tweezer captures the microsphere and moves the microsphere to the vicinity of the corresponding test positioning layer 14 of the microsphere, and under the microscope (with a 60x oil lens), the optical tweezer moves the microsphere up and down, and when the center of the microsphere and the lower or upper surface of the test positioning layer 14 are both clearly focused, it is assumed that the microsphere is above the microwell 131.

[0077] S3: the electric signal is applied to the dielectrophoresis force test chip to generate the dielectrophoresis force, and the microspheres are moved in the optical trap generated by the optical tweezers under the action of the dielectrophoresis force;

[0078] The specific method of step S3 is that when the electrode layer 12 is applied with the electric signal, the microspheres are moved under the action of the dielectrophoresis force, the optical tweezers are moved up and down or the stage carrying the dielectrophoresis force test device is moved, and it is observed that the focal plane of the microspheres is unchanged, which means that the microspheres are closer to the top of the micro-hole 131, and at this time, the dielectrophoresis force in one-dimensional direction is generated.

[0079] S4: the direction of the dielectrophoresis force is observed, and the dielectrophoresis force is obtained by testing and calculating the lateral optical trap force.

[0080] The optical tweezers are established on the principle of laser photon dynamics, and the very strong gradient force is generated near the focusing center by focusing the micro laser beam near the transparent particles to form a three-dimensional optical potential well (referred to as an optical trap) to bind and move the particles. The binding force of the optical trap on the transparent particles in the optical trap is used to realize the laser optical tweezers capture and manipulation of the particles. In a very small range near the center of the optical trap, the lateral optical trap can be regarded as a simple harmonic potential well, and the movement of the particles / cells in this range under the action of other small external forces can also be regarded as simple harmonic vibration. Therefore, when the particles / cells captured by the optical tweezers are affected by the small dielectrophoresis force, the cells will deviate from the center of the optical trap by a displacement, and at this time, the lateral optical trap force pointing to the center of the optical trap and the dielectrophoresis force are in a balanced state on the premise of still stable capture. According to the Hooke's law, the size of the lateral optical trap force is linearly related to the displacement x of the cell from the center of the optical trap, that is, F=k·x, wherein k is the stiffness coefficient of the optical trap. The size of the DEP force can be obtained by calculating the lateral optical trap force of the cell, and the moving state of the particles / cells under the action of the DEP force can be directly observed in the optical tweezers system, so as to judge the type of the DEP force.

[0081] The calculation formula of the lateral optical trap force of the optical tweezers is F=k·x, wherein k is the stiffness coefficient of the optical tweezers, and x is the displacement of the particles / cells in the optical trap under the action of the external force (the dielectrophoresis force in the present application). The size of the calculated lateral optical trap force F is the size of the dielectrophoresis force, and the lateral optical trap force and the dielectrophoresis force are a pair of balanced forces with opposite directions.

[0082] The present application realizes accurate, efficient and comprehensive analysis of the DEP behavior of a single particle / cell or multiple particles / cells at the same time in the optical tweezers assisted analysis chip.

[0083] The above disclosed is only a preferred embodiment of the present application, of course, cannot be limited by this to limit the scope of the present application, the person skilled in the art can understand that the implementation of all or part of the above-mentioned processes, and according to the equivalent changes of the claims of the present application, still belong to the scope covered by the present application.

Claims

1. An optical tweezer test dielectrophoresis force chip, characterized in that, It comprises a bottom substrate, an electrode layer on the bottom substrate, a micropore array layer between the electrode layer and the arrayed test positioning layer; the electrode layer comprises a plurality of pairs of oppositely arranged first interdigital electrodes and second interdigital electrodes, and each pair of first interdigital electrodes and second interdigital electrodes has a gap; the micropore array layer comprises a plurality of arrayed micropores; The micropores are located between the first interdigital electrodes and the second interdigital electrodes, and each row of first interdigital electrodes and second interdigital electrodes has a plurality of micropores; the plurality of micropores in each row are located between the two adjacent test positioning layers in the row, respectively; the lower surface or the upper surface of the test positioning layer is on the same straight line as the center line of the corresponding row of micropores.

