Cellular mechanical force detection system, method, device and method of making same

Abstract

To provide a solution for quantitative measurement and monitoring of cellular mechanical forces that overcomes the shortcomings of existing technologies, requires no microscope, offers real-time, high-throughput, and low-cost capabilities, the inventors provide a cellular mechanical force detection device, comprising: a base, and a micropillar array consisting of multiple micropillars disposed on the base, capable of deformation under cellular mechanical forces. Each micropillar has a light-reflecting layer at its top or upper surface. The inventors also provide a cellular mechanical force detection system including the above-mentioned device, a detection method for detecting cellular mechanical forces using the above-mentioned device, and a method for preparing the above-mentioned device. Unlike existing technologies, the above-mentioned solution offers advantages such as high throughput and low cost, single-cell resolution, real-time monitoring, high sensitivity, and the ability to simulate the cellular microenvironment; it can also simulate the composition and morphology of the extracellular matrix, thus meeting a wider range of technical needs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a cell mechanical force detection system, a cell mechanical force detection method, a cell mechanical force detection device, and a method for preparing the cell mechanical force detection device. Background Technology

[0002] Cells exert minute mechanical forces on their surrounding microenvironment. Cellular forces play crucial roles in processes including adhesion, migration, proliferation, differentiation, and apoptosis. Together with other biochemical signals, they play a vital regulatory role in embryonic development, stem cell differentiation, immune processes, wound repair, and cancer metastasis, thus becoming targets for the treatment of many diseases. For example, previous studies have found significant differences in the maximum cellular mechanical forces of normal cells, benign cells, and malignant tumor cells. Therefore, high-throughput and precise measurement of cellular mechanical forces will become a core technology for many biomedical applications. However, the significance of measuring maximum cellular mechanical forces is limited; high-resolution, real-time, and high-throughput mechanical sensors will be the core requirement for next-generation cellular force measurement tools.

[0003] Current techniques for measuring cellular mechanical forces primarily include force microscopy (TFM), micro / nano cantilever arrays, and micropillar arrays. The main principle is to calculate the cellular mechanical force by measuring the deformation of an elastic substrate caused by the force exerted by the cell on it. To date, force microscopy (TFM) is the most widely used technique for measuring cellular mechanical forces. Its principle is based on a continuous elastic substrate (e.g., a wrinkleable thin silica membrane or a polyacrylamide (PA) gel embedded with fluorescent microbeads). During cell culture on this elastic substrate, the cells exert mechanical forces, causing deformation. During this deformation, the fluorescent microbeads undergo corresponding displacement. The movement of the fluorescent microbeads is tracked using fluorescence microscopy, and substrate strain information is obtained through image processing. Given the substrate elasticity, the cellular mechanical force can be deduced from a mechanical model. However, this method has several drawbacks. First, it relies on fluorescence microscopy imaging and requires complex calculations, resulting in a complex process with low throughput and high cost. Second, TFM does not directly measure cellular mechanical forces; instead, it infers them by observing changes in the position of fluorescent microbeads within the substrate. Although special processes can ensure that most particles are deposited on the surface, prolonged immersion can lead to their escape or descent, ultimately reducing the density of fluorescent particles on the surface. Fluorescence microscopy typically has a large depth of field, potentially capturing particles at different planes, which can cause deviations in subsequent displacement calculations and result in inaccurate measurements. Furthermore, prolonged immersion in culture medium alters the gel's elastic modulus, inevitably affecting the accuracy of cellular mechanical force calculations. Therefore, gel elastic modulus calibration is required during measurement, significantly increasing the workload. In addition, prolonged laser irradiation can cause phototoxicity to cells and trigger quenching of fluorescent microbeads. Therefore, TFM is unsuitable for long-term continuous monitoring of cellular mechanical forces, while studies of cell growth and differentiation and their drug responses typically require extended monitoring periods. Thus, the aforementioned limitations of TFM greatly restrict its application in the biomedical field.

[0004] Compared to TFM (Transient Functional Mechanism), micro / nano sensors (such as microcantilever arrays and micropillar arrays) can also be used to directly measure cellular mechanical forces. For example, in micropillar arrays, cells adhere to the surface above the micropillars; by taking images of the bottom and top of the micropillars under a microscope, the bending deformation of the micropillars can be calculated, thus inferring the magnitude and direction of the cellular mechanical force at that point. Like TFM, micro / nano sensors also rely on high-resolution microscopy imaging, which places high demands on the equipment and is prone to errors during the imaging process, leading to inaccurate results. Furthermore, the images obtained from the microscope require complex image processing, and the magnitude of the cellular mechanical force needs to be calculated based on mechanical models. This process is complex and time-consuming, making it difficult to achieve real-time, high-throughput, low-cost, and long-term cell monitoring. Therefore, most existing technologies are limited to research in the field of biomechanics and are difficult to apply practically. Summary of the Invention

[0005] Therefore, there is a need for a solution that can overcome the shortcomings of existing technologies, provide a real-time, high-throughput, and low-cost quantitative measurement and monitoring solution for cell mechanical forces, and address the core requirements of next-generation cell force measurement tools.

