Cellular single-molecule mechanical force sensor, method of making and use thereof

Abstract

The application discloses a cell monomolecular mechanical force sensor for detecting a cell phagocytosis process, a preparation method and application thereof, and the sensor comprises a microsphere substrate and a plurality of double-stranded DNA mechanical probes modified on the surface of the microsphere substrate; the probes are composed of a first single-stranded DNA and a partially complementary second single-stranded DNA, and form a FRET or fluorescence quenching pair; when a cell exerts a pulling force on the probes through receptor-ligand interaction during phagocytosis or wrapping of the microsphere and the pulling force exceeds a preset melting threshold, the double-stranded DNA is melted or conformationally changed and a fluorescence signal change is generated, so that detection of a single-molecule level mechanical force at a phagocytosis interface is realized. By adjusting a geometric conformation and sequence design of the probes, a probe library with different melting force thresholds can be constructed, quantitative analysis and spatial distribution characterization of mechanical forces during phagocytosis are realized. The application can truly simulate a three-dimensional phagocytosis interface, and provides an effective tool for revealing a mechanism of blood flow mechanics regulating endothelial cell phagocytosis behavior and drug screening related to diseases.

Description

Technical Field

[0001] This application relates to the field of biomedical engineering technology, specifically to a single-molecule cell mechanical force sensor, its preparation method, and its applications. Background Technology

[0002] Atherosclerosis is one of the most common chronic diseases of the cardiovascular system, and its development is closely related to lipid deposition and inflammatory responses. Although the risk factors for this disease have systemic characteristics, the formation of atherosclerotic plaques exhibits significant regional specificity, often preferentially occurring at arterial branches, bends, and bifurcation areas. These areas are usually accompanied by low-amplitude, oscillating perturbation of blood flow shear forces, suggesting that the hemodynamic environment plays an important role in the development of atherosclerosis.

[0003] Existing research indicates that perturbed blood flow shear forces can lead to endothelial cell dysfunction, causing them to shift from a resting state to a pro-inflammatory and pro-atherosclerotic phenotype. Recent studies have further revealed that endothelial cells located in perturbed blood flow regions do not merely function as passive barriers, but exhibit characteristics similar to non-professional phagocytes, actively phagocytosing harmful particles, including low-density lipoprotein (LDL), thereby participating in the early formation of lesions. However, the specific mechanobiological mechanisms by which endothelial cells regulate their own mechanical forces to drive this active phagocytic process after sensing external hemodynamic stimuli remain poorly elucidated.

[0004] The key to a deeper understanding of these issues lies in the precise measurement of the microscopic mechanical forces generated during cellular phagocytosis. Existing cellular mechanical detection techniques mainly include traction force microscopy (TFM) and atomic force microscopy (AFM). TFM relies on substrate deformation to invert cellular traction forces, making it difficult to distinguish mechanical events at the single-molecule scale. While AFM has high mechanical sensitivity, it is complex to operate, has low throughput, and is mainly suitable for measuring surface indentation or pull-off forces, making it difficult to simulate the dynamic encapsulation behavior involved in cellular phagocytosis of three-dimensional particles.

[0005] To overcome the limitations of traditional single-molecule force detection spectroscopy, DNA-based tension probe technology has been proposed and applied to cell mechanics research. This technology utilizes the unwinding mechanics of DNA double strands to achieve visualized detection of mechanical forces. However, most existing DNA tension probes are fixed on two-dimensional planar substrates, making it difficult to realistically simulate the membrane curvature changes and three-dimensional contact interfaces encountered by cells when they engulf three-dimensional particles. Furthermore, existing probe systems generally lack targeted design for specific mechanoreceptors and are susceptible to interference from factors such as degradation by intracellular and extracellular nucleases, resulting in false positives and insufficient stability.

[0006] Therefore, how to provide a new technical solution that can specifically target cell surface mechanoreceptors in three-dimensional space and dynamically and quantitatively detect the strength of single-molecule mechanical forces during cell phagocytosis in a highly sensitive and specific manner, thereby providing an effective tool for revealing the mechanobiological mechanism by which blood flow shear force regulates endothelial cell phagocytosis, and also providing new technical means for the screening and evaluation of anti-atherosclerotic drug candidates, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] In view of the shortcomings of the above-mentioned related technologies, the purpose of this application is to provide a single-molecule mechanical force sensor for cells, its preparation method and application.

[0008] To achieve the above and other related objectives, the first aspect of this application discloses a single-molecule mechanosensor for cells. The single-molecule mechanosensor comprises: a microsphere substrate; a plurality of double-stranded DNA probes modified on the surface of the microsphere substrate; each double-stranded DNA probe includes a first single-stranded DNA and a second single-stranded DNA partially or completely complementary to the first single-stranded DNA; wherein one end of the first single-stranded DNA is covalently coupled to the microsphere substrate, and the other end is modified with a first fluorescent label; the second single-stranded DNA is modified with a targeting ligand for specific binding to mechanosensors on the cell surface, and is modified with a second fluorescent label; the first fluorescent label and the second fluorescent label constitute a fluorescence resonance energy transfer or fluorescence quenching pair, and the double-stranded DNA probe is located at... When the double strands are closed, the distance between the first fluorescent label and the second fluorescent label is within the effective range of fluorescence resonance energy transfer or fluorescence quenching, thus forming a corresponding fluorescence signal state. When the targeting ligand binds to the mechanosensitive receptor on the cell surface and is subjected to mechanical tension applied by the cell, and the mechanical tension is greater than the unwinding force threshold of the double-stranded DNA probe, the double-stranded DNA probe undergoes unwinding, dissociation, or conformational change, causing a change in the spatial distance between the first fluorescent label and the second fluorescent label, and a change or decrease in the fluorescence resonance energy transfer signal, thereby generating a detectable signal change. The signal change is used to characterize the single-molecule-level mechanical force applied by the cell during the process of phagocytosis or encapsulation of the microsphere substrate, and the signal change includes an increase or decrease in fluorescence intensity.

[0009] The second aspect of this application discloses a method for preparing the above-mentioned single-molecule mechanosensitive cell, comprising the following steps: a microsphere substrate activation step: providing a microsphere substrate, chemically modifying the surface of the microsphere substrate to introduce active functional groups capable of covalently coupling with single-stranded DNA onto the surface of the microsphere substrate, thereby obtaining a surface-activated microsphere substrate for stable immobilization of a single-molecule mechanical probe; the active functional groups include maleimide groups; a DNA probe assembly step: providing a first single-stranded DNA and a second single-stranded DNA, wherein the first single-stranded DNA is modified with a first fluorescent label and has end groups for coupling, and the second single-stranded DNA is modified with a second fluorescent label and a targeting ligand; and, assembling the first single-stranded DNA and the second single-stranded DNA in a hybrid... The DNA probes are mixed in a buffer solution and annealed to form a double-stranded DNA probe with a predetermined mechanical response configuration through base complementary pairing. The coupling step involves mixing the double-stranded DNA probe obtained in the DNA probe assembly step with the surface-activated microsphere substrate obtained in the microsphere substrate activation step, and reacting under coupling reaction conditions to covalently link the double-stranded DNA probe to the surface of the microsphere substrate in a controlled orientation via the end groups of the first single-stranded DNA. The end groups are thiol groups, and the coupling reaction includes an addition reaction between thiol groups and maleimide groups to achieve directional covalent linking of the double-stranded DNA probe. The purification step involves removing uncoupled DNA probes and impurities by centrifugation, washing, or filtration to obtain the single-molecule mechanosensor.

[0010] The third aspect of this application discloses a method for detecting single-molecule mechanical forces during cell phagocytosis, using the aforementioned single-molecule mechanical force sensor, comprising the following steps: a co-incubation step: a group of the single-molecule mechanical force sensors with different unwinding force thresholds are co-incubated with test cells in a culture medium, allowing the cells to bind to the sensors via surface receptors and initiate the phagocytosis process; a fixation and imaging step: cells are fixed at predetermined time points, and fluorescence signal images of the single-molecule mechanical force sensors are acquired using a fluorescence microscope or a confocal microscope; a signal extraction step: single-molecule mechanical force sensors that have been phagocytosed or bound by cells are identified, the fluorescence intensity change of each single-molecule mechanical force sensor is measured, and single-molecule mechanical force sensors whose fluorescence intensity enhancement factor exceeds a preset background threshold are determined as positive signals; a force value calculation step: the proportion of positive signals under different unwinding force thresholds is statistically analyzed, the relationship curve between the proportion of positive signals and the unwinding force threshold is fitted, and the half-maximum effective mechanical force threshold is calculated, wherein the half-maximum effective mechanical force threshold is defined as the mechanical force required to unwind 50% of the double-stranded DNA probe, thereby quantifying the single-molecule mechanical force applied by the cell during phagocytosis.

[0011] In summary, the single-molecule mechanosensitive sensor disclosed in this application, its preparation method, and its applications, introduce active functional groups (such as maleimide) that can selectively covalently react with DNA end groups on the surface of a microsphere substrate. Double-stranded DNA threshold probes are then covalently immobilized at a single point, in a directional and stable manner through a process involving annealing assembly, thiol activation, and thiol-maleimide directional coupling. This results in a probe array with consistent orientation, conformation, and dispersion on the three-dimensional surface of the microsphere. Combined with a unified positive criterion (such as intensity enhancement determination based on a background threshold) and a cross-threshold statistical / fitting strategy, positive responses under different unwinding force thresholds can be converted into half-maximal effective mechanosensitive sensors. Comparable quantitative indicators such as the force threshold F50 enable the quantification of the intensity of mechanical forces applied during cell phagocytosis and batch / threshold comparability. Simultaneously, purification after coupling to remove free probes and non-specific adsorbed components can significantly reduce background fluorescence and false positives caused by mismatch / non-specificity, improving the signal-to-noise ratio and measurement robustness. Furthermore, combined with spatial distribution analysis of fluorescence enhancement regions on the microsphere surface, it can also help determine the cell encapsulation state and force direction, thereby improving the spatial resolution capability of the phagocytic mechanics process and can be extended to the screening of mechanical phenotypes and mechanism verification under drug intervention conditions. Attached Figure Description

[0012] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0013] Figure 1 The diagram shown is a structural schematic of the single-molecule mechanical force sensor of this application.

[0014] Figure 2 This is a schematic diagram of confocal microscopy imaging showing the mechanically triggered fluorescence signal and cytoskeleton signal during cell phagocytosis of microspheres.

[0015] Figure 3 The diagram shows a discrete unwinding force threshold probe array formed by different loading geometries and sequence designs.

[0016] Figure 4 The graph shows the statistical relationship between the proportion of positive fluorescent signals and the probe dissociation force threshold, as well as a schematic diagram of F50 fitting.

[0017] Figure 5 The diagram shown is a flowchart of the preparation method of this application in one embodiment.

[0018] Figure 6 The diagram shown is a flowchart of the steps of the single-molecule mechanical force detection method of this application in one embodiment.

[0019] Figure 7 The flowchart shown is a step diagram of the single-molecule mechanical force detection method of this application in another embodiment. Detailed Implementation

[0020] The following specific embodiments illustrate the implementation of this application. Those skilled in the art can easily understand the advantages and technical effects of this application from the content disclosed in this specification. In the following description, some embodiments may refer to the accompanying drawings. It should be understood that other embodiments not shown in the drawings may also be used, and specific steps, modules or units, electrical and operational changes may be made without departing from the spirit and scope of this application. The detailed description below should not be considered limiting, and the scope of the embodiments of this application is limited only by the claims published in this application. The terminology used herein is for describing particular embodiments only and is not intended to limit this application.

[0021] In this application, unless otherwise stated, the following terms have the following meanings:

[0022] The aforementioned "Microsphere DNA-based Force Sensor (MDFS)" refers to a single-molecule mechanical probe system constructed based on a micrometer-scale spherical substrate. It utilizes the three-dimensional curvature of the microspheres to simulate the three-dimensional contact interface during cell phagocytosis of particles, and directionally modulates the surface of the microspheres with double-stranded DNA probes possessing specific mechanical response thresholds. This MDFS can convert receptor-mediated pioton (pN) level mechanical tensile events on the cell surface into detectable changes in fluorescence signals through a "mechanical-chemical-optical" conversion mechanism.

[0023] The "double-stranded DNA probe (dsDNA Force Probe)" refers to a double-helix structure formed by two single-stranded oligonucleotides (a first single-stranded DNA and a second single-stranded DNA) through complementary base pairing. Its structural features include a first single-stranded DNA (anchor strand) anchored covalently to the microsphere surface at one end, and a fluorescent group (or quencher group) modified at the other end; the second single-stranded DNA (ligand strand) is modified with a targeting ligand and a quencher group (or fluorescent group) matching the first single strand. The double-stranded DNA probe operates in the following states: when no force is applied or the force is less than a threshold, the double strands remain closed, and the fluorescent group and quencher group are within the effective distance for Ferrastre resonance energy transfer (FRET) or contact quenching (OFF state); when the force exceeds the threshold, causing the double strands to dissociate, they separate, and fluorescence is restored (ON state). In some embodiments, after unwinding, the second single-stranded DNA can leave the microsphere surface, resulting in a continuous change in signal; however, this application is not limited to this.

[0024] The term "loading geometries" refers to the direction of the applied mechanical force relative to the DNA double helix axis. Different loading modes determine the mechanical threshold required for DNA to unwind, including unzipping mode and shearing mode. In unzipping mode, the force direction is primarily perpendicular to the hydrogen bond alignment of the base pairs. In unzipping mode, the external force acts on one end of the double helix, causing the base pairs to break one by one (similar to unzipping a zipper), requiring a relatively small unwinding force. In shearing mode, the force direction is primarily parallel to the hydrogen bond alignment of the base pairs. In shearing mode, the external force acts on both ends of the double helix, requiring the simultaneous overcoming of hydrogen bond forces and base stacking forces throughout the hybridization region, resulting in a larger unwinding force.

