Systems, methods, and devices for detecting mechanical forces in cells

Abstract

In order to overcome the deficiencies of the current technology and provide a quantitative measurement and monitoring method of cell mechanical force that does not require a microscope, is real-time, high-throughput and low-cost, the inventors have provided a device for detecting cell mechanical force, which includes a base and a micropillar array that is arranged on the base and is composed of a plurality of micropillars that can be deformed by cell mechanical force, and has a light reflecting layer on the top of the micropillar or the upper part of the columnar surface. The inventors have also provided a system for detecting cell mechanical force including the above-mentioned device, a detection method for detecting cell mechanical force using the above-mentioned device, and a manufacturing method for the above-mentioned device. Compared with the prior art, the above-mentioned technical solution has advantages such as high throughput and low cost, single-cell resolution, real-time monitoring, high sensitivity, simulation of cell microenvironment, simulation of composition and morphology of extracellular matrix, etc., and can meet more abundant technical needs.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a system for detecting the mechanical force of a cell, a method for detecting the mechanical force of a cell, an apparatus for detecting the mechanical force of a cell, and a method for manufacturing the same. [Background technology]

[0002] Cells exert minute mechanical forces on their surrounding microenvironment. Cell mechanical forces play important roles in processes such as adhesion, migration, proliferation, differentiation, and apoptosis. Together with other biochemical signals, they also play crucial regulatory roles in embryonic development, stem cell differentiation, immune processes, wound healing, and cancer metastasis, and are therapeutic targets for many diseases. For example, previous studies have shown that there are significant differences in the maximum cellular mechanical forces of normal cells, benign tumor cells, and malignant tumor cells. Therefore, high-throughput and accurate measurement of cellular mechanical forces is a core technology for many biomedical applications. However, the significance of measuring maximum cellular mechanical forces is limited, making high-resolution, real-time, and high-throughput force sensors a core need for next-generation cellular mechanical force measurement tools.

[0003] Currently, the main technological methods for measuring cellular mechanical forces include mechanical force microscopy, micro / nanocantilevers, and micropillar arrays. The main principle is to calculate cellular mechanical forces by measuring the deformation of an elastic substrate caused by the force exerted by the cells. Cell traction force microscopy (TFM) is currently the most widely used cellular mechanical force measurement technique, based on continuous elastic substrates (e.g., wrinkle-prone silicon thin films or polyacrylamide gels embedded with fluorescent beads). When cells are cultured on an elastic substrate, they exert mechanical forces on the substrate, deforming it. During the deformation process, the fluorescent beads undergo a corresponding displacement. The trajectory of the fluorescent beads is tracked using methods such as fluorescence microscopy, and substrate strain information is obtained through image processing and other methods. If the elasticity of the substrate is known, the cellular mechanical forces can be inversely calculated using a mechanical model. However, this measurement method has the following drawbacks. First, it relies on fluorescence microscopy and requires complex calculations, resulting in complex processes, low throughput, and high costs. Second, TFM does not directly measure cellular mechanical forces but instead calculates them by observing the positional changes of fluorescent beads on the substrate. Although a special process can ensure that most particles are deposited on the surface, particles may be released or settle after prolonged immersion, ultimately reducing the density of surface fluorescent particles. Because the focal depth of a fluorescence microscope is typically large, particles may be captured at different planes, resulting in discrepancies in subsequent displacement calculations and inaccurate measurement results. Furthermore, prolonged immersion in culture medium changes the gel's elastic modulus, which inevitably affects the accuracy of cell mechanical force calculations. Therefore, the gel's elastic modulus must be inspected and calibrated during the measurement process, significantly increasing the labor required. Furthermore, prolonged laser irradiation can cause phototoxicity in cells and photobleaching of fluorescent beads, making TFM unsuitable for long-term continuous measurement of cell mechanical forces. However, since studies of cell growth, differentiation, and drug response typically require longer-term monitoring, the aforementioned drawbacks of TFM significantly limit its application in biomedical fields.

[0004] Compared to TFM, micro / nano sensors (e.g., microcantilever arrays and micropillar arrays) can also be used to directly measure cellular mechanical forces. For example, in a micropillar array, cells can adhere to the top surface of a micropillar. By simply capturing images of the base and top of the micropillars with a microscope, the bending deformation of the micropillar can be resolved, allowing the magnitude and direction of the cellular mechanical force at that point to be calculated. Similar to TFM, micro / nano sensors rely on high-resolution imaging via a microscope, which places high demands on the equipment and is prone to errors during the imaging process, leading to inaccurate results. Furthermore, images captured by the microscope require complex image processing, and the magnitude of the cellular mechanical force must be calculated based on a mechanical model. The operation is complex and time-consuming, making it difficult to achieve real-time, high-throughput, low-cost, and long-term cell monitoring. Therefore, most current techniques are limited to scientific research in the field of biomechanics and are difficult to apply in practical applications. Summary of the Invention [Means for solving the problem]

[0005] The present invention aims to overcome the shortcomings of existing technologies and provide a method for quantitatively measuring and monitoring cellular mechanical forces in real time, with high flux and low cost, without the need for a microscope.

[0006] To address the core requirements of next-generation cellular mechanical force measurement tools, the present inventors provide a device for detecting cellular mechanical forces, which includes:

[0007] it is, The base and A device for detecting the mechanical force of a cell, comprising: a micropillar array arranged on the base and consisting of a plurality of micropillars that can be deformed by the mechanical force of the cell; and a light-reflecting layer on the top of the micropillars and / or on the upper part of the cylindrical surface.

[0008] Furthermore, in the device for detecting the mechanical force of a cell, the base is a light-transmitting base, the bodies of the micropillars are light-transmitting, and the tops of the micropillars have a light-reflecting layer.

[0009] Furthermore, in the device for detecting the mechanical force of a cell, the cylindrical surfaces of the micropillars have an anti-reflection layer.

[0010] Furthermore, in the device for detecting the mechanical force of a cell, the light-reflecting layer is a metal foil layer, a metal oxide or metal salt, ultrafine particle beads or microprisms, one type of organic reflective material, or a combination thereof.

[0011] Furthermore, in the device for detecting the mechanical force of a cell, a substance having cell adhesive properties is provided on the top end surfaces of all or some of the micropillars of the micropillar array.

[0012] Furthermore, in the device for detecting the mechanical force of cells, the substance having cell adhesion activity includes one or more of extracellular matrix molecules including collagen, fibronectin, vitronectin, laminin, elastin, etc., extracellular matrix mimics including polypeptides containing an RGD adhesion sequence, substances that promote cell adhesion including polylysine, and substances that interact with cell surface receptors.

[0013] Furthermore, in the device for detecting the mechanical force of a cell of the present invention, a substance having cell adhesive properties is provided on the top end surfaces of some of the micropillars in the predetermined region of the micropillar array.

[0014] Furthermore, in the device for detecting the mechanical force of cells of the present invention, a substance having cell adhesion activity is provided on the top end surface of the micropillars that do not have a substance having cell adhesion activity on the top end surface of the micropillar array, and a substance having cell adhesion inhibitory activity is provided on the top end surface of the micropillars.

[0015] Furthermore, in the device for detecting the mechanical force of a cell, the cross-sectional shape of the micropillars is circular, elliptical, or polygonal.

[0016] Furthermore, in the device for detecting the mechanical force of the cell, the size range of the micropillars and micropillar arrays includes a micropillar height of 10 nm to 500 μm, a micropillar spacing of 10 nm to 50 μm, and a micropillar top diameter of 50 nm to 50 μm.

[0017] Furthermore, the device for detecting the mechanical force of a cell further includes a cell control mechanism, which includes one or more limiting surfaces, each of which is a flat or curved surface disposed perpendicular to the plane on which the base is located and connected to or integrally formed with the base, and which is higher than the height of the micropillars and surrounds a predetermined number of the micropillars.

[0018] The inventors have also provided a system for detecting the mechanical force of a cell, which includes the above-mentioned device for detecting the mechanical force of a cell, an optical signal generating device, and an optical signal detecting device. The optical signal generating device has a light source, and light emitted from the light source is irradiated onto the light reflecting layer of the micropillar through an incident light path; The optical signal detection device is for detecting light reflected from the light reflection layer of the micro-pillar, and the light reflected from the light reflection layer enters the optical signal detection device through a reflection light path.

[0019] Furthermore, in the system for detecting the mechanical force of a cell, the base of the device for detecting the mechanical force of a cell is a light-transmitting base, the body of the micropillar is light-transmitting, and a light-reflecting layer is provided on the top of the micropillar; The light emitted from the light source is irradiated through an incident light path from the base of the device for detecting the mechanical force of the cell to the light reflecting layer of the micropillar; The optical signal detection device is for detecting light reflected from the light reflecting layer on the top of the micro-pillar, and the light reflected from the light reflecting layer enters the optical signal detection device through a reflected light path.

[0020] Furthermore, the system for detecting a mechanical force on a cell further includes an optical signal analyzer for analyzing an optical signal.

[0021] The present inventor also provides a method for detecting the mechanical force of cells according to the above technical solutions. The method includes the steps of generating light using an optical signal generating device in the system for detecting mechanical forces of cells described in the above-mentioned technical solution, and detecting light after the action of the device for detecting mechanical forces of cells using an optical signal detecting device in the system for detecting mechanical forces of cells described in the above-mentioned technical solution.

[0022] Furthermore, the method for detecting the mechanical force of a cell further includes a step of using an optical signal analysis device to compare and analyze the reflected light before and after the action of the mechanical force of the cell generated between the device for detecting the mechanical force of the cell and the test cell, thereby obtaining information about the mechanical force of the cell.