[0006] To achieve the above objectives, the inventors provide a device for detecting cellular mechanical forces, comprising:

[0007] Base, and

[0008] A micropillar array consisting of multiple micropillars that can deform under the action of cellular mechanical forces, set on a base, wherein the top or upper part of the micropillar has a light-reflecting layer.

[0009] Furthermore, in the aforementioned cellular mechanical force detection device, the base is a light-transmitting base, and the column of the micropillar can transmit light; the top of the micropillar has a light-reflecting layer.

[0010] Furthermore, in the aforementioned cellular mechanical force detection device, the surface of the micropillar has an anti-reflective layer.

[0011] Furthermore, in the aforementioned cellular mechanical force detection device, the light-reflecting layer is a combination of one or more of the following: a metal foil layer, a metal oxide or metal salt, an ultrafine glass bead or a microprism, and an organic reflective material.

[0012] Furthermore, in the aforementioned cellular mechanical force detection device, the top end face of all or part of the micropillars of the micropillar array is provided with a substance that has a cell adhesion effect.

[0013] Furthermore, in the aforementioned cellular mechanical force detection device, the substance with cell adhesion function includes one or more of the following substances: extracellular matrix molecules, including collagen, fibronectin, hyalin, laminin, or elastin; extracellular matrix mimicry substances, including polypeptides containing RGD adhesion sequences; substances with cell adhesion promotion mechanisms, including polylysine; and substances that interact with cell surface receptors.

[0014] Furthermore, in the aforementioned cellular mechanical force detection device, a substance with cell adhesion properties is provided on the top end face of a portion of the micropillars in the preset region of the micropillar array.

[0015] Furthermore, in the aforementioned cellular mechanical force detection device, the micropillar array has a substance with cell adhesion inhibition on the top end face of the micropillars that do not have a substance with cell adhesion effect.

[0016] Furthermore, in the aforementioned cellular mechanical force detection device, the cross-sectional shape of the micropillar is circular, elliptical, or polygonal.

[0017] Furthermore, in the aforementioned cellular mechanical force detection device, the size range of the micropillar array includes: pillar height of 10nm to 500μm, pillar spacing of 10nm to 50μm, and pillar surface diameter of 50nm to 50μm.

[0018] Furthermore, the cellular mechanical force detection device also includes a cell restriction mechanism, which includes one or more restriction surfaces. The restriction surface is a plane or curved surface that is perpendicular to the plane of the base, connected to the base, or integrally formed with the base. The height of the restriction surface is higher than that of the micropillars and surrounds a preset number of micropillars.

[0019] The inventors also provide a cell mechanical force detection system, including the cell mechanical force detection device, light signal generator, and light signal detection device described in the above technical solution;

[0020] The optical signal generating device has a light source, and the light emitted by the light source illuminates the light-reflecting layer of the micropillar through the incident light path;

[0021] The optical signal detection device is used to detect the light reflected from the light-reflecting layer of the micropillar. The light reflected from the light-reflecting layer enters the optical signal detection device through the reflected light path.

[0022] Furthermore, in the aforementioned cell mechanical force detection system, the base of the cell mechanical force detection device is a light-transmitting base, and the column of the micropillar can transmit light; the top of the micropillar has a light-reflecting layer;

[0023] The light emitted by the light source shines from the base of the cell mechanical force detection device onto the light-reflecting layer of the micropillar through the incident light path;

[0024] The optical signal detection device is used to detect the light reflected from the light-reflecting layer at the top of the micropillar. The light reflected from the light-reflecting layer enters the optical signal detection device through the reflected light path.

[0025] Furthermore, the cell mechanical force detection system also includes a light signal analysis device for analyzing light signals.

[0026] The inventors also provided a method for detecting cellular mechanical forces, comprising the following steps:

[0027] The light signal generator in the cell mechanical force detection system described above emits light;

[0028] The light signal detection device in the cell mechanical force detection system described above is used to detect the light after it has been acted upon by the cell mechanical force detection device.

[0029] Furthermore, the method for detecting cellular mechanical force further includes the step of: using an optical signal analysis device to compare and analyze the reflected light of the cellular mechanical force detection device and the cell under test before and after the cellular mechanical force is applied, in order to obtain cellular mechanical force information.

[0030] The inventors also provide a method for preparing a cell mechanical force detection device, which includes the following steps: laying a reflective layer on the top or upper half of the micropillar to obtain a micropillar with a reflective layer on the top or upper half of the micropillar.

[0031] Furthermore, in the method for preparing the cell mechanical force detection structure, the step prior to "laying a reflective layer on the top or upper half of the micropillar" includes the following step:

[0032] A uniform anti-reflective layer is coated onto the entire micropillar;

[0033] Remove the anti-reflective layer from the top or upper half of the cylinder.

