WO2023046166A1 - 细胞机械力的检测系统、方法、装置及其制备方法 - Google Patents

细胞机械力的检测系统、方法、装置及其制备方法 Download PDF

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WO2023046166A1
WO2023046166A1 PCT/CN2022/121338 CN2022121338W WO2023046166A1 WO 2023046166 A1 WO2023046166 A1 WO 2023046166A1 CN 2022121338 W CN2022121338 W CN 2022121338W WO 2023046166 A1 WO2023046166 A1 WO 2023046166A1
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cell
mechanical force
microcolumn
light
detection device
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PCT/CN2022/121338
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English (en)
French (fr)
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林哲
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瑞新（福州）科技有限公司
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Publication of WO2023046166A1 publication Critical patent/WO2023046166A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/84—Systems specially adapted for particular applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material

Definitions

  • the invention relates to the field of biotechnology, in particular to a cell mechanical force detection system, a cell mechanical force detection method, a cell mechanical force detection device, and a preparation method for a cell mechanical force detection device.
  • Cells will exert tiny mechanical force on the surrounding microenvironment.
  • Cell force plays a key role in processes including adhesion, migration, proliferation, differentiation, apoptosis, etc., and together with other biochemical signals, it plays a role in embryonic development, stem cell differentiation, and immune processes.
  • Wound repair, cancer metastasis and other processes play a vital regulatory role, so it has also become a target for the treatment of many diseases.
  • the significance of measuring the maximum cellular mechanical force is limited, and high-resolution, real-time, high-throughput mechanical sensors will become the core requirements of the next generation of cellular force measurement tools.
  • the existing main methods of measuring cell mechanical force include "mechanical force microscope", micro-nano cantilever and micro-column array.
  • the main principle is to calculate the mechanical force of the cell by measuring the deformation of the substrate caused by the force exerted by the cell on the elastic substrate. So far, cell mechanical force microscopy (TFM) is the most widely used technical method for measuring cell mechanical force. glue).
  • TFM cell mechanical force microscopy
  • glue glue
  • the mechanical force of the cells can be deduced through the mechanical model.
  • this measurement method has the following disadvantages.
  • the principle of TFM is not to directly measure the mechanical force of the cell, but to reverse the mechanical force of the cell by observing the position change of the fluorescent microbeads in the substrate.
  • the special process can ensure that most of the particles are deposited on the surface, after a long time of immersion, it is very likely to escape or decline and eventually lead to a decrease in the density of the surface fluorescent particles.
  • Fluorescence microscopes usually have a large depth of field, which may capture particles in different planes, resulting in deviations in later displacement calculations and resulting in inaccurate measurement results.
  • long-term immersion in the culture medium will change the elastic modulus of the gel, which will inevitably affect the accuracy of cell mechanical force calculation. Therefore, it is necessary to detect and calibrate the elastic modulus of the gel during the measurement process, which greatly increases the work. quantity.
  • long-term laser irradiation will cause phototoxicity to the cells, and it will also cause the quenching of fluorescent micropearls. Therefore, TFM is not suitable for long-term continuous detection of cell mechanical force, and it usually takes a long time to study cell growth and differentiation and its response to drugs. Therefore, the above-mentioned defects of TFM greatly limit its application in the field of biomedicine.
  • micro-nanosensors can also be used to directly measure cellular mechanical forces.
  • the microcolumn array the cells are attached to the surface above the microcolumn, and the bending deformation of the microcolumn can be calculated by taking pictures of the bottom and top of the microcolumn with a microscope, so as to deduce the magnitude and direction of the mechanical force of the cell at that point.
  • micro-nano sensors also need to rely on microscopes for high-resolution photography, which requires high equipment, and is prone to errors during the photography process, resulting in inaccurate results.
  • the pictures taken by the microscope need to undergo complex image processing, and calculate and solve the mechanical force of cells according to the mechanical model.
  • the operation is complex and time-consuming, and it is difficult to achieve real-time, high-throughput, low-cost, and long-term detection of cells. Therefore, most of the existing technologies are limited to scientific research in the field of biomechanics, and are difficult to be practical.
  • a detection device for cell mechanical force including:
  • the microcolumn array is set on the base and can be deformed by the mechanical force of the cells, and the top or the top of the microcolumn is provided with a light reflection layer.
  • the base is a light-transmitting base, and the columns of the micro-pillars can transmit light; the top of the micro-pillars has a light-reflecting layer.
  • the cylinder surface of the microcolumn has an anti-reflection layer.
  • the light reflection layer is a metal foil layer, a metal oxide or metal salt, ultrafine glass beads or microprisms, and a combination of one or more organic light-reflecting materials .
  • all or part of the microcolumns of the microcolumn array are provided with a substance with cell adhesion on the top end surface.
  • the substances with cell adhesion include one or more of the following substances: extracellular matrix molecules, including collagen, fibronectin, vitronectin, Laminin or tropoelastin; extracellular matrix mimics, including polypeptides containing the RGD adhesion sequence; substances with cell adhesion-promoting mechanisms, including polylysine; substances that interact with cell surface receptors.
  • the top end surfaces of some micropillars in the predetermined area of the micropillar array are provided with substances with cell adhesion function.
  • the microcolumn array is provided with a substance with cell adhesion inhibitory effect on the top end surface of the microcolumn that is not provided with a substance with cell adhesion effect on the top end surface.
  • the cross-sectional shape of the micropillar is circular, elliptical or polygonal.
  • the size range of the micropillar array includes: column height 10nm-500 ⁇ m, column spacing 10nm-50 ⁇ m, column upper surface diameter 50nm-50 ⁇ m.
  • the cell mechanical force detection device also includes a cell restriction mechanism, the cell restriction mechanism includes one or several restriction surfaces, the restriction surfaces are perpendicular to the plane where the base is located, connected to the base or A flat or curved surface integrally formed with the base, and the height of the limiting surface is higher than the micro-columns and encloses a preset number of micro-columns.
  • the inventor also provides a detection system for cell mechanical force, including the detection device for cell mechanical force described in the above technical solution, an optical signal generating device, and an optical signal detection device;
  • the optical signal generating device has a light source, and the light emitted by the light source is irradiated to the light reflection layer of the microcolumn through the incident light path;
  • the light signal detection device is used to detect light reflected from the light reflection layer of the microcolumn, and the light reflected by the light reflection layer enters the light signal detection device through a reflection light path.
  • the base of the cell mechanical force detection device is a light-transmitting base, the column of the microcolumn can transmit light; the top of the microcolumn has light reflection layer;
  • the light emitted by the light source is irradiated from the base of the cell mechanical force detection device to the light reflection layer of the microcolumn through the incident light path;
  • the light signal detection device is used to detect the light reflected from the light reflection layer on the top of the microcolumn, and the light reflected by the light reflection layer enters the light signal detection device through the reflection light path.
  • the system for detecting the mechanical force of cells also includes an optical signal analysis device for analyzing the optical signal.
  • the inventor also provides a method for detecting cell mechanical force, which includes the following steps:
  • the optical signal detection device in the cell mechanical force detection system described in the above technical solution is used to detect the light after the action of the cell mechanical force detection device.
  • the method for detecting cell mechanical force further includes the step of: using an optical signal analysis device to compare and analyze the reflected light of the cell mechanical force detection device and the cells to be tested before and after the cell mechanical force acts, and obtain Cell mechanics information.
  • the inventor also provides a cell state detection method, which includes: using the cell mechanical force detection system described in any of the above schemes or the cell mechanical force detection method described in any of the above schemes to obtain the cell mechanical force force information, analyzing and determining the cell state according to the cell mechanical force information;
  • the cell state specifically includes cell adhesion, cell viability, cell differentiation/activation, cell proliferation and/or cell migration.
  • the cell state is a static cell state or a real-time cell state.
  • the inventor also provides a method for cell recognition, which includes: using the cell mechanical force detection system described in any of the above schemes or the cell mechanical force detection method described in any of the above schemes to obtain the cell mechanical force information, different cell types are distinguished according to the cell mechanical force information.
  • the step "acquires cell mechanical force information by using the cell mechanical force detection system described in any one of the above schemes or the cell mechanical force detection method described in any one of the above schemes, according to The cell mechanical force information distinguishes different cell types” specifically includes:
  • the cell information includes the cell mechanical force information of a certain point in the cell obtained by the detection device based on the cell mechanical force, and the cell mechanical force information includes the size of the cell mechanical force at this point;
  • the structured cell information includes the number of cells, the number of cell characteristics and characteristic information of each cell characteristic;
  • the cell mechanical force information also includes the direction of the cell mechanical force at this point.
  • the cell mechanical force information also includes the change of the magnitude or direction of the cell mechanical force at the point within a certain time interval.
  • the cell information also includes cell shape information.
  • the inventor also provides 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-cylindrical surface of the microcolumn to obtain a microcolumn with a reflective layer on the top or upper half-cylindrical surface .
  • the steps further include:
  • An anti-reflection layer is evenly coated on the micro-column as a whole;
  • the "laying a reflective layer on the top or upper half cylinder of the microcolumn to obtain a microcolumn with a reflective layer on the top or upper half cylinder” specifically The method is: uniformly sputtering a layer of reflective metal on the top or upper half cylinder of the microcolumn to obtain a microcolumn with a metal light reflection layer on the top or upper half cylinder.
  • the cells may be single cells, or multicellular aggregates of any shape formed by two or more cells.
  • the present invention is not limited to the various forms formed by single cells or two or more cells.
  • single cell resolution high resolution, real-time monitoring of each cell, can be combined with other single cell analysis techniques to measure the heterogeneity of cell response to drugs; real-time monitoring: no fluorescence, can avoid laser light on cells Toxicity, so it is suitable for long-term monitoring, and can be used to study the long-term response of cells to drugs; high sensitivity: through the reflected signal, the micro-column deformation signal is amplified to increase the sensitivity of deformation monitoring.
  • the detection of bending deformation of micro- and nano-pillars generally relies on optical systems (such as microscopes) for detection, but the smaller the size of the micro-pillars, the higher the precision and resolution of the optical system.
  • a microcolumn with a width of 2 microns and a height of 6 microns requires an objective lens with a power of 20 times or more and a conjugate focus system for effective observation.
  • the present invention utilizes the principle of specular reflection to detect the attenuation of reflected light, and actually amplifies the signal of the deformation of the micro-column. It has been verified by experiments that the same signal can be observed under a 5x objective lens. With a special reading system, it can effectively detect the deformation of micro/nano-pillars without relying on high-magnification optical objective lenses, thereby greatly reducing system costs and effectively improving throughput.
  • Multilayer cells including cell mechanical forces such as tumor polymers, can be detected, so that they can be applied to drug screening, regenerative medicine, gene editing, precision medicine, organ development, and disease modeling. The scene represented by the body.
  • FIG. 1 is a schematic structural view of a cell mechanical force detection device in the first embodiment of the present invention
  • Fig. 2 is a scanning electron microscope (SEM) image of a microcolumn (real object) of a cell mechanical force detection device in the first embodiment of the present invention; wherein, Fig. 2a is a top view of a cell mechanical force detection device, and Fig. 2b is a cell The side view of the mechanical force detection device;
  • SEM scanning electron microscope
  • FIG. 3 is a schematic structural diagram of a cell mechanical force detection system related to the ninth embodiment of the present invention.
  • FIG. 4 is a schematic structural diagram of a cell mechanical force detection system related to the tenth embodiment of the present invention.
  • Figure 5 is a scanning electron microscope image of a microcolumn (polydimethylsiloxane) that is provided with a light reflection layer (gold) at the top position; wherein, Figure 5a is a scanning electron microscope image of a microcolumn; Figure 5b is a microcolumn The element characterization diagram of the top area; Figure 5c is the element characterization diagram of the micropillar side area (except the top area);
  • Figure 6a is a fluorescence imaging image of cells adhering to a preset pattern formed by microcolumn groups provided with fibronectin on the top;
  • Figure 6b is a cell force distribution diagram calculated from the light reflection signal measured on the microcolumn group with fibronectin attached to the top of the cell;
  • Figure 7a is a schematic diagram of an experiment using the OKT3 antibody as a substance with cell adhesion
  • the upper part of the image in Figure 7b is a fluorescence imaging image of cells adhering to the top of the microcolumn with OKT3 antibody and fibronectin respectively;
  • the lower part of the image in Figure 7b is the size distribution diagram of the cell mechanical force calculated from the light reflection signal measured on the microcolumn;
  • Figure 7c is a comparison diagram of the mechanical size measured on the surface coated with OKT3 antibody and fibronectin respectively;
  • Figure 7d is a diagram of the dynamic changes in cell mechanics after T cells were planted on the surface of the OKT3 antibody (top of the microcolumn);
  • Fig. 8 is a structural schematic diagram a of a detection device for cell mechanical force with a cell restriction mechanism
  • Fig. 9 is a structural schematic diagram b of a cell mechanical force detection device with a cell restriction mechanism
  • Figure 10a is a physical diagram of a cell mechanical force detection device using a silicon thin film as a cell restriction mechanism
  • Fig. 10b is a fluorescent microscope image of a cell mechanical force detection device using a silicon thin film as a cell restriction mechanism under light reflection;
  • Figure 10c is an enlarged view of Figure 10b;
  • Fig. 11 is a fluorescence microscope image of the cell mechanical force detection system of the eleventh embodiment monitoring the cell mechanical force
  • Fig. 12a is a schematic structural diagram of the cell mechanical force detection system of the twelfth embodiment
  • Fig. 12b is an image of the light reflection signal of the cell mechanical force detection device obtained by the optical signal detection device of the twelfth embodiment
  • Fig. 12c is a visualization effect diagram of the mechanical size and distribution after processing by the optical signal analysis device of the twelfth embodiment
  • Figure 13a is a schematic structural view of the cell mechanical force detection device in the microfluidic environment before and after the fluid is turned on;
  • Fig. 13b is a comparison diagram of the bright field microscope image of the microcolumn, the reflected light signal distribution diagram, and the superimposed effect of the two before the fluid is turned on; where the superimposed effect diagram refers to the bright field microscope image of the microcolumn and the reflected light signal.