[0023] The inventors have further provided a method for detecting a cell state, which includes the steps of employing the system for detecting a cellular mechanical force according to any of the above-mentioned embodiments or the method for detecting a cellular mechanical force according to any of the above-mentioned embodiments to obtain cellular mechanical force information, and analyzing and determining a cellular state based on the cellular mechanical force information. The cell state specifically includes cell adhesion, cell activity, cell differentiation / activation, cell proliferation and / or cell migration.

[0024] Furthermore, the cell state may be a static cell state or a real-time cell state.

[0025] The inventors have further provided a method for cell identification, which includes the steps of employing the system for detecting a mechanical force of a cell according to any of the above-mentioned embodiments or the method for detecting a mechanical force of a cell according to any of the above-mentioned embodiments to obtain mechanical force information of a cell, and distinguishing between different cell types based on the mechanical force information of the cell.

[0026] Furthermore, in the cell identification method, the step of "adopting a system for detecting the mechanical force of a cell according to any of the above-mentioned embodiments or a method for detecting the mechanical force of a cell according to any of the above-mentioned embodiments to obtain mechanical force information of a cell, and distinguishing between different cell types based on the mechanical force information of the cell" specifically includes the following steps: A step of acquiring cell information, wherein the cell information includes mechanical force information of the cell at a certain point of the cell acquired based on a device for detecting the mechanical force of the cell, and the mechanical force information of the cell includes the magnitude of the mechanical force of the cell at that point. A step of preprocessing the cell information to form structured cell information, the structured cell information including the number of cells, the number of cell features, and feature information of each cell feature. A step of using the structured cell information as input data to construct a cell feature model by machine learning, such as supervised learning, unsupervised learning, or semi-supervised learning, and applying the cell feature model to classify or cluster cells of unknown type or unknown state.

[0027] Furthermore, in the cell identification method, the mechanical force information of the cell further includes the direction of the mechanical force of the cell at that point.

[0028] Furthermore, in the cell identification method, the mechanical force information of the cell further includes a change in the magnitude or direction of the mechanical force of the cell at that point within a certain time interval.

[0029] Furthermore, in the cell identification method, the cell information further includes cell morphology information.

[0030] The inventors have further provided a method for manufacturing a cellular mechanical force detection device, which includes the following steps: providing a reflective layer on the top or upper half cylindrical surface of a micropillar, thereby obtaining a micropillar having a reflective layer on the top or upper half cylindrical surface.

[0031] Furthermore, in the method for manufacturing the mechanical force detection structure for cells, before the step of "providing a reflective layer on the top or upper half of the micropillar", uniformly coating the entire micropillar with an anti-reflection layer; and removing the anti-reflection layer on the top or upper half cylindrical surface.

[0032] Furthermore, in the manufacturing method of the mechanical force detection structure for cells, the step of "providing a reflective layer on the top or upper half cylindrical surface of the micropillar to obtain a micropillar having a reflective layer on the top or upper half cylindrical surface" specifically means uniformly sputtering a reflective metal on the top or upper half cylindrical surface of the micropillar to obtain a micropillar having a metal light-reflective layer on the top or upper half cylindrical surface.

[0033] Furthermore, in any of the above-described embodiments, the cell may be a single cell or any form of multicellular aggregate formed by two or more cells, and the present invention is not limited to a single cell or various forms formed by two or more multicellular cells. [Effects of the Invention]

[0034] Compared with the prior art, the above technical solution has the following advantages: First, it has high throughput and low cost. Compared with conventional TFM and conventional micropillar arrays, the technical solution of the present invention is independent of the microscope and greatly simplifies the operation flow. This is because high-resolution imaging by a microscope is not required, and high-throughput monitoring of cells can be achieved by simply monitoring the intensity of reflected light. Second, it offers single-cell resolution. Its high resolution allows for real-time monitoring of individual cells, and by combining it with other single-cell analysis techniques, it is possible to measure the heterogeneity of cellular responses to drugs. Because it enables real-time monitoring, does not require fluorescence, and avoids laser-induced phototoxicity, it is suitable for long-term monitoring and can be used to study long-term cellular responses to drugs. Its high sensitivity allows the micropillar deformation signal to be amplified by the reflected signal, thereby increasing the sensitivity of deformation monitoring. Detection of bending deformation of micro- and nanopillars typically relies on optical systems (such as microscopes), but the smaller the micropillar size, the greater the demands on the precision and resolution of the optical system. For example, a micropillar measuring 2 μm wide and 6 μm high can only be effectively observed by combining a 20x or higher objective lens with a confocal system. In contrast, our method utilizes the principle of specular reflection to detect the attenuation of reflected light, effectively amplifying the micropillar deformation signal. Experiments have confirmed that the same signal can be observed under a 5x objective lens. Combined with a special readout system, it can effectively detect the deformation of micro / nanopillars without relying on high-magnification optical objective lenses, greatly reducing the cost of the system and effectively improving throughput. Third, it can simulate the cellular microenvironment, including the composition and morphology of the extracellular matrix, thereby meeting a wider range of technological needs. Fourth, it can detect the mechanical forces of cells, such as multilayered cells and tumor polymers, and can be applied to situations requiring the characterization of multicellular aggregates, such as drug screening, regenerative medicine, gene editing, precision medicine, organ development, and disease modeling. [Brief explanation of the drawings]

[0035] [Figure 1] 1 is a structural schematic diagram of a device for detecting the mechanical force of a cell according to a first embodiment of the present invention; [Figure 2]2A and 2B are scanning electron microscope (SEM) images of the micropillars (actual objects) of the device for detecting the mechanical force of a cell according to the first embodiment of the present invention, where Fig. 2A is a plan view of the device for detecting the mechanical force of a cell, and Fig. 2B is a side view of the device for detecting the mechanical force of a cell. [Figure 3] FIG. 13 is a structural schematic diagram of a system for detecting the mechanical force of a cell according to a ninth embodiment of the present invention. [Figure 4] FIG. 10 is a structural schematic diagram of a system for detecting the mechanical force of a cell related to a tenth embodiment of the present invention. [Figure 5] Figure 5a shows a scanning electron microscope image of a micropillar (polydimethylsiloxane) with a light-reflecting layer (gold) at the top position. Figure 5b shows an elemental characteristic map of the top region of the micropillar, and Figure 5c shows an elemental characteristic map of the side region (excluding the top region) of the micropillar. [Figure 6a] This is a fluorescent image of cells adhering to a predetermined pattern of micropillars with fibronectin on top. [Figure 6b] FIG. 10: Cell force distribution map analyzed from optical reflection signals measured on micropillars with fibronectin on top. [Figure 7a] FIG. 1 is a schematic diagram of a test using OKT3 antibody as a substance having cell adhesion activity. [Figure 7b] The top image is a fluorescent image of cells attached to the top of micropillars, each with OKT3 antibody and fibronectin at the top. [Figure 7b] The bottom image shows the distribution of the optical reflection signal (reflecting the magnitude of the mechanical force of the cell) measured on the micropillar. [Figure 7c] FIG. 1 is a comparison of the magnitude of the forces measured on surfaces coated with OKT3 antibody and fibronectin, respectively. [Figure 7d] Figure 1 shows the dynamic changes in cell dynamics that occurred after seeding T cells onto an OKT3 antibody surface (top of the micropillars). [Figure 8] Figure 1a is a structural schematic diagram of a device for detecting the mechanical force of cells with a cell control mechanism. [Figure 9] Figure 2b is a structural schematic diagram of a device for detecting the mechanical force of cells with a cell control mechanism. [Figure 10a] This is an actual diagram of a device that detects the mechanical force of cells using a silicon thin film as a cell control mechanism. [Figure 10b] This is a fluorescence microscope image of a device that detects the mechanical force of cells using a silicon thin film as a cell control mechanism under light reflection. [Figure 10c] This is an enlarged view of Figure 10b. [Figure 11] 13 is a fluorescence microscope image of the mechanical force of a cell monitored by the system for detecting the mechanical force of a cell according to the eleventh embodiment. [Figure 12a] FIG. 22 is a structural schematic diagram of a cellular mechanical force detection system according to a twelfth embodiment. [Figure 12b] 23 is an image of the optical reflection signal of the mechanical force detection device of the cell acquired by the optical signal detection device of the twelfth embodiment. [Figure 12c] FIG. 23 is a visualization effect diagram of the magnitude and distribution of force processed by the optical signal analysis device of the twelfth embodiment. [Figure 13a] 1A-1C are structural schematic diagrams of a mechanical force detection device for cells in a microfluidic environment before and after the fluid is turned on. [Figure 13b] The bright-field microscope image of the micro-pillars before the fluid was turned on, the reflected light signal distribution map, and a comparison of the superposition effect of the two. The superposition effect map refers to the effect map obtained by superposing the bright-field microscope image of the micro-pillars and the reflected light signal distribution map. [Figure 13c] 1 shows a comparison of the bright-field microscope image of the micro-pillars after the fluid is turned on, the reflected light signal distribution map, and the superposition effect map of the two. The superposition effect map refers to the effect map obtained by superposing the bright-field microscope image of the micro-pillars and the reflected light signal distribution map. [Figure 13d] The intensity values ​​of the light reflection signal before and after the fluid is turned on. [Figure 13e] The linear section of the optical reflection signal attenuation and the displacement of the micropillar apex. [Figure 14a]1 is a structural schematic diagram of a device for detecting the mechanical force of a cell before and after the cell comes into contact with the micropillars. [Figure 14b] 4 is a distribution diagram of a reflected optical signal acquired by an optical signal detection device. [Figure 14c] FIG. 11. Monitoring of cell migration process. [Figure 14d] FIG. 10 is a distribution diagram of reflected light signals during the cell migration process. [Figure 15a] This is a fluorescent image of a mixture of healthy cells and non-small cell lung cancer cells. [Figure 15b] FIG. 10 is a diagram showing the distribution of optical reflection signals of the mechanical force detection device of a cell obtained by the optical signal detection device. [Figure 15c] FIG. 10 is a visualization effect diagram of the magnitude and distribution of force processed by the optical signal analysis device. [Figure 15d] Zoomed-in view of the cell force distributions of representative single cells of healthy cells and non-small cell lung cancer cells in Figure 15c. [Figure 15e] FIG. 1 is a comparison of the cell morphology of healthy cells and non-small cell lung cancer cells. [Figure 15f] FIG. 10 is a comparison of the reflected signal intensity between healthy cells, non-small cell lung cancer cells, and after mixing these two types of cells at different ratios. [Figure 15g] This is a clustering analysis diagram obtained by structuring FIG. 15c and then processing it based on the structured cell information. [Figure 16a] FIG. 1 is a schematic diagram of the operation flow of a method for detecting cell activity. [Figure 16b] FIG. 1 is a comparison diagram of cell activity measured by the MTT method and cell activity reflected by the mechanical force of cells after treating A549 cells with different doses of 5FU for 24 hours. [Figure 16c] FIG. 1 is a comparison diagram of cell activity measured by the MTT method and cell activity reflected by the mechanical force of cells after treating A549 cells with different doses of 5FU for different periods of time. [Figure 17a] FIG. 1 is a diagram illustrating the operation process of a cell state detection method. [Figure 17b] This is a fluorescent micrograph of M0 macrophages differentiated into the M1 state. [Figure 17c] This is a fluorescent micrograph of M0 macrophages differentiated into M2 cells. [Figure 17d] FIG. 1 is a comparison of cell adhesion areas in M0 macrophages, M1 states, and M2 states. [Figure 17e] FIG. 1 is a comparison of cell circularity in M0 macrophages, M1 states, and M2 states. [Figure 17f] FIG. 10 is a comparison of mechanical forces in M0 macrophages, M1 and M2 states. [Figure 18a] The first morphology of tumor cell polymers was characterized in the presence and absence of 5-Fu. From left to right, the figure shows a mixed image of cell membrane fluorescence and reflectance signals (1), optical reflectance signals (2), cell nuclei (3), cell membranes (4), and a visualization image of cell force processed by an optical signal analyzer (ImageJ) (5). [Figure 18b] The second morphology of tumor cell polymers was characterized in the presence and absence of 5-Fu. From left to right, the figure shows a composite image of cell membrane fluorescence and reflectance signals (1), optical reflectance signals (2), cell nuclei (3), cell membranes (4), and a visualization image of cell force processed by an optical signal analyzer (ImageJ) (5). [Explanation of symbols]