[0025] The "half-maximum effective mechanical force threshold (F50)" is defined as the magnitude of the mechanical force required to cause 50% of the double-stranded DNA probes in the sensor population to unwind and generate a positive fluorescence signal in a statistically significant sense. This parameter is obtained by fitting a curve (usually a sigmoid curve) showing the relationship between the percentage of fluorescent positive probes (Positive Counts %) and the probe design unwinding force threshold, and is used to quantitatively characterize the average single-molecule mechanical force level exerted by cells during phagocytosis or specific physiological processes. The positive fluorescence signal can be determined according to a preset judgment rule, for example, using an increase in fluorescence intensity in a local area on the surface of a single probe / microsphere relative to the unforced background exceeding a preset multiple or a preset threshold as a positive judgment criterion; this application does not limit the specific method of selecting the threshold. F50 is the equivalent mechanical force value corresponding to 50% of the probes triggering a force-triggered event for a set of different threshold MDFS; physically, F50 reflects the characteristic force spectrum intensity exerted by cells on a single target receptor under specific physiological / pathological conditions (such as under perturbation flow induction), and an increase in the F50 value represents an enhancement of the cellular phagocytic mechanics function.

[0026] This application provides a single-molecule mechanosensor for cells, comprising: a microsphere substrate and a plurality of double-stranded DNA probes modified on the surface of the microsphere substrate, wherein each double-stranded DNA probe comprises a first single-stranded DNA and a second single-stranded DNA partially or completely complementary to the first single-stranded DNA; wherein one end of the first single-stranded DNA is covalently coupled to the microsphere substrate, and the other end is modified with a first fluorescent label; the second single-stranded DNA is modified with a targeting ligand for specific binding to mechanosensors on the cell surface, and is modified with a second fluorescent label; the first fluorescent label and the second fluorescent label constitute a fluorescence resonance energy transfer or fluorescence quenching pair, and the double-stranded DNA probe is located at... When the double strands are closed, the distance between the first fluorescent label and the second fluorescent label is within the effective range of fluorescence resonance energy transfer or fluorescence quenching. When the targeting ligand binds to the mechanosensitive receptor on the cell surface and is subjected to mechanical tension applied by the cell, and the mechanical tension is greater than the unwinding force threshold of the double-stranded DNA probe, the double-stranded DNA probe undergoes unwinding, dissociation, or conformational change, thereby changing the spatial distance between the first fluorescent label and the second fluorescent label to generate a detectable signal change. The signal change is used to characterize the single-molecule-level mechanical force applied by the cell during phagocytosis or encapsulation of the microsphere substrate, and the signal change includes an increase or decrease in fluorescence intensity.

[0027] Please see Figure 1 The diagram shows the structural schematic of the single-molecule mechanosensitive cell of this application. Figure 1 This schematically illustrates the overall structural framework for constructing a three-dimensional cellular single-molecule mechanosensor using silica microspheres as a substrate, by modifying the surface of the microspheres with double-stranded DNA mechanical probes. Figure 1 As shown, the single-molecule mechanosensor described in this application uses a microsphere substrate 10 with a three-dimensional curved surface as a mechanical sensing carrier to simulate the real spatial geometric interface faced by cells during phagocytosis or encapsulation of particles. In other embodiments of this application, the microsphere substrate 10 can also be made of microspheres made of other inorganic or polymeric materials, specifically, for example, polystyrene (PS), polymethyl methacrylate (PMMA), magnetic nanoparticles, or gold nanoparticles. The microsphere substrate 10 typically has sufficient stiffness to maintain shape stability under culture and imaging conditions, thereby providing stable mechanical support.

[0028] In one embodiment, the microsphere substrate 10 is a commercially available silica microsphere, the particle size of which can be selected according to experimental requirements. The silica microsphere has good biocompatibility, optical transparency, and ease of surface chemical modification.

[0029] For example, in a practical application, considering the size range of lipid particles (such as LDL, VLDL) or apoptotic bodies phagocytosed by vascular endothelial cells, the diameter of the microspheres is preferably set between 1 μm and 15 μm, for example, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, or 15 μm. Preferably, the diameter of the microsphere substrate 10 is on the same order of magnitude as or slightly smaller than the size of the cells to be tested. Specifically, in this embodiment, microspheres with diameters of 2 μm, 5 μm, and 10 μm were used to investigate the effect of different particle sizes on the phagocytic mechanical force of cells. The above particle size range can effectively simulate the membrane curvature changes involved when cells phagocytose lipid particles or pathologically related particles, and is suitable for imaging and signal acquisition under conventional fluorescence microscopy or confocal microscopy.

[0030] Unlike traditional two-dimensional planar substrates, the microsphere substrate 10 has a continuous curved surface structure in space. This allows the cell membrane to bend and encapsulate along the microsphere surface in multiple directions when a cell contacts and phagocytoses or encapsulates the microsphere substrate 10, thus more realistically reproducing the force state experienced by a cell during the phagocytosis of three-dimensional particles. At this three-dimensional curved interface, cell surface mechanoreceptors (e.g., integrins) can interact with mechanical probes modified on the microsphere surface in multiple spatial directions, thereby applying directional and spatially distributed mechanical forces to the mechanical probes during phagocytosis.

[0031] In a preferred embodiment, silica microspheres with a diameter of 5 μm are used as the microsphere substrate 10. Double-stranded DNA mechanical probes are uniformly modified onto the surface of the microsphere substrate 10 to construct a three-dimensionally distributed single-molecule mechanical sensing interface. Experimental results show that microspheres of this size can be effectively recognized by cells and initiate the phagocytic process, while also providing sufficient surface area for spatial distribution analysis of mechanical signals during phagocytosis.

[0032] In one specific embodiment, in order to achieve highly sensitive detection of single-molecule mechanical forces during cell phagocytosis, this application stably fixes the double-stranded DNA probe 11 to the three-dimensional curved surface of the microsphere substrate 10 in a controlled orientation manner, thereby constructing a single-molecule mechanical sensing interface on the microsphere surface 10 with uniform spatial distribution and consistent / controlled orientation relative to the local surface normal.

[0033] In this embodiment, the double-stranded DNA probe 11 is formed by base complementarity pairing of a first single-stranded DNA 111 and a second single-stranded DNA 112. The first single-stranded DNA 111 has a terminal group for covalent coupling at one end, preferably a thiol group; the second single-stranded DNA 112 is modified with a targeting ligand at one end, the targeting ligand being selected from peptides, antibodies, antibody fragments, aptamers, or glycoproteins; the mechanosensitive receptor is selected from integrin, cadherin, selectin, or T-cell receptors (TCRs). In this embodiment, the targeting ligand is preferably a peptide containing an RGD sequence for specifically binding to cell surface integrin receptors, preferably a cyclic RGD peptide (cRGD), and the mechanosensitive receptor is integrin β1.

[0034] The first single-stranded DNA 111 and the second single-stranded DNA 112 are respectively modified with a first fluorescent label and a second fluorescent label, forming a fluorescence quenching pair or a fluorescence resonance energy transfer pair. In this embodiment, the first fluorescent label is a fluorescent donor or a fluorescent group, and the second fluorescent label is a fluorescent acceptor or a quenching group; or, the first fluorescent label is a fluorescent acceptor or a quenching group, and the second fluorescent label is a fluorescent donor or a fluorescent group. Specifically, the fluorescent group is selected from Cy3, Cy3B, Cy5, FITC, Atto series dyes, or Alexa Fluor series dyes; the quenching group is selected from black hole quenchers (BHQ) series, Dabcyl, or gold nanoparticles.

[0035] In some embodiments, the surface of the microsphere substrate 10 is modified with active functional groups selected from amino, carboxyl, hydroxyl, thiol, maleimide, aldehyde, or epoxy groups. Specifically, the surface of the microsphere substrate 10 is modified with amino groups by 3-aminopropyltriethoxysilane (APTES), and maleimide groups are further introduced by a heterobifunctional crosslinking agent SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester). During the microsphere surface immobilization process, it is preferable to pre-introduce maleimide groups on the surface of the microsphere substrate 10, so that the thiol groups at the ends of the first single-stranded DNA 111 can form a single-point, directional covalent connection with the microsphere substrate 10 through a thiol-maleimide addition reaction. This directional immobilization method ensures that the geometric angle of the double-stranded DNA probe 11 relative to the microsphere surface is controlled, thereby guaranteeing the accuracy of subsequent calculations of the mechanical threshold based on geometric conformation (decompression or shearing). Therefore, compared with random multi-point coupling or uncontrollable orientation fixation methods, this application can significantly reduce the threshold dispersion caused by the attitude difference between different probe molecules, making the unwinding force thresholds between different loading geometric conformations (decompression / shearing) comparable and repeatable, thereby improving the reliability of mechanical quantitative results.

[0036] In this manner, the double-stranded DNA probe 11 exhibits an orientation configuration on the surface of the microsphere, with one end anchored to the microsphere and the other end facing the external environment, thereby ensuring that the targeting ligand is fully exposed in space, facilitating specific binding with mechanoreceptors on the cell surface.

[0037] Unlike physical adsorption or random multi-point coupling, the single-point directional coupling achieved through the thiol-maleimide reaction described above ensures that each DNA probe molecule maintains a uniform orientation on the microsphere surface. This allows for precise definition of the angle between the applied force and the DNA helical axis, ensuring that the mechanical models of the decompression and shearing modes hold true at the microscale, thereby guaranteeing the accuracy of force measurements.

[0038] In a preferred embodiment, by controlling the density of active functional groups on the surface of the microspheres and the dosage ratio of the double-stranded DNA probe 11, the double-stranded DNA probe 11 is uniformly distributed on the surface of the microspheres in a dispersed and non-aggregated manner. This distribution method can effectively reduce spatial interference between adjacent probes, avoid fluorescence signal crosstalk or multi-molecule cooperative force phenomena, thereby ensuring that the fluorescence change reported by a single probe corresponds to a single-molecule mechanical event.

[0039] Furthermore, since the double-stranded DNA probe 11 is fixed to the three-dimensional curved surface of the microsphere, it exhibits a multi-oriented distribution in space. During the process of cells phagocytizing or encapsulating the microsphere, the cell membranes and cytoskeleton in different regions can bind to the targeting ligand in different directions and apply mechanical tension to the double-stranded DNA probe 11 at the corresponding positions. This results in the formation of a three-dimensional single-molecule mechanical sensing interface on the surface of the microsphere, capable of spatially resolving the distribution characteristics of the mechanical forces applied by the cells.

[0040] The single-molecule mechanosensitive sensor of this application utilizes fluorescence resonance energy transfer (FRET) or fluorescence contact quenching principles to achieve signal conversion. In one specific embodiment, the single-molecule mechanosensitive sensor of this application converts the mechanical tension exerted by the cell during phagocytosis or encapsulation of microspheres into optically detectable signal changes through a fluorescence-quenching pair disposed on the double-stranded DNA probe 11, thereby achieving visual characterization of single-molecule-level mechanical events.

[0041] In this embodiment, when the double-stranded DNA probe 11 is in an unstressed state, the first single-stranded DNA 111 and the second single-stranded DNA 112 form a stable double-stranded structure through complementary base pairing. At this time, the first fluorescent label modified on the first single-stranded DNA 111 and the second fluorescent label modified on the second single-stranded DNA 112 are spatially adjacent to each other, with the distance between them within the effective range of fluorescence resonance energy transfer or fluorescence quenching. This allows the fluorescence emission of the first fluorescent label to be effectively quenched by the second fluorescent label, and the double-stranded DNA probe 11 is in a low-fluorescence or non-fluorescent OFF state. The OFF state is when there is no stress or the stress is less than the design threshold, the double-stranded DNA maintains a complete double-helix structure, and the spatial distance between the Cy3B fluorescent group and the BHQ2 quenching group is within the effective quenching radius (usually <10nm). At this time, the surface of the microsphere does not emit fluorescence or only has extremely low background fluorescence.

[0042] When the force exceeds a threshold causing double-strand dissociation, the two strands separate, and fluorescence is restored (ON state, or activated state). Specifically, when cells grasp the targeting ligand on the microspheres via surface receptors and apply mechanical tension, and this tension exceeds the unwinding force threshold of double-stranded DNA, the double-stranded structure is disrupted. The targeting ligand strand is endocytosed or removed by the cell along with the receptor, causing the quenching group to physically separate from the fluorescent group on the anchoring strand, thus resolving the quenching effect and generating a significantly enhanced fluorescence signal on the microsphere surface.

[0043] In another embodiment, the first fluorescent label is a fluorescent donor, and the second fluorescent label is a fluorescent acceptor (FRET pair). When the double strands close, the FRET phenomenon occurs, and the acceptor emits light (high signal); when the double strands are unwound, the donor and acceptor separate, the FRET efficiency decreases, and the fluorescence intensity of the acceptor decreases.

[0044] In a preferred embodiment, both the first single-stranded DNA 111 and the second single-stranded DNA 112 are artificially synthesized oligonucleotide sequences; the first single-stranded DNA 111 has a length of 10-100 bases, and the second single-stranded DNA 112 has a length of 10-100 bases.

[0045] In a preferred embodiment, the first fluorescent label is the fluorescent group Cy3B, and the second fluorescent label is the black hole quencher BHQ2. Cy3B and BHQ2 form a highly efficient fluorescence-quenching pair, enabling the double-stranded DNA probe 11 to maintain a stable low background signal under unstressed conditions.

[0046] When cells engulf or encapsulate the microsphere substrate 10, they specifically bind to the target ligand via mechanotropic receptors (e.g., integrins) on the cell surface, and further apply mechanical tension to the double-stranded DNA probe 11 through cytoskeleton contraction or membrane encapsulation. If the magnitude of the mechanical tension exceeds the unwinding force threshold of the double-stranded DNA probe 11, the double-stranded DNA probe 11 will undergo unwinding, dissociation, or conformational change.

[0047] Under this force, the spatial distance between the first and second fluorescent labels changes significantly, causing them to move out of the effective range of fluorescence resonance energy transfer or fluorescence quenching. This results in the cancellation of fluorescence quenching or a reduction in energy transfer efficiency, causing the first fluorescent label to produce a detectable change in fluorescence signal. This signal change may manifest as an increase in fluorescence intensity, or, in other configurations, a decrease in fluorescence intensity.