  • the effect diagram formed by superimposing the signal distribution diagram;
  • Figure 13c is a comparison of the bright field microscope image of the microcolumn, the reflected light signal distribution diagram, and the superimposed effect diagram of the microcolumn after the fluid is turned on; The effect diagram formed by superimposing the optical signal distribution diagram;
  • Figure 13d is the intensity value of the light reflection signal before and after the fluid is turned on
  • Figure 13e is the linear interval of the attenuation of the light reflection signal and the translation of the top of the microcolumn
  • Fig. 14a is a structural schematic view before and after the contact between the microcolumn and the cell of the cell mechanical force detection device;
  • Fig. 14b is a distribution diagram of reflected light signals obtained by the light signal detection device
  • Figure 14c is a monitoring diagram of the cell migration process
  • Figure 14d is a distribution diagram of reflected light signals during cell migration
  • Figure 15a is a fluorescence imaging diagram of a mixed system of healthy cells and lung non-small cell cancer cells
  • Figure 15b is a distribution diagram of the light reflection signal of the cell mechanical force detection device obtained by the optical signal detection device;
  • Fig. 15c is a visualization effect diagram of the mechanical size and distribution after processing by the optical signal analysis device
  • Figure 15d is an enlarged view of representative single-cell cell force distributions of healthy cells and lung non-small cell carcinoma cells in Figure 15c;
  • Figure 15e is a comparison diagram of healthy cells and lung non-small cell carcinoma cells in cell morphology
  • Fig. 15f is a comparison chart of reflection signal intensity of healthy cells, lung non-small cell carcinoma cells and the mixture of these two kinds of cells in different proportions;
  • Fig. 15g is a cluster analysis diagram obtained based on structured cell information processing after Fig. 15c is structured;
  • Figure 16a is a schematic diagram of the operation flow of the cell viability detection method
  • Figure 16b is a comparison chart of the cell viability measured by the MTT method and the cell viability reflected by the cell mechanical force after A549 cells were treated with different doses of 5FU for 24 hours;
  • Figure 16c is a comparison chart of the cell viability measured by the MTT method and the cell viability reflected by the cell mechanical force after A549 cells were treated with different doses of 5FU for different times;
  • Figure 17a is a diagram of the operation process of the cell state detection method
  • Figure 17b is a fluorescence microscope image of M0 macrophages differentiated to M1 state
  • Figure 17c is a fluorescence microscope image of M0 macrophages differentiated to M2 state
  • Figure 17d is a comparison chart of cell adhesion area of M0 macrophages, M1 state and M2 state;
  • Figure 17e is a comparison diagram of cell roundness of M0 macrophages, M1 state and M2 state;
  • Figure 17f is a mechanical comparison diagram of M0 macrophages, M1 state and M2 state;
  • Figure 18a is the characterization diagram of tumor cell multimers with the first form after the presence or absence of 5-Fu; in the figure, from left to right are the mixed images of cell membrane fluorescence and reflection signals (1), light reflection Signal (2), cell nucleus (3), cell membrane (4) and the visualized image of cell force after processing by the optical signal analysis device (ImageJ) (5);
  • Figure 18b is a characterization diagram of tumor cell multimers with the second morphology with or without the action of 5-Fu; in the figure, from left to right are the mixed images of cell membrane fluorescence and reflection signals (1), light reflection Signal (2), cell nucleus (3), cell membrane (4) and the visualized image of cell force after processing by the optical signal analysis device (ImageJ) (5).
  • ImageJ optical signal analysis device
  • FIG. 1 is a schematic structural diagram a of a detection device for cell mechanical force.
  • the detection device for cell mechanical force shown in the figure includes a light-transmitting base 11 and a cell mechanically actuated base 11 arranged on the base 11 .
  • the microcolumn 12 that deforms due to force, the top of the microcolumn 12 is coated with a light reflection layer 13, the thickness of the light reflection layer 13 is 5nm (in some other embodiments, the thickness of the light reflection layer 13 can be 5nm-20nm Between - the thickness of the coating is related to the coating material.
  • the thickness of the coating should be selected to ensure the light transmission effect, the stability of the micro-column column, and the guarantee and micro-column column. The connection does not fall off).
  • the cylinders of the microcolumns 12 can transmit light, and the clusters of arrows in opposite directions in the figure represent incident light and reflected light. (Note: the word "coating" is used in this embodiment, which only means that the light reflection layer 13 in this embodiment can be prepared by a coating process, and does not limit that the light reflection layer 13 must be prepared by a coating process)
  • Fig. 2 is a scanning electron microscope (SEM) image of the microcolumn 12 (real object) of the cell mechanical force detection device of the present embodiment
  • Fig. 2 a is a top view of the cell mechanical force detection device
  • Fig. 2 b is a cell mechanical force detection device side view. It can be seen from Fig. 2 that the microcolumns of the cell mechanical force detection device have uniform microstructure and controllable size. Compared with the existing cell mechanical force detection device, the cell mechanical force detection device based on this embodiment measures the Mechanics are more precise.
  • SEM scanning electron microscope
  • FIG. 3 is a schematic structural diagram of a cell mechanical force detection system related to the ninth embodiment of the present invention; FIG. 3 can be used to understand this embodiment.
  • the system shown in FIG. 3 involves, in addition to the cell mechanical force detection device 1 described in this embodiment, an optical signal generation device 2 with a light source and an optical signal detection device 3 arranged below the base 11.
  • the light emitted by the light source is irradiated from the light-transmitting base 11 of the detection device 1 of the cell mechanical force to the light reflection layer of the microcolumn 12 through the incident optical path; 13 , the light reflected by the light reflection layer 13 enters the optical signal detection device 3 after passing through the reflection optical path and the beam splitter 5 .
  • an optical signal analysis device 4 can compare and analyze the reflected light of the cell mechanical force detecting device 1 and the cells to be tested before and after the cell mechanical force acts on the cells to obtain cell mechanical force information.
  • the microcolumn 12 When the microcolumn 12 is not stressed, the microcolumn should remain upright, so as to reflect the detection light to the greatest extent; and when the microcolumn 12 is in contact with the cell, the microcolumn 12 bends under the mechanical force of the cell , leading to a reduction in the level of light reflection. Therefore, when the cell mechanical force is greater, the obtained light reflection signal should be smaller, so that the size of the cell mechanical force at that point can be easily deduced by observing the intensity of the light reflection signal.
  • the measurement light source in the technical solution of this embodiment may use a certain intensity infrared laser.
  • the micro-column measurement in the traditional technical scheme needs to take high-resolution images. In this process, if the laser is used, it is easy to cause cell phototoxicity or sample fluorescence quenching.
  • the influence of the infrared laser within a certain light intensity on the cells is basically negligible, so it is suitable for long-term monitoring of the cells.
  • the microcolumn 12 not only has a light reflection layer 13 on the top end surface, but also has a light reflection layer 13 on the upper half of the cylinder surface of the microcolumn 12 (that is, the curved surface connecting the two end surfaces of the cylinder).
  • reflective layer 13 In fact, in other embodiments, except that the solution of disposing the light reflection layer 13 on the lower half of the side cylinder surface of the microcolumn 12 is not adopted due to poor practical effect, as long as the light reflection layer 13 is disposed on the microcolumn
  • the upper half of the side of the column 12 can basically achieve the desired detection effect of the present invention.
  • the light reflection layer 13 can even be laid on any partial position of the upper half cylinder or a partial position of the top, and does not necessarily have to cover the entire upper half cylinder or the entire top.
  • the end face can achieve the expected purpose, although the obtained data and the effect of the post-operation may be different.
  • first embodiment and the second embodiment of the present invention present the definition of the "cylindrical surface” and "end surface” of the microcolumn, that is to say, an independent column as we usually understand it should have two end surfaces and the two A curved surface (cylindrical surface) connected by two end faces, and the micro-column in the present invention has only one end face, namely the top end face, due to the existence of the base, and the other end is fixedly connected to the base or integrally formed with the base.
  • the end surface of the top may be a curved surface smoothly connected with the cylindrical surface as a whole, and does not necessarily have intersection lines or obvious boundaries as shown in the first embodiment or the second embodiment.
  • the location of the light reflection layer 13 will also be understood as the upper half of the cylinder, and cannot be limited to "end surface” or "cylindrical surface”.
  • FIG. 4 is a schematic structural diagram of a cell mechanical force detection system in the tenth embodiment of the present invention, which is used to illustrate the cell mechanical force detection device 1 in this embodiment.
  • the difference between this embodiment and the first and second embodiments is that the light transmission properties of the base 11 and the micro-pillars 12 of the micro-pillar array are not required, that is, they can be light-transmitting or opaque. Can also be translucent.
  • the optical signal will change when the microcolumn 12 is upright and not deformed.
  • the relative size of the cell mechanical force can also be obtained. After correction with the standard value, the cell mechanical force can be obtained.
  • an anti-reflection layer for light is provided on the surface of the micropillar 12 except for the area where the light reflection layer 13 is provided.
  • Such a design can reduce the interference of reflected light signals that may be caused by the surface of the cylinder, enhance the signal-to-noise ratio, and make the detection results more accurate.
  • the light reflection layer 13 may be a layer of gold foil. In other embodiments, the light reflection layer 13 may also be other metal layers with light reflection function or other reflective materials. There may be differences in the reflective effect brought by different materials, the difficulty of preparing the reflective layer, and the cost. In actual operation, consideration and selection can be made according to specific conditions.
  • the cross-sectional shape of the micropillar 12 is circular. In other embodiments, the cross-sectional shape of the micropillars 12 may also be oval or polygonal. In various implementations of the present invention, different cross-sections can achieve different purposes. For example, the circular cross-section is isotropic, that is, the mechanical properties of the micropillar itself are not sensitive to the direction.
  • the cross-section is elliptical, it is anisotropic, that is, the mechanical properties of the micropillar itself are sensitive to the direction, which can control the sensitivity of different directions to the force field, and can regulate the tropism of the cell to a certain extent (large
  • the geometry of some cells is actually asymmetrical, and the tropism of cells in the present invention refers to the asymmetry, polarity or directionality in the form that cells show. For example, if an ellipse is used to fit the cell projection The shape of the ellipse, the long axis of the ellipse can be considered as the direction that the cell has).
  • the cross-section is elliptical, the cross-section has a major axis and a minor axis, and it is much easier to push the microcolumn along the minor axis than the major axis, and the deformation is relatively large under the condition of relative force.
  • the size of the micro-column array is: the column height is 10 nm-500 ⁇ m, the column spacing is 10 nm-50 ⁇ m, and the diameter of the upper surface of the column is 50 nm-50 ⁇ m.
  • the micropillars within this size range can meet the basic conditions for use as micropillars used as sensors, that is, at least deformable and not lodging.
  • the regulation and control of different microcolumn array sizes can also realize the following functions: for example, by regulating the aspect ratio AspectRatio of microcolumns (in the level of microcolumns, it can be understood as the ratio of height and cross-sectional diameter/side length/long diameter ) can achieve a certain micro-column deformation performance regulation function, so as to achieve better simulation of the internal organ tissue environment (such as bone tissue and nerve tissue with different hardness).
  • the overall specification of the array or the number of micropillars 12 on a base 11 in a certain area will also affect the Ligand Density, that is, the number of points on the surface where cells can find adhesion. If the array of microcolumns 12 is sparser, the adhesion points that cells can find are smaller, which will have a considerable impact on cell behavior.
  • the size of the cross-sectional area in the shape of the micro-column will also affect the cell adhesion behavior, because a certain area is required for cell adhesion to form the adhesion spot FocalAdhesion. If it is a nano-column, the cross-sectional area of the micro-column is small, which will affect the formation of FocalAdhesion.
  • the characteristics of the material itself and a certain size of the micro-column array can achieve a more satisfactory cell support effect, chip stability, and measurement accuracy.
  • the state of cell attachment can also be regulated and influenced to a certain extent.
  • the material of the micropillars 12 is polydimethylsiloxane (PDMS).
  • the material of the microcolumn 12 can also be some other polymer materials, such as silicon-based polymers, photoresist polymer materials, conductive polymer materials, temperature-sensitive polymer materials, etc. .
  • the reason why the main implementation mode of the present invention mainly adopts polymer materials is that current polymer materials have deformable properties that are more suitable for the application of the present invention, but the implementation of the present invention does not need to limit the microcolumn material to polymer materials, but should And can be extended to all materials with corresponding deformability, the inventive concept of the present invention can be realized.
  • the material of the micro-column must meet the conditions that it has a certain force deformability, and in some embodiments, it needs to have a certain degree of light transmission, which is not a necessary condition for all embodiments.
  • the inventive concept of the present invention can also be realized.
  • the hardness (deformability) of micropillars 12 can be adjusted according to actual needs through multiple technical dimensions such as size (mainly AspectRatio), selection of material type, control of crosslinking degree of polymer materials, chemical or physical surface treatment, etc. to control.
  • size mainly AspectRatio
  • selection of material type mainly AspectRatio
  • control of crosslinking degree of polymer materials mainly AspectRatio
  • Figure 5 is a scanning electron microscope image of a microcolumn (polydimethylsiloxane) with a light reflection layer (gold) at the top position; wherein, Figure 5a is a scanning electron microscope image of a microcolumn; Figure 5b is an elemental characterization diagram of the top area of the microcolumn; Figure 5c is an elemental characterization diagram of the side area of the microcolumn (except the top area).
  • the material composition of the microcolumns is characterized by the scanning electron microscope image in Figure 5, and it can be confirmed that there is Au element at the top of the microcolumn, and Si element exists at the rest of the microcolumn.
  • this embodiment uses the extracellular matrix molecule Collagen, in other embodiments, can also be combined with one or several extracellular matrix molecules including collagen, fibronectin, vitronectin, laminin, and tropoelastin.
  • other types of substances with cell adhesion can also be set on the top end faces of all or part of the microcolumns of the microcolumn 12 array, such as extracellular matrix mimic substances, such as those containing the RGD adhesion sequence.
  • microcolumn 12 Setting such a substance with cell adhesion on the top end surface of the microcolumn 12 can effectively promote the attachment of cells to the microcolumn 12, thereby realizing the regulation of cell attachment, proliferation, migration, state, differentiation, etc.
  • substances with cell adhesion function such as extracellular matrix proteins such as Fibronectin
  • these microcolumns can form a certain shape.
  • cells tend to adhere to micropillars of a specific location and shape, allowing high-throughput mechanometry while controlling cell size, shape, and tropism.
  • the top end faces of some of the micropillars 12 of the micropillar array are provided with substances with cell adhesion function, while in this embodiment, the The part of the microcolumn 12 whose top end surface is not provided with the substance with cell adhesion function (end surface or side surface) is also provided with a substance with cell adhesion inhibitory effect, such as F-127.
  • a substance with cell adhesion inhibitory effect such as F-127.
  • the micro-pillars 12 are provided with a substance with cell adhesion function on the top surface of the array of micro-pillars 12 to form a preset pattern.
  • specific patterned layers of cell adhesion molecules can be printed by microprinting techniques to promote cell attachment in these areas.
  • the so-called preset patterns can be triangles, quadrilaterals, polygons, circles, ellipses and other shapes.
  • the functions of the preset patterns include: first, the cells and intercellular contacts are controlled by the patterns composed of these substances with cell adhesion, so as to It is convenient to realize the demands of high-throughput data acquisition.
  • the dimensionality reduction effect in data processing can be achieved by making the cell shape uniform, thereby reducing the difficulty of analysis.
  • the purpose of controlling cell size, shape, tropism, differentiation state, etc. can be achieved by limiting the cell attachment area, and even the mechanical state of cells can be regulated by controlling actin filaments, so as to meet some special technical requirements scenarios requirements.
  • the part of the preset pattern that is not printed can be made of a substance that inhibits cell attachment, such as BSA (bovine serum albumin) or F127 (polymer non-ionic surface active agent) to inhibit cell attachment in these areas, thereby achieving directional attachment, controlling cell morphology, or simulating a specific cellular microenvironment.
  • BSA bovine serum albumin
  • F127 polymer non-ionic surface active agent
  • the substance having cell adhesion function is selected as fibronectin (FN) as an example, but it is not intended to limit the embodiment of the present invention.
  • fibronectin FN
  • Polydimethylsiloxane micro-stamps with protruding square and rectangular patterns on the surface were used respectively, and fibronectin was adhered on the surface of the micro-stamp, and the fibronectin in the protruding part of the stamp was transferred to the It is located above the metal reflective layer on the top of the micro-column.
  • the microcolumn is immersed in F-127 solution, so that the part without fibronectin has the effect of inhibiting cell adhesion.
  • Figure 6a is a fluorescence imaging image of cells adhering to a preset pattern composed of microcolumns with fibronectin on the top. area; based on this, the cell attachment area can be limited by a preset pattern, and then the cells can be mechanically monitored under the condition of controlling the size, shape, tropism, and differentiation state of the cells.
  • Figure 6b shows the The distribution diagram of the cell mechanical force calculated from the measured light reflection signal.
  • the substance with cell adhesion is selected from OKT3 antibody (that is, a substance that interacts with cell surface receptors) or fibronectin (Fibronectin, FN) as an illustration, but it is not intended to limit the scope of the present invention. implementation.