[0034] Furthermore, in the method for preparing the cell mechanical force detection structure, the step of "laying a reflective layer on the top or upper half of the micropillar to obtain a micropillar with a reflective layer on the top or upper half of the micropillar" specifically means: uniformly sputtering a reflective metal layer on the top or upper half of the micropillar to obtain a micropillar with a metal light reflective layer on the top or upper half of the micropillar.

[0035] The above technical solution has the following advantages, unlike existing technologies:

[0036] First, high throughput and low cost: Compared with existing TFM and ordinary micropillar arrays, the technical solution of this invention eliminates the dependence on microscopes and greatly simplifies the operation process. Because there is no need for high-resolution imaging with a microscope, high-throughput cell monitoring can be achieved simply by monitoring the intensity of reflected light, and the cost is low.

[0037] Secondly, single-cell resolution: high resolution allows for real-time monitoring of each cell, and can be combined with other single-cell analysis techniques to measure the heterogeneity of cellular responses to drugs; real-time monitoring: no fluorescence is required, avoiding the phototoxicity of lasers on cells, making it suitable for long-term monitoring and applicable to studying long-term cellular responses to drugs; high sensitivity: by amplifying the micropillar deformation signal through reflected signals, the sensitivity of deformation monitoring is increased. Detection of bending deformation of micro and nanopillars generally relies on optical systems (such as microscopes), but the smaller the micropillar size, the higher the precision and resolution requirements of the optical system. For example, a micropillar 2 micrometers wide and 6 micrometers high requires a 20x or higher objective lens with a conjugate focal system for effective observation. Utilizing the principle of specular reflection, the signal of micropillar deformation is amplified by detecting the attenuation of reflected light. Experiments have verified that the same signal can be observed under a 5x objective lens. With a special readout system, micro / nanopillar deformation can be effectively detected without relying on a microscope, thereby greatly reducing system costs and effectively increasing throughput.

[0038] Furthermore, it can simulate the cellular microenvironment and the composition and morphology of the extracellular matrix, thus meeting a wider range of technical requirements. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the structure of a cell mechanical force detection device in the first embodiment of the present invention;

[0040] Figure 2 This is a schematic diagram of a cell mechanical force detection system related to the ninth embodiment of the present invention;

[0041] Figure 3 This is a schematic diagram of a cellular mechanical force detection device related to the tenth embodiment of the present invention;

[0042] Figure 4 A schematic diagram (a) of a cellular mechanical force detection device with a cell restriction mechanism;

[0043] Figure 5 Schematic diagram b of a cellular mechanical force detection device with a cell restriction mechanism.

[0044] Explanation of reference numerals in the attached figures:

[0045] 1- Detection device for cellular mechanical forces

[0046] 2-Optical signal generator

[0047] 3-Optical signal detection device

[0048] 4-Optical Signal Analysis Device

[0049] 5-Spectrum Splitter

[0050] 11-Base

[0051] 12-microcolumn

[0052] 13-Light Reflecting Layer

[0053] 15-Recessed Space

[0054] 16-Restriction Surface Detailed Implementation

[0055] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0056] First Embodiment

[0057] Please refer to Figure 1 Figure a is a schematic diagram of a cellular mechanical force detection device. The device includes a light-transmitting base 11 and micropillars 12 disposed on the base 11, which can deform under cellular mechanical forces. The top of each micropillar 12 is coated with a light-reflecting layer 13, the thickness of which is 5 nm (in some other embodiments, the thickness of the light-reflecting layer 13 can be between 5 nm and 20 nm—the coating thickness is related to the coating material; when using the same coating material, the choice of coating thickness should be limited to ensuring light transmission, micropillar stability, and preventing the connection with the micropillars from detaching). The micropillars 12 can transmit light; the cluster of arrows pointing in opposite directions in the figure represents incident and reflected light. (Note: The term "coating" is used in this embodiment only to indicate that the light-reflecting layer 13 in this embodiment can be prepared by a coating process, and does not limit the light-reflecting layer 13 to necessarily being prepared by a coating process.)

[0058] When the cell mechanical force detection device 1 described in this embodiment is put into use, the number of micropillars 12 will be more than one. Please refer to Figure 2 This is a schematic diagram of the cellular mechanical force detection system related to the ninth embodiment of the present invention; Figure 2 This can be used to understand this embodiment. Figure 2The system demonstrated, in addition to the cell mechanical force detection device 1 described in this embodiment, also includes: a light signal generator 2 with a light source and a light signal detection device 3 disposed below the base 11. Light emitted from the light source illuminates the light-reflecting layer of the micropillar 12 through the light-transmitting base 11 of the cell mechanical force detection device 1 via an incident light path. The light signal detection device 3 detects the light reflected from the light-reflecting layer 13 at the top of the micropillar. The light reflected from the light-reflecting layer 13 passes through a reflection light path and a beam splitter 5 before entering the light signal detection device 3. After obtaining the reflected light signal, a light signal analysis device 4 can compare and analyze the reflected light from the cell mechanical force detection device 1 before and after the cell mechanical force is applied to the test cell to obtain cell mechanical force information. When the micropillar 12 is not under force, it should remain upright to maximize the reflection of the detection light; however, when the micropillar 12 comes into contact with the cell, it bends under the action of cell mechanical force, resulting in a decrease in light reflection. Therefore, the greater the cellular mechanical force, the smaller the light reflection signal should be. In this way, the magnitude of the cellular mechanical force at that point can be easily deduced by observing the intensity of the light reflection signal.