[0048] It should be understood that although this embodiment is mainly described using the fluorescence quenching-recovery mode as an example, it is not limited to this. In another embodiment, a combination of FRET fluorescent donor (such as Cy3) and acceptor (such as Cy5) is used. In the initial state, the double strand closure results in a close donor-acceptor distance, and the fluorescence emission of the acceptor is monitored; when a mechanical event causes the double strand to separate, the donor-acceptor distance increases, and the FRET efficiency decreases significantly, manifested as a decrease in the fluorescence intensity of the acceptor channel. This signal reduction mode is also covered by this application.

[0049] Therefore, whether a single double-stranded DNA probe 11 undergoes a change in fluorescence signal corresponds to whether it has experienced a single-molecule mechanical force event exceeding its unwinding force threshold. By statistically analyzing the fluorescence state changes of multiple double-stranded DNA probes 11 on the microsphere surface, quantitative analysis of the distribution and intensity of mechanical forces applied by cells during phagocytosis can be achieved.

[0050] To visually verify the effectiveness of the above signal transduction mechanism in a living cell environment, please refer to [link / reference needed]. Figure 2 This is a schematic diagram of confocal microscopy imaging showing the mechanically triggered fluorescence signal and cytoskeleton signal during cell phagocytosis of microspheres. (See diagram for reference.) Figure 2 As shown, the surface of the microsphere substrate is modified with a single-molecule mechanotropic probe based on double-stranded DNA, wherein the red channel represents the fluorescence signal triggered by the mechanotropic force generated by the double-stranded DNA probe, and the green channel represents the signal of the labeled cytoskeletal actin (F-actin).

[0051] In areas where cells do not come into contact with microspheres, the double-stranded DNA probe remains in a closed double-stranded state, and the fluorescent label and quenching group are within the effective quenching distance, exhibiting only a low background fluorescence signal. However, in the contact interface region formed by cell phagocytosis of microspheres, a significantly enhanced fluorescence signal is observed, indicating that the double-stranded DNA probe in this region is activated by mechanical pulling forces exceeding its unwinding force threshold. Furthermore, the mechanically triggered fluorescence signal and the cytoskeleton F-actin signal exhibit a high degree of spatial co-localization, indicating that the fluorescence signal originates from the mechanical force applied by the cytoskeleton through the targeting ligand-receptor pathway during phagocytosis.

[0052] Figure 2 The images include XY planar views and corresponding YZ cross-sectional views, used to demonstrate the spatial distribution characteristics of mechanical force signals on the three-dimensional curvature interface of the microspheres. This embodiment confirms that the probe's fluorescence signal is indeed triggered by the mechanical force exerted by the cytoskeleton during phagocytosis, and that the sensor can accurately report the spatial distribution of cellular forces at the submicron scale.

[0053] In fluorescence quenching-recovery mode, a positive signal is characterized by enhanced fluorescence in the donor channel; in FRET donor-acceptor mode, a positive signal may be characterized by decreased fluorescence in the acceptor channel or enhanced fluorescence in the donor channel; the signal changes in this application include any of the above-mentioned repeatable fluorescence intensity changes.

[0054] In one specific embodiment, the single-molecule mechanosensitive sensor of this application achieves selective detection of the magnitude and spatial distribution characteristics of single-molecule mechanosensitive force during cell phagocytosis through a force transmission pathway constructed between the targeting ligand, cell surface mechanosensitive receptor, and double-stranded DNA probe 11.

[0055] In one embodiment, the double-stranded DNA probe 11 constructs different mechanical response geometric conformations by adjusting the relative distance and orientation of the anchoring point of the first single-stranded DNA 111 on the microsphere substrate 10 and the connection site of the second single-stranded DNA 112 and the target ligand on the double-stranded helical structure; the mechanical response geometric conformations include an unzipping mode and a shearing mode, as well as an intermediate loading mode in between.

[0056] To explain the influence mechanism of different loading geometries on the unwinding force threshold of double-stranded DNA probes, in this invention, by adjusting the relative positions of the anchoring point of the first single-stranded DNA on the microsphere substrate and the connection site of the second single-stranded DNA and the target ligand on the double-stranded helical structure, different external force loading directions can be constructed, thereby forming different mechanical response modes.

[0057] In decompression mode, the anchoring sites of the first single-stranded DNA and the linking sites of the second single-stranded DNA are located on the same side of the double helix structure of the double-stranded DNA, so that the applied mechanical tension acts approximately perpendicular to the axis of the double-stranded DNA. At this time, the external force mainly breaks the base pairs one by one along a zipper-like path by peeling off the ends one by one. It only needs to overcome the hydrogen bonding forces of local base pairs, thus corresponding to a relatively low unwinding force threshold, for example, in the range of about 10 pN to 15 pN.

[0058] In the shearing mode, the anchoring sites of the first single-stranded DNA and the linking sites of the second single-stranded DNA are located at opposite ends of the double-stranded DNA double helix structure, causing the applied mechanical force to act approximately parallel to the axis of the double-stranded DNA. At this point, the external force acts on the entire hybridization segment in an end-to-end shearing manner, requiring the simultaneous overcoming of hydrogen bonding forces and stacking interactions between multiple base pairs. Therefore, this corresponds to a relatively high unwinding force threshold, for example, in the range of approximately 50 pN to 60 pN.

[0059] Therefore, by changing the relative positions of anchor points and linker sites in the double-stranded DNA structure, different loading geometries can be constructed on the same microsphere platform, forming significantly distinct mechanical response thresholds, which can be used to detect single-molecule mechanical force events of different intensities during phagocytosis.

[0060] Please see Figure 3 The figure shows a schematic diagram of discrete unwinding force threshold probe arrays formed by different loading geometries and sequence designs. As shown, when the target ligand binds to the mechanosensitive receptor on the cell surface and applies mechanical tension, the double-stranded DNA probe can be constructed into unzipping mode, shearing mode, or several intermediate loading modes (Intermediate I–III) depending on the anchoring position of the first single-stranded DNA on the microsphere substrate and the connection mode between the second single-stranded DNA and the target ligand. In unzipping mode, the double-stranded DNA probe has a lower unwinding force threshold (e.g., about 12 pN); in shearing mode, the double-stranded DNA probe has a higher unwinding force threshold (e.g., about 56 pN); by adjusting the GC content, length, or mismatch of the complementary pairing region, double-stranded DNA probes with intermediate unwinding force thresholds (e.g., 23 pN, 33 pN, 43 pN) can be obtained, thereby constructing a single-molecule mechanical sensing probe array with a discrete mechanical threshold distribution.

[0061] In this embodiment, the targeting ligand is preferably a cyclic RGD peptide (cRGD), which can specifically bind to integrin receptors on the cell surface. During the process of phagocytosis or encapsulation of the microsphere substrate 10, the mechanical tension generated by the cytoskeleton contraction or membrane encapsulation behavior is transmitted to the double-stranded DNA probe 11 via the integrin-cRGD binding site along the pathway of the cytoskeleton, mechanoreceptor, targeting ligand, and double-stranded DNA probe 11.

[0062] In one embodiment, when the double-stranded DNA probe 11 is configured in a decompression mode, the anchoring site of the first single-stranded DNA 111 and the targeting ligand linking site of the second single-stranded DNA 112 are located on the same side end of the double helix structure of the double-stranded DNA probe 11. This allows mechanical pulling forces applied by the cell to act on the double-stranded DNA probe 11 in an end-peeling manner, with the direction of the mechanical pulling force primarily perpendicular to the base pair hydrogen bond direction of the double-stranded DNA probe 11, specifically perpendicular to the loading direction of the double-stranded DNA axis. Under this loading geometry, the double-stranded DNA probe 11 has a low unwinding force threshold, preferably 10 pN to 15 pN, thereby enabling selective response to early or low-intensity mechanical pulling events during phagocytosis.

[0063] like Figure 3 The diagram illustrates a "zipper-like" force-bearing structure. In this conformation, the tension is perpendicular to the hydrogen bond axis, and only a small energy barrier needs to be overcome to open each base pair individually. This embodiment utilizes this pattern to construct a low-threshold probe (e.g., 12pN) for detecting weak mechanical signals in the early stages of phagocytosis.

[0064] In another embodiment, when the double-stranded DNA probe 11 is configured in a shearing mode, the anchoring site of the first single-stranded DNA 111 and the targeting ligand linking site of the second single-stranded DNA 112 are located at opposite ends of the double helix structure of the double-stranded DNA probe 11, such that the mechanical force applied by the cell acts on the double-stranded DNA probe 11 in an end-to-end shearing manner. The direction of the mechanical force is mainly parallel to the hydrogen bond direction of the base pairs of the double-stranded DNA probe 11, specifically parallel to the loading direction of the double-stranded DNA axis. Under this loading geometry, the double-stranded DNA probe 11 has a high unwinding force threshold, preferably 50 pN to 60 pN, thereby enabling the detection of high-intensity mechanical forces generated by strong contraction or stable encapsulation of the cytoskeleton during phagocytosis.

[0065] like Figure 3 The diagram illustrates a "shear-like" force-bearing structure. In this conformation, the tension is parallel to the double helix axis, requiring the simultaneous disruption of hydrogen bonds and stacking forces of all base pairs. This embodiment utilizes this pattern to construct a high-threshold probe (e.g., 56 pN) for detecting strong mechanical signals during intense cytoskeleton contraction.

[0066] In one embodiment, in addition to the decompression mode and the shearing mode, an intermediate loading mode located between the decompression mode and the shearing mode can be constructed by adjusting the relative positions of the anchoring point of the first single-stranded DNA on the microsphere substrate and the connection site of the second single-stranded DNA and the target ligand on the double helix structure of the double-stranded DNA.

[0067] The intermediate loading mode alters the position of the anchor point within the double-stranded sequence, causing the applied mechanical force relative to the double-stranded DNA axis to gradually transition from approximately perpendicular to approximately parallel, thus forming a transitional geometric conformation between end-to-end shearing loading and end-to-end exfoliation loading. In this intermediate loading mode, the unwinding force threshold of the double-stranded DNA probe can continuously vary and lies between the thresholds corresponding to the decompression and shearing modes. For example, in one embodiment, by changing the anchoring position of the first single-stranded DNA within the double-stranded sequence, multiple probe configurations with different loading direction angles can be constructed, with unwinding force thresholds ranging from approximately 15 pN to 50 pN. Therefore, by adjusting the relative distance and direction between the anchor point and the connection site, a series of transitional conformation modes with continuously varying mechanical responses can be constructed to achieve graded detection of different mechanical force ranges.

[0068] Furthermore, in one embodiment, the double-stranded DNA probe 11 can also finely control the unwinding force threshold by adjusting the GC content, length, or number of mismatched bases in the complementary pairing regions of the first single-stranded DNA 111 and the second single-stranded DNA 112. In this embodiment, by simultaneously modifying the surface of the same microsphere substrate 10 with double-stranded DNA probes 11 of different unwinding force thresholds, a mechanical probe array with a discrete force threshold distribution can be constructed. The unwinding force threshold can be selected from any one or more of 12pN, 23pN, 33pN, 43pN, and 56pN. By statistically analyzing the spatial location and proportion of fluorescence signal changes of probes with different thresholds on the microsphere surface, the spatial distribution characteristics and intensity gradient of mechanical pulling force during cell phagocytosis can be analyzed on the three-dimensional curvature interface.

[0069] To verify the effectiveness of the above-mentioned mechanical threshold control strategy and to establish quantitative mechanical analysis standards, please refer to [link to relevant documentation]. Figure 4 The graph shows the statistical relationship between the proportion of positive fluorescence signals and the probe melting force threshold, along with a schematic diagram of F50 fitting. Figure 4 As shown, the horizontal axis is labeled "Phagocytosis force (pN)," which represents the unwinding force threshold of the double-stranded DNA probe set through geometric conformation design (decompression mode or shearing mode) and sequence optimization; the vertical axis represents the percentage of probes producing a positive fluorescent signal (Positive Counts %) measured under standardized conditions. Figure 4As can be seen, with the increase of force value, the proportion of positive signals shows a monotonic trend of decreasing positive proportion as the threshold increases, and can be fitted by a sigmoid function / logistic model. Through fitting analysis of the curve, the half-maximum effective mechanical force threshold (F50) of vascular endothelial cells during phagocytosis can be calculated to be approximately 27.89 pN under the experimental conditions of this embodiment. This result indicates that by setting up a double-stranded DNA probe array with different unwinding force thresholds, this application can perform graded response and quantitative analysis of single-molecule mechanical force over a wide dynamic range, thereby providing a unified and reproducible quantitative indicator for subsequent comparison of changes in cell mechanical properties under different physiological states, pathological conditions, or drug interventions. Specifically, by integrating multiple discrete threshold probes (e.g., 12 pN, 23 pN, 33 pN, 43 pN, 56 pN) onto the same sensing interface, this application can map the cell force intensity to a statistical response curve of "positive proportion - threshold," thereby outputting statistically significant equivalent mechanical parameters (e.g., F50), avoiding the problem of only being able to determine presence / absence without quantitative comparison caused by a single threshold probe.

[0070] Under a defined geometric conformation, multiple intermediate gradient thresholds (e.g., 23pN, 33pN, 43pN) can be obtained by adjusting GC content, hybridization length, or introducing mismatches. Thus, by combining the target ligand-receptor-mediated force transmission pathway with double-stranded DNA probes 11 of different loading geometric conformations and unwinding force thresholds, the cellular single-molecule mechanosensor can not only sense whether a single-molecule mechanosensory event has occurred, but also spatially distinguish the areas of action of different intensities of mechanical force during phagocytosis, achieving multi-dimensional characterization of cellular phagocytic mechanical behavior. In a preferred embodiment of this application, the generation and transmission of the single-molecule mechanosensory force depends on the formation of a vitreous adhesion complex mediated by Vinculin, which is regulated by Syk and FAK kinases, thereby transmitting the mechanical work generated by the cytoskeleton to the integrin receptor and the double-stranded DNA probe. By regulating the activity of Arp2 or mDia1, the magnitude of the single-molecule mechanosensory force can be further modulated, thereby achieving selective response to probes with different thresholds.