  • Figure 7a is a schematic diagram of the experiment using OKT3 antibody as a substance with cell adhesion
  • the upper part of Figure 7b is the image of cells adhered to the top of the microcolumn with OKT3 antibody and fibronectin on the top
  • Figure 7b shows the distribution of the light reflection signal (reflecting the size of the cell mechanical force) measured on the microcolumn
  • Figure 7c shows the mechanical force measured on the surface coated with OKT3 antibody and fibronectin.
  • Size comparison diagram Figure 7d is a diagram of the dynamic changes in cell mechanics after T cells were planted on the surface of the OKT3 antibody (top of the microcolumn).
  • OKT3 antibody or fibronectin can be coated on the top of some microcolumns of the same or different cell mechanical force detection devices, and T cells are planted on the surface of the cell mechanical force detection device with cell adhesion substances. It can be seen from Figure 7a- Figure 7d that the cell mechanical force detection device coated with a substance (such as OKT3 antibody) or fibronectin (FN) that interacts with cell surface receptors on the surface of the microcolumn can be used to monitor the cell in real time. The mechanical influence and interaction of substances on cells.
  • a substance such as OKT3 antibody
  • FN fibronectin
  • the cell mechanical force detection device also includes a cell restriction mechanism, and the cell restriction mechanism includes one or several restriction surfaces 16, and the restriction surface 16 is a flat or curved surface perpendicular to the plane where the base 11 is located, connected to the base 11 or integrally formed with the base 11, and the height of the limiting surface 16 is higher than that of the microcolumn 12 and a predetermined number of Micropillars 12 are wrapped inside.
  • the function of the cell restriction mechanism set in this embodiment is to isolate and detect single cells, that is, to avoid contact or adhesion between cells during detection, and to restrict cell morphology, thereby facilitating high-throughput testing.
  • the number or shape of the limiting surfaces 16 in the cell position limiting mechanism may be different.
  • the limiting surface 16 included in the cell position limiting mechanism can be a cylindrical surface, or it can be three planes connected end to end to form a triangular cross-sectional shape and surrounded by a certain number of microcolumns, perpendicular to each other and connected end to end to form a rectangular shape
  • the cross-sectional shape formed by the restriction surface 16 is a controllable closed shape, and its area (or the number of micropillars that can be understood as its space) is also controllable.
  • the cell restriction mechanism can also appear in the following forms:
  • Figure 8 is a schematic structural view of a cell mechanical force detection device with a cell restraint mechanism a, in the figure, the cell restraint mechanism and the base 11 are integrally formed, that is: the cell restraint mechanism is formed
  • the material has several concave spaces 15, the wall surface of the concave spaces 15 is the limiting surface 16, the depth of the concave spaces 15 is the height of the limiting surfaces 16, the bottom of the concave spaces 15 is the base 11, and each concave space 15 There are several micropillars 12 in it.
  • FIG. 9 is a schematic structural diagram b of a cell mechanical force detection device with a cell restriction mechanism.
  • the restriction surface 16 is a structure bonded to the base 11 .
  • the difference between this embodiment and the eighth embodiment lies in that the cell restriction mechanism in this embodiment is a silicon thin film.
  • Fig. 10a is a physical diagram of a cell mechanical force detection device using a silicon thin film as a cell restriction mechanism.
  • the silicon thin film is adhered to the base after being drilled with a laser , each well is equipped with a micro-column, the shape and migration of cells are restricted by the silicon film, and the contact or adhesion between cells is controlled at the same time;
  • Figure 10b shows the cells using the silicon film as the cell restriction mechanism
  • the fluorescent microscope image of the mechanical force detection device, Fig. 10c is an enlarged view of Fig. 10b.
  • the size of each hole of the silicon membrane can be set to match the size of a single cell, suitable for single cell attachment, thereby limiting cell contact, cell shape and its migration range.
  • Embodiment 10 A detection system for cell mechanical force
  • a cell mechanical force detection system comprising the cell mechanical force detection device 1 described in the first or second embodiment, an optical signal generation device 2 and an optical signal detection device 3; the optical signal generation device 2 and the optical signal detection device 3 are located below the base 11 in the cell mechanical force detection device 1, the optical signal generating device 2 has a light source, and the light emitted by the light source passes through the incident optical path (through the light-transmittable base and the The light-transmissible microcolumn cylinder) irradiates the light reflection layer 13 of the microcolumn 12, and reflection occurs, and the reflected light enters the optical signal through the reflection optical path (passing through the light-transmissible microcolumn cylinder and the light-transmissible base successively) Detection device 3.
  • the light signal detection device 3 can acquire reflected light signals before and after the contact between the microcolumn 12 and the cell.
  • the cell mechanical force detection system also includes an optical signal analysis device 4, which can obtain cell mechanical force information by comparing, analyzing, and calculating the reflected light signals before and after the contact between the microcolumn 12 and the cell, including The magnitude, direction, and change of the mechanical force within a certain period of time, etc.
  • Fig. 4 is a cell mechanical force detection system related to the eleventh embodiment of the present invention, including the cell mechanical force detection device 1 described in the third embodiment, and also includes an optical signal generating device 2 and an optical Signal detection device 3; the optical signal generation device 2 and the optical signal detection device 3 are all located above the base 11 in the detection device 1 of the cell mechanical force, the optical signal generation device 2 has a light source, and the light source emits The light is irradiated to the light reflection layer 13 through the incident light path, and is reflected, and the light signal detection device 3 can acquire reflected light signals before and after the contact between the microcolumn 12 and the cell.
  • the optical signal detection device can be a microscope, a charge-coupled device CCD, a complementary metal oxide semiconductor CMOS, a photomultiplier tube PMT and a photoelectric converter PT, a film, or other optical signal detection elements with the same function , the present invention is not specifically limited. It should be noted that, in some embodiments of the present invention, when a microscope is used as the optical signal detection device, there is no need to set up an independent optical signal generation device, and the cell mechanical force detection device of the present invention can be directly placed on the microscope carrier.
  • the light source of the microscope is used as the optical signal generating device, and the objective lens of the microscope (5x objective lens is enough, no need to rely on high-magnification optical objective lens) is used as the optical signal detection device; when using other optical signal detection devices, such as charge When the coupling element CCD is used, it is necessary to set up an independent optical signal generating device.
  • the optical signal generating device may be LED, halogen lamp, laser (for example, infrared laser), or other light sources, or other devices with these light sources, which are not specifically limited in the present invention.
  • FIG. 11 is a fluorescent microscope image of monitoring cell mechanical force using the cell mechanical force detection system of this embodiment.
  • the cells for example, fibroblast used in this embodiment
  • the optical signal detection device for example, a microscope is used in this embodiment
  • the optical signal forms an image for visual observation, and can feed back the change of cell mechanical force in real time.
  • FIG. 4 is a cell mechanical force detection system related to the twelfth embodiment of the present invention, which includes the cell mechanical force detection device 1 described in the third embodiment, and also includes an optical signal generating device 2, An optical signal detection device 3 and an optical signal analysis device 4 .
  • Both the optical signal generating device 2 and the optical signal detecting device 3 are located above the base 11 in the cell mechanical force detecting device 1; the optical signal generating device 2 has a light source, and the light emitted by the light source passes through the incident optical path When it irradiates the light reflection layer 13, reflection occurs; the beam splitter 5 can be a transflective or other equivalent optical element, the main purpose is to simplify the design of the optical path; the optical signal detection device 3 can obtain the contact between the microcolumn 12 and the cell Reflected optical signals before and after; the optical signal analysis device 4 can obtain cell mechanical force information by comparing, analyzing, and calculating the reflected optical signals before and after the contact between the microcolumn 12 and the cell, including the size and direction of the cell mechanical force, and within a certain time range. changes, etc.
  • the optical signal analysis device can be optical image analysis software ImageJ, Matlab, Fluoview, Python, or other optical image analysis components with the same function, or the combined use of these analysis software, the present invention is not specifically limited .
  • Figure 12a is a schematic structural diagram of the cell mechanical force detection system
  • Figure 12b is an image of the light reflection signal of the cell mechanical force detection device obtained by the optical signal detection device
  • Figure 12c is the visualization of the mechanical size and distribution renderings.
  • each microcolumn is provided with a metal reflection layer, and the side is provided with an antireflection layer; when there are no cells, the light irradiates the microcolumn from below, which will be completely reflected It is completely received by the optical signal detection device (such as a CCD camera); but when the cells are attached to the microcolumn, the cell force generated by the cell movement will make the microcolumn tilt, thereby reducing the reflection signal, after the light reflection signal analysis , the strength of the cell force can be calculated.
  • the optical signal detection device such as a CCD camera
  • the image of the light reflection signal of the cell mechanical force detection device and the enlarged image of the local cell adhesion area are collected by an optical signal detection device (such as a CCD camera) (as shown in FIG. 12b ). Then, the image in Fig. 12b is further processed by the optical signal analysis device to transform it into a more intuitive visual effect diagram 12c of mechanical size and distribution.
  • an optical signal detection device such as a CCD camera
  • the specific processing process is as follows: first, based on Figure 12b, obtain the bright field reflection signal map (I, focusing on the cell); then, filter the high-frequency signal after performing Fourier transform on the image, and perform the inverse Fourier transform operation, thereby calculating and obtaining Then, the I and I 0 images are further processed to convert the reflection signal map into a more intuitive cell mechanics map (I 0 signal value minus I Signal value) after normalization to obtain a more intuitive cell mechanical force intensity map j.
  • This embodiment describes the calculation of mechanical force in the embodiment of the present invention in combination with the cell mechanical force detection system described in any one of the tenth to twelfth embodiments or the cell mechanical force detection method described in the fourteenth embodiment method; and use the fluid as an external force to verify the relationship between mechanics and light reflection signals.
  • Fig. 13a is a structural schematic diagram of the cell mechanical force detection device in the microfluidic environment before and after the fluid is turned on;
  • Figure 13d is a graph of the intensity of the light reflection signal before and after the fluid is turned on;
  • Figure 13e is a graph of the linear relationship between the light reflection signal and the offset of the microcolumn.
  • the superimposed effect diagram refers to the effect diagram formed by superimposing the bright-field microscope image of the microcolumn and the reflected light signal distribution image.
  • the cell mechanical force detection device is integrated in the microfluidic channel.
  • the microcolumn will be displaced and the angle of the reflective layer on the surface of the microcolumn will be changed at the same time ( Figure 13a- Figure 13c).
  • the light reflection signal changes from strong to weak before and after the fluid is turned on.
  • the flow velocity is used to change the offset of the microcolumn, and the displacement of the top of the microcolumn relative to the bottom of the microcolumn is photographed using a confocal microscope, and the mechanical force on each microcolumn can be calculated by the following formula:
  • F represents the mechanical force that causes the micropillar to deflect by an angle ⁇
  • E represents the Young's modulus
  • k bend represents the ideal spring constant of an isolated nanopillar
  • D represents the diameter of the micropillar
  • L represents the height of the micropillar.
  • a method for detecting cell mechanical force comprising the steps of:
  • the optical signal generating device 2 in the cell mechanical force detection system described in any one of the tenth to the twelfth embodiments emits light.
  • the optical signal detection device 3 in the cell mechanical force detection system described in any one of the tenth to the twelfth embodiment is used to detect the light after the action of the cell mechanical force detection device 1 .
  • the optical signal detection device 3 can acquire reflected light signals before and after the contact between the microcolumn 12 and the cell in the cell mechanical force detection device 1 .
  • the optical signal analysis device 4 in the cell mechanical force detection system obtains the cell mechanical force information, including the size of the cell mechanical force, by comparing, analyzing, and calculating the reflected light signals before and after the microcolumn 12 contacts the cells. , direction, changes in a certain time range, etc.
  • Fig. 14a-Fig. 14d Fig. 14a and Fig. 14b are the structure schematic diagrams before and after contact between the microcolumn of the cell mechanical force detection device and the cell;
  • Fig. 14b is the light signal detection device (CCD electronic photosensitive element) The reflected light signal of the cell can be clearly attenuated in the larger force field area around the cell;
  • Figure 14c is a monitoring map of the cell migration process (after the cell membrane is stained, it is excited with a fluorescent light source, and its migration is recorded with a CCD electronic photosensitive element process);
  • Figure 14d is a distribution diagram of reflected light signals during cell migration (using a CCD electronic photosensitive element to record changes in reflected light signals during the migration process). It can be seen from Fig.
  • Figure 14d shows the reflected light signal monitored in real time during the cell migration process, and the mechanical force in the migration process is fed back in real time through the reflected light signal. Feedback changes in cell mechanical forces during cell migration.
  • a method for preparing a cell mechanical force detection device comprising the steps of:
  • a layer of light reflection layer 13 is laid on the top or upper half cylinder of the microcolumn 12 to obtain the microcolumn 12 with a reflective layer on the top or upper half cylinder.
  • a method for preparing a cell mechanical force detection device comprising the steps of:
  • An anti-reflection layer is evenly coated on the microcolumn 12 as a whole;
  • a layer of light reflection layer 13 is laid on the top or upper half of the microcolumn 12 .
  • step "laying a layer of light reflection layer 13 on the top or upper half of the microcolumn 12" is specific to: on the top or on the microcolumn A layer of reflective metal is uniformly sputtered on the half-cylindrical surface to obtain a microcolumn with a metal light reflection layer on the top or upper half-cylindrical surface.
  • This embodiment provides a method for cell recognition, which includes: using the cell mechanical force detection system described in any of the above embodiments or the cell mechanical force detection method described in any of the above schemes to obtain cell mechanical force information, different cell types are distinguished according to the cell mechanical force information.
  • the step "use the cell mechanical force detection system described in any one of the above embodiments or the cell mechanical force detection method described in any one of the above schemes to obtain the cell mechanical force Force information, distinguishing different cell types according to the cell mechanical force information” specifically includes:
  • the cell information includes the cell mechanical force information of a certain point in the cell obtained by the detection device based on the cell mechanical force, the cell mechanical force information includes the magnitude of the cell mechanical force at this point, specifically: using light
  • the signal detection device or used in conjunction with the optical signal analysis device collects cell information on multiple cells on the cell mechanical force detection device, including collecting information on the size of the cell mechanical force at multiple points of each cell, so as to obtain multiple Multi-point cell mechanical force size data in cells;
  • the structured cell information includes the number of cells, the number of cell characteristics, and the characteristic information of each cell characteristic.
  • the cell mechanical force information also includes the direction of the cell mechanical force at this point.
  • the cell mechanical force information also includes changes in the magnitude or direction of the cell mechanical force at a certain time interval.
  • the cell information further includes cell shape information.
  • This embodiment specifically provides the cell mechanical force information obtained by the cell mechanical force detection system described in any one of the tenth to twelfth embodiments or the cell mechanical force detection method described in the fourteenth embodiment, which is applied to Methods for identifying cells.
  • Figure 15a is the fluorescence imaging diagram of the mixed system of healthy cells and lung non-small cell cancer cells
  • Figure 15b is the distribution diagram of the light reflection signal of the cell mechanical force detection device obtained by the optical signal detection device
  • Figure 15c It is a visualization effect diagram of mechanical size and distribution
  • Figure 15d is an enlarged view of the representative single-cell force distribution of healthy cells and lung non-small cell cancer cells in Figure 15c
  • Figure 15e is a healthy cell and lung non-small cell cancer cells
  • Figure 15f is a comparison chart of the reflection signal intensity of healthy cells, lung non-small cell carcinoma cells, and the mixture of these two cells in different proportions
  • Figure 15g is the structural treatment of Figure 15c , a cluster analysis graph based on structured cell information processing.