[0059] Furthermore, the measurement light source in this embodiment can be an infrared laser of a certain intensity. Traditional microcolumn measurements require high-resolution imaging, and using lasers during this process can easily cause cell phototoxicity or quenching of sample fluorescence. However, in this embodiment, since only the reflected signal needs to be measured, the effect of infrared lasers within a certain intensity on cells is negligible, making it suitable for long-term cell monitoring.

[0060] Second Embodiment

[0061] The difference from the first embodiment is that the micropillar 12 not only has a light-reflecting layer 13 on its top end face, but also on the upper half of the cylindrical surface (i.e., the curved surface connecting the two end faces of the pillar). In fact, in other embodiments, except for the scheme of setting the light-reflecting layer 13 on the lower half of the side cylindrical surface of the micropillar 12 due to its poor practical effect, as long as the light-reflecting layer 13 is set on the upper half of the side surface of the micropillar 12, the detection effect desired by the present invention can basically be achieved. That is to say, in some other embodiments, the light-reflecting layer 13 can even be laid at any local position on the upper half of the side cylindrical surface or at a local position on the top, without necessarily covering the entire upper half of the cylindrical surface or the entire top end face, and the expected purpose can still be achieved, although the acquired data and the effect of subsequent calculations may differ.

[0062] Furthermore, the first and second embodiments of the present invention define the "cylindrical surface" and "end face" of the micropillar. That is, a normally understood independent column should have two end faces and a curved surface (cylindrical surface) connecting the two end faces. However, the micropillar in the present invention, due to the presence of the base, only has one end face, namely the top end face, while the other end is fixedly connected to the base or integrally formed with the base. However, in other embodiments, the top end face may be a smooth, integrally connected curved surface with the cylindrical surface, and does not necessarily have an intersection line or clear boundary as shown in the first or second embodiments. In this case, the location of the light-reflecting layer 13 will also be understood as the upper half of the column, and cannot be limited to an "end face" or "cylindrical surface."

[0063] Third Embodiment

[0064] Please see Figure 3 , Figure 3 This is a schematic diagram of a cellular mechanical force detection device according to the third embodiment of the present invention, used to illustrate this embodiment. This embodiment differs from the first and second embodiments in that the light transmittance of the base 11 and the micropillars 12 of the micropillar array is not required; they can be transparent, opaque, or semi-transparent. In this case, only the positions of the light signal generator 2 and the light signal detection device 3 need to be changed, placing them above the base 11. Thus, each time a micropillar bends, the light signal received by the light signal detection device 3 will change relative to when the micropillar 12 is upright and undeformed. By analyzing the changes in the light signal before and after bending, the relative magnitude of the cellular mechanical force can also be obtained. After correction with a standard value, the absolute magnitude of the cellular mechanical force can be obtained.

[0065] Fourth embodiment

[0066] The difference between this embodiment and the first to third embodiments is that, on the surface of the micropillar 12, in areas other than those with the light-reflecting layer 13, an anti-reflective layer is provided. This design reduces interference from reflected light signals that may be present on the surface of the pillar, enhances the signal-to-noise ratio, and makes the detection results more accurate.

[0067] In some embodiments, the light-reflecting layer 13 may be a layer of gold foil. In other embodiments, the light-reflecting layer 13 may also be other metal layers or other reflective materials with light-reflecting functions. The reflective effect, ease of preparation of the reflective layer, and cost may vary depending on the material, and the choice can be made based on specific conditions in actual operation.

[0068] In the first to fourth embodiments, the cross-sectional shape of the micropillar 12 is circular. In other embodiments, the cross-sectional shape of the micropillar 12 can also be elliptical or polygonal. Different cross-sections in various specific embodiments of the present invention can achieve different purposes. For example, a circular cross-section is isotropic, meaning the mechanical properties of the micropillar itself are not sensitive to direction. However, an elliptical cross-section is anisotropic, meaning the mechanical properties of the micropillar itself are sensitive to direction. This allows control over the sensitivity of different directions to the force field and can, to some extent, regulate the tropism of cells (most cells are actually asymmetrical in geometry; in this invention, cell tropism refers to the morphological asymmetry, polarity, or directionality exhibited by the cell. For example, if an ellipse is used to fit the shape of a cell projection, the major axis of the ellipse can be considered as the direction the cell possesses). Because if the cross-section is elliptical, it has a major axis and a minor axis, making it much easier to push the micropillar along the minor axis than along the major axis, resulting in greater deformation under relative stress. In some extended embodiments, if cells are seeded on these micropillars, the anisotropic mechanical interaction between the cells and the micropillars will cause the cells to grow along one side. When applied to fluids, this can be used to determine the direction of the fluid.