[0071] To ensure that the measured mechanical force originates from a specific biological process (such as endothelial cell phagocytosis associated with atherosclerosis) rather than non-specific cell adhesion, the single-molecule mechanosensor provided in this application is modified with a cRGD peptide at the end of the ligand chain. cRGD can specifically bind to integrin receptors on the surface of vascular endothelial cells, which are key nodes mediating phagocytosis and mechanotransduction. Simultaneously, this application also includes a control probe without cRGD in the above embodiments to monitor and eliminate background signal interference, ensuring the biological specificity of the detection results.

[0072] In one specific embodiment, to improve the reliability and specificity of the detection results of the cell single-molecule mechanomechanical sensor, the cell single-molecule mechanomechanical sensor further includes a control double-stranded DNA probe. The control double-stranded DNA probe is modified on the surface of the microsphere substrate 10, and the control double-stranded DNA probe is not modified with the targeting ligand, or the control double-stranded DNA probe has a non-destructible structure, used to exclude non-specific signals or monitor false positive signals caused by nuclease degradation. By setting the control double-stranded DNA probe, this application can distinguish between receptor-mediated mechanomechanical dissociation signals and signal changes caused by non-mechanical factors such as non-specific adsorption, nuclease degradation, fluorescent label shedding, or photobleaching fluctuations, thereby significantly improving the specificity and reliability of the detection conclusions.

[0073] In this embodiment, a control double-stranded DNA probe is simultaneously or in parallel disposed on the surface of the microsphere substrate 10 to exclude non-specific signals and monitor false positive signals caused by nuclease degradation or optical artifacts. The control double-stranded DNA probe and the functional double-stranded DNA probe 11 are consistent in molecular composition and immobilization method, both formed by complementary pairing of a first single-stranded DNA 111 and a second single-stranded DNA 112, and are directionally covalently modified on the surface of the microsphere substrate 10 through the end group of the first single-stranded DNA 111; the only difference lies in whether it has receptor-mediated force transmission capability or whether it has a helical mechanical response.

[0074] In one embodiment, the control double-stranded DNA probe is not modified with a targeting ligand. Specifically, the second single-stranded DNA 112 is not linked to a ligand for specific binding to cell surface mechanosensitive receptors, and the remaining structure (including the fluorescent label, complementary pairing segments, and immobilization method) remains identical to the functional double-stranded DNA probe 11. With this configuration, when cells come into contact with or are phagocytosed by the microsphere substrate 10, the lack of specific receptor-ligand binding prevents the cells from applying effective mechanical pull to the control probe via the receptor-mediated pathway. If a change in fluorescence signal is still observed under this control condition, it can be determined that the signal originates from non-specific adsorption, optical background fluctuations, or imaging noise, rather than a genuine receptor-mediated single-molecule mechanosensitive event.

[0075] In another embodiment, the control double-stranded DNA probe is constructed with a non-dehiscent structure. For example, by increasing the GC content of the complementary pairing region between the first single-stranded DNA 111 and the second single-stranded DNA 112, the double-strand stability can be significantly improved; or by extending the length of the complementary pairing region or introducing chemical cross-linking, the double-stranded DNA probe 11 can be prevented from undergoing mechanotropic dehiscence under experimental conditions; or other structural designs capable of inhibiting dehiscence or conformational changes of double-stranded DNA under stress can be employed. Under this control condition, even if cells bind to the mechanoreceptor via the targeting ligand and apply mechanical tension, the control probe will not undergo dehiscence or conformational change, and therefore should not produce a mechanically triggered change in fluorescence signal. If a change in fluorescence signal is observed under this control condition, it may indicate a false positive signal caused by nuclease degradation, fluorescent label shedding, or other non-mechanical factors.

[0076] In this embodiment, the control double-stranded DNA probe can be modified in parallel with the functional double-stranded DNA probe 11 on the surface of different microsphere substrates 10, or different microsphere populations can be used to carry the functional probe and the control probe respectively in the same experimental system, so as to perform comparative analysis under the same imaging and culture conditions. By comparing the fluorescence signal change results of the functional double-stranded DNA probe 11 with the signal change results of the control double-stranded DNA probe, non-specific signals and false positive signals caused by nuclease degradation can be effectively eliminated, thereby ensuring that the detected fluorescence signal changes truly reflect the single-molecule mechanical events mediated by cell receptors.

[0077] In the above embodiments, this application has been combined with Figures 1 to 4 The overall structure, mechanical response mechanism, and signal conversion mechanism of the single-molecule cellular mechanical force sensor are described. To enable the sensor to be reproducibly constructed under different experimental conditions and to ensure the comparability between probes with different mechanical thresholds, this application further provides a method for preparing the single-molecule cellular mechanical force sensor, which stably fixes the double-stranded DNA probe on the surface of a microsphere substrate in a controlled orientation manner, thereby obtaining a surface-functionalized microsphere sensor for single-molecule mechanical force detection.

[0078] Please see Figure 5 The diagram shows a flowchart of the preparation method of this application in one embodiment. Figure 5 The sequence of steps S11 (microsphere substrate activation), S12 (DNA probe assembly), S13 (coupling), and S14 (purification) is shown in the figure. The preparation method of the cell single-molecule mechanosensor includes steps S11-S14.

[0079] Through the combination of steps S11 to S14 described above, this application can form a stable, orientation-controlled, and dispersed double-stranded DNA probe array on the three-dimensional curved surface of microspheres. This ensures consistency in the loaded geometric conformation (decompression / shearing and its intermediate conformations) at the microscale, thereby achieving comparability and reproducibility between probes with different unwinding force thresholds. Furthermore, by removing free probes and non-specifically adsorbed components during the coupling and purification stages, background fluorescence and false positive signals can be significantly reduced, improving the signal-to-noise ratio and quantitative analysis reliability of the sensor during live cell phagocytosis or encapsulation. Therefore, the preparation method described in this application can transform the "structure / mechanism" described in the first set of embodiments into a standardized and executable process, providing a stable and consistent sensor foundation for subsequent spatial distribution imaging and mechanical threshold statistical analysis of single-molecule mechanical forces in cells.

[0080] In step S11, a microsphere substrate is first provided, and the surface of the microsphere substrate is chemically modified to introduce active functional groups that can covalently couple with single-stranded DNA onto the surface of the microsphere substrate, thereby obtaining a surface-activated microsphere substrate for stable immobilization of a single-molecule mechanical probe.

[0081] In one specific embodiment, to achieve stable characterization of single-molecule-level mechanical forces during cell phagocytosis or encapsulation, this application preferably uses a microsphere substrate with a three-dimensional curved surface as a sensing carrier. A subsequent surface activation step introduces active functional groups (preferably maleimide groups) capable of covalently coupling with single-stranded DNA onto the surface of the microsphere substrate, thereby obtaining a surface-activated microsphere substrate for stable immobilization of double-stranded DNA probes. In this application, the material of the microsphere substrate can be selected from silica, polystyrene, polymethyl methacrylate (PMMA), magnetic nanomaterials, gold nanoparticles, or combinations thereof, and its particle size is preferably in the range of 1 μm to 15 μm, for example, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, or 15 μm, to adapt to the membrane curvature changes in the cell phagocytosis scenario. By selecting microsphere substrates with controllable particle size and good monodispersity, the influence of geometric differences between different particles on the force distribution and threshold statistics can be reduced, thereby improving the repeatability and comparability of subsequent mechanical threshold comparison analysis.

[0082] In one embodiment, the microsphere substrate is a commercially available microsphere substrate. Preferably, the microsphere substrate is a commercially available silica microsphere, which has good biocompatibility, optical transparency, and ease of surface chemical modification. The commercially available microspheres can be selected according to application requirements, such as 2 μm, 5 μm, or 10 μm, and are provided in aqueous or buffer form. To ensure the uniformity of the subsequent surface activation reaction and the stability of the coupling density, the commercially available microspheres are preferably pre-cleaned and dispersed before surface activation. For example, this can be achieved by repeatedly washing with deionized water or ethanol using centrifugation, discarding the supernatant, and resuspending to remove preservatives, surface dispersants, or trace organic residues. Short-term ultrasonic dispersion can be used to reduce agglomeration. For silica microspheres, after washing with deionized water / ethanol, the microspheres can be dispersed in deionized water by stirring and allowed to stand for equilibration to expose the surface silanol groups and improve the consistency of the silanization reaction. The above pretreatment can reduce the background signal and coupling site unevenness caused by non-specific adsorption, which is beneficial to obtaining a sensor substrate with low background and batch consistency.

[0083] In another embodiment, the microsphere substrate can be prepared by a reverse microemulsion method. The reverse microemulsion method utilizes nano / micron-sized water cores formed in the continuous oil phase as a confined reactor, allowing the silicon source precursor to undergo hydrolysis and condensation within the water cores, thereby forming silica microspheres with controllable particle size and good monodispersity. In one embodiment, the self-made route includes the following steps:

[0084] 1) Construction of microemulsion system: Provide oil phase solvent and surfactant, dissolve the surfactant in oil phase to form a homogeneous solution; then add aqueous phase (which may contain basic catalytic components) to form a stable reverse microemulsion system, wherein the aqueous phase is dispersed in oil phase in the form of tiny water nuclei;

[0085] 2) Silicon source introduction and reaction: Add a silicon source precursor (e.g., tetraethoxysilane TEOS or other hydrolyzable silanes) to the reverse microemulsion system. Under stirring conditions, the silicon source is hydrolyzed at the water core interface and further condensed to generate a silica network, which gradually grows to form microspheres.

[0086] 3) Termination and demulsification: After the target particle size or reaction time is reached, the microemulsion structure is destroyed by adding a polar solvent (such as alcohols) or changing the ionic strength / solvent composition of the system to terminate the reaction;

[0087] 4) Washing and purification: The oil phase, surfactants and unreacted precursor residues were removed by centrifugation, washing and resuspension to obtain the self-made microsphere suspension;

[0088] 5) Grading and sieving and monodispersity optimization: Differential centrifugation, filtration grading or density gradient separation can be used to further narrow the particle size distribution in order to obtain a microsphere population with higher monodispersity.

[0089] To control the microsphere size and monodispersity, the water core size is preferably controlled by adjusting the molar ratio of water to surfactant, thereby controlling the final microsphere size. After obtaining and pretreating the microsphere substrate, the surface activation reaction begins.

[0090] In another alternative embodiment, the microsphere substrate can be prepared by a reverse microemulsion method, and the particle size and monodispersity of the microspheres can be controlled by adjusting the molar ratio of water to surfactant. Specifically, in the reverse microemulsion method, the aqueous phase is dispersed in the continuous oil phase in the form of nano / micron-sized water cores, and the silicon source precursor is hydrolyzed and condensed in the water core confinement environment to generate microsphere particles. Preferably, the final microsphere particle size is controlled by changing the water core size by adjusting the molar ratio of water to surfactant (which can also be characterized as the WO parameter); by controlling the water core size through the WO parameter, the particle size can be controlled and reproducible, thereby reducing the systematic deviation of the phagocytic force statistical results caused by the curvature difference of microspheres with different particle sizes, and improving the comparability between different experimental batches.

[0091] After preparation, the reaction can be terminated by demulsification, and the oil phase and surfactant residues can be removed by centrifugation / washing. If necessary, differential centrifugation, filtration fractionation, or density gradient separation can be used to narrow the particle size distribution and improve monodispersity. Final washing and fractionation purification can reduce the cytotoxicity and nonspecific adsorption caused by surfactant residues, and reduce the interference of microsphere aggregation on co-incubation phagocytosis behavior and imaging resolution, thereby improving the stability and signal-to-noise ratio of the sensor during application.

[0092] In an optional embodiment, to introduce amino sites for subsequent coupling reactions on the surface of the microsphere substrate and provide precursor functional groups capable of reacting with the crosslinking agent, this application performs APTES amination treatment on the pretreated microsphere substrate. Specifically, the microsphere substrate is dispersed in an anhydrous alcohol solvent or other solvent system suitable for silanization reaction, 3-aminopropyltriethoxysilane (APTES) is added, and the reaction is carried out under stirring for a preset time, allowing APTES to silanize on the microsphere surface and form stable siloxane bonds, thereby introducing -NH2 functional groups on the microsphere surface. After the reaction is completed, unreacted APTES and byproducts are removed by centrifugation and solvent washing to obtain the surface-aminated microsphere substrate.

[0093] It should be understood that the purpose of the APTES amination treatment is to provide reactive amino sites for the subsequent introduction of maleimide groups onto the microsphere surface using the heterobifunctional crosslinking agent SMCC, enabling the maleimide groups to be stably present on the microsphere surface in a covalent manner, thereby establishing a reaction interface for thiol-maleimide addition coupling. Furthermore, by forming a controllable density amino layer on the microsphere surface, the surface coverage and uniformity of the maleimide groups introduced by SMCC can be improved, thereby increasing the effective coupling density and coupling stability of the double-stranded DNA probe, reducing the risk of probe detachment during culture / washing, and ultimately facilitating the acquisition of mechanical threshold response results with lower background fluorescence and better batch-to-batch consistency.

[0094] In one specific embodiment, to give the surface of the microsphere substrate active functional groups capable of covalently coupling with the end groups of single-stranded DNA, this application further introduces maleimide groups onto the surface of the surface-aminated microsphere substrate. Specifically, a heterobifunctional crosslinking agent SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester) is provided. SMCC is added to the suspension system of the surface-aminated microsphere substrate and reacted under preset conditions, causing the succinimide active ester (NHS ester) in the SMCC molecule to undergo amidation coupling with the amino groups on the surface of the microspheres, thereby covalently fixing the maleimide groups at the other end of the SMCC molecule onto the surface of the microspheres, thus obtaining a surface-maleiminated microsphere substrate.

[0095] After the reaction, to reduce background interference in subsequent coupling steps and avoid non-specific reactions caused by residual crosslinking agents, the reaction system was purified in this embodiment. Purification methods may include centrifugation, resuspension and repeated washing, and / or filtration. In some embodiments, centrifugal ultrafiltration or gel filtration / desalting column buffer replacement may be used to further reduce free fluorescence background. Preferably, multiple centrifugation-resuspension washing is performed to remove unreacted SMCCs and their hydrolysis byproducts, and finally, the system is resuspended in a physiological buffer system (e.g., PBS or other buffers) to obtain a maleimide-modified microsphere suspension suitable for subsequent coupling reactions.