[0036] 1...Device for detecting mechanical force of cell, 2...Optical signal generating device, 3...Optical signal detecting device, 4...Optical signal analyzing device, 5...Beam splitter, 11...Base, 12...Micropillar, 13...Light reflecting layer, 15...Recess space, 16...Limiting surface. DETAILED DESCRIPTION OF THE INVENTION

[0037] The technical contents, structural features, objectives to be achieved, and effects of the present invention will be described in detail below with reference to specific examples. First embodiment: Device for detecting mechanical forces in cells

[0038] See Figure 1. This is a schematic diagram of the structure of a device for detecting the mechanical force of cells. As shown in the figure, the device for detecting the mechanical force of cells includes a light-transmitting base 11 and micropillars 12 mounted on the base 11, which are deformable by the mechanical force of cells. A light-reflecting layer 13 is provided on the top of the micropillars 12, and the light-reflecting layer 13 has a thickness of 5 nm. (In some other embodiments, the thickness of the light-reflecting layer 13 may be between 5 nm and 20 nm. This is related to the coating material. When using the same coating material, the thickness of the coating layer should be selected to ensure light transmittance, ensure the stability of the micropillars, and prevent the connection with the micropillars from peeling off.) The micropillars 12 are light-transmitting, and the reverse arrows in the figure represent incident and reflected light. (Note: Although the term "coating layer" is used in this embodiment, this only indicates that the light-reflecting layer 13 in this embodiment can be manufactured by a coating process, but does not necessarily mean that the light-reflecting layer 13 must be manufactured by a coating process.)

[0039] Please refer to Figure 2, which shows scanning electron microscope (SEM) images of the micropillars 12 (actual objects) of the cellular mechanical force detection device of this embodiment, where Figure 2a is a plan view of the cellular mechanical force detection device and Figure 2b is a side view of the cellular mechanical force detection device. As can be seen from Figure 2, the micropillars of this cellular mechanical force detection device have an orderly and uniform microstructure and are controllable in size. Compared with conventional cellular mechanical force detection devices, the force values ​​measured by the cellular mechanical force detection device of this embodiment are more accurate.

[0040] When the device 1 for detecting mechanical force of cells described in this embodiment is put into use, the number of micropillars 12 is not limited to one. Please refer to FIG. 3, which is a structural schematic diagram of a system for detecting mechanical force of cells related to the ninth embodiment of the present invention. FIG. 3 can be used to understand this embodiment. The system shown in FIG. 3 not only relates to the device 1 for detecting mechanical force of cells described in this embodiment, but also to an optical signal generator 2 having a light source installed below the base 11 and an optical signal detector 3. Light emitted from the light source is irradiated through the incident optical path from the transparent base 11 of the device 1 for detecting mechanical force of cells to the optical reflective layer of the micropillar 12. The optical signal detector 3 detects light reflected from the optical reflective layer 13 at the top of the micropillar. The light reflected from the optical reflective layer 13 passes through the reflected optical path and enters the optical signal detector 3 after passing through the beam splitter 5. After obtaining the reflected light signal, the optical signal analyzer 4 compares and analyzes the reflected light before and after the action of the mechanical force generated between the cell's mechanical force detection device 1 and the test cell, thereby obtaining information about the cell's mechanical force. When the micropillars 12 are not subjected to force, they should maintain an upright state, thereby maximizing the reflection of the detected light. On the other hand, when the micropillars 12 come into contact with the cell, they bend under the action of the cell's mechanical force, reducing the level of light reflection. Therefore, the greater the cell's mechanical force, the smaller the resulting optical reflection signal should be. In this way, the magnitude of the cell's mechanical force at that point can be easily calculated by observing the intensity of the optical reflection signal.

[0041] In addition, the measurement light source in the technical solution of this embodiment can also be an infrared laser with a certain intensity. In the conventional technical solution, micropillar measurement requires capturing high-resolution images, but using a laser in the process can easily cause phototoxicity in cells and quench the fluorescence of the sample. In contrast, the technical solution of this embodiment only requires measuring the reflected signal, so the impact of an infrared laser within a certain light intensity on cells is essentially negligible, making it suitable for long-term cell monitoring. Second embodiment: Device for detecting mechanical force of cells

[0042] The difference from the first embodiment is that the micropillars 12 not only have a light-reflecting layer 13 on their top end surfaces, but also have a light-reflecting layer 13 on the upper half of the cylindrical surface of the micropillars 12 (i.e., the curved surface connecting the two end surfaces of the pillar). In fact, in some other embodiments, the method of providing a light-reflecting layer 13 on the lower half of the side cylindrical surface of the micropillars 12 is not adopted because the actual effect is poor. However, as long as the light-reflecting layer 13 is provided on the upper half of the side of the micropillars 12, the detection effect intended by the present invention can be achieved. That is, in some other embodiments, the light-reflecting layer 13 can be provided at any local position on the upper half side cylindrical surface or at a local position on the top, and does not necessarily have to be provided on the entire upper half cylindrical surface or the entire top end surface. Although the expected purpose can be achieved, there may be differences between the obtained data and the subsequent calculation effect.

[0043] Furthermore, in the first and second embodiments of the present invention, the definitions of the "cylindrical surface" and "end surface" of the micropillar are used. In other words, while an independent pillar as we normally understand it has two end surfaces and a curved surface (cylindrical surface) connecting the two end surfaces, the micropillars of the present invention have only one end surface, i.e., the top surface, due to the presence of a base, and the other end is fixed to the base or integrally formed with the base. However, in some other embodiments, the top surface may be a smoothly connected curved surface integral with the pillar surface, and does not necessarily need to have an intersection or a clear boundary line as in the first or second embodiments. In this case, the location of the light-reflecting layer 13 should also be understood to be the upper half of the pillar and should not be limited to the "end surface" or "cylindrical surface." Third embodiment: Device for detecting mechanical force of cells

[0044] Please refer to FIG. 4. FIG. 4 is a structural schematic diagram of a system for detecting cellular mechanical force according to a tenth embodiment of the present invention, which is used to explain the device 1 for detecting cellular mechanical force in this embodiment. The difference between this embodiment and the first and second embodiments is that no requirements are imposed on the optical transparency of the base 11 and the micropillars 12 of the micropillar array. That is, they may be optically transparent, non-transparent, or semi-transparent. In this case, simply by changing the positions of the optical signal generator 2 and the optical signal detector 3, they can be installed above the base 11. In this way, each time the micropillars bend, the optical signal received by the optical signal detector 3 will change compared to when the micropillars 12 are upright and undeformed. By analyzing the changes in the optical signal before and after bending, the relative magnitude of the cellular mechanical force can be obtained. After calibration with a standard value, the absolute magnitude of the cellular mechanical force can be obtained. Fourth embodiment: Device for detecting mechanical force of cells

[0045] The difference between this embodiment and the first to third embodiments is that an anti-reflection layer against light rays is provided on the surface of the micropillar 12 in areas other than the area where the light-reflecting layer 13 is provided. This design reduces the interference of the reflected light signal that may be caused by the pillar surface layer, improves the S / N ratio, and makes the detection results more accurate.