[0069] In the first to fourth embodiments, the dimensions of the micropillar array are: pillar height 10 nm to 500 μm, pillar spacing 10 nm to 50 μm, and pillar surface diameter 50 nm to 50 μm. Micropillars within this size range can meet the basic usage requirements for micropillars used as sensors, i.e., they must be deformable without collapsing. Furthermore, adjusting the dimensions of different micropillar arrays can achieve the following functions: for example, by adjusting the aspect ratio of the micropillars (which can be understood at the micropillar level as the ratio of height to cross-sectional diameter / side length / long-diameter ratio), a certain degree of micropillar deformation performance regulation can be achieved, thereby better simulating the in vivo organ and tissue environment (e.g., bone and nerve tissues of different hardness).

[0070] Furthermore, the overall size of the array, or the number of micropillars 12 on a given area of ​​base 11, also affects the ligand density, i.e., the number of adhesion points that cells can find on the surface. If the array of micropillars 12 is sparser, the adhesion points that cells can find are smaller, which will have a significant impact on cell behavior.

[0071] The cross-sectional area of ​​the micropillars also affects cell adhesion behavior, as cell adhesion requires a certain area to form focal adhesion. In the case of nanopillars, the smaller cross-sectional area will influence the formation of focal adhesion.

[0072] In summary, by combining the inherent properties of the material with a specific micropillar array size, it is possible to achieve cell support, chip stability, and measurement accuracy that better meet the requirements. Furthermore, by controlling the distribution of the micropillar array, the cell attachment state can be regulated and influenced to some extent.

[0073] In the first to fourth embodiments, the micropillar 12 is made of polydimethylsiloxane (PDMS). In other main embodiments of the present invention, the micropillar 12 can also be made of other polymeric materials, such as silicon-based polymers, photoresistive polymers, conductive polymers, and thermosensitive polymers. The main reason for using polymeric materials in the main embodiments of the present invention is that polymeric materials currently have deformable properties suitable for the application of the present invention. However, the implementation of the present invention does not require limiting the micropillar material to polymeric materials, but can be extended to all materials with corresponding deformability, all of which can realize the inventive concept of the present invention. In short, the material of the micropillar must meet the following conditions: it must have a certain degree of deformability under stress, and in some embodiments, it must have a certain degree of light transmittance. The latter is not a necessary condition for all embodiments. In the case of using materials with limited light transmittance to prepare micropillars, the inventive concept of the present invention can also be realized as long as the positions of the light signal generating device and the light signal detecting device are appropriately set.

[0074] Overall, the hardness (deformability) of the micropillar 12 can be controlled according to actual needs through multiple technical dimensions such as size (mainly aspect ratio), selection of material type, control of the degree of cross-linking of polymer materials, and chemical or physical surface treatment.

[0075] Fifth embodiment

[0076] The difference between this embodiment and the first to fourth embodiments is that the top end faces of some of the micropillars 12 in the micropillar array are provided with substances that promote cell adhesion. In this embodiment, collagen from the extracellular matrix is ​​used. In other embodiments, collagen, as well as one or more of the extracellular matrix molecules such as fibronectin, hyalin, laminin, and elastin, may also be used. In other embodiments, other types of substances that promote cell adhesion may be provided on the top end faces of all or part of the micropillars in the micropillar array, such as extracellular matrix mimics, such as peptides containing RGD adhesion sequences; or substances with cell adhesion-promoting mechanisms, including polylysine; or substances that interact with cell surface receptors.

[0077] Applying cell adhesion-enhancing substances to the top surface of micropillars 12 can effectively promote cell adhesion, thereby regulating cell adhesion, proliferation, migration, state, and differentiation. Furthermore, if cell adhesion-enhancing substances, such as extracellular matrix proteins like Fibronectin, are applied to the top surface of some micropillars within a predetermined region of the micropillar array, these micropillars can form specific shapes. This allows cells to tend to adhere to micropillars of specific locations and shapes, enabling high-throughput mechanical measurements while controlling cell size, shape, and tropism.

[0078] Sixth Embodiment

[0079] The difference between this embodiment and the fifth embodiment is that, as described in the fifth embodiment, some of the micropillars 12 in the micropillar array have a substance with cell adhesion properties on their top end faces. However, in this embodiment, the micropillars 12 whose top end faces do not have this cell adhesion property have a substance with cell adhesion inhibition properties (such as F-127) on their cylindrical surfaces (end faces or side faces). This makes cells more inclined to adhere to micropillars of specific locations and shapes, thus enabling high-throughput mechanical measurements while controlling cell size, shape, and tropism.