[0096] It should be understood that the formation of a maleimide active layer on the microsphere surface by SMCC enables the subsequent covalent coupling of the first single-stranded DNA carrying thiol end groups through a thiol-maleimide addition reaction under mild aqueous conditions, thus providing a reaction basis for the controlled orientation and fixation of double-stranded DNA probes on the microsphere surface. Furthermore, the washing and purification step significantly reduces the risk of non-specific cross-linking and increased background fluorescence caused by free SMCC or its byproducts, reducing the probability of random multi-point coupling or bridging aggregation of probes on the microsphere surface. This improves the controllability of coupling sites and batch consistency, thereby enhancing the comparability and signal-to-noise ratio between probes with different melting force thresholds.

[0097] Thus, a surface-activated microsphere substrate with maleimide groups introduced on its surface is obtained, which can selectively covalently couple with a first single-stranded DNA with thiol end groups in a subsequent coupling step to achieve stable immobilization of a single-molecule mechanical probe on the surface of the microsphere substrate.

[0098] In an optional embodiment, to introduce maleimide groups capable of covalently coupling with thiol groups onto the surface of the microsphere substrate, in addition to SMCC, other equivalent crosslinking agents can be used to construct a maleimide active layer on the microsphere surface. The equivalent crosslinking agent is preferably a heterobifunctional crosslinking agent with a "first reactive group-spacer arm-maleimide group" structure, wherein the first reactive group is used to react with the preceding functional groups on the microsphere surface. For example, when the microspheres have amino groups after APTES treatment, the first reactive group can be an NHS active ester; when the microsphere surface has carboxyl or hydroxyl functional groups, a matching reactive group can also be selected to achieve covalent fixation. Through this type of equivalent crosslinking agent, the maleimide groups are stably presented on the microsphere surface, obtaining a surface-activated microsphere substrate. After the reaction, free crosslinking agents and byproducts can be removed by centrifugation-resuspending washing or filtration to obtain a maleimide-modified microsphere suspension suitable for subsequent coupling. Using an equivalent crosslinking agent can improve the exposure and coupling uniformity of active sites without changing the nature of the "maleimide-thiol selective addition" reaction, thereby improving probe coupling density and batch stability.

[0099] In step S12, a DNA probe assembly step is performed: a first single-stranded DNA and a second single-stranded DNA are provided, wherein the first single-stranded DNA is modified with a first fluorescent label and has end groups for coupling, and the second single-stranded DNA is modified with a second fluorescent label and a targeting ligand; and the first single-stranded DNA and the second single-stranded DNA are mixed in a hybridization buffer and annealed to form a double-stranded DNA probe with a preset mechanical response configuration through complementary base pairing.

[0100] In one specific embodiment, to obtain a double-stranded DNA probe with a predetermined mechanical response configuration, this application provides a first single-stranded DNA and a second single-stranded DNA, which are then mixed and annealed in a salt-containing hybridization buffer to form a stable double-stranded structure through complementary base pairing. The first single-stranded DNA is modified with a first fluorescent label and has end groups (preferably thiol end groups) for subsequent coupling, while the second single-stranded DNA is modified with a second fluorescent label and a targeting ligand (e.g., cRGD or other ligands that specifically bind to mechanoreceptors on the cell surface).

[0101] In this embodiment, the first single-stranded DNA and the second single-stranded DNA are mixed at a preset molar ratio; preferably, the molar ratio of the first single-stranded DNA to the second single-stranded DNA is 2:1. More preferably, annealing is performed in a hybridization buffer containing NaCl to promote complementary base pairing and suppress the decrease in hybridization efficiency caused by charge repulsion; for example, the hybridization buffer can be a NaCl-containing buffer system (such as a salt-containing buffer in a Tris or PBS system), and its NaCl concentration can be selected and adjusted according to the oligonucleotide length and GC content.

[0102] Annealing can employ a temperature program of denaturation by heating and annealing by slow cooling. For example, the mixture can be heated to 80°C–95°C and held for 1–10 minutes, then cooled to room temperature or 25°C at a rate of, for example, 0.1°C / s–1°C / s and held for 5–30 minutes to complete annealing. Specifically, the mixture can be heated to a temperature range sufficient to completely anneal the single strands and held for a predetermined time to eliminate any possible mispairing or localized secondary structures. Subsequently, the temperature is lowered to room temperature or a target temperature at a controlled rate, allowing the first and second single-stranded DNAs to gradually anneal along complementary regions to form a double-stranded DNA probe. This annealing program can improve the consistency of hybridization pairing at the molecular level, making the loaded geometry of the double-stranded DNA probe more statistically consistent with the complementary pairing regions, thereby providing better reproducibility and comparability for probes designed with different batches and thresholds.

[0103] Specifically, the 2:1 molar ratio configuration enables the second single-stranded DNA to become the limiting component in the hybridization system, thereby reducing self-aggregation or non-target hybridization caused by excess second single-stranded DNA while forming the target double-stranded structure. Furthermore, the combination of the NaCl buffer system and the annealing process can reduce the proportion of mismatched bases or non-specific hybridization, thereby reducing the risk of false positive signals caused by premature unwinding at low thresholds or spontaneous background dequenching due to mispairing.

[0104] After annealing, a double-stranded DNA probe solution for subsequent directional coupling is obtained. The double-stranded DNA probe has a preset complementary pairing segment and a preset mechanical response configuration, and can further enter the steps of thiol end-group activation (e.g., TCEP reduction) and directional covalent coupling with the surface-activated microsphere substrate.

[0105] In one specific embodiment, to ensure that the double-stranded DNA probe can undergo a highly efficient and controllable orientation covalent coupling reaction with the surface-activated microsphere substrate through the end group of the first single-stranded DNA, a TCEP (tris(2-carboxyethyl)phosphine) reduction treatment is performed on the assembled double-stranded DNA probe before the coupling step to activate the thiol groups on the end group of the first single-stranded DNA.

[0106] Specifically, the terminal group of the first single-stranded DNA is preferably a thiol group (-SH). During probe preparation and storage, the thiol group may oxidize to form a disulfide bond (-SS-), thereby reducing the number of effective reaction sites for the addition reaction between the thiol group and the maleimide group. Therefore, in this embodiment, tris(2-carboxyethyl)phosphine (TCEP) is added to the double-stranded DNA probe solution for reduction treatment, so that the disulfide bonds that may be formed are reduced and broken, restoring them to free thiol groups (-SH), thereby obtaining an active thiol terminal group.

[0107] In this embodiment, the TCEP reduction process can be carried out at room temperature for a preset time (e.g., 10-30 minutes). Subsequently, the residual TCEP can be removed or diluted as needed by means of buffer replacement, centrifugation ultrafiltration or column purification to avoid unnecessary impact on the subsequent coupling reaction system.

[0108] In this embodiment, by introducing a TCEP reduction and activation step, the effective exposure ratio and chemical reactivity of thiol end groups can be significantly improved, thereby enhancing the reaction efficiency of thiol-maleimide addition coupling. Furthermore, this reduction and activation step can reduce coupling failures or uneven coupling density caused by disulfide bond formation, and reduce probe modification differences between different microspheres and between different regions of the same microsphere. Therefore, in subsequent directional coupling, the double-stranded DNA probe selectively adds to the maleimide groups on the microsphere surface through a single-point thiol end group, which is more conducive to obtaining probe arrays with consistent orientation and more uniform spatial distribution, thereby improving the consistency and batch-to-batch reproducibility of directional coupling.

[0109] In one embodiment, the assembled double-stranded DNA probe (e.g., 100 μL) is preferably reacted with TCEP solution at room temperature in the dark for 15 minutes to fully reduce and activate the thiol groups at the ends of the first single-stranded DNA. The activated double-stranded DNA probe should be used immediately for subsequent coupling reactions with maleimide-modified microspheres to prevent the thiol groups from being re-oxidized. By strictly controlling the activation time and reaction environment, it can be ensured that the probe is stably immobilized on the surface of the microspheres through single-point directional covalent coupling, thereby ensuring the conformational consistency of the sensor in subsequent force spectroscopy detection.

[0110] After the TCEP reduction and activation process is completed, the double-stranded DNA probe can be used for subsequent directional covalent coupling with the surface-activated microsphere substrate to construct a single-molecule mechanosensor.

[0111] In an optional embodiment, to enable the second single-stranded DNA to specifically bind to cell surface mechanosensitive receptors, the targeting ligand is linked to the second single-stranded DNA before or after the DNA probe assembly step. The targeting ligand may be selected from peptides (e.g., peptides containing RGD sequences or cyclic RGD peptides cRGD), antibodies, antibody fragments, aptamers, or glycoproteins; the linkage method includes click chemistry, active ester-amine coupling, maleimide-thiol coupling, or combinations thereof.

[0112] In one embodiment, the targeting ligand can be ligated to the second single-stranded DNA before annealing assembly, i.e., a "ligand-second single-stranded DNA" covalently coupled product is first obtained, and then annealed and hybridized with the first single-stranded DNA to form a double-stranded DNA probe carrying the targeting ligand. This method is beneficial to complete the quantitative modification of the ligand before hybridization, reduce the fluctuation of the ligand efficiency caused by steric hindrance after hybridization, and thus improve the consistency and comparability of ligand density of different batches of probes.

[0113] In another embodiment, the targeting ligand can also be linked to the second single-stranded DNA after annealing assembly, that is, a double-stranded DNA probe is formed first, and then the ligand is linked without destroying the stability of the double strand. This method is beneficial to introduce the ligand after confirming that the double strand conformation and threshold design are correct, reducing the risk of mismatch or conformational shift caused by the introduction of the ligand, thereby reducing non-specific signals and false positive probability.

[0114] Specifically, in a preferred embodiment, the click chemistry reaction is a copper-catalyzed azido-alkynyl cycloaddition reaction (CuAAC). For example, azido / alkynyl reactive groups can be introduced onto the second single-stranded DNA or the targeting ligand, and the CuAAC reaction can be carried out in the presence of copper ions and a reducing agent, thereby achieving site-specific ligand-second single-stranded DNA ligand connection. Through the site-specific and highly selective ligand connection characteristics of click chemistry, non-specific cross-linking and random multi-site ligand connection can be reduced, making the exposure orientation of the targeting ligand more controllable, thereby improving the effectiveness and reproducibility of the receptor-ligand mediated force transmission pathway.

[0115] In another embodiment, an active ester-amine coupling method can be used, where an NHS active ester group is provided on the targeting ligand or linker arm, which undergoes an amidation reaction with an amino group pre-introduced on the second single-stranded DNA; or a maleimide-thiol coupling method can be used, where a maleimide group is introduced on the targeting ligand or linker arm, which undergoes an addition reaction with a thiol group introduced on the second single-stranded DNA. It should be understood that the above coupling methods can be used alone or in combination to adapt to the chemical structures of different targeting ligands and the requirements of reaction conditions. Through the above coupling methods, the mechanotransduction link of "targeting ligand-second single-stranded DNA-double-stranded DNA probe-microsphere substrate" can be standardized, thereby improving the receptor-mediated specificity of the sensor during cell phagocytosis and reducing background signals caused by non-specific adsorption.

[0116] In one specific embodiment, to stably fix the double-stranded DNA probe on the surface of a microsphere substrate in a controlled orientation manner, thereby obtaining a cell single-molecule mechanical force sensor that can be used for single-molecule mechanical detection, this application selectively adds a double-stranded DNA probe, which has been annealed and assembled and whose thiol end groups have been activated by TCEP reduction, to a surface-activated microsphere substrate that has undergone maleimide conversion. Specifically, in coupling step S13, the double-stranded DNA probe obtained in the DNA probe assembly step is mixed with the surface-activated microsphere substrate obtained in the microsphere substrate activation step, and the reaction is carried out under coupling reaction conditions, so that the double-stranded DNA probe is covalently linked to the surface of the microsphere substrate in a controlled orientation manner through the end groups of the first single-stranded DNA; the end groups are thiol groups, and the coupling reaction includes an addition reaction between the thiol group and the maleimide group to achieve directional covalent connection of the double-stranded DNA probe;

[0117] In this embodiment, maleimide groups are introduced onto the surface of the surface-activated microsphere substrate; the first single-stranded DNA of the double-stranded DNA probe has a free thiol end group. After mixing, the two are reacted under coupling conditions suitable for thiol-maleimide addition reactions, allowing the double-stranded DNA probe to undergo an addition reaction with the maleimide groups through the thiol end groups, thereby achieving single-point, directional covalent linkage of the double-stranded DNA probe on the microsphere surface.

[0118] In one embodiment, the coupling reaction can be carried out in phosphate-buffered saline (PBS), HEPES buffer, or Tris buffer; preferably, a buffer system free of high concentrations of primary amine interfering groups. The ionic strength of the buffer can be set as needed, for example, it can contain NaCl to maintain nucleic acid structural stability. The pH of the coupling reaction can be 6.5-8.0, preferably 6.8-7.5, to balance maleimide reactivity and nucleic acid stability. The reaction temperature can be carried out at 4-37°C, preferably room temperature (e.g., 20-25°C) to obtain better reaction efficiency and reproducibility.

[0119] In a preferred embodiment, the TCEP-treated double-stranded DNA probe is buffered or diluted with PBS, then mixed with maleimide-modified microspheres and gently shaken and incubated at room temperature to promote full contact between the probe and the surface of the microspheres and complete addition coupling.

[0120] In one embodiment, the coupling reaction can last for 30-180 minutes, preferably 60-120 minutes, and more preferably about 90 minutes. The amount of double-stranded DNA probe and microspheres can be adjusted according to the microsphere particle size, surface functional group density, and target probe coverage density; for example, by controlling the final probe concentration and the microsphere mass concentration / particle number concentration, the probe can be dispersed and covered on the microsphere surface rather than crowded and aggregated.

[0121] In the above embodiments, by placing the thiol end group at the connection end between the first single-stranded DNA and the microsphere and employing selective coupling with maleimide, this application can achieve single-point oriented fixation of the probe on the microsphere surface, thereby facilitating the obtaining of probes with more consistent orientation. Furthermore, orientation consistency can reduce conformational dispersion caused by random multi-point coupling, making the loading geometry of probes from the same batch more controllable, thereby improving the "threshold comparability" between probes with different mechanical thresholds and reducing threshold drift or false positive signals caused by conformational deviations. Simultaneously, covalent linkages are less prone to detachment under culture / imaging conditions compared to physical adsorption, thereby improving the stability of sensor construction and long-term imaging consistency.