[0046] In some embodiments, the light-reflecting layer 13 may be a gold foil layer. In other embodiments, the light-reflecting layer 13 may be another metal layer or other reflective material having a light-reflecting function. Different materials may have different reflective effects, manufacturing difficulties and costs for the reflective layer, etc., and the selection may be made based on specific conditions in actual operation.

[0047] In the first to fourth embodiments, the cross-sectional shape of the micropillars 12 is circular. In other embodiments, the cross-sectional shape of the micropillars 12 may be elliptical or polygonal. Different cross-sectional shapes can achieve different objectives in various specific situations of the present invention. For example, a circular cross-section has isotropic characteristics, meaning that the mechanical properties of the micropillars themselves are not sensitive to direction. On the other hand, an elliptical cross-section is anisotropic, meaning that the mechanical properties of the micropillars themselves are sensitive to direction, thereby controlling their sensitivity to force fields in different directions and, to some extent, regulating cell tropism. (Most cells have asymmetric geometric shapes. In this invention, cell tropism refers to the morphological asymmetry, polarity, or directionality exhibited by the cells. For example, when fitting the shape of a cell projection using an ellipse, the major axis of the ellipse can be considered the direction of the cell.) This is because, when the cross-section is elliptical, the cross-section has a major axis and a minor axis, and therefore, pushing the micropillar along the minor axis is much easier than pushing along the major axis, resulting in greater deformation under relative force conditions. In some advanced embodiments, when cells are seeded on such micropillars, anisotropic mechanical interactions exist between the cells and the micropillars, causing the cells to grow along one side, while in fluidic applications they can be used to measure the direction of the fluid.

[0048] In the first to fourth embodiments, the dimensions of the micropillar array range from 10 nm to 500 μm in pillar height, 10 nm to 50 μm in pillar spacing, and 50 nm to 50 μm in pillar top diameter. Micropillars within this size range can meet the basic requirements for use as sensors, i.e., at least the requirement that they be deformable and not collapse. Based on this, the following functions can also be achieved by adjusting the size of the micropillar array. For example, by adjusting the aspect ratio of the micropillar (which can be understood as the ratio of height to cross-sectional diameter / side length / major axis at the micropillar level), the deformation performance of the micropillar can be adjusted to a certain extent, thereby better simulating the internal organ tissue environment (e.g., bone tissue and nervous tissue, which have different hardnesses).

[0049] The overall size of the array, i.e., the number of micropillars 12 on a given area of ​​the base 11, also affects the ligand density, i.e., the number of adhesion points that cells can find on the surface. The sparser the array of micropillars 12, the fewer adhesion points cells can find, which has a significant impact on cell behavior.

[0050] The cross-sectional area of ​​the micropillar shape also affects cell adhesion behavior because focal adhesions formed by cell adhesion require a certain area. In the case of nano-micropillars, the small cross-sectional area of ​​the micropillars affects the formation of focal adhesions.

[0051] In summary, by combining the properties of the material itself with a certain size of the micropillar array, it is possible to achieve more tailored cell support, chip stability, and measurement accuracy. Adjusting the distribution of the micropillar array can also influence the cell adhesion to some extent.

[0052] In the first through fourth embodiments, the micropillars 12 are made of polydimethylsiloxane (PDMS). In other major embodiments of the present invention, the micropillars 12 may be made of other polymeric materials, such as silicone-based polymers, photoresist polymers, conductive polymers, and temperature-sensitive polymers. The reason that the major embodiments of the present invention primarily use polymeric materials is that polymeric materials currently have relatively suitable deformation characteristics for the application of the present invention. However, the present invention does not necessarily limit the micropillar material to polymeric materials; it can be applied to any material with comparable deformation characteristics, and the inventive concept of the present invention can be realized in either case. Simply put, the micropillar material must have certain mechanical deformation characteristics, and in some embodiments, it must also have certain optical transparency. The latter is not a requirement for all embodiments. When fabricating micropillars using materials with limited optical transparency, the inventive concept of the present invention can be realized simply by appropriately positioning the optical signal generator and optical signal detector.

[0053] Overall, the hardness (deformability) of the micropillars 12 can be adjusted from multiple technical aspects, such as size (mainly aspect ratio), selection of material type, and control of the cross-linking degree of polymer materials, chemical or physical surface treatment, according to actual needs. See Figure 5. Figure 5 is a scanning electron microscope image of a micropillar (polydimethylsiloxane) with a light-reflecting layer (gold) at its top. Figure 5a is a scanning electron microscope image of the micropillar, Figure 5b is an elemental mapping image of the top region of the micropillar, and Figure 5c is an elemental mapping image of the side region (other than the top region) of the micropillar. Characterizing the material composition of the micropillar using the scanning electron microscope image in Figure 5 confirms the presence of Au elements at the top of the micropillar and Si elements at other positions on the micropillar. Fifth embodiment: Device for detecting mechanical force of cells

[0054] The difference between this embodiment and the first to fourth embodiments is that a substance with cell adhesion properties is provided on the apical end surfaces of some of the micropillars 12 in the micropillar array. While collagen is used in this embodiment, in other embodiments, one or more types of extracellular matrix molecules including collagen can be used in combination. Examples include fibronectin, vitronectin, laminin, and elastin. In some other embodiments, other types of substances with cell adhesion properties can be provided on the apical end surfaces of all or some of the micropillars in the micropillar array 12. Examples include extracellular matrix mimics (e.g., polypeptides containing an RGD adhesion sequence), substances with cell adhesion-promoting mechanisms (e.g., polylysine), or substances that interact with cell surface receptors.

[0055] By providing such a substance with cell adhesion properties on the apical end surfaces of the micropillars 12, cell adhesion to the micropillars 12 can be effectively promoted, thereby enabling the adjustment of cell adhesion, proliferation, migration, condition, differentiation, etc. Furthermore, by providing a substance with cell adhesion properties (extracellular matrix proteins such as fibronectin) on the apical end surfaces of some micropillars in a predetermined region of the micropillar array, these micropillars can be configured to form a specific shape. This makes it easier for cells to adhere to micropillars with specific positions and shapes, enabling high-throughput mechanical measurements while controlling the size, shape, and tropism of cells. Sixth embodiment: Device for detecting mechanical force of cells

[0056] The difference between this example and the fifth example is that, as described in the fifth example, a substance having cell adhesion properties is provided on the top end surfaces of some of the micropillars 12 in the micropillar array, whereas in this example, a substance having cell adhesion inhibitory properties (such as F-127) is further provided on the cylindrical surfaces (end faces or side faces) of the micropillars 12 in the parts where the substance having cell adhesion properties is not provided on the top end surfaces. This makes it easier for cells to adhere to micropillars in specific positions and shapes, enabling high-throughput mechanical measurements to be performed while controlling the size, shape, and tropism of cells. Seventh embodiment: Device for detecting mechanical force of cells

[0057] The difference between this embodiment and the first to fourth embodiments is that the micropillars containing a cell adhesive material are arranged in a predetermined pattern on the top surface of the micropillar array 12. Specifically, a specific pattern of cell adhesion molecule layer can be printed using microcontact printing technology to promote cell adhesion in these areas. The predetermined pattern can be triangular, rectangular, polygonal, circular, elliptical, or other shapes. The purpose of the predetermined pattern is, first, to control cell-to-cell contact through these cell adhesive material patterns, thereby facilitating high-throughput data acquisition. Second, by standardizing cell shapes, dimensionality reduction can be achieved in data processing, thereby reducing the difficulty of analysis. Furthermore, by limiting the cell adhesion area, cell size, shape, tropism, differentiation state, etc. can be controlled. Furthermore, by controlling actin filaments, the mechanical state of cells can be adjusted, thereby meeting the needs of certain technical scenarios.

[0058] In another embodiment broadly similar to this embodiment, substances with cell adhesion inhibitory properties, such as BSA (bovine serum albumin) or F127 (polymeric nonionic surfactant), can be used in the unprinted portions of the predetermined pattern to inhibit cell adhesion in these areas, thereby enabling directional adhesion, control of cell morphology, or simulation of specific cellular microenvironments.

[0059] In some other examples, fibronectin (FN) is used as an example of a cell adhesion material, but this is not intended to limit the scope of the present invention. Polydimethylsiloxane microstamps with protruding square and rectangular patterns are used, and fibronectin is attached to the microstamp surface. Microcontact printing is then used to transfer the fibronectin from the protruding portions of the stamp onto the metal reflective layer at the top of the micropillars. The micropillars are then immersed in F-127 solution, allowing the areas without fibronectin to inhibit cell adhesion. Finally, after thoroughly rinsing the micropillars with saline, fibroblasts with stained cell membranes are seeded on the micropillar surface, and fluorescent imaging of the cells is performed (see Figure 6a). Simultaneously, the force field within the cells is measured with high resolution (see Figure 6b). See Figures 6a and 6b. Figure 6a shows a fluorescent image of cells adhering to a specific pattern of micropillars with fibronectin at their tops. It can be seen that the cell adhesion area is limited to the fibronectin-containing regions. This allows us to monitor cell mechanics while controlling the cell size, shape, tropism, and differentiation state by restricting the cell adhesion area through a specific pattern. Figure 6b shows the distribution of the mechanical force of the cell analyzed from the optical reflection signal measured on the micropillars.