2. The optical tweezer test dielectrophoresis force chip of claim 1, wherein, The micropores have a pore diameter of 10-20 microns; the center distance of the micropores in two adjacent rows is 50-200 microns; the height of the first interdigital electrodes and the height of the second interdigital electrodes are both 100-300 nanometers; the spacing between each pair of first interdigital electrodes and second interdigital electrodes is 4-8 microns; the width of the first interdigital electrodes and the width of the second interdigital electrodes are both 15-25 microns.

3. The optical tweezer test dielectrophoresis force chip of claim 1, wherein, The lower surface or the upper surface of the test positioning layer is on the same straight line as the center line of the corresponding row of micropores.

4. A method for manufacturing an optical tweezer test dielectrophoresis force chip, characterized by, The optical tweezer test dielectrophoresis force chip according to any one of claims 1 to 3 comprises the following steps: S1: cleaning the bottom substrate; S2: forming an electrode protection layer on the bottom substrate; S3: forming the first interdigital electrodes and the second interdigital electrodes of the electrode layer; S4: forming the micropore array layer between the first interdigital electrodes and the second interdigital electrodes; S5: forming the arrayed test positioning layer, and the micropores in each row are located between the two test positioning layers in the row.

5. A method for testing dielectrophoretic force by using the chip for testing dielectrophoretic force by optical tweezers according to claim 1, characterized in that, The optical tweezer test dielectrophoresis force chip is fixed in a device, comprising the following steps: S1: placing a solution with microspheres in the device; S2: capturing at least one microsphere with the optical tweezer so that the microsphere corresponds to a micropore outside, and the center of the microsphere and the lower surface or the upper surface of the corresponding test positioning layer of the micropore are on the same focal plane; S3: applying an electrical signal to the optical tweezer test dielectrophoresis force chip to generate dielectrophoresis force, and the microsphere moves laterally in the optical trap generated by the optical tweezer under the action of the dielectrophoresis force; S4: observing the direction of the dielectrophoresis force, and obtaining the size of the dielectrophoresis force by testing and calculating the lateral optical trap force; The lateral optical trap force is calculated according to the following formula to obtain the size of the dielectrophoresis force; The calculation formula of the lateral optical trap force of the optical tweezer is F=k·x, where k is the stiffness coefficient of the optical tweezer, and x is the displacement of the microsphere / cell in the optical trap under the action of external force (i.e. dielectrophoresis force in the present application), and the size of the calculated lateral optical trap force F is the size of the dielectrophoresis force.

6. The optical tweezer test method for testing dielectrophoretic force according to claim 5, wherein, In step S2, whether the center of the microsphere and the lower surface or the upper surface of the corresponding test positioning layer of the micropore are on the same focal plane is observed under a microscope.

7. The optical tweezer test method for testing dielectrophoretic force according to claim 6, wherein, The specific method of step S2 is as follows: after the microspheres are placed in the device, the optical tweezers capture the microspheres and move the microspheres to the vicinity of the test positioning layer corresponding to the micropore, and under the microscope, the optical tweezers move the microspheres up and down, and when the center of the microspheres and the lower surface or the upper surface of the test positioning layer are both focused, it is determined that the microspheres are in the vicinity of the micropore.

8. The optical tweezer test method for testing dielectrophoretic force according to claim 5, wherein, The step S2 adopts laser to capture the microspheres.

9. The optical tweezer test method for testing dielectrophoretic force according to claim 5, wherein, The specific method of step S3 is as follows: when the electrode layer of the dielectrophoresis force chip is electrified by the optical tweezers, the microspheres are moved by the dielectrophoresis force, and the optical tweezers are moved up and down or the stage of the device for testing the dielectrophoresis force is moved, and when the focal plane of the microspheres is unchanged, it is determined that the microspheres are closer to the micropore, and at this time, the dielectrophoresis force in one-dimensional direction is generated.

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