[0080] Seventh Embodiment

[0081] The difference between this embodiment and the first to fourth embodiments lies in that the top end face of the micropillar array 12 is provided with micropillars forming a preset pattern of substances with cell adhesion properties. Specifically, cell adhesion molecular layers with specific patterns can be printed using micron-printing technology to promote cell adhesion in these areas. The preset pattern can be triangular, quadrilateral, polygonal, circular, elliptical, or other shapes. The functions of the preset pattern include: first, controlling cell-cell contact through patterns formed by these substances with cell adhesion properties to facilitate high-throughput data acquisition; second, achieving dimensionality reduction in data processing by unifying cell shapes, thereby reducing the difficulty of analysis; and third, controlling cell size, shape, orientation, differentiation state, etc., by limiting the cell adhesion area, and even regulating cell mechanical state by controlling actin filaments to meet the requirements of certain special technical scenarios.

[0082] In another embodiment that is substantially similar to this embodiment, the unprinted portions of the preset pattern can be treated with substances that inhibit cell adhesion, such as BSA or F127, to suppress cell adhesion in these areas, thereby enabling directional adhesion, control of cell morphology, or simulation of a specific cellular microenvironment.

[0083] Eighth embodiment

[0084] The difference between this embodiment and the first to seventh embodiments is that the cell mechanical force detection device further includes a cell restriction mechanism. The cell restriction mechanism includes one or more restriction surfaces 16. The restriction surface 16 is a plane or curved surface that is perpendicular to the plane where the base 11 is located, connected to the base 11, or integrally formed with the base 11. The height of the restriction surface 16 is higher than that of the micropillars 12 and surrounds a preset number of micropillars 12.

[0085] The cell restriction mechanism in this embodiment serves to isolate and detect single cells, preventing contact or adhesion between cells during detection and restricting cell morphology to facilitate high-throughput testing. Depending on different needs, the number or shape of the restriction surfaces 16 in the cell location restriction mechanism can vary. For example, the restriction surfaces 16 in the cell location restriction mechanism can be a cylindrical surface, or three planes forming a triangular cross-section and surrounding a certain number of micropillars, four planes perpendicular to each other and forming a rectangular shape and surrounding a certain number of micropillars, N planes forming an N-sided polygon, or a curved surface with a near-circular cross-section. In other words, the cross-sectional shape formed by the restriction surfaces 16 is a controllable closed shape, and its area (or the number of micropillars it can accommodate within its space) is also controllable.

[0086] In practical implementations, depending on the manufacturing process, the cell confinement mechanism can also appear in the following forms:

[0087] A, please refer to Figure 4 , Figure 4 The diagram a shows the structure of a cell mechanical force detection device with a cell restriction mechanism. In the diagram, the cell restriction mechanism and the base 11 are integrally formed. That is, the material forming the cell restriction mechanism has several recessed spaces 15, the wall of the recessed space 15 is the restriction surface 16, the depth of the recessed space 15 is the height of the restriction surface 16, the bottom of the recessed space 15 is the base 11, and each recessed space 15 contains several micropillars 12.

[0088] B, please refer to Figure 5 , Figure 5 This is a schematic diagram (b) of a cellular mechanical force detection device with a cell restriction mechanism. In the diagram, the restriction surface 16 is a structure bonded to the base 11.

[0089] Ninth Embodiment

[0090] A cell mechanical force detection system includes a cell mechanical force detection device 1, a light signal generator 2, and a light signal detection device 3 as described in the first or second embodiment. Both the light signal generator 2 and the light signal detection device 3 are located below the base 11 of the cell mechanical force detection device 1. The light signal generator 2 has a light source. The light emitted by the light source passes through an incident light path (passing successively through a light-transmitting base and a light-transmitting micropillar) and irradiates the light-reflecting layer 13 of the micropillar 12, where it is reflected. The reflected light then passes through a reflected light path (passing successively through the light-transmitting micropillar and the light-transmitting base) and enters the light signal detection device 3. The light signal detection device 3 can acquire the reflected light signals before and after the micropillar 12 comes into contact with the cell. In other embodiments, this cell mechanical force detection system further includes a light signal analysis device 4, which can obtain cell mechanical force information, including the magnitude, direction, and changes in cell mechanical force over a certain time range, by comparing, analyzing, and calculating the reflected light signals before and after the micropillar 12 comes into contact with the cell.

[0091] Tenth Embodiment

[0092] Please see Figure 3 , Figure 3 This is a schematic diagram of a cellular mechanical force detection device related to the tenth embodiment of the present invention; Figure 3 A cell mechanical force detection system is demonstrated, comprising the cell mechanical force detection device 1 described in the third embodiment, and further comprising a light signal generator 2 and a light signal detection device 3. Both the light signal generator 2 and the light signal detection device 3 are located above the base 11 of the cell mechanical force detection device 1. The light signal generator 2 has a light source, and the light emitted by the light source illuminates the light reflector layer 13 through an incident light path, resulting in reflection. The light signal detection device 3 can acquire the reflected light signals before and after the micropillar 12 comes into contact with the cell. In other embodiments, this cell mechanical force detection system further includes a light signal analysis device 4, which can obtain cell mechanical force information, including the magnitude, direction, and changes within a certain time range of the cell mechanical force, by comparing, analyzing, and calculating the reflected light signals before and after the micropillar 12 comes into contact with the cell.