[0060] In some other examples, OKT3 antibody (a substance that interacts with cell surface receptors) or fibronectin (FN) are described as examples of substances having cell adhesion activity, but these examples are not intended to limit the scope of the present invention. See Figures 7a to 7d. Figure 7a is a schematic diagram of a test using OKT3 antibody as a substance having cell adhesion activity. The upper image in Figure 7b is a fluorescent image of cells adhering to the top of micropillars with OKT3 antibody or fibronectin on the top, respectively. The lower image in Figure 7b is a distribution diagram of the optical reflection signal (reflecting the magnitude of the mechanical force of the cell) measured on the micropillar. Figure 7c is a comparison diagram of the magnitude of force measured on surfaces coated with OKT3 antibody and fibronectin, respectively. Figure 7d is a diagram showing the dynamic changes in cell mechanics that occurred after T cells were seeded on the OKT3 antibody surface (top of the micropillar). Specifically, the above test involves coating the apex of some micropillars of the same or different cellular mechanical force detection device with OKT3 antibody or fibronectin, and seeding T cells onto the surface of the cellular mechanical force detection device's cell adhesion material. Figures 7a to 7d show that a cellular mechanical force detection device with a micropillar surface coated with a material that interacts with cell surface receptors (such as OKT3 antibody) or fibronectin (FN) can be used to monitor the effect of mechanical force and interaction of the material on cells in real time. Eighth embodiment: Device for detecting mechanical force of cells

[0061] The difference between this embodiment and the first to seventh embodiments is that the device for detecting the mechanical force of the cell further includes a cell control mechanism, which includes one or more limiting surfaces 16, which are flat or curved surfaces perpendicular to the plane on which the base 11 is located, connected to the base 11, or integrally formed with the base 11, and are higher than the micropillars 12 and surround a predetermined number of the micropillars 12.

[0062] The role of the cell control mechanism installed in this embodiment is to isolate and detect single cells, i.e., to avoid contact or adhesion between cells during detection and to restrict the cell morphology, thereby facilitating high-throughput testing. The number of restriction surfaces 16 included in the cell positioning mechanism or the surrounding shape may vary depending on different requirements. For example, the restriction surface 16 included in the cell positioning mechanism may be a single cylindrical surface, three planes whose ends connect to form a triangular cross-section and surround a certain number of micropillars, four planes whose ends are perpendicular to each other and connect to form a rectangular shape and surround a certain number of micropillars, N planes whose ends connect to form an N-sided polygon, or a single curved surface whose cross-section is quasi-circular. In other words, the cross-sectional shape formed by the restriction surface 16 is a controllable closed shape, and its area (which can be understood as the number of micropillars that can be accommodated within that space) is also controllable.

[0063] In practical embodiments, due to differences in the manufacturing process, the cell control mechanism can also appear in the following forms: A. Please refer to Figure 8. Figure 8 is a structural schematic diagram a of a device for detecting the mechanical force of a cell having a cell control mechanism, in which the cell control mechanism is integrally molded with a base 11. That is, the material forming the cell control mechanism has a plurality of recessed spaces 15, the wall surfaces of the recessed spaces 15 are limiting surfaces 16, the depth of the recessed spaces 15 is the height of the limiting surfaces 16, the bottom of the recessed spaces 15 is the base 11, and a plurality of micropillars 12 are present within each recessed space 15. B. Please refer to Figure 9. Figure 9 is a structural schematic diagram b of a device for detecting the mechanical force of a cell having a cell control mechanism, in which the limiting surface 16 is a structure attached to the base 11. Ninth embodiment: Device for detecting mechanical force of cells

[0064] The difference between this embodiment and the eighth embodiment is that the cell control mechanism in this embodiment is a silicon thin film.

[0065] Specifically, see Figures 10a to 10c. Figure 10a is an actual diagram of a device for detecting the mechanical force of cells using a silicon membrane as a cell control mechanism. In Figure 10a, the silicon membrane is laser-drilled and then attached to a base, with micropillars in each hole. This restricts cell morphology and migration through the silicon membrane, while simultaneously controlling cell-to-cell contact or adhesion. Figure 10b is a fluorescent microscope image of the device for detecting the mechanical force of cells using a silicon membrane as a cell control mechanism under light reflection, and Figure 10c is an enlarged view of Figure 10b. In some embodiments, the size of each hole in the silicon membrane is designed to match the size of a single cell, making it suitable for single-cell adhesion and thereby restricting cell contact, cell morphology, and the range of its migration. Tenth embodiment: System for detecting mechanical forces of cells

[0066] A system for detecting cellular mechanical force includes the device 1 for detecting cellular mechanical force described in the first or second embodiment, an optical signal generator 2, and an optical signal detector 3, wherein the optical signal generator 2 and the optical signal detector 3 are both located below the base 11 of the device 1 for detecting cellular mechanical force. The optical signal generator 2 has a light source, and light emitted from the light source is irradiated onto the light-reflecting layer 13 of the micropillar 12 through an incident light path (sequentially passing through the optically transparent base and the optically transparent micropillar pillar), causing reflection. The reflected light enters the optical signal detector 3 through a reflected light path (sequentially passing through the optically transparent micropillar pillar and the optically transparent base). The optical signal detector 3 can acquire reflected optical signals before and after contact between the micropillar 12 and the cell. In some other embodiments, such a system for detecting cellular mechanical force further includes an optical signal analyzer 4, which can compare, analyze, and calculate the reflected optical signals before and after contact between the micropillar 12 and the cell to obtain cellular mechanical force information (such as the magnitude, direction, and change in the cellular mechanical force within a certain time range). Eleventh embodiment: System for detecting mechanical force of cells

[0067] Please refer to Figure 4. Figure 4 shows a system for detecting the mechanical force of cells according to an eleventh embodiment of the present invention, which includes the device 1 for detecting the mechanical force of cells described in the third embodiment, an optical signal generating device 2, and an optical signal detecting device 3, wherein the optical signal generating device 2 and the optical signal detecting device 3 are all located above the base 11 of the device 1 for detecting the mechanical force of cells, the optical signal generating device 2 has a light source, and the light beam emitted from the light source is irradiated onto the light reflecting layer 13 through the incident light path, causing reflection, and the optical signal detecting device 3 can obtain reflected optical signals before and after contact between the micropillars 12 and the cells.

[0068] In embodiments of the present invention, the optical signal detection device may be a microscope, a charge-coupled device (CCD), a complementary metal-oxide semiconductor (CMOS), a photomultiplier tube (PMT), a photoelectric converter (PT), a film, or other optical signal detection devices with similar functions, without any specific limitations of the present invention. It should be noted that in some embodiments of the present invention, when a microscope is used as the optical signal detection device, a separate optical signal generator is not required; the cell mechanical force detection device of the present invention can be placed directly on the microscope stage, and the microscope's light source can be used as the optical signal generator, with the microscope's objective lens (a 5x objective lens is sufficient; there is no need to rely on a high-magnification optical objective lens) being used as the optical signal detection device. When other optical signal detection devices, such as a charge-coupled device (CCD), are used, a separate optical signal generator is required. In embodiments of the present invention, the optical signal generator may be an LED, a halogen lamp, a laser (e.g., an infrared laser), or other light sources, or other devices having such light sources, without any specific limitations of the present invention.

[0069] Hereinafter, a visualization process for monitoring the mechanical force of cells using a system for detecting the mechanical force of cells related to this embodiment will be specifically introduced.

[0070] See Figure 11. Figure 11 shows a fluorescence microscope image of cell mechanical force monitoring using the cell mechanical force detection system of this example. Specifically, a cell (e.g., a fibroblast cell in this example) is placed on the micropillar of the cell mechanical force detection device, and an optical signal detection device (e.g., a microscope in this example) converts the cell's mechanical force information into an optical signal, forming an image for visualization observation, and providing feedback on changes in the cell's mechanical force in real time. Twelfth embodiment: System for detecting mechanical forces of cells

[0071] Please refer to FIG. 4. FIG. 4 shows a twelfth embodiment of the present invention, illustrating a system for detecting the mechanical force of a cell, which includes the device 1 for detecting the mechanical force of a cell described in the third embodiment, an optical signal generator 2, an optical signal detector 3, and an optical signal analyzer 4. The optical signal generator 2 and the optical signal detector 3 are both located above the base 11 of the device 1 for detecting the mechanical force of a cell. The optical signal generator 2 has a light source, and light emitted from the light source is irradiated onto the light-reflecting layer 13 through an incident light path, causing reflection. The beam splitter 5 may be a semi-transmissive, semi-reflective, or other equivalent optical element, the main purpose of which is to simplify the design of the light path. The optical signal detector 3 can acquire reflected light signals before and after contact between the micropillars 12 and the cell. The optical signal analyzer 4 can compare, analyze, and calculate the reflected light signals before and after contact between the micropillars 12 and the cell to obtain information about the mechanical force of the cell (such as the magnitude, direction, and changes in the mechanical force of the cell within a certain time range).

[0072] In an embodiment of the present invention, the optical signal analysis device may be optical image analysis software such as ImageJ, Matlab, Fluoview, Python, or other optical image analysis elements with similar functions, or a combination of these analysis software, and the present invention is not specifically limited thereto.

[0073] The detection and analysis process of the system for detecting the mechanical force of cells related to this example will be specifically introduced below.

[0074] Please refer to Figures 12a to 12c. Figure 12a is a structural schematic diagram of the cell mechanical force detection system. Figure 12b is an image of the optical reflection signal of the cell mechanical force detection system acquired by an optical signal detection device. Figure 12c is a visualization effect diagram of the force magnitude and distribution.

[0075] As shown in Figure 12a, in the cell mechanical force detection device, each micropillar has a metal reflective layer on the top and an anti-reflective layer on the side. When there are no cells, light shines on the micropillar from below, is completely reflected, and is completely received by the optical signal detection device (e.g., a CCD camera). However, when a cell adheres to the micropillar, the cellular force generated by the cell's movement causes the micropillar to tilt, resulting in a decrease in the reflected signal. The intensity of the cellular force can be calculated by analyzing the optical reflected signal.

[0076] Furthermore, an optical signal detection device (e.g., a CCD camera) collects an image of the optical reflection signal of the cell's mechanical force detection device and a magnified image of the local cell adhesion area (as shown in Figure 12b).Then, the image in Figure 12b is further processed by an optical signal analysis device and converted into a more intuitive visualization effect of the force magnitude and distribution (Figure 12c).