[0122] After the coupling reaction is complete, the reaction system can be purified to remove uncoupled double-stranded DNA probes, residual small molecule reagents, and possible non-specific adsorbed impurities. In purification step S14, uncoupled DNA probes and impurities are removed by centrifugation, washing, or filtration to obtain the cell single-molecule mechanosensor.

[0123] In one embodiment, the microspheres may be washed 1-6 times, preferably 3-5 times, to remove the free probe while avoiding excessive loss of microspheres. Washing buffers may include PBS, Hank's buffer, HEPES buffer, or cell culture-related buffers; if necessary, a low concentration of salt may be added to the washing solution to maintain the stability of the double-stranded DNA. Preferably, in a preferred embodiment, washing is performed by centrifugation to allow the microspheres to settle and discarding the supernatant, repeated 3 times or more, until the background of free fluorescence / free nucleic acid in the supernatant is significantly reduced.

[0124] The purification steps described above can significantly reduce background fluorescence and non-specific adsorption caused by uncoupled probes, thereby reducing background noise and improving the signal-to-noise ratio (SNR) of the force-triggered signal. Furthermore, removing the free probe helps avoid its non-specific binding on the cell surface or in the culture system, thus reducing false positive signals caused by non-mechanical factors.

[0125] After purification, the microsphere sensor coupled with the double-stranded DNA probe is resuspended in the final use or storage buffer to obtain the finished cell single-molecule mechanical force sensor.

[0126] In one embodiment, the final resuspension system may be PBS, Hank's buffer, HEPES buffer, serum-free culture medium, or a combination thereof; appropriate ionic components may also be added as needed for imaging. The resulting sensor can be used directly for cell incubation and imaging; or it can be stored for a short period at low temperature and its dispersibility restored by gentle mixing before use.

[0127] In the above embodiments, using Hank's buffer or a cell culture-compatible buffer system as the final resuspension system can improve the dispersibility and biocompatibility of the sensor in the cellular environment, reduce the interference of microsphere aggregation on phagocytic behavior and imaging resolution, thereby improving experimental repeatability and the accuracy of spatial force distribution resolution.

[0128] This application further provides a method for detecting single-molecule mechanical forces during cell phagocytosis. This method converts the fluorescence response indicating whether double-stranded DNA unwinding occurs into a quantifiable single-molecule mechanical force index during cell phagocytosis or encapsulation of a microsphere sensor. This allows for the characterization of the intensity and spatial distribution of the mechanical force applied to the cell. By setting probe libraries with different unwinding force thresholds and employing a unified positive criterion and fitting strategy, this single-molecule mechanical force detection method achieves cross-threshold and cross-batch force comparability and reduces false positives caused by background drift, non-specific adsorption, or mismatch.

[0129] Please see Figure 6The figure shows a flowchart of the steps of the single-molecule mechanical force detection method of this application in one embodiment. As shown in the figure, the method may include at least the S21 co-incubation step, the S22 fixation and imaging step, the S23 signal extraction step, and the S24 force value calculation step. The order of each step and the data flow are used to convert the mechanical action in the cell phagocytosis process into quantitative indicators such as the half-maximum effective mechanical force threshold F50 (also known as the equivalent half-maximum force threshold).

[0130] In co-incubation step S21, a group of single-molecule mechanosensitive sensors with different unwinding force thresholds are co-incubated with the target cells in a culture medium, allowing the cells to bind to the sensors via surface receptors and initiate phagocytosis. The group of sensors with different unwinding force thresholds can be understood as: double-stranded DNA probes with different mechanical thresholds immobilized on the surface of a microsphere substrate to form a probe library for force threshold statistics; each threshold group can be set as an independent sample for parallel co-incubation, or they can be mixed and co-incubated within the same sample under the premise of distinguishable identification (e.g., different channels / different codes). For example, they can be distinguished by different fluorescent codes, different particle size codes, or different identifiable markers.

[0131] In one embodiment, the test cells may be phagocytosis-related cells or cells exhibiting encapsulation / endocytosis behavior; preferably, the test cells are vascular endothelial cells. The co-incubation can be performed in a cell culture incubator to maintain the cells in a physiologically phagocytic / encapsulated state. During co-incubation, the cells specifically bind to the targeting ligands on the surface of the microsphere sensor via their surface receptors, thereby establishing a "receptor-ligand-double-stranded DNA probe" mechanotransduction link and applying mechanical action to the double-stranded DNA probe during the phagocytosis / encapsulation process. In one embodiment, the temperature of the incubator environment can be 35-39°C, preferably about 37°C; the CO2 volume fraction in the incubator gas environment can be 3%-8%, preferably about 5%, to maintain pH stability in conjunction with the culture medium containing a bicarbonate buffer system. The culture medium can be serum-containing medium, low-serum medium, or serum-free medium, specifically selected according to cell state, receptor-ligand binding requirements, and background fluorescence control.

[0132] To ensure the statistical stability of the threshold fitting and reduce the phagocytic burden and occlusion interference caused by excessive particles, the amount of the sensor added can be set according to the particle number / cell number or particle concentration. The feasible range for the particle number / cell number ratio is, for example, 0.5:1-50:1, preferably 2:1-20:1, and more preferably 5:1-10:1; equivalently, the feasible range for the particle concentration is, for example, 10... 4 -10 7 Cells / mL, preferably 10 5 -10 6The co-incubation time can range from, for example, 5 minutes to 4 hours; preferably, for mechanical characterization of the phagocytic initiation and early encapsulation phases, sampling can be performed from 10 minutes to 60 minutes; for the more stable encapsulation / endocytosis phase, sampling can be performed from 1 hour to 4 hours. In some embodiments, to improve the uniformity of sensor-cell contact and reduce random bias caused by particle sedimentation, gentle mixing, intermittent gentle shaking, or pre-dispersing the sensor in the culture medium can be used during co-incubation.

[0133] In this embodiment, by introducing sensors with different unwinding force thresholds under the same cell type and culture conditions, and allowing them to bind to cell receptors and enter the phagocytic process, the difference in the "threshold-positive ratio" curve can be attributed primarily to the distribution of mechanical force applied by the cells, rather than to differences in particle contact opportunities or reaction systems. This improves the comparability and fitting stability between different threshold groups. Simultaneously, by controlling the amount of particles added and the co-incubation time window, the cell stress, non-specific adsorption, and increased background signal caused by excessive particle addition can be reduced while ensuring the statistical sample size. This provides low-background, reproducible starting conditions for subsequent fixation imaging, positive determination, and calculation of the half-maximum effective mechanical force threshold (F50).

[0134] In some embodiments, when the method is used to screen anti-atherosclerotic drugs, perturbation flow or oscillatory shear force conditions can be applied to the test cells before or during the co-incubation step S21, and the candidate drug can be brought into contact with the test cells to activate the corresponding signaling pathways in a simulated pathological mechanical environment. Subsequently, following the co-incubation, imaging, extraction and fitting process described in this application, the half-maximum effective mechanical force threshold or the proportion of positive signals at different thresholds of the drug-treated group and the untreated control group can be obtained to quantify and compare the mechanical phenotypic regulatory effects of the candidate drugs.

[0135] In step S22, the sample co-incubated with cells is fixed at a predetermined time point, and fluorescence signal images of the cell single-molecule mechanical force sensor are acquired using a fluorescence microscope or confocal microscope. This freezes the receptor-mediated mechanical state within the target time window of the phagocytosis / encapsulation process, and the fluorescence distribution on the microsphere surface is obtained under repeatable optical acquisition conditions, thereby providing a consistent data basis for subsequent positive determination and force value fitting. The predetermined time point can be set according to the progress of the phagocytosis / encapsulation process; in one embodiment, when the cells to be tested are vascular endothelial cells, the predetermined time point can be within the range of 10 minutes to 4 hours after the start of co-incubation.

[0136] In one embodiment, the fixation can be performed chemically, and the fixative can be selected from paraformaldehyde (PFA), formaldehyde, glutaraldehyde, methanol, ethanol, or combinations thereof; preferably, paraformaldehyde fixation is used to balance cell morphology preservation and fluorescence signal stability. The concentration of the fixative can be 1%-8% (w / v), preferably 2%-4%; the fixation time can be 5-30 minutes, preferably 10-20 minutes; the fixation temperature can be 4℃ to room temperature (20-25℃), preferably room temperature fixation to obtain better morphological fidelity and reproducibility. After fixation, the cells can be washed 1-5 times with buffer (e.g., PBS or Hank's buffer), preferably 2-3 times, to remove residual fixative and reduce background fluorescence.

[0137] In another embodiment, if higher structural stability is required (e.g., for subsequent immunofluorescence co-localization or longer imaging time), a mixed immobilization system containing a small amount of glutaraldehyde can be used without significantly affecting the stability of the fluorescent group; or cold methanol / ethanol immobilization can be used to obtain higher membrane permeability. It should be understood that different immobilization systems can be selected based on the target readout (fluorescence readout only / fluorescence and protein co-localization / scaffold staining, etc.).

[0138] In the embodiments, to ensure comparability between different unwinding force threshold sensors, it is preferable to use the same type, concentration, fixation time and number of washes for each threshold group in the same batch of experiments; and to keep the culture medium / buffer system as consistent as possible before and after fixation to reduce the impact of pH and ionic strength changes on fluorescence background.

[0139] When performing fluorescence imaging after fixation, the acquisition objects should include at least the fluorescence signal of the single-molecule mechanosensitive cell that has been phagocytosed or bound by the cell; and the cell structure reference signal can be acquired simultaneously as needed so that the fluorescence enhancement area can be correlated with the cell-microsphere contact relationship in subsequent analysis.

[0140] In one embodiment, the imaging can be performed using a wide-field fluorescence microscope or a confocal microscope; when it is necessary to analyze the spatial distribution of fluorescence signals on the microsphere surface and determine the cell encapsulation state and the direction of force, a confocal microscope is preferred to acquire the three-dimensional surface signal of the microsphere. The objective lens magnification can be 20×-100×, preferably 40×-60×; the numerical aperture (NA) can be 0.8-1.49, preferably a higher NA to improve the resolution and signal-to-noise ratio of the microsphere surface signal. The imaging channels can include at least channels corresponding to the first fluorescent label and the second fluorescent label; if necessary, cytoskeleton or cell membrane labeling channels can be added for structural reference.

[0141] In one embodiment, a Z-Stack method is used to acquire three-dimensional fluorescence signal images of the single-molecule mechanosensor. The Z-Stack step size can be 0.1-1.0 μm, preferably 0.2-0.5 μm; the Z-axis scanning range preferably covers the upper to lower hemispheres of the microsphere (or at least the area where the microsphere contacts the cell and its surroundings) to ensure that the fluorescence distribution on the microsphere surface can be reconstructed. The acquisition exposure time / laser power can be set to the lowest level within the detectable range according to the signal intensity to reduce photobleaching and maintain acquisition consistency between different threshold groups; preferably, the exposure time (or laser power and gain parameters) is fixed within the same experimental batch, and the same imaging setting template is used for different threshold groups to reduce intensity shifts caused by differences in acquisition parameters.

[0142] In a preferred embodiment, the distribution of fluorescence signals on the microsphere surface in three-dimensional space is acquired using confocal Z-Stack imaging. The fluorescence-enhanced regions are then compared with cellular structural references (e.g., cell membrane or actin ring markers) to determine the encapsulation state of the microspheres and the main direction of force application. For example, when fluorescence enhancement is mainly concentrated at the microsphere-cell interface and distributed in a ring / arc shape, it indicates that the cytoskeleton is forming an encapsulation or contraction ring structure on the microspheres and generating directional traction. When fluorescence enhancement is asymmetrically polarized along the microsphere surface, it indicates that the main direction of force application has a spatial bias. Therefore, fixation and three-dimensional imaging not only provide a basis for subsequent positive counts but also provide a feasible data source for encapsulation determination and force direction analysis based on spatial distribution.

[0143] Furthermore, to support the observation of the spatial distribution of fluorescence signals on the surface of microspheres, in a preferred embodiment, confocal imaging can be used to acquire the distribution of fluorescence signals at different orientations on the surface of microspheres, thereby providing an image basis for subsequent determination of the encapsulation state of the cytoskeleton (e.g., actin rings) on the microspheres and the direction of force.

[0144] In step S23, signal extraction is performed on the fluorescence image obtained in step S22 to identify single-molecule mechanosensitive cells that have been phagocytosed or bound by cells. The fluorescence intensity change of each single-molecule mechanosensitive cell is measured, and single-molecule mechanosensitive cells whose fluorescence intensity enhancement factor exceeds a preset background threshold are identified as positive signals. The preset background threshold can be, for example, set to a fluorescence intensity change greater than 5 times the background. The fluorescence intensity change can be characterized as an enhancement factor relative to the background or an enhancement amount after background correction.

[0145] In one embodiment, the single-molecule mechanosensitive cell that has been phagocytosed or bound by the cell can be identified by a morphological and signal co-occurrence method: the outline of the microsphere or its surface fluorescent ring is located in the fluorescence channel, and the state of the microsphere in contact with, attachment to or endocytosis of the cell is confirmed by combining bright field / phase contrast or other structural reference channels (if collected).

[0146] For each microsphere sensor, a region of interest (ROI) for measuring fluorescence intensity is defined. The ROI can be: a ring-shaped region on the microsphere surface, a partitioned region on the microsphere surface, or a projected region containing the main fluorescence signal from the microsphere surface; or, under confocal imaging conditions, a surface signal region obtained based on an optical slice set of the microsphere surface. Preferably, consistent ROI definition rules (e.g., the same ring width rule / the same partitioning method / the same threshold segmentation strategy) are used for the same batch of experiments, the same threshold group, and different threshold groups to reduce systematic errors introduced by differences in ROI selection. This consistency control helps reduce human selection bias and improves the comparability and statistical stability of positive rates between different threshold groups.