[0093] Eleventh Embodiment

[0094] A method for detecting cellular mechanical forces includes the following steps:

[0095] Light is emitted using the light signal generator 2 in the cell mechanical force detection system as described in the ninth or tenth embodiment;

[0096] The light signal detection device 3 in the cell mechanical force detection system described in the ninth or tenth embodiment is used to detect the light after it has been acted upon by the cell mechanical force detection device 1. The light signal detection device 3 is able to acquire the reflected light signals before and after the micropillar 12 in the cell mechanical force detection device 1 comes into contact with the cell. In other embodiments, the light signal analysis device 4 in the cell mechanical force detection system obtains cell mechanical force information, including the magnitude, direction, and changes of the cell mechanical force within a certain time range, by comparing, analyzing, and calculating the reflected light signals before and after the micropillar 12 comes into contact with the cell.

[0097] Twelfth Embodiment

[0098] A method for preparing a cellular mechanical force detection device includes the following steps:

[0099] A light-reflecting layer 13 is laid on the top or upper half of the micropillar 12 to obtain a micropillar 12 with a reflective layer on the top or upper half of the micropillar.

[0100] Thirteenth Embodiment

[0101] A method for preparing a cellular mechanical force detection device includes the following steps:

[0102] A uniform anti-reflective layer is coated on the entire micropillar 12;

[0103] Remove the anti-reflective layer from the top or upper half of the cylinder;

[0104] A light-reflecting layer 13 is laid on the top or upper half of the micropillar 12.

[0105] Fourteenth Embodiment

[0106] The difference between this embodiment and the twelfth and thirteenth embodiments is that the step "laying a light-reflecting layer 13 on the top or upper half of the micro-pillar 12" specifically means: uniformly sputtering a layer of reflective metal on the top or upper half of the micro-pillar to obtain a micro-pillar with a metal light-reflecting layer on the top or upper half of the micro-pillar.

[0107] The above technical solution has the following advantages, unlike existing technologies:

[0108] First, high throughput and low cost: Compared with existing TFM and ordinary micropillar arrays, the technical solution of this invention eliminates the dependence on microscopes and greatly simplifies the operation process. Because there is no need for high-resolution imaging with a microscope, high-throughput cell monitoring can be achieved simply by monitoring the intensity of reflected light.

[0109] Secondly, single-cell resolution: high resolution allows for real-time monitoring of each cell, and can be combined with other single-cell analysis techniques to measure the heterogeneity of cellular responses to drugs; real-time monitoring: no fluorescence is required, avoiding the phototoxicity of lasers on cells, making it suitable for long-term monitoring and applicable to studying long-term cellular responses to drugs; high sensitivity: by amplifying the micropillar deformation signal through reflected signals, the sensitivity of deformation monitoring is increased. Detection of bending deformation of micro and nanopillars generally relies on optical systems (such as microscopes), but the smaller the micropillar size, the higher the precision and resolution requirements of the optical system. For example, a micropillar 2 micrometers wide and 6 micrometers high requires a 20x or higher objective lens with a conjugate focal system for effective observation. Utilizing the principle of specular reflection, the signal of micropillar deformation is amplified by detecting the attenuation of reflected light. Experiments have verified that the same signal can be observed under a 5x objective lens. With a special readout system, micro / nanopillar deformation can be effectively detected without relying on a microscope, thereby greatly reducing system costs and effectively increasing throughput.

[0110] Furthermore, it can simulate the cellular microenvironment and the composition and morphology of the extracellular matrix, thus meeting a wider range of technical requirements.

[0111] It should be noted that although the above embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included within the scope of patent protection of the present invention.

Claims

1. A device for detecting cellular mechanical force, characterized in that, include: Base, and A micropillar array consisting of multiple micropillars that can deform under the action of cellular mechanical forces, set on a base, wherein the top or upper part of the micropillar has a light-reflecting layer. The light-reflecting layer is configured to acquire cellular mechanical forces based on light reflection signals; The intensity of light reflected from the light-reflecting layer has a quantitative relationship with the magnitude of the cellular mechanical force or the amount of micropillar displacement caused by the cellular mechanical force; The base of the cell mechanical force detection device is a light-transmitting base, and the column of the micropillar can transmit light. The light emitted by the light source shines from the base onto the light-reflecting layer of the micropillar through the incident light path.

2. The cellular mechanical force detection device as described in claim 1, characterized in that, The surface of the micropillar has an anti-reflective layer.

3. The cellular mechanical force detection device as described in claim 1, characterized in that, The light-reflecting layer is one or more of the following: a metal foil layer, a metal oxide or metal salt, an ultrafine glass bead or a microprism, or an organic reflective material.