  • this embodiment takes healthy cells (Normal) and lung non-small cell carcinoma cell lines (Cancer) as detection objects, uses two different fluorescent dyes (Dil&DIO) to pre-stain the cell membranes of healthy cells and lung cancer cells, and uses a certain After proportional mixing, add to the same cell mechanical force detection device (in other embodiments, it may be added to different independent cell mechanical force detection devices).
  • healthy cells Normal
  • lung non-small cell carcinoma cell lines Cancer
  • Dil&DIO fluorescent dyes
  • the image of the light reflection signal of the cell mechanical force detection device (as shown in Figure 15b) was collected through the optical signal detection device (a microscope was used in this embodiment), and the high-resolution force field distribution in the two cells was detected by the optical signal
  • the detection device can directly render and convert it into a readable light intensity attenuation signal (reflecting the strength of the cell force) and display it in the picture (as shown in FIG. 15c ).
  • the two cells can be visually distinguished by visual observation (qualitative analysis).
  • the optical reflection signal in Fig. 15c is further processed by an optical signal analysis device.
  • this embodiment uses an optical signal analysis device (this embodiment uses ImageJ and Python analysis software, and other image analysis software can also be used in other embodiments) to collect information on the obtained cell force field in Figure 15c, wherein It includes collecting information on the size of cell mechanical force at multiple points of each cell, so as to obtain data on the size of cell mechanical force at multiple points in multiple cells; preprocessing the information on the size of cell mechanical force obtained to form structured cell information; and according to The structured cell information analysis obtained a comparison result map of healthy cells and lung non-small cell cancer cells in terms of cell morphology (as shown in FIG. 15e ).
  • Feature Matrix feature matrix
  • a supervised machine compared with pre-staining of two cell lines with different cell membrane dyes (Dil&DIO) is used to learn to build a cell feature model, and use the structure of a large number of cells
  • the cell characteristic model is trained by using the chemicalized cell information to obtain a cluster analysis diagram as shown in FIG.
  • an optical signal analysis device this embodiment uses ImageJ and Python analysis software, in other implementations
  • other cluster analysis software can also be used to cluster and type normal healthy cells and cancer cells, so as to realize the identification of unknown cell types.
  • Figure 15e shows that there is no statistically significant difference in the morphology (including cell adhesion area and cell roundness) of different cells
  • Figure 15f shows the obvious difference in the reflected signal intensity (reflecting cell force) between normal cells and tumor cells , and after mixing normal cells and tumor cells in a certain proportion, the reflected signal intensity and the mixing proportion have a certain linear relationship. It can be seen that, compared with other characteristics of cells (such as cell adhesion area, cell roundness and other morphological information in Fig. type for more intuitive and precise identification (quantitative and qualitative analysis).
  • tumor cells showed higher mechanical force magnitudes than normal cells, and the distribution was more uneven. It can be seen that after the cell mechanical force is visualized in the form of an image, the force field characteristics of different cells can be seen intuitively by the naked eye; and further, the force field size of each point of different cells is structured through image analysis software Afterwards, the cell morphology information in Fig. 15e, the reflected signal intensity (reflecting the cell force) in Fig. 15f and the cluster analysis diagram in Fig. 15g are obtained through comprehensive analysis.
  • the present invention can cluster, type and quantitatively analyze different cells (such as healthy cells and non-small cell lung cancer cells in this embodiment) through comprehensive analysis of the force field structured information at each point of the cell, thereby realizing accurate cell types identify.
  • the cell mechanical force detection device of the present invention not only can visual distinction be made by the naked eye for qualitative analysis, but also the state and type of cells can be more intuitively and accurately identified (quantitative and qualitative) based on the measured cell mechanical characteristics. analysis), and confirmed that the cell force field can be used as a marker to better distinguish cell types.
  • This embodiment specifically provides the cell mechanical force information obtained by the cell mechanical force detection system described in any one of the tenth to twelfth embodiments or the cell mechanical force detection method described in the fourteenth embodiment, which is applied to Monitor cell viability.
  • Figure 16a is a schematic diagram of the operation flow of the cell viability detection method
  • Figure 16b is the cell viability measured by the MTT method after A549 cells were treated with different doses of 5FU for 24 hours, and the device or system or method of the present invention.
  • Figure 16c is a comparison chart of the cell viability measured by the MTT method and the cell mechanical force measured by the device or system or method of the present invention after A549 cells were treated with different doses of 5FU for different times.
  • non-small cell lung cancer cells A549 were cultured on multiple cell mechanical force detection devices, treated with different doses of the drug 5-fluorouracil (5-FU) that inhibits cell proliferation, and passed the tenth to
  • the cell mechanical force detection system described in any one of the twelfth embodiment or the cell mechanical force detection method described in the fourteenth embodiment monitors the cell mechanical force at different time points, and monitors different time points through the CCK-8 kit
  • the cell proliferation and cytotoxicity were measured, and the cell viability measured by the MTT assay was used as a control group to obtain the data in Figure 16b and Figure 16c.
  • the cell viability measured by the MTT assay and the cell viability reflected by the cell mechanical force are in a dose-dependent manner.
  • the decrease in force specifically, a significant decrease trend can be seen at 6 h at a treatment dose of 0.5 ⁇ M, and a significant decrease trend can be seen at 3 h at a treatment dose of 1 ⁇ M, so that the decrease in cell viability can be more sensitively characterized.
  • direct detection of cell mechanical force by the cell mechanical force detection device is a highly sensitive and effective method for evaluating the response activity of cells to drugs.
  • This embodiment specifically provides the cell mechanical force information obtained by the cell mechanical force detection system described in the twelfth embodiment or the cell mechanical force detection method described in the fourteenth embodiment, and according to the cell mechanical force information Analysis determines cell state.
  • Figure 17a is a diagram of the operation process of the cell state detection method
  • Figure 17b is a fluorescence microscope image of M0 macrophages differentiated to M1 state
  • Figure 17c is a fluorescence microscope image of M0 macrophages differentiated to M2 state
  • Fig. 17d is a comparison diagram of cell adhesion area of M0 macrophages, M1 state and M2 state
  • Fig. 17e is a comparison diagram of cell roundness of M0 macrophages, M1 state and M2 state
  • Fig. 17f is a comparison diagram of M0 macrophages, Mechanical force comparison diagram of M1 state and M2 state.