[0147] In one embodiment, for each microsphere sensor, the target fluorescence intensity value within the microsphere ROI and the corresponding background fluorescence intensity value are obtained. In specific implementations, the selection of the background fluorescence intensity value can be achieved using a local background method, selecting a background ROI within the same field of view that is close to the microsphere but does not contain the microsphere signal, avoiding areas with strong cell autofluorescence or significantly non-uniform illumination; or using a global background method, obtaining the background mean / median from multiple regions without microsphere signals under the same threshold group and imaging conditions as a unified background; or using a negative reference method, using the fluorescence level corresponding to a microsphere region that has not contacted cells, or a negative system (if any) that does not undergo unwinding, as a background reference to improve the robustness of the background definition.

[0148] In one embodiment, the fluorescence intensity statistics may use the mean, integral intensity, or median intensity as the target fluorescence intensity value; preferably, a statistic that is insensitive to outliers (e.g., median intensity) is used to reduce the impact of occasional noise on the determination. More preferably, the image is preprocessed before calculation, including but not limited to: removing significant hot pixels / isolated noise, background subtraction, and (if applicable) correction for illumination non-uniformity between different fields of view; however, it should be ensured that the preprocessing rules remain consistent across different threshold groups.

[0149] The above-mentioned method of extracting intensity using local background and robust statistics can reduce the interference of cell autofluorescence, uneven illumination, and occasional noise on the determination, thereby reducing false positives and improving the reliability of positive counts.

[0150] In one embodiment, the fluorescence intensity change of each microsphere sensor is represented by an enhancement factor. The enhancement factor is calculated by comparing the fluorescence intensity of the microsphere ROI with the background fluorescence intensity. A positive result is obtained when the enhancement factor is greater than 5. In another optional embodiment, without changing the essential meaning of the 5-fold background threshold, the intensity change can be expressed as a difference or an enhancement amount after background correction, and converted into an equivalent judgment condition consistent with the 5-fold background threshold.

[0151] Preferably, to avoid false positives caused by edge artifacts or non-specific adsorption, a consistency constraint can be introduced when determining a positive result. For example, the enhanced signal may be required to be continuously / patterned on the surface of the microsphere rather than isolated single points, or the two channels (in the case of a FRET / dual-dye system) may be required to meet the expected synchronous change trend. This constraint helps to further reduce false positives caused by random noise, free fluorescent probes, or non-specific adsorption, thereby improving the stability of the signal-to-noise ratio and threshold fitting.

[0152] In step S24, based on the positive determination results obtained in step S23, the proportion of positive signals (Positive Counts %) under different unwinding force thresholds is statistically analyzed, the relationship curve between the proportion of positive signals and the unwinding force threshold is fitted, and the half-maximum effective mechanical force threshold F50 is calculated. The half-maximum effective mechanical force threshold F50 is defined as the mechanical force required to unwind 50% of the double-stranded DNA probes. This quantifies the single-molecule mechanical force applied by cells during phagocytosis and can be used to compare the mechanical phenotypes of the drug-treated group and the untreated control group in drug screening.

[0153] Specifically, based on the positive determination results obtained in step S23, statistical analysis is performed on multiple groups of single-molecule cellular force sensors with different unwinding force thresholds: For each unwinding force threshold group, the total number of microspheres included in the analysis is counted, and the number of microspheres determined to be positive signals is also counted; the proportion of positive microspheres to the total number of microspheres included in the analysis for that threshold group is defined as the positive signal proportion for that threshold group. The microspheres included in the analysis can be limited to those that have been phagocytosed or bound by cells and whose image quality meets preset consistency conditions (e.g., excluding microspheres with obvious aggregation, severe defocus, signal saturation, or inability to reliably determine the background region), to ensure consistency in statistical standards between different threshold groups. By unifying statistical standards and quality control, systematic errors introduced by sampling bias or imaging anomalies can be reduced, making the positive proportions of different threshold groups comparable, thereby improving the repeatability of the mechanical quantification results.

[0154] In this embodiment, the proportion of positive signals corresponding to each threshold group is arranged in ascending order of the unwinding force threshold, thus obtaining the relationship between the proportion of positive signals and the unwinding force threshold. As the unwinding force threshold increases, the difficulty of triggering unwinding increases, and the proportion of positive signals exhibits a transitional response that changes with the threshold. The direction of its change depends on the definition of the fluorescence event corresponding to the positive criterion (e.g., enhancement / dequenching triggered by unwinding). However, under the same criterion and the same system, this response has a fittable transitional characteristic within the threshold range. To transform the statistical results of discrete threshold points into directly comparable mechanical indicators, this application performs curve fitting on the above-mentioned "proportion of positive signals - unwinding force threshold" relationship. The fitting process can employ an S-shaped response model, a piecewise monotonic model, or other fitting methods suitable for reflecting the transitional change in the proportion of positive signals caused by the increase of the threshold, which can characterize the threshold response characteristics. Preferably, an S-shaped response fitting method with upper and lower plateaus and a transition region is used to stably determine the position of the half-point. During fitting, methods such as least squares, weighted fitting, or maximum likelihood can be used. When the statistical microsphere counts differ significantly among different threshold groups, a weighted strategy related to the sample size is preferred, giving threshold points with larger sample sizes a more reasonable weight in the fitting process, thereby improving the robustness of the fit. Fitting can smooth the random fluctuations of discrete points into a continuous response curve, reducing the impact of occasional errors on threshold determination, thus making the subsequent solution of the half-effective mechanical force threshold F50 more stable and consistent across batches.

[0155] In the above embodiments, after obtaining the fitted curve, the half-maximal effective mechanical force threshold F50 is calculated. The half-maximal effective mechanical force threshold F50 is defined as the mechanical force required to unwind 50% of the double-stranded DNA probes. Within the statistical framework of this application, the half-maximal effective mechanical force threshold F50 can be equivalently expressed as the unwinding force threshold corresponding to a 50% positive signal ratio. Therefore, this application determines the half-maximal effective mechanical force threshold F50 in one of the following ways: first, if the fitted model parameters directly include a half-maximal point threshold parameter, then the force threshold corresponding to that half-maximal point is directly read as F50; second, if the fitted model does not explicitly include a half-maximal point parameter, then the unwinding force threshold corresponding to a 50% positive signal ratio is determined on the fitted curve, and this threshold is used as the half-maximal effective mechanical force threshold F50. Preferably, fitting quality evaluation information (optional, such as residual trend or goodness-of-fit index) and the confidence interval of the half-maximal effective mechanical force threshold F50 or the dispersion of repeated experiments (optional) are simultaneously output to characterize the reliability of the mechanical quantification result. By using the half effective mechanical force threshold F50 as a unified quantitative indicator, the positive proportion distribution under multi-threshold probes can be compressed into a single, comparable mechanical phenotypic parameter, which facilitates cross-sectional comparison and statistical testing between different cell types, different time points, or different treatment conditions.

[0156] When the single-molecule mechanical force detection method is used to screen candidate drugs for anti-atherosclerosis, steps S21-S24 are performed on the treated group and the untreated control group respectively to obtain the half-maximum effective mechanical force threshold F50 for each group, and the results are compared: if the half-maximum effective mechanical force threshold F50 of the treated group is lower than that of the control group, it indicates that the level of effective single-molecule mechanical force exerted by cells during phagocytosis is reduced under the drug administration conditions; and / or, if the proportion of positive signals at multiple unwinding force threshold points in the treated group is lower or shifted downward overall compared to the control group, it indicates that the proportion of force-triggered unwinding events is reduced. Candidate drugs that meet the criteria of "reducing the half-maximum effective mechanical force threshold and / or reducing the proportion of positive signals" can be identified as candidate drugs for anti-atherosclerosis. This comparison strategy can transform the influence of drugs on force transmission-related processes into quantifiable and repeatable evaluation indicators, thereby improving the objectivity and feasibility of the screening process.

[0157] In one embodiment, based on the image data obtained in step S22, the spatial distribution of fluorescence signals on the surface of the microspheres is analyzed to determine the encapsulation state of the microspheres by the cells during phagocytosis and the main direction of force. Specifically, for microspheres that have been phagocytosed or bound by cells, the outline and center position of the microspheres are first determined in the image, and the fluorescence signals on the surface of the microspheres are spatially mapped: for example, polar / azimuth coordinates can be established along the surface of the microspheres, or the fluorescence enhancement region can be segmented and labeled on an equivalent spherical projection map, thereby obtaining parameters such as the area ratio, spatial position, continuity (whether a ring band is formed), and intensity distribution of the fluorescence enhancement region.

[0158] In one embodiment, if the fluorescence enhancement is mainly distributed in a specific area on the surface of the microsphere and exhibits a clear directional bias (e.g., mainly concentrated on the contact interface area facing the cell, while the enhancement is weaker on the side away from the cell), it can be determined that the cellular force is mainly concentrated in the radial direction pointing towards the microsphere or along the specific contact interface direction. Correspondingly, if the fluorescence enhancement area presents an approximately ring-shaped or arc-shaped band distribution along the surface of the microsphere and spatially corresponds to cytoskeleton markers (e.g., immunofluorescence or fluorescent protein markers of actin rings), it can be determined that the cytoskeleton (e.g., actin rings) forms a wrapping / contraction structure around the microsphere and applies a mechanical force with a significant circumferential or tangential component to the microsphere, thereby characterizing the formation and contraction propulsion process of the phagocytic cup. Furthermore, the evolution trend of the phagocytic process stage and the direction of force application can be inferred based on the position and morphological changes of the ring-shaped enhancement area on the surface of the microsphere (e.g., from local arc segments gradually closing into complete rings, or the position of the ring migrating along the surface of the microsphere).

[0159] In a preferred embodiment, to improve the comparability of spatial distribution determinations between different experiments and different threshold groups, consistency control rules can be set for spatial distribution analysis. These rules include, for example, unifying the imaging mode (wide field or confocal), unifying the Z-Stack stepping and reconstruction method (e.g., using the same stepping spacing and the same projection method), unifying the microsphere surface projection or segmentation threshold strategy, and unifying the definition of enhanced regions (e.g., still using the background threshold rule of step S23 to determine pixels / regions). Furthermore, negative control or non-targeted ligand control microspheres are introduced in the same batch of analyses to correct non-specific distribution patterns. Through spatial mapping and consistency control of the microsphere surface signal, intensity changes can be further enhanced into spatial mechanical spectrum information. This enhances the ability to resolve the position and direction of force action during phagocytosis without changing the core detection process, and reduces distribution misjudgments caused by differences in imaging parameters.

[0160] In the above embodiments, steps S21-S24 achieve quantitative characterization of single-molecule mechanical forces during phagocytosis. To further expand the application value of the detection method in disease intervention and candidate drug screening, this application also provides a single-molecule mechanical force detection method for screening anti-atherosclerotic drugs. This method can quantitatively evaluate the inhibitory effect of candidate drugs on mechanical force transmission under the background of mechanical stress induced by turbulent flow or oscillatory shear force in vascular endothelial cells, and can further confirm its influence on molecular events related to mechanical force pathways through mechanism verification methods.

[0161] In one specific embodiment, the screening method includes the following steps S25 to S27. Figure 7 The diagram shows a flowchart of the single-molecule mechanical force detection method in another embodiment of this application. The single-molecule mechanical force detection method further includes a screening method step:

[0162] Mechanical stress modeling and drug administration step S25: The test cells are cultured under turbulent flow or oscillating shear stress conditions, and the candidate drug is brought into contact with the test cells. Preferably, the test cells are vascular endothelial cells (e.g., human umbilical vein endothelial cells HUVEC or other endothelial cell lines). The turbulent flow or oscillating shear stress can be achieved through a fluid shearing device, a swing / shaking shear culture device, a microfluidic chip, or an equivalent system. The candidate drug can be added to the culture system before, during, or after modeling, and multiple drug concentrations and treatment durations can be set to form a dose-response relationship. Specifically, the parameters of the oscillating shear stress are set to ±4 dyn / cm², with a frequency of 1 Hz; or set to a low oscillating shear stress in the range of 0.5-5 yn / cm² to simulate the hemodynamic characteristics of areas prone to atherosclerosis.

[0163] Step S26 for single-molecule mechanical force detection and index output: In the single-molecule mechanical force detection method described in the above embodiments, co-incubation, fixation imaging, signal extraction and force value calculation steps are performed on the drug-treated group and the untreated control group respectively to obtain the half effective mechanical force threshold F50 of the drug-treated group and the control group, and / or to obtain the positive signal ratio corresponding to each unwinding force threshold.

[0164] Screening and determination step S27: Candidate drugs that can reduce the half of the effective mechanical force threshold and / or reduce the proportion of positive signals are identified as anti-atherosclerotic candidate drugs.

[0165] In a preferred embodiment, the screening determination can be carried out by comparing and analyzing the same experimental batch, the same threshold probe group, the same background threshold rule, and the same fitting / statistical caliber, so as to reduce the impact of batch differences on the screening conclusion.

[0166] Through the above screening process, this application can use receptor-mediated single-molecule force-triggered response as a functional readout to directly reflect the effect of candidate drugs on the mechanical force application ability and mechanical force transmission efficiency during endothelial cell phagocytosis / encapsulation, thereby achieving quantitative screening of candidate drugs to inhibit abnormal force transmission; and by simultaneously outputting two types of indicators, namely the half-maximum effective mechanical force threshold F50 and the proportion of positive signals, it can take into account the characterization of changes in the overall mechanical level and changes in the response spectrum of different threshold intervals, thereby improving the robustness and interpretability of the screening results.

[0167] In one embodiment, to verify the mechanism by which the candidate drug leads to a decrease in the half-maximal effective mechanical force threshold (F50) and / or a decrease in the proportion of positive signals, and to exclude artifacts caused solely by changes in cell number, viability, or nonspecific adhesion, the screening method further includes a mechanism verification step S28. In step S28, molecular events related to mechanotransmission are verified. Specifically, immunofluorescence, Western blot, or co-localization analysis is performed on cells in the drug-treated group and the untreated control group to assess changes in the expression or recruitment of mechanotransmission-related proteins. In one embodiment, the mechanotransmission-related protein is selected from at least one of integrin β1, Vinculin, spleen tyrosine kinase (Syk), focal adhesion kinase (FAK), actin-associated protein 2 (Arp2), or mDia1.