4. The cellular mechanical force detection device according to any one of claims 1 to 3, characterized in that, The top end face of all or part of the micropillars in the micropillar array is provided with a substance that has a cell adhesion effect.

5. The cellular mechanical force detection device as described in claim 4, characterized in that, The substances that have cell adhesion effects include one or more of the following: extracellular matrix molecules, including collagen, fibronectin, hyalin, laminin, or elastin; extracellular matrix mimics, including polypeptides containing RGD adhesion sequences; substances with cell adhesion-promoting mechanisms, including polylysine; and substances that interact with cell surface receptors.

6. The cellular mechanical force detection device as described in claim 4, characterized in that, Some micropillars have a group of micropillars with a substance that has cell adhesion function on the top end face, forming a pre-set pattern.

7. The cellular mechanical force detection device as described in claim 6, characterized in that, The top end face of some micropillars in the preset region of the micropillar array is provided with a substance that has a cell adhesion effect.

8. The cellular mechanical force detection device as described in claim 1, characterized in that, The cross-sectional shape of the micropillar is circular, elliptical, or polygonal.

9. The cellular mechanical force detection device as described in claim 1, characterized in that, The dimensions of the micropillars and micropillar arrays include: pillar height of 10 nm to 500 μm, pillar spacing of 10 nm to 50 μm, and pillar surface diameter of 50 nm to 50 μm.

10. The cellular mechanical force detection device as described in claim 1, characterized in that, It also includes a cell restriction mechanism, which includes one or more restriction surfaces. The restriction surface is a plane or curved surface that is perpendicular to the plane of the base, connected to the base, or integrally formed with the base. The height of the restriction surface is higher than the micropillars and surrounds a predetermined number of micropillars.

11. A system for detecting cellular mechanical forces, characterized in that, Includes the cell mechanical force detection device, the light signal generating device, and the light signal detection device as described in any one of claims 1-10; The optical signal generating device has a light source, and the light emitted by the light source illuminates the light-reflecting layer of the micropillar through the incident light path; The optical signal detection device is used to detect the light reflected from the light-reflecting layer of the micropillar. The light reflected from the light-reflecting layer enters the optical signal detection device through the reflected light path.

12. The cellular mechanical force detection system as described in claim 11, characterized in that, The base of the cell mechanical force detection device is a light-transmitting base, and the column of the micropillar can transmit light; the top of the micropillar has a light-reflecting layer. The light emitted by the light source shines from the base of the cell mechanical force detection device onto the light-reflecting layer of the micropillar through the incident light path; The optical signal detection device is used to detect the light reflected from the light-reflecting layer at the top of the micropillar. The light reflected from the light-reflecting layer enters the optical signal detection device through the reflected light path.

13. The cellular mechanical force detection system as described in claim 11, characterized in that, It also includes an optical signal analysis device for analyzing optical signals.

14. A method for detecting cellular mechanical force, characterized in that, Includes the following steps: The light is emitted using the light signal generator in the cell mechanical force detection system as described in any one of claims 11-13; The light after being acted upon by the cellular mechanical force detection device is detected using the light signal detection device in the cellular mechanical force detection system as described in any one of claims 11-13.

15. The method for detecting cellular mechanical force as described in claim 14, characterized in that, The method also includes the step of: using a light signal analysis device to compare and analyze the reflected light of the cell mechanical force detection device and the cell under test before and after the cell mechanical force is applied, and to obtain cell mechanical force information, wherein the cell mechanical force information includes the magnitude, direction or frequency of change of the cell mechanical force.

16. A method for preparing a cellular mechanical force detection device, characterized in that, Includes the following steps: A light-reflecting layer is laid on the top or upper half of the micropillar to obtain a micropillar with a light-reflecting layer on the top or upper half of the micropillar. The light-reflecting layer is configured to acquire cellular mechanical forces based on light reflection signals; The intensity of light reflected from the light-reflecting layer has a quantitative relationship with the magnitude of the cellular mechanical force or the amount of micropillar displacement caused by the cellular mechanical force; The base of the cell mechanical force detection device is a light-transmitting base, and the column of the micropillar can transmit light. The light emitted by the light source shines from the base onto the light-reflecting layer of the micropillar through the incident light path.

17. The method for preparing the cell mechanical force detection device as described in claim 16, characterized in that, The step preceding "depositing a reflective layer on the top or upper half of the micropillar" also includes the following step: A uniform anti-reflective layer is coated onto the entire micropillar; Remove the anti-reflective layer from the top or upper half of the cylinder.

18. The method for preparing the cell mechanical force detection device as described in claim 16 or 17, characterized in that, The phrase "laying a reflective layer on the top or upper half of the micro-pillar to obtain a micro-pillar with a reflective layer on the top or upper half of the micro-pillar" specifically refers to: uniformly sputtering a reflective metal layer on the top or upper half of the micro-pillar to obtain a micro-pillar with a metal light-reflecting layer on the top or upper half of the micro-pillar.

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