  • macrophages are used as detection objects, which are respectively added to microcolumns of different independent cell mechanical force detection devices, and endotoxin LPS and interleukin IL4 are used to guide macrophages to differentiate from M0 to M1 and M2 states, with M0 state as the control group.
  • endotoxin LPS and interleukin IL4 are used to guide macrophages to differentiate from M0 to M1 and M2 states, with M0 state as the control group.
  • collect Figure 17b and Figure 17c through an optical signal detection device (a microscope is used in this embodiment), and perform further image processing and data analysis on Figure 17b and Figure 17c through optical signal analysis software (ImageJ and Python), This was transformed into structured information and analyzed to generate the data of Figure 17d-17f. From the data in Figures 17a to 17f, it can be seen that M0 macrophages and their differentiated M1 and M2 states are There are obvious differences between them.
  • This embodiment specifically provides the cell mechanical force information obtained by the cell mechanical force detection system described in the twelfth embodiment or the cell mechanical force detection method described in the fourteenth embodiment, and analyzed according to the cell mechanical force information Determine cell state.
  • the multicellular aggregate provided in this example is combined with the cell mechanical force detection device in various ways, and this example provides two specific ways of combining:
  • the first combination method set the culture medium on the microcolumn of the cell mechanical force detection device, and transplant the cells into the culture medium on the microcolumn to obtain multicellular aggregates; in other embodiments, this combination method, With the output of cell mechanical force information in a visualized form, the cell culture process can be monitored in real time to apply to the influence of chemical, biological and physical external stimuli such as culture medium and drugs on cell growth;
  • the second combination method directly attach the cultured multicellular aggregate to the microcolumn of the cell mechanical force detection device for detection.
  • this embodiment provides a tumor cell multimer cultured on a cell mechanical force detection device, which is applied to a drug sensitivity test, including the following steps:
  • Figure 18a is a characterization diagram of tumor cell multimers with the first morphology with or without 5-Fu;
  • Figure 18b is a tumor cell multimer with the second morphology in Characterization diagram with/without 5-Fu effect; from left to right are the mixed image of cell membrane fluorescence and reflection signal (1), light reflection signal (2), cell nucleus (3), cell membrane (4) and light signal The visualized image of cell force processed by the analysis device (ImageJ) (5). Due to the heterogeneity of cells, tumor cells are different, and multimers also have various morphologies, so cell multimers will stick together in different morphologies.
  • two representative forms are selected for cell morphology detection.
  • the first form refers to the cell form in which two larger cells stick together;
  • the second form refers to a group of small cells sticking together. cell morphology together.
  • the cell mechanical force detection device of the present invention can measure the cell mechanical force of multicellular aggregates (such as tumor polymers), and can be used to monitor the viability of cell polymers through cell mechanical force, and can distinguish different cell shapes.
  • cellular multimer refers to:
  • Cells are the basic structural and functional units of organisms. Cells usually multiply or differentiate to form two or more cells that aggregate together to form a cell population, namely: multicellular aggregates; multicellular aggregates include tumor polymers and other in vitro or in vivo cultured cells. Obtained cell groups.
  • the cell detection device of the present invention has very high sensitivity and effectiveness for the detection of cell mechanical force, cell identification, cell state detection, and cell viability detection, and can realize real-time monitoring, and can be applied to drug treatment, cell Response to medication.
  • the technical solution of the present invention gets rid of the dependence on the microscope and greatly simplifies the operation process, because there is no need for high-resolution imaging through the microscope, only By monitoring the intensity of reflected light, high-throughput monitoring of cells can be achieved.
  • single cell resolution high resolution, real-time monitoring of each cell, can be combined with other single cell analysis techniques to measure the heterogeneity of cell response to drugs; real-time monitoring: no fluorescence, can avoid laser light on cells Toxicity, so it is suitable for long-term monitoring, and can be used to study the long-term response of cells to drugs; high sensitivity: through the reflected signal, the micro-column deformation signal is amplified to increase the sensitivity of deformation monitoring.
  • the detection of bending deformation of micro- and nano-pillars generally relies on optical systems (such as microscopes) for detection, but the smaller the size of the micro-pillars, the higher the precision and resolution of the optical system.
  • a microcolumn with a width of 2 microns and a height of 6 microns requires an objective lens with a power of 20 times or more and a conjugate focus system for effective observation.
  • the present invention utilizes the principle of specular reflection to detect the attenuation of reflected light, and actually amplifies the signal of the deformation of the micro-column. It has been verified by experiments that the same signal can be observed under a 5x objective lens. With a special reading system, it can effectively detect the deformation of micro/nano-pillars without relying on high-magnification optical objective lenses, thereby greatly reducing system costs and effectively improving throughput.

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Abstract

一种细胞机械力的检测装置（1），包括：基座（11），以及设置于基座（11）上的，可受细胞机械力作用而产生形变的多个微柱（12）构成的微柱阵列，微柱（12）的顶部或柱面上部具有光线反射层（13）。同时提供了检测系统、检测方法以及制备方法。方案具有高通量低成本、单细胞分辨率、实时监测、高灵敏度、可模拟细胞微环境、可模拟细胞外基质的组分和形态等优势，可应付更丰富的技术需求场景。

Description

细胞机械力的检测系统、方法、装置及其制备方法 技术领域
本发明涉及生物技术领域，尤其涉及一种细胞机械力的检测系统、细胞机械力的检测方法、细胞机械力的检测装置、细胞机械力的检测装置的制备方法。
背景技术
细胞对周边微环境会施加微小的机械力，细胞力在包括粘附，迁移，增殖，分化，凋亡等过程中发挥关键作用，并且与其它生化信号一起，在胚胎发育、干细胞分化、免疫过程、伤口修复、癌症转移等过程中起到至关重要的调控作用，因此也成为不少疾病治疗的靶点。例如在之前的研究中发现，正常细胞，良性及恶性肿瘤细胞的最大细胞机械力存在巨大差异，因此对细胞机械力进行高通量且精准的测量将成为多项生物医学应用的核心技术。但测量最大细胞机械力的意义有限，高解析度、实时、高通量的力学传感器将成为下一代细胞力测量工具的核心需求。
现有对细胞机械力的测量的技术主要方法包括“机械力显微镜”，微纳米悬臂及微柱阵列。主要原理是通过测量细胞对弹性基底施力引起基底的形变来计算细胞的机械力。迄今为止细胞机械力显微镜(TFM)是运用最广的细胞机械力测量的技术方法，其原理是基于连续弹性基底(例如可起皱的薄硅膜或镶嵌带荧光微珠的聚丙烯酰胺PA凝胶)。在弹性基底上进行细胞培养时，细胞会对基底施加机械力使基底发生变形，变形过程中荧光微珠产生相应的位移；利用荧光显微镜等方法跟踪荧光微珠的运动轨迹，通过图像处理等方法获得基底应变信息。基底弹性已知的情况下可通过力学模型，反推出细胞的机械力。然而这种测量方法存在以下缺陷，首先其依赖荧光显微镜成像并需要进行复杂的计算，过程复杂，通量低且成本高。其次TFM的原理不是对细胞机械力进行直接测量，而是通过观察基底中荧光微珠的位置变化反推细胞机械力。尽管通过特殊工艺可保证大部分颗粒沉积在表面，但是在长时间浸泡后，其极有可能逸出或下降最终导致表面荧光颗粒的密度降低。荧光显微镜通常景深较大，可能会拍摄到处于不同平面的颗粒，从而导致后期位移计算的偏差，导致测量结果不准确。最后，另外，长期浸泡在培养液中会改变凝胶的弹性模量，势必影响细胞机械力计算的准确性，因此在测量过程中需要对凝胶弹性模量进行检测校准，极大增加了工作量。此外长期激光照射会对细胞产生光毒性，也会引发荧光微珠光淬灭，因此TFM难以胜任细胞机械力的长期连续检测，而研究细胞生长和分化及其对药物的反应过程通常都需要较长时间的监测，因此以上所述TFM的缺陷极大地限制了其在生物医学领域方面的应用。
与TFM相比，微纳米传感器(例如微悬臂阵列和微柱阵列)也可用于直接测量细胞机械力。例如微柱阵列，细胞贴附于与微柱上方表面，通过显微镜拍摄微柱底部和顶部的图片即可解算出微柱的弯曲变形，从而反推出该点细胞机械力的大小与方向。和TFM一样，微纳米传感器也需要依赖显微镜高解析度拍照对设备要求高，且容易在拍照过程中产生误差造成结果不准确。此外显微镜拍摄得到的图片需要经过复杂的图像处理，并根据力学模型来计算求解细胞机械力的大小。操作复杂且耗时，难以实现实时性，高通量，低成本，及对细胞的长期检测。因此现有技术大都仅限于生物力学领域科研，难以实用化。
发明内容
为此，需要提供一种能够克服现有技术的不足，无需显微镜、实时、高通量及低成本的细胞机械力定量测量及监测方案，来解决下一代细胞力测量工具的核心需求。
为实现上述目的，发明人提供了一种细胞机械力的检测装置，包括：
基座，以及
设置于基座上的，可受细胞机械力作用而产生形变的多个微柱构成的微柱阵列，所述微柱的顶部或柱面上部具有光线反射层。
进一步地，所述的细胞机械力的检测装置中，所述基座为透光基座，所述微柱的柱体可透射光线；所述微柱顶部具有光线反射层。
进一步地，所述的细胞机械力的检测装置中，所述微柱的柱面具有抗反射层。
进一步地，所述的细胞机械力的检测装置中，所述光线反射层为金属箔层，金属氧化物或金属盐，超细玻珠或微棱镜，有机反光材料一种或一种以上的组合。
进一步地，所述的细胞机械力的检测装置中，所述微柱阵列的全部或部分微柱的顶部端面上设有具有细胞黏附作用的物质。