[0168] The mechanism validation can target at least one of the following categories of indicators: cytoskeleton remodeling-related indicators, such as actin (F-actin) structure, the formation and integrity of ring-like encapsulation structures, and the recruitment of cytoskeleton proteins related to phagocytosis / encapsulation; receptor and adhesion complex-related indicators, such as receptor enrichment at the contact interface and the recruitment and aggregation status of adhesion-related molecules in the interfacial region; signaling pathway activation indicators, such as changes in phosphorylation levels of signaling molecules related to mechanotransduction or changes in the expression of downstream marker proteins; and spatial colocalization indicators, which determine whether the candidate drug alters the spatial consistency between the force-acting region and the cytoskeleton / receptor recruitment region through spatial correlation analysis with the fluorescence-enhanced region on the microsphere surface.

[0169] By incorporating the aforementioned mechanism verification steps, this application can establish a correlation between changes in mechanical readout (F50 / Positive Counts%) and changes in molecular events (expression / recruitment / colocalization), thereby improving the credibility of the screening conclusions and supporting the explanation of the mechanism of action. For example, when a candidate drug simultaneously causes a decrease in the half-maximal effective mechanical force threshold (F50) accompanied by weakened recruitment of mechanotransmission-related proteins at the contact interface or inhibition of the cytoskeleton ring structure, it can support its potential anti-atherosclerotic effect by inhibiting mechanotransmission pathways; conversely, if only changes in mechanical readout occur but no corresponding differences in molecular events are observed, it may indicate the need to further investigate factors such as cell state, non-specific adsorption, or imaging threshold settings.

[0170] In an optional embodiment, to reduce the impact of factors such as non-specific adsorption, residual free probes, imaging drift, or photobleaching on positive determination and force statistics, this application sets up a control system and implements quality control during steps S21 to S24: for example, at least one set of negative control sensors can be set up, including (i) sensor microspheres not connected to the target ligand, and / or (ii) microspheres not carrying double-stranded DNA probes or only carrying single-stranded DNA, and / or (iii) microspheres that have been blocked to reduce non-specific adsorption, and the negative controls and the test cells are treated under the same culture and imaging conditions as the experimental group to obtain a reference distribution of background fluorescence and non-specific signals; in the image During acquisition and signal extraction, it is preferable to use consistent microscope settings (including excitation intensity, exposure time / scanning power, gain, Z-Stack layer spacing and number of layers) for different unbearing force threshold groups. Furthermore, it is preferable to limit the acquisition of samples from the same batch to the same time window to reduce systematic errors caused by instrument drift and photobleaching. During signal extraction, it is preferable to only count microspheres that meet preset quality criteria, such as excluding microspheres that show significant aggregation, exceed the linear dynamic range with saturated pixels, or are in defocused or edge-occluded areas. It is also preferable to select microspheres or cell-free areas that are not bound / phagocytosed by cells within the same field of view or imaging batch as background references to unify background selection rules. Through the above control and quality control measures, false positives and batch differences can be further reduced, and the comparability of the statistical results of positive signal proportions between different unbearing force threshold groups can be improved, thereby enhancing the stability and repeatability of the inference of the half-maximum effective mechanical force threshold (F50).

[0171] In summary, the single-molecule mechanosensitive sensor disclosed in this application, its preparation method, and its applications, introduce active functional groups (such as maleimide) that can selectively covalently react with DNA end groups on the surface of a microsphere substrate. Double-stranded DNA threshold probes are then covalently immobilized at a single point, in a directional and stable manner through a process involving annealing assembly, thiol activation, and thiol-maleimide directional coupling. This results in a probe array with consistent orientation, conformation, and dispersion on the three-dimensional surface of the microsphere. Combined with a unified positive criterion (such as intensity enhancement determination based on background threshold) and a cross-threshold statistical / fitting strategy, different unwinding force thresholds can be identified. Positive responses at certain values ​​are converted into comparable quantitative indicators such as the half-maximal effective mechanical force threshold (F50), enabling the quantification of the intensity of mechanical forces applied during cell phagocytosis and batch / threshold comparability. Simultaneously, purification after coupling removes free probes and non-specific adsorbed components, significantly reducing background fluorescence and false positives caused by mismatch / non-specificity, thus improving the signal-to-noise ratio and measurement robustness. Furthermore, combined with spatial distribution analysis of fluorescence enhancement regions on the microsphere surface, it can also help determine the cell encapsulation state and force direction, thereby enhancing the spatial resolution capability of the phagocytic mechanics process and can be extended to mechanical phenotypic screening and mechanism verification under drug intervention conditions.

[0172] The above embodiments are merely illustrative of the inventive essence and beneficial effects of this application, and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the principles and scope of this application. Therefore, all equivalent modifications or alterations achieved by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A method for detecting single-molecule mechanical forces during phagocytosis in the cell process for screening anti-atherosclerotic drugs, characterized in that, A single-molecule mechanosensor is used, comprising: a microsphere substrate and a plurality of double-stranded DNA probes modified on the surface of the microsphere substrate; each double-stranded DNA probe comprises a first single-stranded DNA and a second single-stranded DNA partially or entirely complementary to the first single-stranded DNA; wherein one end of the first single-stranded DNA is covalently coupled to the microsphere substrate, and the other end is modified with a first fluorescent label; the second single-stranded DNA is modified with a targeting ligand for specific binding to mechanosensors on the cell surface and is also modified with a second fluorescent label; the surface of the microsphere substrate is modified with active functional groups selected from amino, carboxyl, hydroxyl, thiol, maleimide, aldehyde, or epoxy groups; wherein the first fluorescent label and the second fluorescent label constitute a fluorescence resonance energy transfer or fluorescence quenching pair, and the double-stranded DNA... When probe A is in a double-stranded closed state, the distance between the first fluorescent label and the second fluorescent label is within the effective range of fluorescence resonance energy transfer or fluorescence quenching, thus forming a corresponding fluorescence signal state. When the targeting ligand binds to the mechanosensitive receptor on the cell surface and is subjected to mechanical tension applied by the cell, and the mechanical tension is greater than the unwinding force threshold of the double-stranded DNA probe, the double-stranded DNA probe undergoes unwinding, dissociation, or conformational change, causing a change in the spatial distance between the first fluorescent label and the second fluorescent label, resulting in a change or decrease in the fluorescence resonance energy transfer signal, thereby generating a detectable signal change. The signal change characterizes the single-molecule-level mechanical force applied by the cell during phagocytosis or encapsulation of the microsphere substrate, and the signal change includes an increase or decrease in fluorescence intensity. The single-molecule mechanical force detection method includes the following steps: Co-incubation step: A set of the cell monomolecular mechanosensors having at least three different unwinding force thresholds are co-incubated with vascular endothelial cells in the drug-treated group and the untreated control group in a culture medium, allowing the vascular endothelial cells to bind to the cell monomolecular mechanosensors through surface receptors and initiate a phagocytic process; wherein the at least three different unwinding force thresholds are selected from 12pN, 23pN, 33pN, 43pN, and 56pN; during the co-incubation step, the vascular endothelial cells are cultured under turbulent flow or oscillating shear stress conditions, and the candidate drug is contacted with the vascular endothelial cells to form the drug-treated group, and an untreated control group is set up; Fixation and imaging steps: Vascular endothelial cells in the drug-treated group and the untreated control group are fixed at predetermined time points, and fluorescence signal images of the single-molecule mechanical force sensors of the cells are acquired using a fluorescence microscope or a confocal microscope; Signal extraction steps: Identify single-molecule mechanosensitive cells that have been phagocytosed or bound by the vascular endothelial cells, measure the fluorescence intensity change of each single-molecule mechanosensitive cell, and determine single-molecule mechanosensitive cells whose fluorescence intensity enhancement factor exceeds a preset background threshold as positive signals; wherein, the preset background threshold is set to a fluorescence intensity change greater than 5 times the background; the fluorescence intensity change can be characterized as the enhancement factor relative to the background or the enhancement amount after background correction; Force value calculation steps: The proportion of positive signals in the drug-treated group and the untreated control group under at least three different unwinding force thresholds are statistically analyzed. The relationship curve between the proportion of positive signals and the unwinding force threshold is fitted. The half-maximum effective mechanical force threshold F50 of the drug-treated group and the untreated control group is calculated respectively. The half-maximum effective mechanical force threshold F50 is defined as the mechanical force required to unwind 50% of the double-stranded DNA probe, or as the unwinding force threshold corresponding to the proportion of positive signals reaching 50% on the relationship curve. This is used to quantify the single-molecule mechanical force applied by the vascular endothelial cells during phagocytosis. Screening and determination steps: The half-maximal effective mechanical force threshold F50 of the administered group is compared with that of the unadministered control group, and / or the proportion of positive signals in the administered group at the at least three different disintegration force thresholds is compared with that of the unadministered control group at the corresponding disintegration force thresholds; when the candidate drug can reduce the half-maximal effective mechanical force threshold F50 of the administered group relative to the unadministered control group, and / or reduce the proportion of positive signals in the administered group at the at least three different disintegration force thresholds relative to the unadministered control group, the candidate drug is identified as an anti-atherosclerotic candidate drug.

2. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The predetermined time point is from 10 minutes to 4 hours; the parameter of the oscillating shear force is set to ±4 dyn / cm², and the frequency is 1 Hz; or, the oscillating shear force is a low oscillating shear force in the range of 0.5-5 dyn / cm².

3. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, By observing the spatial distribution of fluorescence signals on the surface of microspheres, the encapsulation state of the cytoskeleton on the microspheres and the direction of the force can be determined.

4. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The method for screening anti-atherosclerotic drugs also includes further steps of evaluating changes in the expression or recruitment of mechanotransmission-related proteins by immunofluorescence, Western blot, or colocalization analysis.

5. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The microsphere substrate is made of materials selected from silica, polystyrene, polymethyl methacrylate (PMMA), magnetic nanomaterials, gold nanoparticles, or combinations thereof.

6. The method for detecting single-molecule mechanical force according to claim 5, characterized in that, The microsphere substrate is silica microspheres.

7. The method for detecting single-molecule mechanical force according to claim 6, characterized in that, The surface of the microsphere substrate is modified with amino groups by 3-aminopropyltriethoxysilane (APTES), and maleimide groups are further introduced by the heterobifunctional crosslinking agent SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester).

8. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The diameter of the microsphere substrate ranges from 1 μm to 15 μm.

9. The method for detecting single-molecule mechanical force according to claim 8, characterized in that, The diameter of the microsphere substrate is 2 μm, 5 μm or 10 μm.

10. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, Both the first single-stranded DNA and the second single-stranded DNA are artificially synthesized oligonucleotide sequences; the length of the first single-stranded DNA is 10-100 bases, and the length of the second single-stranded DNA is 10-100 bases.

11. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The double-stranded DNA probe constructs different mechanical response geometric conformations by adjusting the relative distance and orientation of the anchoring point of the first single-stranded DNA on the microsphere substrate and the connection site of the second single-stranded DNA with the target ligand on the double-stranded helix structure. These mechanical response geometric conformations include a decompression mode, a shearing mode, and an intermediate loading mode. In the decompression mode, the anchoring point of the first single-stranded DNA on the microsphere substrate and the connection site of the second single-stranded DNA with the target ligand are located on the same side of the double-stranded DNA probe's double helix structure, with the applied tension perpendicular to the base pair hydrogen. The direction of the bond acts on the double-stranded DNA probe; the shearing mode is that the anchoring point of the first single-stranded DNA and the linking site of the second single-stranded DNA are located at opposite ends of the double helix structure of the double-stranded DNA probe, so that the applied tensile force acts on the double-stranded DNA probe in a direction parallel to the base pair hydrogen bond; the intermediate loading mode is that by adjusting the relative distance and direction of the anchoring point of the first single-stranded DNA and the linking site of the second single-stranded DNA on the double helix structure, the applied tensile force relative to the axis of the double-stranded DNA is in a loading direction between the decompression mode and the shearing mode, thereby forming a transitional conformation with different unwinding force thresholds.

12. The method for detecting single-molecule mechanical force according to claim 11, characterized in that, In the decompression mode, the direction of the mechanical pulling force is mainly perpendicular to the base pair hydrogen bond direction of the double-stranded DNA probe, the unwinding force threshold is 10pN to 15pN, and the decompression mode corresponds to the end-peeling loading geometry.

13. The method for detecting single-molecule mechanical force according to claim 11, characterized in that, In the shearing mode, the direction of the mechanical tension is mainly parallel to the base pair hydrogen bond direction of the double-stranded DNA probe, the unwinding force threshold is 50 pN to 60 pN, and the shearing mode corresponds to an end-to-end shearing loading geometry.

14. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The double-stranded DNA probe adjusts the unwinding force threshold by regulating the GC content, length, or number of mismatched bases in the complementary pairing regions of the first single-stranded DNA and the second single-stranded DNA.

15. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The first fluorescent label is a fluorescent donor or a fluorescent group, and the second fluorescent label is a fluorescent acceptor or a quencher; or, the first fluorescent label is a fluorescent acceptor or a quencher, and the second fluorescent label is a fluorescent donor or a fluorescent group.

16. The method for detecting single-molecule mechanical force according to claim 15, characterized in that, The fluorescent group is selected from Cy3, Cy3B, Cy5, FITC, Atto series dyes or Alexa Fluor series dyes; the quenching group is selected from black hole quencher (BHQ) series, Dabcyl or gold nanoparticles.

17. The method for detecting single-molecule mechanical force according to claim 16, characterized in that, The first fluorescent label is Cy3B, and the second fluorescent label is BHQ2.

18. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The targeting ligand is selected from peptides, antibodies, antibody fragments, aptamers, or glycoproteins; the mechanoreceptor is selected from integrins, cadherins, selectins, or T-cell receptors (TCRs).

19. The method for detecting single-molecule mechanical force according to claim 18, characterized in that, The targeting ligand is a polypeptide containing an RGD sequence; the mechanosensitive receptor is integrin β1.

20. The method for detecting single-molecule mechanical force according to claim 1, characterized in that, The single-molecule mechanosensor also includes a control double-stranded DNA probe, which is modified on the surface of the microsphere substrate and is not modified with the target ligand, or has a non-destructible structure, used to exclude non-specific signals or monitor false positive signals caused by nuclease degradation.

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