[0077] The specific processing process is as follows: First, based on Figure 12b, a bright-field reflection signal map (I, focused on the cell) is obtained. Next, the image is Fourier transformed, and the high-frequency signal is filtered and an inverse Fourier transform operation is performed to calculate and obtain a micropillar reflection signal image (I0) under non-biased conditions. Next, the I and I0 images are further processed to convert the reflection signal map into a more intuitive cell mechanics map (I0 signal value minus I signal value), and then normalized to obtain a more intuitive cell mechanical force intensity map j. 13th embodiment: Method for calculating mechanical force, correlation between mechanics and optical reflection signal

[0078] This embodiment describes a method for calculating mechanical force in an embodiment of the present invention in combination with a system for detecting mechanical force of cells described in any one of the tenth to twelfth embodiments or a method for detecting mechanical force of cells described in the fourteenth embodiment, and uses a fluid as an external force to verify the correlation between mechanics and optical reflection signals.

[0079] Please refer to Figure 13. In Figure 13, Figure 13a is a structural schematic diagram of a mechanical force detection device for cells in a microfluidic environment before and after fluid is turned on. Figures 13b and 13c are a comparison of bright-field microscope images of micropillars, reflected light signal distribution maps, and superposition effect maps of the two before and after fluid is turned on. Figure 13d is a diagram of the intensity of the reflected light signal before and after fluid is turned on. Figure 13e is a diagram of the linear relationship between the reflected light signal and the shift of the micropillars. Here, the superposition effect map refers to the effect map obtained by superposing the bright-field microscope image of the micropillars and the reflected light signal distribution map.

[0080] First, as shown in Figure 13, a cell mechanical force detection device was integrated into a microfluidic channel. As the external flow rate increased, the micropillars were displaced, and the angle of the reflective layer on the micropillar surface changed (Figures 13a to 13c). Figure 13d shows that the optical reflection signal changed from strong to weak before and after the fluid was turned on. Specifically, the amount of micropillar displacement was changed by varying the flow rate, and a confocal microscope was used to capture the displacement of the top of the micropillar relative to the bottom of the micropillar. The mechanical force experienced by each micropillar could be calculated using the following equation:

[0081] JPEG0007821451000001.jpg826

[0082] where F represents the mechanical force that deflects the micropillar by an angle δ, E represents the Young's modulus, kbend represents the ideal spring constant of an isolated nanopillar, D represents the diameter of the micropillar, and L represents the height of the micropillar.

[0083] At the same time, the optical reflection signal from the top of the micropillar was recorded, and by plotting the optical reflection signal and the displacement of the micropillar in Figure 13e, the linear relationship diagram and linear range between the optical reflection signal (reflecting the mechanical force of the cell) and the displacement of the micropillar could be obtained. Fourteenth embodiment: Method for detecting mechanical force of cells

[0084] A method for detecting mechanical forces in a cell, comprising the steps of: A light beam is emitted from the optical signal generating device 2 of the system for detecting the mechanical force of a cell according to any one of the tenth to twelfth embodiments. The optical signal detection device 3 of the system for detecting the mechanical force of a cell described in any one of the tenth to twelfth embodiments is used to detect light rays after the action of the device 1 for detecting the mechanical force of a cell. The optical signal detection device 3 can acquire reflected light signals before and after the cell comes into contact with the micropillars 12 of the device 1 for detecting the mechanical force of a cell. In some other embodiments, the optical signal analysis device 4 of the system for detecting the mechanical force of a cell can obtain information about the mechanical force of the cell (such as the magnitude, direction, and change in the mechanical force of the cell within a certain time range) by comparing, analyzing, and calculating the reflected light signals before and after the cell comes into contact with the micropillars 12.

[0085] Please refer to Figures 14a to 14d. Figures 14a and 14b are schematic diagrams of the structure of the device for detecting the mechanical force of a cell before and after contact between the micropillar and the cell. Figure 14b shows the reflected light signal acquired by the optical signal detection device (CCD electronic photodetector), and obvious attenuation of the reflected signal can be seen in the area where the force field around the cell is large. Figure 14c shows the monitoring diagram of the cell migration process (the cell membrane is stained, excited with a fluorescent light source, and the migration process is recorded by the CCD electronic photodetector). Figure 14d shows the distribution of the reflected light signal during the cell migration process (the CCD electronic photodetector records the changes in the reflected light signal during the migration process). Figures 14c and 14d show that the reflected light signal is obviously attenuated in the area where the cell applies force. Figure 14d shows the reflected light signal monitored in real time during the cell migration process. By feeding back the mechanical force during the migration process in real time through the reflected light signal, it can be seen that monitoring the reflected light signal during the cell migration process using an optical signal detection device can feed back the changes in the mechanical force of the cell during the cell migration process in real time. Fifteenth embodiment: Method for manufacturing a device for detecting mechanical force of a cell

[0086] A method for manufacturing a cellular mechanical force detection device, comprising the steps of: A layer of light-reflecting layer 13 is provided on the top or upper half-cylindrical surface of the micropillar 12. This results in a micropillar 12 having a reflective layer on the top or upper half-cylindrical surface. Sixteenth embodiment: Method for manufacturing a device for detecting mechanical force of a cell

[0087] A method for manufacturing a cellular mechanical force detection device, comprising the steps of: The entire micropillar 12 is uniformly coated with an anti-reflection layer. The anti-reflection layer on the top or upper half-cylindrical surface is removed. A light-reflecting layer 13 is provided on the top or upper half surface of the micropillar 12 . Seventeenth embodiment: Method for manufacturing a device for detecting mechanical force of a cell

[0088] The difference between this embodiment and the fifteenth and sixteenth embodiments is that the step of "providing a layer of light-reflecting layer 13 on the top or upper half-cylindrical surface of the micropillar 12" is specifically as follows: By uniformly sputtering a reflective metal on the top or upper half-cylindrical surface of the micropillar, a micropillar having a metal light-reflecting layer on the top or upper half-cylindrical surface is obtained. 18th embodiment: Cell identification method

[0089] This embodiment provides a method for cell identification, which includes acquiring cellular mechanical force information using the system for detecting cellular mechanical force described in any of the above embodiments or the method for detecting cellular mechanical force described in any of the above embodiments, and distinguishing between different cell types based on the cellular mechanical force information.

[0090] In some embodiments, in the cell identification method, the step of "obtaining mechanical force information of cells using a system for detecting mechanical force of cells described in any of the above-mentioned embodiments or a method for detecting mechanical force of cells described in any of the above-mentioned embodiments, and distinguishing between different cell types based on the mechanical force information of cells" specifically includes the following steps: S1: Acquire cell information. The cell information includes cell mechanical force information at a certain point on the cell acquired based on a device for detecting the mechanical force of the cell, and the cell mechanical force information includes the magnitude of the mechanical force of the cell at that point. Specifically, an optical signal detection device (or used in combination with an optical signal analysis device) is used to collect cell information for multiple cells on the device for detecting the mechanical force of the cell. This includes collecting information on the magnitude of the mechanical force of the cell at multiple points on each cell, thereby obtaining data on the magnitude of the mechanical force of the cell at multiple points on multiple cells. S2: Preprocess the acquired cell information to form structured cell information. The structured cell information includes the number of cells, the number of cell features, and feature information for each cell feature. At this time, the structured cell information can be regarded as a two-dimensional feature matrix, where N is the number of cells and P is the number of cell features, where P=1, i.e., the cell feature is the magnitude of the mechanical force of the cell. S3: Using the structured cell information as input data, construct a cell feature model through supervised, unsupervised or semi-supervised machine learning, and apply the cell feature model to classify or cluster cells of unknown type or unknown state.

[0091] In some other embodiments, in the cell identification method, the mechanical force information of the cell also includes the direction of the mechanical force of the cell at that point.

[0092] In some other embodiments, in the cell identification method, the mechanical force information of the cell also includes a change in the magnitude or direction of the mechanical force of the cell at that point within a certain time interval.

[0093] In some other embodiments, in the cell identification method, the cell information also includes cell morphology information. 19th Example: Methods for Cell Identification (including Visual Qualitative Identification, and Accurate Qualitative and Quantitative Identification)

[0094] This embodiment specifically provides a method for applying cell mechanical force information obtained by the system for detecting the mechanical force of a cell described in any one of the tenth to twelfth embodiments or the method for detecting the mechanical force of a cell described in the fourteenth embodiment to cell identification.

[0095] Please refer to Figures 15a to 15g. Figure 15a is a fluorescent image of a mixture of healthy cells and non-small cell lung cancer cells. Figure 15b is a diagram of the optical reflection signal distribution of a mechanical force detection device for cells acquired by an optical signal detection device. Figure 15c is a diagram of the visualization effect of force magnitude and distribution. Figure 15d is an enlarged diagram of the cell force distribution of representative single cells of healthy cells and non-small cell lung cancer cells in Figure 15c. Figure 15e is a diagram comparing the cell morphologies of healthy cells and non-small cell lung cancer cells. Figure 15f is a diagram comparing the reflection signal intensity of healthy cells, non-small cell lung cancer cells, and after mixing these two types of cells at different ratios. Figure 15g is a clustering analysis diagram obtained by structuring Figure 15c and then processing it based on the structured cell information.

[0096] Specifically, in this embodiment, healthy cells (Normal) and a non-small cell lung cancer cell line (Cancer) are used as detection targets, and the cell membranes of the healthy cells and lung cancer cells are pre-stained using two different fluorescent dyes (Dil & DIO), mixed in a certain ratio, and then added to a mechanical force detection device for the same cell (in some other embodiments, they can also be added to mechanical force detection devices for different independent cells).