进一步地，所述的细胞机械力的检测装置中，所述具有细胞黏附作用的物质包括如下物质中的一种或多种：细胞外基质分子，包括胶原蛋白、纤粘连蛋白、玻璃粘连蛋白、层粘连蛋白或弹性蛋白原；细胞外基质的模拟物质，包括含有RGD粘附序列的多肽；具有细胞黏附促进机制的物质，包括聚赖氨酸；与细胞表面受体具有相互作用的物质。
进一步地，所述的细胞机械力的检测装置中，所述微柱阵列的预设区域的部分微柱的顶部端面上设有具有细胞黏附作用的物质。
进一步地，所述的细胞机械力的检测装置中，所述微柱阵列在顶部端面上未设有具有细胞黏附作用的物质的微柱的顶部端面上设有具有细胞黏附抑制作用的物质。
进一步地，所述的细胞机械力的检测装置中，所述微柱的横截面形状为圆形、椭圆形或多边形。
进一步地，所述的细胞机械力的检测装置中，所述微柱阵列的尺寸范围包括：柱高10nm～500μm，柱间距10nm～50μm，柱上表面直径50nm～50μm。
进一步地，所述的细胞机械力的检测装置还包括细胞限制机构，所述细胞限制机构包括一个或若干个限制面，所述限制面为与基座所在平面垂直、连接于所述基座或与所述基座一体成型的平面或曲面，且所述限制面的高度高于微柱并将预设数量的微柱包绕在内。
发明人同时提供了一种细胞机械力的检测系统，包括上述技术方案所述的细胞机械力的检测装置、光信号发生装置和光信号检测装置；
所述光信号发生装置具有光源，所述光源发出的光线通过入射光路照射到微柱的光线反射层；
所述光信号检测装置用于检测从微柱的光线反射层反射的光线，所述光线反射层反射的光线经过反射光路进入光信号检测装置。
进一步地，所述的细胞机械力的检测系统中，所述细胞机械力的检测装置的基座为透光基座，所述微柱的柱体可透射光线；所述微柱顶部具有光线反射层；
所述光源发出的光线通过入射光路从细胞机械力的检测装置的基座照射到微柱的光线反射层；
所述光信号检测装置用于检测从微柱顶部的光线反射层反射的光线，所述光线反射层反射的光线经过反射光路进入光信号检测装置。
进一步地，所述的细胞机械力的检测系统中，还包括用于分析光信号的光信号分析装置。
发明人同时还提供了一种细胞机械力的检测方法，包括如下步骤：
使用如上技术方案所述的细胞机械力的检测系统中的光信号发生装置发出光线；
使用如上技术方案所述的细胞机械力的检测系统中的光信号检测装置检测经所述细胞机械力的检测装置作用后的光线。
进一步地，所述的细胞机械力的检测方法中，还包括步骤：使用光信号分析装置对所述细胞机械力的检测装置与待测细胞发生细胞机械力作用前后的反射光线进行对比分析，获取细胞机械力信息。
发明人同时还提供了一种细胞状态的检测方法，其包括：采用上述任意一项方案中所述细胞机械力的检测系统或上述任意一项方案中所述细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息分析并确定细胞状态；
所述细胞状态，具体包括细胞黏附，细胞活力，细胞分化/活化，细胞增殖和/或细胞迁移。
进一步地，所述细胞状态为静止细胞状态或实时细胞状态。
发明人同时还提供了一种细胞识别的方法，其包括：采用上述任意一项方案中所述细胞机械力的检测系统或上述任意一项方案中所述细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息区分不同的细胞种类。
进一步地，所述细胞识别的方法中，步骤“采用上述任意一项方案中所述细胞机械力的检测系统或上述任意一项方案中所述细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息区分不同的细胞种类”具体包括：
获取细胞信息，所述细胞信息包括基于细胞机械力的检测装置获取的细胞中某点的细胞机械力信 息，所述细胞机械力信息包括该点细胞机械力的大小；
对细胞信息做预处理，形成结构化细胞信息；所述结构化细胞信息包括细胞数目、细胞特征数目和各细胞特征的特征信息；
以结构化细胞信息作为输入数据，利用有监督、无监督或半监督的机器学习建立细胞特征模型，并将所述细胞特征模型应用于未知类型或未知状态的细胞的分类或聚类。
进一步地，所述的细胞识别的方法中，所述细胞机械力信息还包括该点细胞机械力的方向。
进一步地，所述的细胞识别的方法中，所述细胞机械力信息还包括该点细胞机械力的大小或方向在一定时间间隔内的变化。
进一步地，所述的细胞识别的方法中，所述细胞信息还包括细胞形貌信息。
发明人同时还提供了一种细胞机械力检测装置的制备方法，包括如下步骤：在微柱的顶部或上半柱面铺设一层反射层，获得顶部或上半柱面具有反射层的微柱。
进一步地，所述的细胞机械力检测结构的制备方法中，在步骤“在微柱的顶部或上半柱面铺设一层反射层”之前还包括步骤：
在微柱整体均匀镀一层抗反射层；
将顶部或上半柱面的抗反射层去除。
进一步地，所述的细胞机械力检测结构的制备方法中，所述“在微柱的顶部或上半柱面铺设一层反射层，获得顶部或上半柱面具有反射层的微柱”具体为：在微柱的顶部或上半柱面均匀溅射一层反射金属，获得顶部或上半柱面具有金属光线反射层的微柱。
进一步地，上述任一方案中，细胞可以是单细胞，或者两个以上细胞形成的任何形态的多细胞聚合体。本发明对单细胞或两个以上多细胞形成的各种形态无限定。
区别于现有技术，上述技术方案具有如下优点：
首先，高通量低成本：与现有TFM及普通微柱阵列相比，本发明的技术方案摆脱了对显微镜的依赖，极大地简化了操作流程，因为无需通过显微镜高分辨率成像，只需通过对反射光的强度进行监测，即可实现高通量对细胞进行监测，且成本低廉。
其次，单细胞分辨率：分辨率高，可对每只细胞进行实时监测，可结合其他单细胞分析技术测量细胞对药物反应的异质性；实时监测：无需荧光，可避免激光对细胞的光毒性作用，因此适合长期监测，可用于研究细胞对药物的长期反应；高灵敏度：通过反射讯号，将微柱形变信号放大，增加对形变监测的灵敏度。检测微、纳米柱弯曲形变一般依赖光学系统(如显微镜)进行检测，但微柱尺寸越小，对光学系统的精密度和解析度要求越高。例如2微米宽，6微米高的微米柱，需要20倍以上的物镜搭配共轭焦系统才可有效观测。而本发明利用镜面反射原理通过探测反射光的衰减，实际使微柱形变的信号得以放大，经实验验证，同样的讯号在5倍物镜下即可观测。搭配特殊的读取系统，不需依赖高倍数的光学物镜即可有效检测微/纳米柱形变，从而极大降低系统成本，并有效提升通量。
再次：可模拟细胞微环境；可模拟细胞外基质的组分和形态，可应付更丰富的技术需求场景。
再次：可以对多层细胞，包括肿瘤多聚体等细胞机械力进行检测，使其可以应用于药物筛选、再生医学、基因编辑、精准医疗、器官发育、疾病建模中的需要对多细胞聚合体表征的场景。
附图说明
图1为本发明第一实施例中一种细胞机械力的检测装置的结构示意图；
图2为本发明第一实施例中一种细胞机械力的检测装置的微柱(实物)的扫描电子显微镜(SEM)图像；其中，图2a为细胞机械力检测装置的俯视图，图2b为细胞机械力检测装置的侧视图；
图3为本发明第九实施例相关的一种细胞机械力的检测系统的结构示意图；
图4为本发明第十实施例相关的一种细胞机械力的检测系统的结构示意图；
图5为在顶部位置设有光线反射层(金)的微柱(聚二甲基硅氧烷)的扫描电子显微镜图像；其中，图5a为微柱的扫描电子显微镜图像；图5b为微柱顶部区域的元素表征图；图5c为微柱侧面区域(除顶部区域外)的元素表征图；
图6a为细胞粘附于顶部设有纤连蛋白的微柱群构成的预设图案的荧光成像图；
图6b为细胞粘附于顶部设有纤连蛋白的微柱群上测得的光反射信号解算出来的细胞力分布图；
图7a为采用OKT3抗体作为具有细胞黏附作用的物质的试验示意图；
图7b的上部分图像为细胞分别粘附于顶部设有OKT3抗体、纤连蛋白的微柱顶部的荧光成像图；
图7b的下部分图像为在微柱上测得的光反射信号解算出的细胞机械力大小分布图；
图7c为分别在OKT3抗体、纤连蛋白涂敷表面测得的力学大小对比图；
图7d为将T细胞种植于OKT3抗体表面(微柱顶部)后发生的细胞力学动态变化图；
图8为具有细胞限制机构的细胞机械力的检测装置的结构示意图a；
图9为具有细胞限制机构的细胞机械力的检测装置的结构示意图b；
图10a为采用硅薄膜作为细胞限制机构的细胞机械力的检测装置的实物图；
图10b为在光反射下采用硅薄膜作为细胞限制机构的细胞机械力的检测装置的荧光显微镜图；
图10c为图10b的放大图；
图11第十一实施例的细胞机械力的检测系统监测细胞机械力的荧光显微镜图像；
图12a为第十二实施例的细胞机械力检测系统的结构示意图；
图12b为第十二实施例的光信号检测装置获取得到的细胞机械力检测装置光反射讯号的图像；
图12c为第十二实施例的光信号分析装置处理后的力学大小及分布的可视化效果图；
图13a为流体开启前后微流体环境中细胞机械力检测装置的结构示意图；
图13b为流体开启前微柱的明视场显微镜图、反射光信号分布图以及二者叠合效果的对比图；其中，叠合效果图是指将微柱的明视场显微镜图和反射光信号分布图叠合而成的效果图；
图13c为流体开启后微柱的明视场显微镜图、反射光信号分布图以及二者叠合效果图的对比图；其中，叠合效果图是指将微柱的明视场显微镜图和反射光信号分布图叠合而成的效果图；
图13d为流体开启前后光反射信号的强度值；
图13e为光反射信号衰减和微柱顶部平移的线性区间；
图14a为细胞机械力的检测装置的微柱与细胞发生接触前后的结构示意图；
图14b为光信号检测装置获取的反射光信号分布图；
图14c为细胞迁移过程的监测图；
图14d为细胞迁移过程中的反射光信号分布图；
图15a为健康细胞和肺非小细胞癌细胞混合体系的荧光成像图；
图15b为光信号检测装置获取得到的细胞机械力检测装置光反射讯号分布图；
图15c为光信号分析装置处理后的力学大小及分布的可视化效果图；
图15d为图15c中的健康细胞和肺非小细胞癌细胞的代表性单细胞细胞力分布的放大图；
图15e为健康细胞和肺非小细胞癌细胞在细胞形态上的对比图；
图15f为健康细胞、肺非小细胞癌细胞以及这两种细胞以不同比例混合后的反射信号强弱的对比图；
图15g为将图15c经过结构化处理后，基于结构化细胞信息处理得到的聚类分析图；
图16a为细胞活力检测方法的操作流程示意图；
图16b为A549细胞在不同剂量的5FU处理24h后，MTT法测定得到的细胞活力以及细胞机械力所反映的细胞活力的对比图；
图16c为A549细胞在不同剂量的5FU处理不同时间后，MTT法测定得到的细胞活力以及细胞机械力所反映的细胞活力的对比图；
图17a为细胞状态检测方法的操作过程图；
图17b为M0巨噬细胞分化至M1状态的荧光显微镜图；
图17c为M0巨噬细胞分化至M2状态的荧光显微镜图；
图17d为M0巨噬细胞、M1状态以及M2状态的细胞黏附面积对比图；
图17e为M0巨噬细胞、M1状态以及M2状态的细胞圆度对比图；
图17f为M0巨噬细胞、M1状态以及M2状态的机械力对比图；
图18a为具有第一种形态的肿瘤细胞多聚体在有/无5-Fu作用后的表征图；图中，从左到右分别是细胞膜荧光和反射讯号的混合图像(1)、光反射讯号(2)、细胞核(3)、细胞膜(4)及经过光信号分析装置(ImageJ)处理后的细胞力可视化图像(5)；
图18b为具有第二种形态的肿瘤细胞多聚体在有/无5-Fu作用后的表征图；图中，从左到右分别是细胞膜荧光和反射讯号的混合图像(1)、光反射讯号(2)、细胞核(3)、细胞膜(4)及经过光信号分析装置(ImageJ)处理后的细胞力可视化图像(5)。
附图标记说明：
1-细胞机械力的检测装置；2-光信号发生装置；3-光信号检测装置；4-光信号分析装置
5-分光器；
11-基座；12-微柱；13-光线反射层；15-凹陷空间；16-限制面
具体实施方式
为详细说明技术方案的技术内容、构造特征、所实现目的及效果，以下结合具体实施例并配合附图详予说明。
第一实施例 一种细胞机械力的检测装置
请参照图1，为一种细胞机械力的检测装置的结构示意图a，图中展示的细胞机械力的检测装置包括透光的基座11以及设置在该基座11上的、可受细胞机械力作用而产生形变的微柱12，微柱12的顶部涂覆有光线反射层13，光线反射层13的厚度为5nm(在其他一些实施方式中，光线反射层13的厚度可以在5nm-20nm之间——涂层的厚度和涂层材料有关，在应用同种涂层材料的前提下，涂层厚度的选择应以保证透光效果、微柱柱体稳定性、保证与微柱柱体的连接不脱落为限)。微柱12的柱体可以透射光线，图中方向相反的箭头簇表示入射光线和反射光线。(注意：本实施例中用了“涂层”一词，仅表示本实施例中的光线反射层13可以是涂覆工艺制备的，并不限定光线反射层13一定是涂覆工艺制备的)
请参照图2，为本实施例的细胞机械力检测装置的微柱12(实物)的扫描电子显微镜(SEM)图像，图2a为细胞机械力检测装置的俯视图，图2b为细胞机械力检测装置的侧视图。由图2可以看出，该细胞机械力检测装置的微柱的微结构整齐均一，尺寸可控，相比现有的细胞机械力检测装置，基于本实施例的细胞机械力检测装置测量得到的力学值更为精准。
当本实施方式所述的细胞机械力的检测装置1在投入使用时，微柱12的数量将不止一个。请参阅图3，为本发明第九实施例相关的细胞机械力的检测系统的结构示意图；图3可用于对本实施例的理解。图3展示的系统除了涉及本实施例阐述的细胞机械力的检测装置1之外，还涉及：在基座11的下方设置的具有光源的光信号发生装置2以及光信号检测装置3，所述光源发出的光线通过入射光路从细胞机械力的检测装置1的透光的基座11照射到微柱12的光线反射层；所述光信号检测装置3用于检测从微柱顶部的光线反射层13反射后的光线，所述光线反射层13反射的光线经过反射光路并经过分光器5作用之后进入光信号检测装置3。在获得反射光信号之后，可以由一个光信号分析装置4对所述细胞机械力的检测装置1与待测细胞发生细胞机械力作用前后的反射光线进行对比分析，获取细胞机械力信息。当微柱12未受力的情况下微柱理应保持直立状态，从而能最大限度地反射探测光；而当微柱12与细胞相接触时，在细胞机械力的作用下，微柱12发生弯曲，导致光反射水平降低。所以，当细胞机械力越大时，所得到的光反射信号就应越小，这样藉由对光反射信号强度的观测即可轻易反推该点细胞机械力的大小。
此外，本实施例的技术方案中的测量光源可以采用一定强度的红外激光。传统技术方案中的微柱测量需要拍高解析度图像，在该过程中若使用激光容易造成细胞光毒性或是样品荧光光淬灭。而本技术方案中若只要测反射信号，因而一定光强度以内的红外激光对细胞影响基本可以忽略不计，因此适合对细胞进行长期监测。
第二实施例 一种细胞机械力的检测装置
与第一实施例不同之处在于，所述微柱12不仅顶部端面具有光线反射层13，在微柱12的柱面(即连接柱体两个端面的曲面)的上半部分也设有光线反射层13。实际上，在其他实施方式中，除了在微 柱12的侧柱面的下半部分设置光线反射层13的方案因实际效果较差而不予采用之外，只要将光线反射层13设置于微柱12的侧面的上半部分，基本都能够实现本发明所想达到的检测效果。也就是说，在某些其他实施方式中，光线反射层13甚至可以铺设在上半侧柱面的任一局部位置或顶部的局部位置而并不一定要铺满整个上半柱面或整个顶部端面，都能达到预期目的，虽然获取的数据和后期运算的效果上可能有所差别。
此外，本发明的第一实施例和第二实施例出现了对微柱“柱面”和“端面”的定义，也就是说，通常我们理解的独立柱体，应有两个端面以及将两个端面连接的曲面(柱面)，而本发明中的微柱由于基座的存在而只具有一个端面即顶端面，另一端则固定连接于基座或与基座一体成型。然而，在另外一些实施方式中，顶部的端面可能与柱面为一个整体光滑连接的曲面，并不必然如第一实施例或第二实施例所示的这样有交线或明显分界。在这种情况下，光线反射层13的设置位置也将理解为柱体的上半部分，而并不可被限定为“端面”或“柱面”。
第三实施例 一种细胞机械力的检测装置
请参阅图4，图4为本发明第十实施例中一种细胞机械力的检测系统的结构示意图，用于说明本实施例中的细胞机械力的检测装置1。本实施例与第一、第二实施例不同之处在于，对所述基座11和所述微柱阵列的微柱12柱体的透光性能不作要求，即既可透光也可不透光也可半透光。此时，只需要改变光信号发生装置2和光信号检测装置3的位置，将二者设置于基座11的上方，这样每次当微柱发生弯曲的时候，光信号检测装置3所接收到的光信号都将相对于微柱12直立不形变时发生变化，通过对前后光信号的变化情况进行分析同样也可以得到细胞机械力的相对大小情况，在与标准值经过校正之后可以得到细胞机械力的绝对大小数值。
第四实施例 一种细胞机械力的检测装置
本实施例与第一至第三实施例不同之处在于，在微柱12表面，除设有光线反射层13区域之外的区域，设置有对光线的抗反射层。这样的设计可以减少柱体表层可能带来的反射光信号的干扰，增强信噪比，使检测结果更加精确。
在某些实施例中，所述光线反射层13可以是一层金箔。在其他实施例中，光线反射层13还可以是其他具有光线反射功能的金属层或其他反光材料。不同材料带来的反光效果、反射层制备难易程度以及成本等可能存在差异，在实际操作中可以根据具体条件进行考量和抉择。
在第一至第四实施例中，所述微柱12的横截面形状为圆形。在其他实施方式中，所述微柱12的横截面形状还可以是椭圆形或多边形。在本发明的各种不同具体情况的实施方式中，横截面的不同可以达到不同的目的，例如圆形横截面具有各向同性的特点，即微柱本身的力学特性对方向不敏感。而如截面为椭圆形的情形则为各项异性，即微柱本身的力学特性对方向敏感，可以此控制不同方向对力场的敏感度，并且能在一定程度上调控细胞的向性(大部分细胞的几何形态其实都是不对称的，本发明中细胞的向性指的是细胞表现出的形态上的不对称性、极性或方向性。例如，假若用一个椭圆来拟合细胞投影的形状，椭圆的长轴可以认为是细胞具有的方向)。因为如截面是椭圆形的话，截面具有长轴和短轴，则沿着短轴比长轴要推动微柱要容易得多，则相对受力条件下形变也大。在一些延伸实施方式中，如果把细胞种在这种微柱上，细胞和微柱存在各向异性的力学交互，将会导致细胞沿着某一侧生长。而应用在流体上，则可用来测定流体的方向。
在第一至第四实施例中，所述微柱阵列的尺寸为：柱高10nm～500μm，柱间距10nm～50μm，柱上表面直径50nm～50μm。该尺寸范围内的微柱可以满足作为传感器使用的微柱的基本使用条件，即至少可形变且不倒伏。在此基础上，不同微柱阵列尺寸的调控还可以实现如下功能：例如，通过调控微柱的纵横比AspectRatio(在微柱的层面可以理解为高度和横截面直径/边长/长径之比)可以实现一定的微柱形变性能调控功能，从而对体内器官组织环境(例如不同硬度的骨组织和神经组织)实现更好的模拟。
此外，阵列的整体规格或者说一定面积基座11上的微柱12数量也会影响配体密度Ligand Density，即细胞在表面能找到可粘附的点的数量。如果微柱12阵列越稀疏，则细胞能找到的粘附点越小，对细胞行为会产生不小的影响。
微柱的形状中的截面积大小也将影响细胞黏附行为，因为细胞黏附形成黏附斑FocalAdhesion是需 要一定面积的。如果是纳米微柱，则微柱截面面积小，对FocalAdhesion的形成会产生影响。
总而言之，结合材料本身特性和一定的微柱阵列尺寸，能够达到更符合需求的细胞支撑效果、芯片稳定性、测量精度。通过调控微柱阵列的分布还可以在一定程度上对细胞附着状态进行调控和影响。
在第一至第四实施例中，所述微柱12的材质为聚二甲基硅氧烷(PDMS)。在其他一些本发明的主要实施方式中，微柱12的材质还可以是其他一些高分子材料，例如硅基高分子聚合物、光阻高分子材料、导电高分子材料、温敏高分子材料等。本发明主要实施方式主要采用高分子材料的原因是当前高分子材料具有比较合适于本发明应用的可形变性能，但本发明的实施并不需要把微柱材质限定为高分子材料，而完全应当和可以扩展至所有具有相应可形变性的材料，均可实现本发明的发明构思。简言之，微柱的材质必须满足的条件是具有一定的受力可形变性，以及在一些实施方式中，需要有一定的透光性，后者并非所有实施方式的必要条件，在选用透光性能受限的材料制备微柱的情形下，只要适当设置光信号发生装置和光信号检测装置的位置，同样也能够实现本发明的发明构思。
总体而言，微柱12的硬度(可形变性)可根据实际需求，通过尺寸(AspectRatio为主)、材料类型的选择以及高分子材料交联程度的控制、化学或物理表面处理等多重技术维度来进行调控。
请参照图5，图5为在顶部位置设有光线反射层(金)的微柱(聚二甲基硅氧烷)的扫描电子显微镜图像；其中，图5a为微柱的扫描电子显微镜图像；图5b为微柱顶部区域的元素表征图；图5c为微柱侧面区域(除顶部区域外)的元素表征图。通过图5的扫描电镜图像表征微柱的物质成分构成，可以确认微柱的顶部位置存在有Au元素，微柱的其余位置存在有Si元素。
第五实施例 一种细胞机械力的检测装置
本实施例与第一至第四实施例的不同之处在于，微柱阵列的部分微柱12的顶部端面上设有具有细胞黏附作用的物质，本实施方式采用的是细胞外基质分子中的胶原蛋白，在其他实施方式中还可以采用包括胶原蛋白在内、以及纤粘连蛋白、玻璃粘连蛋白、层粘连蛋白以及弹性蛋白原这些细胞外基质分子中的一种或若干种的结合。在另外一些实施方式中，还可以在微柱12阵列的全部或部分微柱的顶部端面上设其他类别的具有细胞黏附作用的物质，例如细胞外基质的模拟物质，如含有RGD粘附序列的多肽；或具有细胞黏附促进机制的物质，包括聚赖氨酸；又或者是与细胞表面受体具有相互作用的物质。
在微柱12顶部端面上设置这样的具有细胞黏附作用的物质可以有效促进细胞对微柱12的贴附，从而实现对细胞贴附、增殖、迁移、状态、分化等的调控。此外，如在微柱阵列的预设区域的部分微柱的顶部端面上设有具有细胞黏附作用的物质，例如Fibronectin等细胞外基质蛋白，则这些微柱可组成一定的形状。从而细胞倾向于粘附在特定位置和形状的微柱上，从而在控制细胞大小，形状及向性特征的情况下进行高通量力学测量。
第六实施例 一种细胞机械力的检测装置
本实施例与第五实施例不同之处在于，如第五实施例所述，微柱阵列的部分微柱12的顶部端面上设有具有细胞黏附作用的物质，而本实施例中，所述顶部端面未设有具有细胞黏附作用的物质的那部分微柱12的柱面(端面或侧面)还设有具有细胞黏附抑制作用的物质，例如F-127。从而细胞更倾向于粘附在特定位置和形状的微柱上，从而在控制细胞大小，形状及向性特征的情况下进行高通量力学测量。
第七实施例 一种细胞机械力的检测装置
本实施例与第一至第四实施例的不同之处在于，微柱12阵列的顶部端面上设有具有细胞黏附作用的物质的微柱构成预设的图案。具体而言，可通过微米印刷技术来印刷特定图案的细胞粘附分子层促进细胞在这些区域的贴附。所谓的预设图案可以是三角形、四边形、多边形、圆形，椭圆形等形状，预设图案的作用包括：首先，通过这些具有细胞黏附作用的物质构成的图案来控制细胞和细胞间接触，以方便实现高通量数据获取的诉求。其次，可通过使细胞形状统一达到数据处理中的降维效果，从而降低分析难度。再次，通过限制细胞贴附区域来达到控制细胞的大小，形状、向性、分化状态等的目的，甚至还可以通过控制肌动蛋白丝Actin filament调控细胞力学状态，从而达到某些特殊技术要求场景的要求。
在与本实施例大致相似的另一实施例中，未印刷的所述预设图案的部分可用具有细胞贴附抑制作用的物质如BSA(牛血清白蛋白)或F127(高分子非离子型表面活性剂)来抑制细胞在这些区域的贴附，从而实现定向贴附、对细胞形态的控制或模拟特定的细胞微环境。
在其它一些实施例中，具有细胞黏附作用的物质选用纤维粘连蛋白(Fibronectin，FN)作为示例性说明，但不用以限制本发明的实施方式。分别使用表面带有凸出正方形及长方形图案的聚二甲基矽氧烷微印章，并在微印章表面黏附纤连蛋白，采用微接触印刷的方式，将印章凸出部分的纤连蛋白转移到位于微柱的顶端金属反射层上方。接着，将微柱浸入F-127溶液，使得未设有纤连蛋白的部分具备抑制细胞黏附的效果。最后，将微柱用生理盐水彻底清洗后，将带有细胞膜染色成纤维细胞种植于微柱表面，再对细胞进行荧光成像(如图6a所示)，同时对细胞内的力场进行高分辨率的测量(如图6b所示)。请参照图6a和图6b，图6a为细胞粘附于顶部设有纤连蛋白的微柱群构成的预设图案的荧光成像图，可以看出细胞的黏附范围被限制于有纤连蛋白的区域；基于此，可以通过预设的图案来限制细胞贴附区域，进而对细胞的大小，形状、向性、分化状态等进行控制的情况下对细胞进行力学监测，图6b为在微柱上测得的光反射信号解算出来的细胞机械力大小分布图。
在其它一些实施例中，具有细胞黏附作用的物质选用OKT3抗体(即与细胞表面受体具有相互作用的物质)或纤连蛋白(Fibronectin，FN)作为示例性说明，但不用以限制本发明的实施方式。请参阅图7a-图7d，图7a为采用OKT3抗体作为具有细胞黏附作用的物质的试验示意图；图7b的上部分图像为细胞分别粘附于顶部设有OKT3抗体、纤连蛋白的微柱顶部的荧光成像图；图7b的下部分图像为在微柱上测得的光反射信号(反映细胞机械力大小)分布图；图7c为分别在OKT3抗体、纤连蛋白涂敷表面测得的力学大小对比图；图7d为将T细胞种植于OKT3抗体表面(微柱顶部)后发生的细胞力学动态变化图。具体的，上述试验可以在同一或不同细胞力检测装置的部分微柱顶端涂敷OKT3抗体或纤连蛋白，并将T细胞种植于细胞机械力检测装置的具有细胞黏附作用物质的表面。由图7a-图7d可知，在微柱表面涂敷与细胞表面受体有相互作用的物质(例如OKT3抗体)或纤连蛋白(Fibronectin，FN)的细胞机械力检测装置，可用于实时监测该物质对细胞的机械力影响及相互作用。
第八实施例 一种细胞机械力的检测装置
本实施例与第一至第七实施例的不同之处在于，所述的细胞机械力的检测装置还包括细胞限制机构，所述细胞限制机构包括一个或若干个限制面16，所述限制面16为与基座11所在平面垂直、连接于所述基座11或与所述基座11一体成型的平面或曲面，且所述限制面16的高度高于微柱12并将预设数量的微柱12包绕在内。
本实施例设置的细胞限制机构的作用是单细胞隔离检测，即避免检测时细胞与细胞之间存在接触或黏连，以及限制细胞形态，从而方便高通量测试。根据不同的需求，细胞位置限定机构中的限制面16的数量或者围成的形状可以是不同的。例如，细胞位置限定机构包括的限制面16可以是一个圆柱面，也可以是首尾相接形成三角形截面形状并将一定数量微柱包围在内的3个平面、彼此垂直并首尾相接形成矩形形状将一定数量微柱包围在内的4个平面，首尾相接包围形成N边形的N个平面、或者横截面为类圆形的一个曲面等。也就是说，限制面16构成的横截面形状是可控的封闭形状，且其面积(或可理解为其空间内可容纳的微柱数量)也是可控的。
在实际的实施方式中，根据制程工艺的不同，细胞限制机构还可以通过以下的形式出现：
A，请参照图8，图8为具有细胞限制机构的细胞机械力的检测装置的结构示意图a，图中，所述细胞限制机构与基座11是一体成型的，即：在形成细胞限制机构的物质具有若干个凹陷空间15，其凹陷空间15的壁面即为限制面16，凹陷空间15的深度即为限制面16的高度，凹陷空间15的底部即为基座11，每个凹陷空间15中有若干个微柱12。
B，请参照图9，图9为具有细胞限制机构的细胞机械力的检测装置的结构示意图b，图中，限制面16是粘结在基座11上的结构。
第九实施例 一种细胞机械力的检测装置
本实施例与第八实施例的不同之处在于，本实施例中的细胞限制机构为硅薄膜。
具体的，请参照图10a-图10c，图10a为采用硅薄膜作为细胞限制机构的细胞机械力的检测装置的实物图，图10a中，硅薄膜在使用激光打孔后，黏附在基座上，每个孔中均设有微柱，通过硅薄膜限制细胞的形态和迁移，同时控制细胞与细胞之间存在接触或黏连；图10b为在光反射下采用硅薄膜作为细胞限制机构的细胞机械力的检测装置的荧光显微镜图，图10c为图10b的放大图。在一些实施例中，硅 薄膜的每个孔的大小可以设置为与单细胞大小相适配，适合单一细胞贴附，从而限制细胞接触、细胞形态及其迁移范围。
第十实施例 一种细胞机械力的检测系统
一种细胞机械力的检测系统，包括第一或第二实施例所述的细胞机械力的检测装置1、光信号发生装置2和光信号检测装置3；所述光信号发生装置2和光信号检测装置3均位于所述细胞机械力的检测装置1中的基座11的下方，所述光信号发生装置2具有光源，所述光源发出的光线通过入射光路(相继穿过可透光的基座和可透光的微柱柱体)照射到微柱12的光线反射层13，发生反射，反射光经过反射光路(相继通过可透光的微柱柱体以及可透光的基座)进入光信号检测装置3。光信号检测装置3能够获取到微柱12与细胞发生接触前后的反射光信号。在另外一些实施方式中，这种细胞机械力的检测系统还包括光信号分析装置4，可通过对比、分析、计算微柱12与细胞发生接触前后的反射光信号得到细胞机械力信息，包括细胞机械力的大小、方向、在一定时间范围内的变化情况等。
第十一实施例 一种细胞机械力的检测系统
请参阅图4，图4为本发明第十一实施例相关的一种细胞机械力的检测系统，包括第三实施例所述的细胞机械力的检测装置1，还包括光信号发生装置2和光信号检测装置3；所述光信号发生装置2和光信号检测装置3均位于所述细胞机械力的检测装置1中的基座11的上方，所述光信号发生装置2具有光源，所述光源发出的光线通过入射光路照射到光线反射层13，发生反射，光信号检测装置3能够获取到微柱12与细胞发生接触前后的反射光信号。
在本发明的实施例中，光信号检测装置可以是显微镜，电荷耦合元件CCD，互补金属氧化物半导体CMOS，光电倍增管PMT及光电转换器PT，胶片，或者其它具有相同功能的光信号检测元件，本发明不作具体限制。需要说明的是，在本发明的一些实施例中，当采用显微镜作为光信号检测装置时，则无需设置独立的光信号发生装置，可以直接将本发明的细胞机械力检测装置放置在显微镜的载物台上，以显微镜的光源作为光信号发生装置，以显微镜的物镜(5倍物镜即可，不需依赖高倍数的光学物镜)作为光信号检测装置；当采用其它光信号检测装置，例如电荷耦合元件CCD时，则需要设置独立的光信号发生装置。在本发明的实施例中，光信号发生装置可以是LED，卤素灯，激光(例如，红外激光)，或其他光源，或其他具有这些光源的装置，本发明不作具体限制。
下面对本实施例相关的一种细胞机械力的检测系统用于细胞机械力监测的可视化过程作具体介绍。
请参阅图11为采用本实施例的细胞机械力检测系统监测细胞机械力的荧光显微镜图像。具体的，将细胞(例如，本实施例采用成纤维细胞Fibroblast)放置于细胞机械力检测装置的微柱上，光信号检测装置(例如，本实施例采用显微镜)可以将细胞机械力信息转化为光学信号并形成图像，以供可视化观测，并且能够实时反馈细胞机械力的变化。
第十二实施例 一种细胞机械力的检测系统
请参阅图4，图4为本发明第十二实施例相关的一种细胞机械力的检测系统，包括第三实施例所述的细胞机械力的检测装置1，还包括光信号发生装置2、光信号检测装置3和光信号分析装置4。所述光信号发生装置2和光信号检测装置3均位于所述细胞机械力的检测装置1中的基座11的上方；所述光信号发生装置2具有光源，所述光源发出的光线通过入射光路照射到光线反射层13，发生反射；分光器5可以为半透半反或其它等效的光学元件，主要目的是简化光路的设计；光信号检测装置3能够获取到微柱12与细胞发生接触前后的反射光信号；光信号分析装置4可通过对比、分析、计算微柱12与细胞发生接触前后的反射光信号得到细胞机械力信息，包括细胞机械力的大小、方向、在一定时间范围内的变化情况等。
在本发明的实施例中，光信号分析装置可以是光学图像分析软件ImageJ，Matlab，Fluoview，Python，或者其它具有相同功能的光学图像分析元件，或者这些分析软件的联合使用，本发明不作具体限制。
下面对本实施例相关的一种细胞机械力的检测系统的检测、分析过程作具体介绍。
请参阅图12a-图12c，图12a为细胞机械力检测系统的结构示意图；图12b为光信号检测装置获取得到的细胞机械力检测装置光反射讯号的图像；图12c为力学大小及分布的可视化效果图。
如图12a所示，在细胞机械力检测装置上，每根微柱的顶部均设有金属反射层，侧面则设有抗反射 层；在无细胞时，光线由下方照射微柱，会完全反射而被光信号检测装置(例如CCD相机)完全接收；但当细胞贴附在微柱上时，细胞移动产生的细胞力会使得微柱发生倾斜，从而降低了反射讯号，经过光反射讯号分析后，即可计算出细胞力强度。
进一步地，通过光信号检测装置(例如CCD相机)采集细胞机械力检测装置光反射信号的图像以及局部细胞黏附区域放大图像(如图12b所示)。接着，通过光信号分析装置对图12b的图像进行进一步的处理，使其转化为更为直观的力学大小及分布的可视化效果图12c。
具体处理过程如下：首先，基于图12b，获得明视野反射信号图(I，对焦于细胞上)；接着，通过对图像进行傅立叶变换后过滤高频信号，并进行逆傅立叶变换操作，从而计算获得到未偏移情况下的微柱反射讯号图像(I 0)；接着，将I和I 0图像进一步处理，以将反射讯号图转为更为直观的细胞力学图(I 0讯号值减去I讯号值)后标准化获得更为直观的细胞机械力强度图j。
第十三实施例 机械力的计算方法，力学与光反射信号的相互关系
本实施例结合第十至第十二实施例中任意一个所述的细胞机械力的检测系统或第十四实施例所述的细胞机械力的检测方法，说明本发明实施例中机械力的计算方法；并利用流体当作外加力，以验证力学与光反射信号的相互关系。
请参阅图13，其中，图13a为流体开启前后微流体环境中细胞机械力检测装置的结构示意图；图13b和12c为流体开启前后微柱的明视场显微镜图、反射光信号分布图以及二者叠合效果图的对比图；图13d为流体开启前后光反射信号强度图；图13e为光反射信号与微柱偏移的线性关系图。其中，叠合效果图是指将微柱的明视场显微镜图和反射光信号分布图叠合而成的效果图。
首先，如图13所示，将细胞机械力检测装置整合于微流体通道中，当外加流速增加，微柱产生位移，同时改变微柱表面反射层的角度(图13a-图13c)，由图13d可以看出，在流体开启前后，光反射信号由强转弱。具体的，利用流速强弱来改变微米柱的偏移量，使用共聚焦显微镜拍摄微柱顶部相对于微柱底部的位移，通过以下公式即可计算出每个微柱所受的机械力：
Figure PCTCN2022121338-appb-000001
式中，F代表引起微柱偏转δ角度的机械力，E代表杨氏模量，k bend代表孤立纳米柱的理想弹簧常数，D代表微柱的直径，L代表微柱的高度。
同时，纪录微柱顶部的光反射信号，将光反射信号与微柱偏移量做图13e，即可得到光反射信号(反映细胞机械力)与微柱偏移的线性关系图及线性范围。
第十四实施例 一种细胞机械力的检测方法
一种细胞机械力的检测方法，包括如下步骤：
使用如第十至第十二实施例中任意一个所述的细胞机械力的检测系统中的光信号发生装置2发出光线。
使用如第十至第十二实施例中任意一个所述的细胞机械力的检测系统中的光信号检测装置3检测经所述细胞机械力的检测装置1作用后的光线。所述光信号检测装置3能够获取到细胞机械力的检测装置1中的微柱12与细胞发生接触前后的反射光信号。在另外一些实施方式中，细胞机械力的检测系统中的光信号分析装置4通过对比、分析、计算微柱12与细胞发生接触前后的反射光信号得到细胞机械力信息，包括细胞机械力的大小、方向、在一定时间范围内的变化情况等。
请参照图14a-图14d，图14a和图14b为细胞机械力的检测装置的微柱与细胞发生接触前以及发生接触后的结构示意图；图14b为光信号检测装置(CCD电子感光元件)获取的反射光信号，在细胞周边力场较大区域可以看到明显的反射讯号衰减；图14c为细胞迁移过程的监测图(对细胞膜染色之后，使用荧光光源激发，用CCD电子感光元件记录其迁移的过程)；图14d为细胞迁移过程中的反射光信号分布图(用CCD电子感光元件记录其迁移过程中的反射光信号变化)。由图14c和图14d可以看出，在细胞施力的部分反射光信号明显衰减。图14d示出了在细胞迁移过程中实时监测的反射光信号，并通过反射光信号实时反馈迁移过程中的机械力，可见通过光信号检测装置对细胞迁移过程中的反射光信号 进行监测能够实时反馈细胞迁移过程中的细胞机械力的变化。
第十五实施例 一种细胞机械力检测装置的制备方法
一种细胞机械力检测装置的制备方法，包括如下步骤：
在微柱12的顶部或上半柱面铺设一层光线反射层13，获得顶部或上半柱面具有反射层的微柱12。
第十六实施例 一种细胞机械力检测装置的制备方法
一种细胞机械力检测装置的制备方法，包括如下步骤：
在微柱12整体均匀镀一层抗反射层；
将顶部或上半柱面的抗反射层去除；
在微柱12的顶部或上半柱面铺设一层光线反射层13。
第十七实施例 一种细胞机械力检测装置的制备方法
本实施例与第十五、十六实施例不同之处在于，所述步骤“在微柱12的顶部或上半柱面铺设一层光线反射层13”具体到：在微柱的顶部或上半柱面均匀溅射一层反射金属，获得顶部或上半柱面具有金属光线反射层的微柱。
第十八实施例 一种细胞识别的方法
本实施例提供了一种细胞识别的方法，其包括：采用上述任意一项实施例中所述细胞机械力的检测系统或上述任意一项方案中所述细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息区分不同的细胞种类。
在一些实施例中，所述细胞识别的方法中，步骤“采用上述任意一项实施例中所述细胞机械力的检测系统或上述任意一项方案中所述细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息区分不同的细胞种类”具体包括：
S1、获取细胞信息，所述细胞信息包括基于细胞机械力的检测装置获取的细胞中某点的细胞机械力信息，所述细胞机械力信息包括该点细胞机械力的大小，具体为：使用光信号检测装置(或者和光信号分析装置一起联用)对细胞机械力的检测装置上的多个细胞进行细胞信息采集，其中包括对各个细胞的多点进行细胞机械力大小信息采集，从而获取多个细胞中的多点细胞机械力大小数据；
S2、对获取细胞信息做预处理，形成结构化细胞信息；所述结构化细胞信息包括细胞数目、细胞特征数目和各细胞特征的特征信息，此时结构化细胞信息可以视为一个的二维特征矩阵(Feature matrix)，其中N为细胞数目，P为细胞特征数目，此处P＝1，即细胞特征为细胞机械力大小；