[0097] Furthermore, an optical signal detection device (a microscope is used in this example) collects images of the optical reflection signals of the mechanical force detection device of the cells (as shown in Figure 15b). The high-resolution force field distributions within the two types of cells are then directly rendered by the optical signal detection device and converted into readable optical intensity attenuation signals (reflecting the cell force intensity), which are then displayed in the image (as shown in Figure 15c). Based on the difference in the optical attenuation of the two types of cells displayed in Figure 15c, the two types of cells can be intuitively distinguished by visual observation (qualitative analysis).

[0098] The optical reflection signal in Figure 15c is further processed by an optical signal analyzer. Specifically, in this embodiment, the optical signal analyzer (ImageJ and Python analysis software is used in this embodiment; other image analysis software can be used in other embodiments) collects information on the acquired cell force field in Figure 15c. This includes collecting information on the magnitude of the cell's mechanical force at multiple points on each cell, thereby obtaining data on the magnitude of the cell's mechanical force at multiple points on multiple cells. The acquired cell mechanical force magnitude information is preprocessed to form structured cell information. Analysis is performed based on the structured cell information to obtain a comparison diagram of the cell morphology between healthy cells and non-small cell lung cancer cells (as shown in Figure 15e).

[0099] The structured cell information includes the number of cells, the number of cell features, and feature information for each cell feature (e.g., cell adhesion area and cell circularity in this example). At this time, the structured cell information can be regarded as a two-dimensional feature matrix, where N is the number of cells and P is the number of cell features, where P=2. That is, the cell features are the magnitude of the mechanical force of the cell and the distribution of the mechanical force within the cell.

[0100] Furthermore, using the above-mentioned structured cell information as input data, a cell feature model was constructed using supervised machine learning (two cell lines were pre-stained with different cell membrane dyes (Dil & DIO) and compared). The cell feature model was then trained using a large amount of structured cell information to obtain a clustering analysis diagram as shown in Figure 15g. The resulting cell feature model was then applied to classify and identify cells of unknown type or state. This demonstrates that by using structured cell feature data (the magnitude of the cell's mechanical force and the distribution of the cell's mechanical force within the cell) as input data, an optical signal analyzer (ImageJ and Python analysis software was used in this example; other clustering analysis software can also be used in other examples) can cluster and classify normal healthy cells and cancer cells, thereby enabling the identification of unknown cell types.

[0101] Figure 15e shows that there is no statistically significant difference in the morphology of different cells (including morphological information such as cell adhesion area and cell circularity). Figure 15f shows a clear difference in the reflected signal intensity (reflecting cell force) between normal cells and tumor cells, as well as a certain linear relationship between the reflected signal intensity and the mixing ratio after normal cells and tumor cells are mixed at a certain ratio. This shows that, compared with other cell characteristics (e.g., morphological information such as cell adhesion area and cell circularity in Figure 15e), the cell mechanical characteristics measured by the cell mechanical force detection device of the present invention can more intuitively and accurately identify the state and type of cells (quantitative and qualitative analysis).

[0102] Furthermore, the data in Figures 15d and 15f show that tumor cells exhibit higher mechanical force magnitudes and more heterogeneous distributions than normal cells. After visualizing the mechanical forces of cells, it is possible to intuitively see with the naked eye that the force field characteristics of different cells have clear differences. Furthermore, after using image analysis software to perform a structuring process on the force field magnitudes at each point of different cells, the cell morphology information in Figure 15e, the reflected signal intensity (reflecting the cell force) in Figure 15f, and the clustering analysis diagram in Figure 15g are comprehensively analyzed. Through the comprehensive analysis of the structured information of the force fields at each point of cells, the present invention can perform clustering and quantitative analysis of different cells (e.g., healthy cells and non-small cell lung cancer cells in this example), thereby enabling accurate identification of cell types.

[0103] As described above, the cell mechanical force detection device of the present invention not only enables qualitative analysis through intuitive visual discrimination, but also enables more intuitive and accurate identification of cell states and types (quantitative and qualitative analysis) based on the measured cell mechanical characteristics. Furthermore, it has been demonstrated that cell types can be better distinguished by using the cell force field as a marker. 20th embodiment: Method for detecting cell vitality

[0104] This embodiment provides a specific method for applying cellular mechanical force information obtained by the system for detecting cellular mechanical force described in any one of the tenth to twelfth embodiments or the method for detecting cellular mechanical force described in the fourteenth embodiment to monitoring cellular vitality.

[0105] Please refer to Figures 16a to 16c. Figure 16a is a schematic diagram of the operation flow of the cell vitality detection method. Figure 16b is a comparative diagram of the cell vitality measured by the MTT method and the cellular mechanical force measured by the device, system, or method of the present invention after treating A549 cells with different doses of 5FU for 24 hours. Figure 16c is a comparative diagram of the cell vitality measured by the MTT method and the cellular mechanical force measured by the device, system, or method of the present invention after treating A549 cells with different doses of 5FU for different periods of time.

[0106] Specifically, in this example, non-small cell lung cancer cells A549 were cultured on multiple cell mechanical force detection devices and treated with different doses of the cell proliferation-suppressing drug 5-fluorouracil (5-FU). The cell mechanical force was monitored at different time points using the cell mechanical force detection system described in any one of Examples 10 to 12 or the cell mechanical force detection method described in Example 14, and cell proliferation and cytotoxicity were monitored at different time points using a CCK-8 reagent kit. At the same time, cell vitality measured by the MTT assay was used as a control group, and the data in Figures 16b and 16c were obtained.

[0107] As shown in Figures 16b and 16c, after measurements using the conventional MTT measurement method and the device, system, or method described in the present invention, the cell vitality measured by the MTT measurement method and the cell vitality reflected by the cell mechanical force both tend to gradually decrease in a dose-dependent manner, that is, there is a positive correlation between the cell mechanical force and the cell vitality.

[0108] Furthermore, as shown in Figure 16b, after 24 hours of treatment with different doses of 5FU, mechanical force reflected a greater decrease in cell vitality compared to the control group (DMSO), allowing for a more intuitive assessment of cell vitality. As shown in Figure 16c, after 12 hours of treatment with 5FU, no significant change in cell vitality was observed as measured by the MTT method. However, by measuring mechanical force, a decrease in cell mechanical force could be observed at an earlier time point, before the decrease in cell metabolic activity was detected by the MTT method. Specifically, a clear decrease was observed at 6 hours with a treatment dose of 0.5 μM, and at 3 hours with a treatment dose of 1 μM, allowing for a more sensitive assessment of the decrease in cell vitality.

[0109] As described above, this example demonstrates that directly detecting the mechanical force of cells using a cell mechanical force detection device is a highly sensitive and effective method for evaluating the drug response activity of cells. 21st Embodiment: Method for detecting cell state

[0110] This embodiment provides a specific method for applying cellular mechanical force information obtained by the system for detecting cellular mechanical force described in the twelfth embodiment or the method for detecting cellular mechanical force described in the fourteenth embodiment to analyzing and determining the state of cells.

[0111] Please refer to Figures 17a to 17f. Figure 17a is a diagram of the operation process of the cell state detection method. Figure 17b is a fluorescent microscope image of M0 macrophages differentiated into the M1 state. Figure 17c is a fluorescent microscope image of M0 macrophages differentiated into the M2 state. Figure 17d is a comparison diagram of the cell adhesion area of ​​M0 macrophages, the M1 state, and the M2 state. Figure 17e is a comparison diagram of the cell circularity of M0 macrophages, the M1 state, and the M2 state. Figure 17f is a comparison diagram of the mechanical force of M0 macrophages, the M1 state, and the M2 state.

[0112] Specifically, in this example, macrophages were used as the detection target. They were placed on the micropillars of a mechanical force detection device for different individual cells, and the macrophages were induced to differentiate from M0 to M1 and M2 states using the endotoxin LPS and interleukin IL4, respectively. The M0 state served as the control. After cell differentiation, images (Figures 17b and 17c) were collected using an optical signal detection device (a microscope was used in this example). Images (Figures 17b and 17c) were further processed and analyzed using optical signal analysis software (ImageJ and Python), which converted the data into structured information and analyzed to produce the data shown in Figures 17d to 17f. From the data shown in Figures 17a to 17f, clear differences were observed between M0 macrophages and the differentiated M1 and M2 states, both intuitively (Figures 17b and 17c) and quantitatively (Figures 17d to 17f). 22nd Example: Method for detecting multicellular polymers

[0113] This embodiment provides a specific method for applying cellular mechanical force information obtained by the system for detecting cellular mechanical force described in the twelfth embodiment or the method for detecting cellular mechanical force described in the fourteenth embodiment to analyzing and determining the state of cells.

[0114] Specifically, in this embodiment, the multicellular polymer is provided to be attached to the cellular mechanical force detection device in several ways, of which two specific attachment methods are provided in this embodiment. First, a culture medium is placed on the micropillars of the cell mechanical force detection device, and cells are transplanted into the culture medium on the micropillars and cultured to obtain a multicellular polymer. In some other embodiments, this combination method allows real-time monitoring of the cell culture process while visualizing the mechanical force information of the cells, which can be applied to the effects of chemical, biological, and physical external stimuli such as culture medium and drugs on cell growth. Second, the cultured multicellular polymer is directly attached onto the micropillars of the mechanical force sensing device for cells to detect the force.