S3、以结构化细胞信息作为输入数据，利用有监督、无监督或半监督的机器学习建立细胞特征模型，并将所述细胞特征模型应用于未知类型或未知状态的细胞的分类或聚类。
在其它一些实施例中，所述的细胞识别的方法中，所述细胞机械力信息还包括该点细胞机械力的方向。
在其它一些实施例中，所述的细胞识别的方法中，所述细胞机械力信息还包括该点细胞机械力的大小或方向在一定时间间隔内的变化。
在其它一些实施例中，所述的细胞识别的方法中，所述细胞信息还包括细胞形貌信息。
第十九实施例 一种细胞识别的方法(包括可视化定性识别，以及精确的定性、定量识别)
本实施例具体提供第十至第十二实施例中任意一个所述的细胞机械力的检测系统或第十四实施例所述的细胞机械力的检测方法获取得到的细胞机械力信息，应用于识别细胞的方法。
请参照图15a至图15g，图15a为健康细胞和肺非小细胞癌细胞混合体系的荧光成像图；图15b为光信号检测装置获取得到的细胞机械力检测装置光反射讯号分布图；图15c为力学大小及分布的可视化效果图；图15d为图15c中的健康细胞和肺非小细胞癌细胞的代表性单细胞细胞力分布的放大图；图15e为健康细胞和肺非小细胞癌细胞在细胞形态上的对比图；图15f为健康细胞、肺非小细胞癌细胞以及这两种细胞以不同比例混合后的反射信号强弱的对比图；图15g为将图15c经过结构化处理后，基于结构化细胞信息处理得到的聚类分析图。
具体的，本实施例以健康细胞(Normal)和肺非小细胞癌细胞系(Cancer)作为检测对象，使用两种不同荧光染料(Dil&DIO)对健康细胞和肺癌细胞的细胞膜预染色，并以一定比例混合后添加到同一 个细胞机械力检测装置(在另一些实施方式中，可以是添加到不同的独立的细胞机械力检测装置)上。
进一步地，通过光信号检测装置(本实施例使用显微镜)采集了细胞机械力检测装置光反射讯号的图像(如图15b所示)，而两种细胞内高分辨率的力场分布被光信号检测装置直接渲染即可转化成可读取的光强度衰减信号(反映细胞力强度)并显示在图片内(如图15c所示)。根据图15c图片显示的两种细胞的光衰减程度的不同，可以通过肉眼观察对两种细胞进行直观的区分(定性分析)。
进一步地，通过光信号分析装置对图15c中的光反射讯号进行进一步处理。具体地，本实施例通过光信号分析装置(本实施例使用ImageJ及Python分析软件，在其它实施例中还可以使用其他图像分析软件)对所获得的细胞力场的图15c进行信息采集，其中包括对各个细胞的多点进行细胞机械力大小信息采集，从而获取多个细胞中的多点细胞机械力大小数据；对获取的细胞机械力大小信息做预处理，形成结构化细胞信息；并根据结构化细胞信息分析得到健康细胞和肺非小细胞癌细胞在细胞形态上的对比结果图(如图15e所示)。
所述结构化细胞信息包括细胞数目、细胞特征数目和各细胞特征的特征信息(例如本实施例中的细胞黏附面积及细胞圆度)，此时结构化细胞信息可以视为一个特征矩阵(Feature Matrix)的二维矩阵，其中N为细胞数目，P为细胞特征数目，此处P＝2，即细胞特征为：细胞机械力大小、细胞机械力在细胞内的分布。
进一步地，以上述的结构化细胞信息作为输入数据，利用有监督机器(与不同的细胞膜染料(Dil&DIO)对两种细胞系进行预染色进行对比)学习建立细胞特征模型，并利用大量细胞的结构化细胞信息对所述细胞特征模型进行训练，得到如图15g所示的聚类分析图，然后将所获得的细胞特征模型应用于未知类型或未知状态的细胞的分类识别。由此可知，根据结构化的细胞特征数据(细胞机械力大小、细胞机械力在细胞内的分布)作为输入数据，可以利用光信号分析装置(本实施例使用ImageJ及Python分析软件，在其它实施例中还可以使用其他聚类分析软件)对正常的健康细胞及癌症细胞进行聚类分型，从而实现对未知细胞种类的识别。
图15e示出了不同细胞在形态(包括细胞黏附面积及细胞圆度)上没有统计学意义上的明显区别；图15f示出了正常细胞及肿瘤细胞反射讯号强度(反映细胞力)的明显差异，以及把正常细胞及肿瘤细胞按一定比例混合在一起后反射讯号强度和混合比例呈一定线性关系。由此可知，相比于细胞的其它特征(例如图15e中的细胞黏附面积、细胞圆度等形态信息)，基于本发明的细胞机械力检测装置测得的细胞力学特征可对细胞的状态及类型进行更直观精确的识别(定量和定性分析)。
此外，从图15d和图15f的数据得出，肿瘤细胞比正常细胞显示出更高的机械力大小，并且分布更不均匀。可见，细胞机械力以图像方式可视化后，即可通过肉眼直观地看出不同细胞的力场特征具有明显区别；并且进一步地，通过图像分析软件对不同细胞各点的力场大小进行结构化处理后，综合分析得到图15e的细胞形态信息，图15f的反射讯号强度(反映细胞力)以及图15g的聚类分析图。本发明通过细胞各点的力场结构化信息的综合分析可以对不同细胞(例如本实施例中健康细胞以及非小细胞肺癌细胞)进行聚类分型及定量分析，从而实现对细胞种类的精确识别。
综上可知，基于本发明的细胞机械力检测装置，不仅可以通过肉眼直观区分以进行定性分析，还可以基于测得的细胞力学特征对细胞的状态及类型进行更直观精确的识别(定量和定性分析)，并且证实了以细胞力场作为标志物能够更好地对细胞类型进行区分。
第二十实施例 一种细胞活力的检测方法
本实施例具体提供第十至第十二实施例中任意一个所述的细胞机械力的检测系统或第十四实施例所述的细胞机械力的检测方法获取得到的细胞机械力信息，应用于监测细胞的活力。
请参阅图16a-图16c，图16a为细胞活力检测方法的操作流程示意图；图16b为A549细胞在不同剂量的5FU处理24h后，MTT法测定得到的细胞活力以及本发明装置或系统或方法测定得到的细胞机械力的对比图；图16c为A549细胞在不同剂量的5FU处理不同时间后，MTT法测定得到的细胞活力以及本发明装置或系统或方法测定得到的细胞机械力的对比图。
具体地，本实施例将非小细胞肺癌细胞A549培养在多个细胞机械力检测装置上，分别加入不同剂量的抑制细胞增殖的药物5-氟尿嘧啶(5-FU)进行处理，并通过第十至第十二实施例中任意一个所述的细胞机械力的检测系统或第十四实施例所述的细胞机械力的检测方法监测不同时间点的细胞机械力，通过CCK-8试剂盒监测不同时间点的细胞增殖和细胞毒性，同时以MTT测定法测定的细胞活力作为对 照组，得到图16b和图16c的数据。
从图16b和图16c显示，通过传统MTT测定法和本发明所述装置或系统或方法进行测定后，MTT测定法测定的细胞活力以及细胞机械力所反映的细胞活力都是以剂量依赖性呈逐渐降低的趋势，即细胞机械力与细胞活力呈正相关。
此外，如图16b所示，在用不同剂量的5FU处理24h后，与对照组DMSO相比，通过机械力可以以更大的降低幅度反映细胞活力的降低，从而更加直观地对细胞活力进行评估。如图16c所示，在用5FU处理12h内，MTT法测定的细胞活力变化不明显；而通过测定机械力，可以在MTT法检测到细胞代谢活性降低之前的更早时间点即观测到细胞机械力的降低，具体的在0.5μM处理剂量下即可在6h时出现明显的降低趋势，在1μM处理剂量下即可在3h时出现明显的降低趋势，从而可以更加灵敏地表征细胞活力的降低。
综上可知，本实施例通过细胞机械力检测装置直接检测细胞机械力是评估细胞对药物反应活力的一种高度敏感且有效的方法。
第二十一实施例 一种细胞状态的检测方法
本实施例具体提供第十二实施例所述的细胞机械力的检测系统或第十四实施例所述的细胞机械力的检测方法获取得到的细胞机械力信息，并根据所述细胞机械力信息分析确定细胞状态。
请参阅图17a至图17f，图17a为细胞状态检测方法的操作过程图；图17b为M0巨噬细胞分化至M1状态的荧光显微镜图；图17c为M0巨噬细胞分化至M2状态的荧光显微镜图；图17d为M0巨噬细胞、M1状态以及M2状态的细胞黏附面积对比图；图17e为M0巨噬细胞、M1状态以及M2状态的细胞圆度对比图；图17f为M0巨噬细胞、M1状态以及M2状态的机械力对比图。
具体的，本实施例以巨噬细胞作为检测对象，将其分别添加到不同的独立的细胞机械力检测装置的微柱上，并分别采用内毒素LPS和白介素IL4引导巨噬细胞从M0分化为M1和M2状态，以M0状态作为对照组。在细胞分化后，通过光信号检测装置(本实施例采用显微镜)采集图17b和图17c，并通过光信号分析软件(ImageJ及Python)对图17b和图17c作进一步的图像处理和数据分析，将其转化为结构化信息，并分析制得图17d-图17f的数据。由图17a至图f的数据可知，不管是从直观的观测(图17b和图17c)还是经过结构的数据量化处理(图17d-图17f)，M0巨噬细胞以及分化后的M1和M2状态之间都具有明显的区别。
第二十二实施例 一种多细胞聚合体的检测方法
本实施例具体提供第十二实施例所述的细胞机械力的检测系统或第十四实施例所述的细胞机械力的检测方法获取得到的细胞机械力信息，根据所述细胞机械力信息分析确定细胞状态。
具体地，本实施例提供的多细胞聚合体以多种方式结合在细胞机械力检测装置上，本实施例提供其中的两种具体结合方式：
第一种结合方式：在细胞机械力检测装置的微柱上设置培养基，将细胞移植到微柱上的培养基中培养得到多细胞聚合体；在另一些实施方式中，这种结合方式，在细胞机械力信息以可视化形式输出下，可以实时监控细胞的培养过程，以应用于如培养基、药物等化学、生物和物理外界刺激对细胞生长的影响；
第二种结合方式：直接将培养好的多细胞聚合体粘附于细胞机械力检测装置的微柱上，进行检测。
更具体地，本实施例提供一种肿瘤细胞多聚体在细胞机械力检测装置上培养，应用于药物敏感试验，包括以下步骤：
S1、肿瘤细胞多聚体的生成：在细胞机械力检测装置的微柱的顶部涂FN 50μg/mL，并紫外线杀菌30分钟；取乳腺癌细胞MCF-7(大约1×10 5～9×10 5个)种植于细胞机械力的检测装置的微柱(无限定微柱的数量)的顶部表面，再将细胞机械力监测装置浸置在3dGRO TM Spheroid Medium(S3077)培养液中培养3天以上，引导肿瘤细胞多聚体生成；
S2、将上述生成的肿瘤细胞多聚体应用于5-Fu药物敏感实验中：在上述肿瘤细胞多聚体加入200μM的5-Fu后培养1天；试验中，在加5-Fu前和加5-Fu后培养一天的细胞机械力检测装置(生成有肿瘤细胞多聚体，可以连同培养液一起)，通过光信号检测装置(CCD感光元件)获取光反射讯号，并使用光信号分析装置(Image J)获得细胞力分布图像，如图18所示。
请参阅图18a-图18b，图18a为具有第一种形态的肿瘤细胞多聚体在有/无5-Fu作用后的表征图； 图18b为具有第二种形态的肿瘤细胞多聚体在有/无5-Fu作用后的表征图；从左到右分别是细胞膜荧光和反射讯号的混合图像(1)、光反射讯号(2)、细胞核(3)、细胞膜(4)及经过光信号分析装置(ImageJ)处理后的细胞力可视化图像(5)。由于细胞具有异质性，肿瘤细胞各不相同，多聚体也有各种各样的形态，因此细胞多聚体会以不同的形态黏附在一起。本实施例选取具有代表性的两种形态进行细胞形态检测，所述第一种形态系指较大的两个细胞黏在一起的细胞形态；所述第二种形态系指一群小细胞黏在一起的细胞形态。
由图18a-图18b可以看出，经抗肿瘤药物处理细胞使之活力降低后，反射讯号明显减弱；并且，从5-Fu作用前后的细胞机械力的改变可以看出两种不同形态的肿瘤细胞多聚体对药物的敏感性也有明显差异。由此可知本发明的细胞机械力检测装置能对多细胞聚合体(如肿瘤多聚体)进行细胞机械力测定，并可通过细胞机械力用于监测细胞多聚体的活力状态，并可区分不同的细胞形态。
在本发明的定义中，细胞多聚体指的是：
细胞是生物体基本的结构和功能单位，细胞通常会繁殖或分化形成两个以上细胞团聚一起形成细胞群体，即：多细胞聚合体；多细胞聚合体包括肿瘤多聚体等体外或体内培养所得到的细胞团体。
综上可知，本发明细胞检测装置用于细胞机械力的检测、细胞识别、细胞状态检测、细胞活力检测具有非常高的灵敏度及有效性，并且可以实现实时监测，可以应用于药物处理下，细胞对药物的反应情况。
区别于现有技术，上述技术方案具有如下优点：
首先，高通量低成本：与现有TFM及普通微柱阵列相比，本发明的技术方案摆脱了对显微镜的依赖，极大地简化了操作流程，因为无需通过显微镜高分辨率成像，只需通过对反射光的强度进行监测，可实现高通量对细胞进行监测。
其次，单细胞分辨率：分辨率高，可对每只细胞进行实时监测，可结合其他单细胞分析技术测量细胞对药物反应的异质性；实时监测：无需荧光，可避免激光对细胞的光毒性作用，因此适合长期监测，可用于研究细胞对药物的长期反应；高灵敏度：通过反射讯号，将微柱形变信号放大，增加对形变监测的灵敏度。检测微、纳米柱弯曲形变一般依赖光学系统(如显微镜)进行检测，但微柱尺寸越小，对光学系统的精密度和解析度要求越高。例如2微米宽，6微米高的微米柱，需要20倍以上的物镜搭配共轭焦系统才可有效观测。而本发明利用镜面反射原理通过探测反射光的衰减，实际使微柱形变的信号得以放大，经实验验证，同样的讯号在5倍物镜下即可观测。搭配特殊的读取系统，不需依赖高倍数的光学物镜即可有效检测微/纳米柱形变，从而极大降低系统成本，并有效提升通量。
再次：可模拟细胞微环境；可模拟细胞外基质的组分和形态，可应付更丰富的技术需求场景。
需要说明的是，尽管在本文中已经对上述各实施例进行了描述，但并非因此限制本发明的专利保护范围。因此，基于本发明的创新理念，对本文所述实施例进行的变更和修改，或利用本发明说明书及附图内容所作的等效结构或等效流程变换，直接或间接地将以上技术方案运用在其他相关的技术领域，均包括在本发明的专利保护范围之内。

Claims (21)

  1. 一种细胞机械力的检测装置，其特征在于，包括：
    基座，以及
    设置于基座上的，可受细胞机械力作用而产生形变的多个微柱构成的微柱阵列，所述微柱的顶部或柱面上部具有光线反射层。
  2. 如权利要求1所述的细胞机械力的检测装置，其特征在于，所述基座为透光基座，所述微柱的柱体可透射光线；所述微柱顶部具有光线反射层。
  3. 如权利要求2所述的细胞机械力的检测装置，其特征在于，所述微柱的柱面具有抗反射层。
  4. 如权利要求1所述的细胞机械力的检测装置，其特征在于，所述光线反射层为金属箔层，金属氧化物或金属盐，超细玻珠或微棱镜，有机反光材料一种或一种以上的组合。
  5. 如权利要求1至4任意一项所述的细胞机械力的检测装置，其特征在于，所述微柱阵列的全部或部分微柱的顶部端面上设有具有细胞黏附作用的物质。
  6. 如权利要求5所述的细胞机械力的检测装置，其特征在于，所述具有细胞黏附作用的物质包括如下物质中的一种或多种：细胞外基质分子，包括胶原蛋白、纤粘连蛋白、玻璃粘连蛋白、层粘连蛋白或弹性蛋白原；细胞外基质的模拟物质，包括含有RGD粘附序列的多肽；具有细胞黏附促进机制的物质，包括聚赖氨酸；与细胞表面受体具有相互作用的物质。
  7. 如权利要求5所述的细胞机械力的检测装置，其特征在于，部分微柱顶部端面上设有具有细胞黏附作用的物质的微柱群构成预设的图案。
  8. 如权利要求7所述的细胞机械力的检测装置，其特征在于，所述微柱阵列的预设区域的部分微柱的顶部端面上设有具有细胞黏附作用的物质。
  9. 如权利要求1所述的细胞机械力的检测装置，其特征在于，所述微柱的横截面形状为圆形、椭圆形或多边形。
  10. 如权利要求1所述的细胞机械力的检测装置，其特征在于，所述微柱和微柱阵列的尺寸范围包括：柱高10nm～500μm，柱间距10nm～50μm，柱上表面直径50nm～50μm。
  11. 如权利要求1所述的细胞机械力的检测装置，其特征在于，还包括细胞限制机构，所述细胞限制机构包括一个或若干个限制面，所述限制面为与基座所在平面垂直、连接于所述基座或与所述基座一体成型的平面或曲面，且所述限制面的高度高于微柱并将预设数量的微柱包绕在内。
  12. 一种细胞机械力的检测系统，其特征在于，包括如权利要求1-11任意一项所述的细胞机械力的检测装置、光信号发生装置和光信号检测装置；
    所述光信号发生装置具有光源，所述光源发出的光线通过入射光路照射到微柱的光线反射层；
    所述光信号检测装置用于检测从微柱的光线反射层反射的光线，所述光线反射层反射的光线经过反射光路进入光信号检测装置。
  13. 如权利要求12所述的细胞机械力的检测系统，其特征在于，所述细胞机械力的检测装置的基座为透光基座，所述微柱的柱体可透射光线；所述微柱顶部具有光线反射层；
    所述光源发出的光线通过入射光路从细胞机械力的检测装置的基座照射到微柱的光线反射层；
    所述光信号检测装置用于检测从微柱顶部的光线反射层反射的光线，所述光线反射层反射的光线经过反射光路进入光信号检测装置。
  14. 如权利要求12所述的细胞机械力的检测系统，其特征在于，还包括用于分析光信号的光信号分析装置。
  15. 一种细胞机械力的检测方法，其特征在于，包括如下步骤：
    使用如权利要求12-14中任一项所述的细胞机械力的检测系统中的光信号发生装置发出光线；
    使用如权利要求12-14中任一项所述的细胞机械力的检测系统中的光信号检测装置检测经所述细胞机械力的检测装置作用后的光线。
  16. 如权利要求15所述的细胞机械力的检测方法，其特征在于，还包括步骤：使用光信号分析装置对所述细胞机械力的检测装置与待测细胞发生细胞机械力作用前后的反射光线进行对比分析，获取细胞机械力信息，所述细胞机械力信息包括细胞机械力的大小、方向或变化频率。
  17. 一种细胞状态的检测方法，其特征在于，包括：通过如权利要求12-14中任意一项所述的细胞机械力的检测系统或权利要求中任意一项15-16所述的细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息分析确定细胞状态；
    所述细胞状态包括：细胞黏附，细胞活力，细胞分化/活化，细胞增殖和/或细胞迁移。
  18. 一种细胞识别的方法，其特征在于，包括：通过如权利要求12-14中任意一项所述的细胞机械力的检测系统或权利要求15-16中任意一项所述的细胞机械力的检测方法获取细胞机械力信息，根据所述细胞机械力信息区分细胞种类。
  19. 一种细胞机械力检测装置的制备方法，其特征在于，包括如下步骤：
    在微柱的顶部或上半柱面铺设一层反射层，获得顶部或上半柱面具有反射层的微柱。
  20. 如权利要求19所述的细胞机械力检测装置的制备方法，其特征在于，在步骤“在微柱的顶部或上半柱面铺设一层反射层”之前还包括步骤：
    在微柱整体均匀镀一层抗反射层；
    将顶部或上半柱面的抗反射层去除。
  21. 如权利要求19或20所述的细胞机械力检测装置的制备方法，其特征在于，所述“在微柱的顶部或上半柱面铺设一层反射层，获得顶部或上半柱面具有反射层的微柱”具体为：在微柱的顶部或上半柱面均匀溅射一层反射金属，获得顶部或上半柱面具有金属光线反射层的微柱。
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