[0115] More specifically, this example provides a method for applying tumor cell polymers cultured on a cell mechanical force detection device to drug sensitivity testing, which includes the following steps: S1. Tumor cell polymer generation: Apply 50 μg / mL of FN to the top of the micropillars of the cell mechanical force detection device and sterilize with UV light for 30 minutes. Seed MCF-7 breast cancer cells (approximately 1 × 105 to 9 × 105 cells) on the top surface of the micropillars of the cell mechanical force detection device (the number of micropillars is not limited). Immerse the cell mechanical force monitoring device in 3d GRO™ Spheroid Medium (S3077) culture medium and culture for at least 3 days to induce tumor cell polymer generation. S2. The tumor cell polymers prepared above were used in a 5-Fu drug sensitivity test: 200 μM 5-Fu was added to the tumor cell polymers and cultured for one day. For the test, the cells cultured for one day before and after 5-Fu addition were subjected to a mechanical force detection device (tumor cell polymers were generated and combined with the culture medium). An optical signal detection device (CCD photodetector) was used to capture optical reflection signals, and an optical signal analysis device (Image J) was used to obtain images of the cell force distribution. The results are shown in Figure 18.

[0116] See Figures 18a and 18b. Figure 18a shows the characteristics of a first morphology of tumor cell polymers with and without the action of 5-Fu. Figure 18b shows the characteristics of a second morphology of tumor cell polymers with and without the action of 5-Fu. From left to right, the images show a mixed image of cell membrane fluorescence and reflectance signals (1), an optical reflectance signal (2), a cell nucleus (3), a cell membrane (4), and a cell force visualization image (5) processed by an optical signal analyzer (ImageJ). Because cells are heterogeneous and tumor cells are different from one another, cell polymers also have various morphologies, and therefore, cell polymers adhere together in different morphologies. In this example, two representative morphologies are selected for cell morphology detection. The first morphology refers to the cell morphology of two relatively large cells adhered together. The second morphology refers to the cell morphology of a group of small cells adhered together.

[0117] 18a and 18b show that the reflected signal becomes significantly weaker after the cell vitality is reduced by treatment with an antitumor drug. Furthermore, the change in the cell mechanical force before and after the action of 5-Fu reveals a clear difference in the drug sensitivity of the two different types of tumor cell polymers. This demonstrates that the cell mechanical force detection device of the present invention can measure the cell mechanical force of multicellular polymers (e.g., tumor polymers), can be used to monitor the vitality state of cell polymers through the cell mechanical force, and can distinguish between different cell morphologies.

[0118] In the present definition, cellular polymers refer to: Cells are the basic structural and functional units of living organisms, and cells typically proliferate or differentiate to form clusters of two or more cells together to form cell colonies, or multicellular polymers. Multicellular polymers include cell populations obtained by in vitro or in vivo culture, such as tumor polymers.

[0119] As described above, the detection of mechanical force of cells, cell identification, cell state detection, and cell vitality detection using the cell detection device of the present invention has very high sensitivity and effectiveness, and can realize real-time monitoring, which can be applied to the response status of cells to drugs under drug treatment.

[0120] Compared with the prior art, the above technical solution has the following advantages: First, it has high throughput and low cost. Compared with conventional TFM and conventional micropillar arrays, the technical solution of the present invention is independent of the microscope and greatly simplifies the operation process. This is because it does not require high-resolution imaging by a microscope, and can achieve high-throughput cell monitoring by simply monitoring the intensity of reflected light, at a low cost. Second, it offers single-cell resolution. Its high resolution allows for real-time monitoring of individual cells, and by combining it with other single-cell analysis techniques, it is possible to measure the heterogeneity of cellular responses to drugs. Because it enables real-time monitoring, does not require fluorescence, and avoids laser-induced phototoxicity, it is suitable for long-term monitoring and can be used to study long-term cellular responses to drugs. Its high sensitivity allows the micropillar deformation signal to be amplified by the reflected signal, thereby increasing the sensitivity of deformation monitoring. Detection of bending deformation of micro- and nanopillars typically relies on optical systems (such as microscopes), but the smaller the micropillar size, the greater the demands on the precision and resolution of the optical system. For example, a micropillar measuring 2 μm wide and 6 μm high can only be effectively observed by combining a 20x or higher objective lens with a confocal system. In contrast, our method utilizes the principle of specular reflection to detect the attenuation of reflected light, effectively amplifying the micropillar deformation signal. Experiments have confirmed that the same signal can be observed under a 5x objective lens. Combined with a special readout system, it can effectively detect the deformation of micro / nanopillars without relying on high-magnification optical objective lenses, greatly reducing the cost of the system and effectively improving throughput. Third, it can simulate the cellular microenvironment, including the composition and morphology of the extracellular matrix, thereby meeting a wider range of technological needs.

[0121] It should be noted that although the above embodiments have been described in detail herein, they are not intended to limit the scope of patent protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the creative concept of the present invention, or any conversion of equivalent structures or processes made by utilizing the contents of the present specification and accompanying drawings, including the direct or indirect application of the above technical solutions to other related technical fields, are all included in the scope of patent protection of the present invention.

Claims

1. 1. A device for detecting mechanical forces on a cell, comprising: The base and a micropillar array disposed on the base and consisting of a plurality of micropillars deformable by the mechanical force of cells; the base is a light-transmitting base, the columnar body of the micropillar is light-transmitting, the top of the micropillar has a light-reflecting layer, and the columnar surface other than the top of the micropillar has an anti-reflection layer; A device for detecting the mechanical force of a cell, characterized in that the base and the micropillars form an optical path in which a light beam emitted from a light source passes through the base and the micropillar body and is reflected by the light-reflecting layer, and the reflected light passes through the micropillar body and the base.

2. The device for detecting the mechanical force of a cell according to claim 1, characterized in that the light-reflecting layer is a metal foil layer, a metal oxide or metal salt, ultrafine particle beads or microprisms, one type of organic reflective material, or a combination thereof.

3. 2. The device for detecting the mechanical force of a cell according to claim 1, wherein a substance having cell adhesion properties is disposed on the top end surfaces of all or some of the micropillars of the micropillar array.

4. The device for detecting the mechanical force of a cell described in claim 3, characterized in that the substance having cell adhesion activity includes one or more of extracellular matrix molecules including collagen, fibronectin, vitronectin, laminin, and elastin, extracellular matrix mimics including polypeptides containing an RGD adhesion sequence, substances that promote cell adhesion including polylysine, and substances that interact with cell surface receptors.

5. 4. The device for detecting the mechanical force of a cell according to claim 3, characterized in that a predetermined pattern consisting of a group of micropillars made of a material having cell adhesive properties is arranged on the top end surfaces of some of the micropillars.

6. 6. A device for detecting the mechanical force of a cell as described in claim 5, characterized in that a substance having cell adhesion inhibitory properties is disposed on the top end surfaces of some of the micropillars in a predetermined region of the micropillar array.

7. The device for detecting the mechanical force of a cell according to claim 1 , characterized in that the cross-sectional shape of the micropillars is circular, elliptical, or polygonal.

8. The device for detecting the mechanical force of a cell described in claim 1, characterized in that the size range of the micropillars and micropillar arrays is a micropillar height of 10 nm to 500 μm, a micropillar spacing of 10 nm to 50 μm, and a micropillar top diameter of 50 nm to 50 μm.

9. The device for detecting the mechanical force of a cell described in claim 1, further comprising a cell control mechanism, wherein the cell control mechanism includes one or more limiting surfaces, which are arranged perpendicular to the plane on which the base is located, are connected to the base, or are flat or curved surfaces integrally formed with the base, and the height of the limiting surfaces is higher than that of the micropillars and surrounds a predetermined number of micropillars.

10. A system for detecting a mechanical force of a cell, comprising: the device for detecting a mechanical force of a cell according to claim 1; an optical signal generating device; and an optical signal detecting device, The optical signal generating device has a light source, and light emitted from the light source is irradiated onto the light reflecting layer of the micropillar through an incident light path; A system for detecting the mechanical force of a cell, characterized in that the optical signal detection device is for detecting light reflected from the optical reflection layer of the micropillar, and the light reflected from the optical reflection layer enters the optical signal detection device through a reflected optical path.

11. the base of the device for detecting the mechanical force of the cell is a light-transmitting base, the columnar bodies of the micropillars are light-transmitting, and the tops of the micropillars have a light-reflecting layer; The light emitted from the light source is irradiated through an incident light path from the base of the device for detecting the mechanical force of the cell to the light reflecting layer of the micropillar; The system for detecting the mechanical force of a cell described in claim 10, characterized in that the optical signal detection device is for detecting light reflected from an optical reflection layer on the top of the micropillar, and the light reflected from the optical reflection layer enters the optical signal detection device through a reflection optical path.

12. The system for detecting a mechanical force of a cell according to claim 10, further comprising an optical signal analyzer for analyzing the optical signal.

13. 1. A method for detecting mechanical forces in a cell, comprising: generating light using an optical signal generating device in the system for detecting mechanical force of a cell according to claim 10; A method for detecting the mechanical force of a cell, comprising the step of detecting light after the action of the device for detecting the mechanical force of a cell using an optical signal detection device in the system for detecting the mechanical force of a cell described in claim 10.

14. The method for detecting the mechanical force of a cell described in claim 13 further includes a step of using an optical signal analysis device to compare and analyze the reflected light before and after the action of the mechanical force of the cell generated between the device for detecting the mechanical force of the cell and the test cell, and obtaining the mechanical force information of the cell, wherein the mechanical force information of the cell includes the magnitude, direction or frequency of change of the mechanical force of the cell.

15. A method for detecting a state of a cell, comprising the steps of acquiring mechanical force information of a cell through the system for detecting a mechanical force of a cell according to claim 10 or the method for detecting a mechanical force of a cell according to claim 13, and analyzing and determining a state of the cell based on the mechanical force information of the cell, A method for detecting a cell state, wherein the cell state comprises cell adhesion, cell activity, cell differentiation / activation, cell proliferation and / or cell migration.

16. 1. A method for cell identification, comprising: A method for cell identification, comprising a step of acquiring mechanical force information of cells through a system for detecting mechanical force of cells described in claim 10 or a method for detecting mechanical force of cells described in claim 13, and distinguishing cell types based on the mechanical force information of cells.

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